Gene expression analysis identifies the Activin/Inhibin...

129
Gene expression analysis identifies the Activin/Inhibin signaling pathway as a target of CSN5/JAB1 in colorectal epithelial cancer cells Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation Vorgelegt von Diplom Biologe Thomas Wilhelm Hennes aus Erkelenz Berichter: Universitätsprofessor Dr. rer. nat. Jürgen Bernhagen Universitätsprofessor Dr. rer. nat. Joost T. van Dongen Tag der mündlichen Prüfung: 28.09.2017 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.

Transcript of Gene expression analysis identifies the Activin/Inhibin...

Page 1: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

Gene expression analysis identifies the

Activin/Inhibin signaling pathway as a target of

CSN5/JAB1 in colorectal epithelial cancer cells

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH

Aachen University zur Erlangung des akademischen Grades eines Doktors der

Naturwissenschaften genehmigte Dissertation

Vorgelegt von

Diplom Biologe

Thomas Wilhelm Hennes

aus Erkelenz

Berichter: Universitätsprofessor Dr. rer. nat. Jürgen Bernhagen

Universitätsprofessor Dr. rer. nat. Joost T. van Dongen

Tag der mündlichen Prüfung: 28.09.2017

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.

Page 2: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and
Page 3: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

I

Acknowledgements

This thesis was conducted and written at the Institute of Biochemistry and Molecular

Cell Biology.

I want to thank Univ.-Prof. Dr. Jürgen Bernhagen for giving me the opportunity to work

on this interesting and challenging topic. His guidance, scientific advices and trust

during the whole progress of this thesis was vital for the successful completion.

I thank Univ.-Prof. Dr. Jost van Dongen for reviewing my thesis and accepting the Co-

Referat. His ideas and comments were always very helpful and contributed to a

successful completion of my thesis.

I want to thank Dr. rer. nat. Anke Schütz for the supervision of my work and reviewing

of my thesis. Furthermore, I want to thank the whole CSN5 group. Birgitt Lennartz for

all the technical support and know-how she shared with me. Sandra Jumpertz for

sharing and solving the problems of our topic, the useful discussions and the helping

hands whenever I needed them. I appreciate the time working in this group.

Also I would like to thank Dr. rer. nat. Bernd Denecke and the IZKF chip facility for

performing the GeneChip microarray and helping with some first analysis of the data.

I want to thank Dr. rer. nat. Ivan Gesteira Costa Filho from the Division of

Computational Biology for the help with the further bioinformatical analysis of the

microarray data sets.

I want to thank all my colleagues at the Bernhagen lab for their support during the last

years. Especially I would like to thank Josefine Soppert, Sandra Krämer, Setareh

Alampour Rajabi, Sebastian Borosch and all the other temporary members of our little

office community. When things did not work out as planned, they helped with technical

support, good advices and helpful discussions or when everything failed even with their

humor. They and all the other members of the lab ensured that I had a great time,

which I will enjoy looking back.

Furthermore, I want to thank my parents, brothers and sister. Without them I would not

be able to go my way. Their support during the whole time was fantastic and made this

thesis possible.

At the end I want to thank especially Sarah Lenz for encouraging and motivating me

even in hard times. Her constant support was invaluable for the successful completion

of this thesis.

Page 4: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

II

Page 5: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

III

Table of contents

Acknowledgements ................................................................................................... I

Table of contents ..................................................................................................... III

Abbreviations ........................................................................................................... VI

1) Introduction ....................................................................................................... 1

1.1) The human digestive system ......................................................................... 1

1.2) The colorectal carcinoma .............................................................................. 4

1.3) The constitutive photomorphogenesis 9 signalosome (CSN) ........................ 6

1.3.1) CSN5/ JAB1 ............................................................................................ 9

1.3.2) CSN(5) as regulator of protein degradation .......................................... 10

1.3.3) The involvement of the CSN in posttranslational modifications............. 12

1.3.4) CSN in cellular homeostasis and cancer .............................................. 12

1.4) Activins/Inhibins ........................................................................................... 15

1.4.1) Activin/Inhibin expression and secretion ............................................... 15

1.4.2) Activin/Inhibin signaling pathways ......................................................... 17

1.4.3) Activin/Inhibin in cellular homeostasis and cancer ................................ 20

1.5) Aim of the study ........................................................................................... 22

2) Material and methods ...................................................................................... 23

2.1) Material ........................................................................................................ 23

2.1.1) Chemicals and reagents ....................................................................... 23

2.1.2) Buffer and solutions .............................................................................. 26

2.1.3) Cell lines ............................................................................................... 27

2.1.4) Equipment and consumables ................................................................ 27

2.1.4.1) Equipment ...................................................................................... 27

2.1.4.2) Consumables ................................................................................. 29

2.2) Methods ....................................................................................................... 30

Page 6: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

IV

2.2.1) Cell culture ............................................................................................ 30

2.2.1.1) Cultivation of mammalian cells ....................................................... 30

2.2.1.2) Cell counting ................................................................................... 30

2.2.1.3) Freezing and thawing of mammalian cells ...................................... 31

2.2.1.4) Knockdown of protein expression in mammalian cells ................... 31

2.2.1.5) Overexpression of proteins in mammalian cells ............................. 32

2.2.2) Molecular biological methods ................................................................ 32

2.2.2.1) Ribonucleic acid (RNA)-Isolation .................................................... 32

2.2.2.2) Complementary DNA (cDNA)-synthesis ......................................... 33

2.2.2.3) Quantitative real-time polymerase chain reaction (RT-qPCR) ........ 34

2.2.2.4) RNA-Isolation and expression analysis using microarray approach 35

2.2.2.5) Protein-Isolation out of whole cell lysates and sample preparation for

gelelectrophoresis .......................................................................... 36

2.2.2.6) Protein-Isolation out of supernatants using TCA-precipitation and

sample preparation ......................................................................... 37

2.2.2.7) SDS-Polyacrylamid (SDS-PAA) gel electrophoresis and Western blot

....................................................................................................... 38

2.2.3) Cell biological assays ............................................................................ 40

2.2.3.1) Proliferation assay .......................................................................... 40

2.2.3.2) Stimulation of CaCo2 cells with supernatants and/or recombinant

protein ............................................................................................ 41

2.2.4) Bioinformatical GeneChip Analysis ....................................................... 42

3) Results ............................................................................................................. 44

3.1) siRNA-knockdown of CSN5 changes the gene expression pattern in SW480

cells ............................................................................................................. 44

3.2) CSN5 influences the expression of activin/inhibin subunits ......................... 53

3.3) Knockdown of CSN5 do not alter the protein level of inhibin A subunits ... 55

3.4) Overexpression of CSN5 does not affect the expression of activin/inhibin

subunits ....................................................................................................... 56

Page 7: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

V

3.5) The knockdown of CSN1 or CSN2 does not alter the expression of inhibin A

.................................................................................................................... 57

3.6) Knockdown of different CSN subunits influences the protein stability of the

CSN ............................................................................................................. 60

3.7) Cullin1 seems to compensate the effect of CSN5 on inhibin A but not

inhibin α mRNA expression ......................................................................... 61

3.8) The influence of CSN5 on activin/inhibin subunits seems to be mediated by

transcriptional regulation ............................................................................. 63

3.9) Inhibition of proteasomal degradation reduces inhibin A protein level ....... 65

3.10) Increased expression of inhibin A seems to increase the secretion of

activins/inhibins ........................................................................................ 66

3.11) The influence of CSN5 on CRC cell proliferation seems to be at least partly

mediated via activin/inhibin signaling ....................................................... 68

3.12) Transfer of supernatants of SW480 cells on CaCo2 cells seems to lead to

an increased Smad2 phosphorylation ...................................................... 71

3.13) Effects of CSN5 on inhibin A mRNA expression seem to depend on the

p53 status in HCT116 cells ...................................................................... 72

4) Discussion ....................................................................................................... 74

4.1) CSN5 influences the gene expression in SW480 cells ................................ 74

4.2) CSN5 and the COP9-signalosome modulate the expression of Inhibin A in

SW480 cells ................................................................................................ 79

4.3) CSN5 does not seem to alter Inhibin A protein levels................................ 84

4.4) Enhanced Activin A secretion after CSN5 knockdown can inhibit the

proliferation of colorectal cancer cells .......................................................... 86

4.5) The p53 status in the cell influences the expression of Inhibin A .............. 88

4.6) CSN5 seems to influence Inhibin α expression via Cullin1 RING E3 Ubiquitin-

Ligases ........................................................................................................ 89

5) Summary .......................................................................................................... 92

6) Zusammenfassung .......................................................................................... 95

References .............................................................................................................. 99

Page 8: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

VI

Abbreviations

53BP1 p53 binding protein 1

5/6 kinase 1, 3, 4 trisphosphate 5/6 kinase

ACTBL2 -actin-like protein 2

ACVR activin receptor

ALK activin receptor like kinase

AMH anti-Müllerian hormone

ADP adenosin diphosphate

AP-1 activating protein 1

APC adenomatous polyposis coli

APS ammonium persulfate

ARL14EPL ADP-ribosylation factor-like 14 effector protein-like

ATF activating transcription factor

BAMBI BMP and activin membrane bound inhibitor

BAX Bcl-2-associated X protein

BCL10 b-cell lymphoma/leukemia 10

Bcr-Abl break point cluster-Abelson fusion protein

BMP bone morphogenetic protein

BSA bovine serum albumin

C/EBP- CCAAT/enhancer binding protein

cAMP cyclic adenosine monophosphate

CAND1 cullin-associated NEDD8-dissociated protein

CBCCs crypt based columnar cells

CDK cyclin dependent kinases

cDNA complementary DNA

ChIP chromatin Immunoprecipitation

CIN chromosomal instability

COP1 constitutive photomorphogenecic 1

COP9 constitutive photomorphogenesis 9

CRE cAMP response element

CREBP cAMP response element-binding protein

CRL cullin E3 RING ligases

CSN constitutive photomorphogenesis 9 signalosome

CSN1-8 constitutive photomorphogenesis 9 signalosome subunits 1-8

CUL1 cullin1

CycD1 cyclin D1

DCC deleted in colorectal cancer gene

DMEM Dulbeccos’s modified Eagle’s medium

DMSO dimethyl sulfoxide

DNA desoxyribonucleic acid

dNTP deoxynucleotide triphosphate

DTT dithiothreitol

Page 9: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

VII

DUBs deubiquitinating enzymes

ECL enhanced chemiluminescence

EDTA ethylene diaminetetraacetic acid

eIF3 eucaryotic initiation factor 3

EMSA electrophilic mobility shift assay

ER endoplasmic reticulum

ERK extracellular signal-regulated kinase

FAP familial adenomatous polyposis

FBS Fetal bovine serum

FBXW7 f-box/wd repeat-containing protein 7

FGF-2 fibroblast growth factor 2

FKBP12 12 kDa Fk506 binding protein

FSH follicle-stimulating hormone

FST follistatin

GMFG glia maturation factor gamma

GDP/GTP guanosin di-/triphosphate

GS glycine- and serin-rich

HAND2 heart and neural crest derivate expressed 2

hESC human embryonic stem cell

HIF-1α hypoxia inducible transcription factor 1α

HNPCC hereditary nonpolyposis colorectal cancer

HRP horse radish peroxidase

IBD inflammatory bowel diseases

Iκ-Bα inhibitor of NF-κB α

IL-1 interleukin 1

INHA inhibin α

INHBA inhibin A

INHBB inhibin B

IP immunoprecipitation

Jab1 c-Jun activation domain binding protein

JAMM-motif jab1/MPN/Mov34-motif

JBD jun binding domain

KRAS V-ki-ras2 kirsten rat sarcoma viral oncogene homolog

KRTAP3-1 keratin associated protein 3-1

LCE1F late cornified envelope protein 1F

LDS lithium dodecyl sulfate

LFA-1 leukocyte functional antigen 1

LPS lipopolysaccharide

MAPK mitogen activated protein kinases

MDM2 mouse double minute 2 homolog

MEK mitogen-activated protein kinase kinase

MIF macrophage migration inhibitory factor

MLH1 mutL homolog 1

MPN MPR1/PAD1 amino terminal domain

Page 10: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

VIII

mRNA messenger RNA

MS mass spectrometry

MSH2 mutS homolog 2

MSI microsatellite instability

MYC v-myc avian myelocytomatosis viral oncogene homolog

NEDD8 Neural precursor cell developmentally down-regulated 8

NES nuclear export sequence

NF-κB nuclear factor-κB

OCT4 octamer-binding transcription factor 4

PBD p27 binding domain

PBS Phosphate buffered saline

PCI proteasome, COP9, initiationfactor 3

PCR polymerase chain reaction

Pen/Strep penicillin/streptomycine

PI3K phosphoinositide 3-kinase

PVDF polyvinylidene fluoride

R/I-SMADs receptor activated/Inhibitory-SMADs

RB retinoblastoma

RBX ring box protein

RING really interesting new gene

RNA ribonucleic acid

RPMI Roswell park memorial institute

RT-qPCR real-time quantitative polymerase chain reaction

RUNX runt-related transcription factor

SARA SMAD anchor for receptor activation

SBE SMAD binding element

SCF skp1-cullin-F box

scrRNA scramble RNA

SDS sodium dodecylsulfate

SDS-PAA SDS-Polyacrylamide

SIAH1 seven in absentia homolog 1

siRNA silencing ribonucleic acid

SMAD small mothers against decapentaplegic

STAT signal transducer and activator of transcription

TBS tris buffered saline

TCA trichloroacetic acid

TEMED N,N,N´,N´-tetramethylethylendiamin

TGF- transforming growth factor

TGFBR3 TGF- receptor γ

TNF-α tumor necrosis factor α

TNM tumor node metastasis

TRE 12-O-tetradecanoylphorbol-13-acetate response element

Tris tris(hydroxymethyl)aminomethane

USP15 ubiquitin-specific-processing protease 15

Page 11: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

IX

WNT wingless-related integration site

Page 12: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and
Page 13: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

1

1) Introduction

1.1) The human digestive system

The function of the digestive system is the uptake of nutrients and water. It can be

divided into two parts, the upper and the lower digestive tract. The upper part consists

of the oral cavity and the esophagus. The lower part of the digestive system consists

of the stomach, which is followed by the small intestine, the colon and the rectum.

Additionally the liver and the pancreas contribute to the lower digestive system1,2.

The main part of the digestion and the uptake of nutrients and water take place in the

small intestine. In humans it is about 3 to 5 meters long and can be divided into three

sections, the duodenum, the jejunum and the ileum. To promote the digestion,

enzymes which can degrade carbohydrates, fat and proteins/peptides are secreted by

the pancreas into the duodenum. Besides the processing of food, the uptake of

nutrients is the second important function of the small intestine.

Figure 1.1: Structure of the different layers of the intestinal tract (2402 Layers of the Gastrointestinal Tract by OpenStax College, Rice University. Licensed under CC BY 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:2402_Layers_of_the_Gastrointestinal_Tract.jpg#/media/File:2402_Layers_of_the_Gastrointestinal_Tract.jpg (05.01.2017))

The small intestine constitutes of an inner mucosa with an epithelial layer, the lamina

propria and the muscularis mucosae. The mucosa is surrounded by the submucosa,

which is surrounded by two layers of muscles. In the submucosa lie the blood vessels

for the supply of the cells and the transport of the nutrients. The first muscle layer

Page 14: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

2

surrounding the submucosa consists of circular muscle and the second layer consists

of longitudinal muscle. These muscle layers are important for the intermingling of the

pulp of food with enzymatic liquids and for transportation through the intestine. The

whole intestine is surrounded by the serosa and connects via mesenteries to the

peritoneum (see Figure 1.1). To ensure an optimal uptake, the surface of the small

intestine is enlarged. For this purpose, the submucosa builds up circular folds, so called

Kerckring folds, which project into the lumen of the small intestine. To further enlarge

the surface, the mucosa is organized in crypts (see Figure 1.2). Finally the enterocytes,

which are the most important cells for the uptake, have microvilli at their luminal side

to enlarge the surface of each cell and create the brush border1,2.

Figure 1.2: Comparison of the structure of the small and large intestine. The main difference between the small and large intestine is the structure of the surface. In the small intestine the epithelium is composed of crypts and villi. In the large intestine the villi are missing. (by Dr. Thomas Caceci, Virginia-Maryland College of Veterinary Medicine, http://www.doctorc.net/Labs/Lab19/Lab19.htm (05.01.2017))

Next the colon follows to the ileum. The colon has a length of 1.5 to 1.8 m and consists

of the cecum with the appendix vermiformis, the colon ascendens, the colon

transversum, the colon descendens, the colon sigmoideum and the rectum. The main

function of the colon is the resorption of the residual water in the food. It harbors a

multitude of different bacteria. They can digest food material (like cellulose) which

Page 15: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

3

cannot be digested by human itself. While doing so, they can provide nutrients which

can be used by humans, e.g. vitamin K.

In contrast to the small intestine, the surface of the colon is relatively smooth and the

crypts are invaginated and unbranched (see Figure 1.2). Although the composition of

the different layer is nearly the same, a main difference is the arrangement of the

longitudinal muscles in three stripes along the colon. Also, the circular muscles are

only contracted in sections which lead to the formation of the plicae semilunares.

Between these sections there are evaginations of the colon, so called Haustren1,2.

Figure 1.3: Structure of a crypt and villus in the intestine and cell types of the intestinal epithelium (A) Structure of acolonic crypt and a small intestine crypt and villus and allocation of the different cell types along them. Crypt base columnar cells (CBCC) and +4 cells are both in the discussion to be the intestinal stem cells (Modified after Medema et al., Nature review 2011). (B) Intestinal stem cell and the various differentiated cells in the intestinal epithelium (Modified after Crosnier et al., Nature review genetics 2006)

The epithelium of the small and large intestine is highly regenerative, the average time

for a complete regeneration is about 7 days. The regeneration starts at the bottom of

the crypts, where the stem cells reside. From the bottom to the top of a crypt, the

proliferative potential of cell decreases and the state of differentiation increases (see

Figure 1.3 A)3-5.

Beside the stem cells there are other different types of cells in the intestinal epithelium

(see Figure 1.3 B). These are the secretory cells like the Paneth cells, the goblet cells

or the enteroendocrine cells. The Paneth cells also reside at the bottom of the crypts.

Their main function is the protection of the epithelium by secreting anti-microbial

Page 16: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

4

peptides. The function of the goblet cells is the secretion of the mucus. The mucus is

important as a barrier against mechanical forces due to the transportation of the food

and also as a barrier against bacterial invasion. Beside this it fosters the transportation

of the food. Another main cell type is the enterocyte, which is important for the

absorption of the nutrients3,5. Besides the other cell types, immune cells are localized

in the intestinal epithelium. They are important as the digestive system is a first line of

defense against pathogens, due to the contact with the environment.

1.2) The colorectal carcinoma

Around 223000 people died in Germany due to malignant tumor growth in 2013.

Consequently, cancer is with around 25%, the second most cause of death in

Germany, after diseases of the coronary system. With around 70000 (ca. 30% of all

cancer types) causes, cancer of the digestive organs is the most prominent cancer

related cause of death. Most lethal cases in this field, are related to malignant

neoplasm of the colon or the pancreas. There were around 60,000 new incidences of

colorectal cancer in Germany in 2010. The incidence and death rate between female

and male is nearly equal. Thus colorectal cancer is the second most prominent form of

cancer in female and the third most prominent form of cancer in male. Most patients

are people at age of 50 years or older. But this could be also due to enhanced

screening for colorectal cancer starting at this age.

The cause of development of colorectal cancer is not fully understood so far. There

are external factors, as also genetic factors which contribute to the development.

Colorectal cancer can be classified into three types6. The sporadic form accounts for

60-80 % of colorectal cancers. This form develops spontaneously without any known

genetic predisposition. Therefore, environmental factors seem to be more important,

but also some genetic prerequisites are likely to play a role. Some of the environmental

factors are physical activity, diet and obesity, alcohol and smoking. Most of the factors

are influenced by the so called “western lifestyle”. Due to changes in the work profile

of most western societies there is a lack of physical activity. It could be shown, that

reduced physical activity leads to an increase risk of colorectal cancer7,8. Due to the

lower physical activity and high fat food, there is also enhanced obesity observed in

western countries. This fatty diet, especially eating more meat and less fibers, and an

eventually resulting obesity are risk factors for colorectal cancer7,9,10. Another important

Page 17: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

5

risk factor for the development of colorectal cancer is an unhealthy lifestyle consuming

nicotine or alcohol11,12.

The second form of colorectal cancer is the family type colorectal cancer. It has been

observed, that the chance to develop tumors increases two to three fold, if family

members developed spontaneous colon cancer. This fact supports that genetic

predisposition plays a role if individuals develop spontaneous colorectal cancer. But

also similar lifestyles within the family, like high fat diet, smoking and less physical

activity, could contribute to the phenomena.

The third and rarest form is the hereditary colon cancer. Here a familiar predisposition

to develop adenomatous polyps is given. In this form it can be distinguished between

familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer

(HNPCC).

Another risk factor for development of colorectal cancer is inflammation of the intestinal

tract. Here are to mention the chronic inflammatory bowel diseases (IBDs) like Crohn´s

disease or ulcerative colitis13.

In the carcinogenesis of colorectal cancer two major classes are distinguished. The

first is the chromosomal instability (CIN) to which the most sporadic tumors, as also

familial adenomatous polyposis belongs. Here, stepwise mutations in different tumor

suppressor genes and/or proto-oncogenes take place and lead to a malignant

transformation of colonic cells. This progression is also named adenoma-carcinoma

sequence14,15. Mostly this type is initiated by mutations in the adenomatous polyposis

coli (APC) gene16. This mutation is found in about 80 % of the tumors in colorectal

cancers. It is an important protein in the Wnt (wingless Ingt-1)/ -catenin signaling

pathway. Mutations mostly lead to a permanent activation, which leads to enhanced

proliferative potential in the colon. Subsequent mutations are often in the Ras

(KRAS)17, the p53 (TP53)18 or the deleted in colorectal carcinoma (DCC)19 gene. Ras

has many cellular functions mediated through effectors like the Raf-MEK (mitogen-

activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway

or phosphoinositide 3-kinase (PI3K). Therefore, it also influences the cell cycle and

proliferation20. P53 is a transcription factor which acts as a tumor suppressor. It is a

central regulator of cellular response to stress, like DNA-damage or oxidative stress.

As so called “guardian of the genome” it is involved in the regulation and transcription

of hundreds of genes, e.g. p21, 14-3-3, Bcl-2-associated X protein (BAX) or FAS21,22.

These genes for example lead to growth arrest or apoptosis. In most colorectal cancers

Page 18: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

6

p53 is mutated. This can lead to a nonfunctional protein as also to a hyperfunctional

protein. Also the loss of only some functions due to mutations is imaginable. The

changes in the functionality can lead to an escape of the cells from apoptosis and cell

cycle arrest. The mutations in the p53 gene have an important role in the transition

from adenoma to carcinoma.

Figure 1.4: The adenoma-carcinoma sequence of colorectal cancer Schematic representation of the different developmental stages of colorectal cancer and the corresponding genetic and epigenetic events. Mentioned underneath the most frequent mutated genes during the development of colorectal cancer. (Shiller et al., Clin Colon Rectal Surg 2015)

The second form of colorectal cancer is the microsatellite instability (MSI) form23. In

this form mutations in genes, which are important for the DNA mismatch repair, like

mutS homolog 2 (MSH2)24 or mutL homolog 1 (MLH1)25 occurs. Often this leads to

mutations in repetitive DNA sequences (so called microsatellites) which lead to

insertion or deletion of repetitive units. This can lead to frameshifts and inactivation of

genes and consequently to malignant transformation.

The progression of colorectal cancer can be classified after the tumor node metastasis

(TNM) - or Dukes-classification. Using the TNM-classification the cancer states are

distinguished due to the size of the tumor and the number and localization of the

metastases. The Dukes-classification distinguishes between the different depth of

tumor infiltration into the colon wall and the number of metastases.

1.3) The constitutive photomorphogenesis 9 signalosome (CSN)

The constitutive photomorphogenesis 9 (COP9) signalosome is a potential key player

in tumorigenesis. An altered expression of the CSN and its subunits has been detected

in a variety of cancer entities26. For example, CSN activation has been linked to

Page 19: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

7

progression in breast cancer27. In oral squamous carcinoma or in epithelial ovarian

tumor the overexpression of CSN5 is associated with a poor outcome for the

patients28,29.

The COP9 signalosome was first described as a regulator of photomorphogenesis in

plants30. Mutations in the CSN lead to an induction of growth without light stimulus in

Arabidopsis thaliana. Later on this complex could be also purified out of red blood cells

and spleens of pigs31,32. These were the first hints that this complex had more functions

beside the light induced growth of plants. Until now the COP9 signalosome could be

discovered in a variety of eukaryotic organisms, like fungi (e.g. Saccharomyces

cerevisiae33,34), worms (Caenorhabditis elegans35) or flies (Drosophila

melanogaster36). Therefore, it seems to be clear that the CSN plays an important role

in many eukaryotic organisms.

The COP9 signalosome is a multi-protein complex of 450-550 kDa in weight. It consists

of 8 subunits, which are named according to their size, from large to small, CSN1 to

CSN8 (constitutive photomorphogenesis 9 signalosome subunits 1-8)37. Beside this

complex, also subcomplexes, consisting only of some subunits and monomeric forms

of some subunits are described in literature38. But their physiological occurrence and

functionality are to some extent unknown and discussed controversially.

Figure 1.5: 2D electron microscopic image of the CSN and model of the subunit interactions (A) 2D electron microscopic image of purified CSN. The putative subunit arrangement is based on subunit-subunit interaction studies. The central groove is marked by a black arrow. (Modified after Bech-Otschir et al., J Cell Sci. 2002) (B) Subunit interaction model based on the mass distribution of the topview of electron microscopic images and summarized subunit-subunit interaction studies (Modified after Kapelari et al., J Mol Biol. 2000)

Page 20: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

8

First hint about the architecture of the COP9 signalosome was given by electron

microscopy and interaction studies of the subunits39 (see Figure 1.5). These first

structures revealed homology of this complex with 19S lid complex of the 26S

Proteasome and the eukaryotic initiation factor 3 (eIF3) complex40-42. The subunits can

be clustered due to their specific domain. The subunits CSN1-4, 7 and 8 exhibit a so

called PCI (proteasome, COP9, initiation factor 3) domain. This domain, which resides

at the C-Terminus of the protein, is composed of two sub domains: a winged-helix

domain and a helix bundle. Because of the winged-helix domain, which is also existent

in proteins, which can bind to nucleic acids, it is discussed that the CSN could also

bind to nucleic acids.

The other signature domain in the CSN subunits is the MPR1/PAD1 amino terminal

(MPN) domain at the N-Terminus of CSN5 and 6. This domain has a metalloprotease

fold.

Figure 1.6: Overall architecture of the CSN (A) Cartoon representation of the CSN based on the crystal structures. (Modified after Lingaraju et al., Nature 2014) (B) Schematic representation of the three dimensional structure of the CSN. (Modified after Deshaies et al., Nature 2014)

Recently a 3.8 Å resolution crystal structure could be done and published43. Therefore,

new insights into the structure and organization of the COP9 signalosome could be

achieved. The structure shows a complex which is dominated by two organizational

centers. The PCI domains of the subunits form an open ring through association of

their winged-helix domains. The helical bundles of the PCI subunits lie on top of this

ring like structure. The N-terminus of the largest PCI subunits radiate from this central

structure in arm like protrusions. The CSN5 and CSN6 heterodimer lies above the

organizational center and therefor build up a three-layered structure (see Figure 1.6)43.

Page 21: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

9

1.3.1) CSN5/ JAB1

CSN5 is the fifth subunit of the COP9 signalosome. It was first discovered as

transcriptional co-activator of c-Jun44. Because of this it was first named c-Jun

activation domain binding protein 1 (Jab1). Later on more interaction partners, e.g.

p2745, p5346, leukocyte functional antigen 1 (LFA-1)47, macrophage migration inhibitory

factor (MIF)48, small mothers against decapentaplegic 4(SMAD4)49, SMAD750 or

hypoxia inducible transcription factor 1α (HIF-1α)51, were discovered and verified. The

CSN5 consists of 334 amino acids and has a molecular mass of 38 kDa. Like the CSN

it is evolutionary conserved in plants, yeast, mice and humans52.

Figure 1.7: Structure of the CSN5 gene and domains of the protein (A) Schematic of the CSN5 gene localized on chromosome 8q13.2 in human. The gene is 33 kb long and consists of 8 exons. (B) The 8 exons within the 334 amino acid sequence of the protein. (C) Schematic of the CSN5 protein with the Jab1/MPN/Mov34 (JAMM) motif containing Mpr1-Pad1-N-terminal (MPN) domain, the nuclear export sequence (NES) close to the p27 binding domain (PBD) and the Jun binding domain (JBD). (Modified after Shackleford et al., Cell Div. 2010)

Page 22: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

10

The characteristic domain of the CSN5 is the MPN-domain (amino acids 51-230), but

in contrast to CSN6 it contains a Jab1/MPN/Mov34 (JAMM) motif. This is the zinc

dependent isopeptidase catalytic center53. The MPN domain containing the JAMM

motif is important to mediate the deneddylase activity of the CSN. But this function of

the CSN5 requires the complete assembled COP9-signalosome53,54. Beside the MPN

domain, CSN5 contains two binding domains, one for p27 (p27 binding domain (PBD))

and another one for c-Jun (Jun binding domain (JBD)). Also a nuclear export sequence

(NES) can be found in CSN5 (see Figure 1.7).

This provides evidence that CSN5 has, beside the function as catalytic active subunit

in the CSN, other functions as monomer or in subcomplexes. CSN5 can directly bind

to p27 and mediate its nuclear export and degradation45,55. Also for p53 and SMAD7

the nuclear export via CSN5 and the following degradation is described50,56,57. Therefor

CSN5 has influence on the cell cycle regulation and gene transcription. Another

possibility of CSN5 to influence the gene transcription is the stabilization of the

transcription factor activator protein 1 (AP-1)44,47,48. The AP-1 consists of members of

the Jun and Fos protein family. CSN5 can stabilize the protein-DNA complex consisting

of c-Jun or JunD (but not other members of the Jun family). Due to this it potentiates

the transcriptional activity, but also the specificity by affecting the interaction between

c-Jun/ JunD and other binding partners. CSN5 as transcriptional coactivator has also

been shown for other transcription factors like nuclear factor-kappa B (NF-κB)58, p53

binding protein 1 (53BP1)59 or heart and neural crest derivate expressed 2 (HAND2)60.

1.3.2) CSN(5) as regulator of protein degradation

The best known function of the CSN is the regulation of cullin E3 RING ligases (CRLs).

That are multi-protein complexes (around 200 complexes are known) which regulate

via ubiquitination the proteasomal degradation of proteins. They are the largest known

class of Ubiquitin E3-ligases and account for around 20 % of the protein ubiquitination.

The regulation of the activity occurs through posttranslational modification via adding

or removing of the protein neural precursor cell developmentally down-regulated 8

(NEDD8)61. The isopeptidase activity of the CSN, mediated through CSN5, is able to

remove NEDD8 from the CRLs. The deNEDDylation leads to disassembly of the CRL

complex and to an dissociation of the substrate-binding module62. Following this the

CRL can be build up again with other substrate-binding modules and therefore get new

Page 23: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

11

substrate specificity. The subsequent NEDDylation leads to an activation of the CLRs

and is required for an optimal activity63-65.

Figure 1.8: Model of the dynamic remodeling of CRLs DeNEDDylated cullins can be bound by CAND1, which prevents the NEDDylation of cullins. In this form the different substrate receptors, can dissociate respectively associate with the cullin. Due to dissociation of CAND1, the formed cullin ubiquitin ligases can be NEDDylated and subsequently activated again. The activated cullin ubiquitin ligases can now ubiquitinate their specific substrates. The COP9 signalosome can deNEDDylate cullins again and thus initiate a new cycle of CAND1 binding and substrate receptor exchange. CUL = Cullin, SR = substrate receptor, S = substrate, RBX = ring box protein, U = Ubiquitin, N = NEDD8, E2 = Ubiquitin E2-ligase, CSN = COP9 signalosome, CAND1 = cullin-associated NEDD8-dissociated protein 1 (Modified after Lydeard et al., EMBO reports 2013)

The dynamic recurrent assembly, activation, deactivation and disassembly facilitate

the cell to adapt their protein pool depending on the needs62,66 (see Figure 1.8).

Although the NEDDylation is important for the activation of the CRLs, the persistent

NEDDylation of CRLs leads to inactivation, autoubiquitination and degradation of the

complex. Therefore, the dynamic dis- and reassembly of the components is

prerequisite for the efficient function of this ubiquitination pathway67,68. Beside the

deNEDDylase activity to facilitate the disassembly, the CSN-associated deubiquitinase

ubiquitin-specific-processing protease 15 (USP15) can also contribute to the

stabilization of the CRL subunits by deubiquitination and thereby prevent the

subsequent degradation69,70. This is another mechanism how the CSN can ensure an

efficient function of the CRLs.

Page 24: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

12

1.3.3) The involvement of the CSN in posttranslational modifications

Beside this deNEDDylase activity other functions of the CSN has been described.

These functions are mostly mediated through associated proteins of the CSN. The

CSN can directly regulate other E3 ubiquitin ligases, like mouse double minute

homolog 2 (MDM2) or constitutively photomorphogenic 1 (COP1), besides the

CRLs57,71. In contrast the CSN can also mediate deubiquitination activity through

associated deubiquitinating enzymes (DUBs). With the help of this DUBs, the CSN can

prevent the excessive auto-ubiquitination of CRLs and enhance their stability69,70. But

also other proteins like inhibitor of NF-κB (Iκ-Bα) or b-cell lymphoma/leukemia (BCL10)

can be stabilized by deubiquitination directly through the CSN. Another activity of the

CSN is mediated through kinases. In literature the phosphorylation of different proteins,

by CSN preparations, has been described in vitro. For example, p53 gets

phosphorylated via the CSN, this is followed by the proteasomal degradation. A

described associated kinase of the CSN is the 1, 3, 4-trisphosphate 5/6 kinase

(5/6 kinase). This kinase phosphorylates inositol 1, 3, 4-trisphosphate to form 1, 3, 4,

5 or 6 phosphate. It interacts directly with CSN1 and it has been shown that this kinase

also can phosphorylate other substrates, like p53 or Iκ-Bα. These activities indicate

that the CSN seem to have many different functions in cell, which are mediated through

the CSN itself or associated proteins.

1.3.4) CSN in cellular homeostasis and cancer

The CSN can modulate different biological processes. It has been shown that the CSN

has an influence on the cell cycle regulation as a negative regulator in the G1 to S

phase progression, via enhancing the degradation of p27. The importance of the CSN

for the embryonic development could be shown in mice. The knockout of the subunits

2, 3, 5 or 8 is lethal already in the early embryonic development72-75. Other biological

responses which are influenced by the CSN are, for example T-cell homeostasis75,

signal transduction or autophagy.

Nowadays the relevance and importance of some subunits of the COP9-signalosome

in cancer are emerging. However, the mechanism, how the CSN influences the

carcinogenesis, remain unclear. The expression patterns of the different CSN subunits

are very diverse through the different cancer types. For instance, CSN5

overexpression has been detected in many tumor entities like hepatocellular

Page 25: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

13

carcinoma or colorectal carcinoma26,76. CSN4 overexpression was found in prostate

cancer and CSN6 overexpression was detected in breast tumors77,78.

Because of the influence, of the CSN, on many cellular functions, like cell cycle control,

signal transduction or gene expression it is likely that the CSN or some subunits play

a crucial role in carcinogenesis. Also many oncogenes and tumor suppressors like p27,

p53 or runt-related transcription factor (RUNX) 3 are direct or indirect influenced by the

CSN.

Figure 1.9: Proteins and their associated cellular processes affected by the CSN in gastrointestinal cancer Proteins arranged according to their related processes, divided into six categories which are reflecting the hallmarks of cancer to some extent. Proteins located at the border of two categories were found to play a role in both cellular processes. Proteins which play a role in multiple cellular processes are depicted twice. CycD1 = Cyclin D1; TopoIIα = Topoisomerase IIα; COX-2 = Cyclooxygenase 2; NF-κB = Nuclear factor κB; MYC = v-myc avian myelocytomatosis viral oncogene homolog; SIAH1 = Seven in absentia homolog-1; SMAD2/3/4 = Small mothers against decapentaplegic 2/3/4; RUNX3 = Runt-related transcription factor 3 (Modified after Jumpertz et al., J Carcinog Mutagen 2015)

An obvious link between CSN and carcinogenesis is the regulation of the ubiquitin

dependent degradation of tumor suppressors like p53. The protein p53 is activated

upon DNA damage and induces apoptosis. As described before the CSN promotes the

export of p53 out of the nucleus, the subsequent phosphorylation and degradation56.

Another example for one of the various influences of the CSN in carcinogenesis is the

involvement in cell cycle progression. The cyclin dependent kinase (CDK)-inhibitor p27

gets exported out of the nucleus by CSN5 and subsequently degraded, which

influences the G1 progression45,55. In many tumor entities an overexpression of CSN5

Page 26: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

14

could be detected. High levels of CSN5 and resulting low levels of p27 correlate in

many cancer entities with a bad prognosis for the patient.

Figure 1.10: Overexpression of CSN5 in different tumor entities Transcriptome analysis of CSN5 expression in human cancer patients. Data sets were obtained from Oncomine and Gene Expression Omnibus. Data were analyzed using Oncomine analysis tools and Nexus Expression 2.0. Only patients with more than 40% increase in CSN5 mRNA levels compared with normal tissues were scored as “CSN5 overexpression” or “CSN6 overexpression,” respectively. N represented the total number of patients analyzed in each type of cancer. (Modified after Lee et al., Cell Cycle, 2011)

Despite CSN5 overexpression, the CSN5 gene is in most cases, apart from some gene

amplifications, not mutated in cancer tissues76,79. This suggests that mutations in

upstream regulators of CSN5 seem to mediate the overexpression. The Ras pathway

could be one possibility for an upstream regulator. The reduction of CSN subunits in

tumor cells containing Ras mutations could inhibit the growth80,81. In chronic myeloid

leukemia cells CSN is located downstream of break point cluster/Abelson fusion

protein (Bcr-Abl). Bcr-Abl mediates via mitogen-activated protein kinases (MAPK) and

phosphoinositide 3-kinase the enrichment of CSN5 and a resulting decrease in p2782.

In colorectal cancer there are first hints that CSN5 might contribute to the “hallmarks”

of colorectal cancer development. It could be shown that nuclear CSN5 directly

enhances the transcriptional activity of unphosphorylated signal transducer and

activator of transcription 3 (STAT3) dimers83. This indicates that overexpressed CSN5

can directly influence the carcinogenesis by gene regulation. The development of

colorectal cancer is often associated with mutations in the Wnt signaling pathway6,84.

Under normal conditions the Wnt pathway is tightly regulated by degradation of -

catenin through the 26S proteasome. In most colorectal cancer cells the protein APC

Page 27: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

15

is mutated, which is part of the destruction complex of -catenin. This results in a loss

of degradation of -catenin and a constitutive active signaling. The result is enhanced

cell proliferation among other effects. It could be shown that the CSN affects the level

of -catenin in colorectal cancer cells85. This could be due to regulation of the

degradation of -catenin via CRLs or by influencing the stability of APC by CRLs or

associated DUBs86. The recently discovered impact of CSN5 on alternative

degradation of -catenin via seven in absentia homolog 1 (SIAH1) could be another

possibility how the CSN influences the tumorigenesis in colorectal cancer87.

1.4) Activins/Inhibins

The activins and inhibins are proteins of the transforming growth factor- (TGF- )

superfamily. Other members of this family are TGF- s, nodal, bone morphogenetic

proteins (BMPs) or anti-Müllerian hormone (AMH). Today more than 40 members of

this superfamily are described. The first reports about activins and inhibins were from

the 1930s. In this report a nonsteriodal substance in testis was described, which could

regulate the pituitary function in rats88. Later at the end of the 1980s the activin/inhibin

family was described to regulate the release of the follicle-stimulating hormone (FSH)

out of the anterior pituitary89-92. Until today activins and inhibins could be detected in

various other tissues including testis93, ovary94, adrenal gland95, pancreas96, liver97 or

the bone marrow98. Accompanied with these findings also the possible functions of

activins and inhibins have been expanded.

1.4.1) Activin/Inhibin expression and secretion

Activin/Inhibin are dimeric signaling molecules, like other ligands of the TGF-

superfamily. They are composed of the α-subunit and/or the -subunits. There is only

one α-subunit, but four -subunits ( A, B, C and E) described in humans so far. If

the molecule consists of two -subunits, it is called activin. For example, the ligand

consisting of two A-subunits is called activin A, the homodimer consisting of two B-

subunits is called activin B or the heterodimer consisting of a A- and a B-subunit is

called activin AB (see Figure 1.11). The C- and E-subunits were recently identified

in the liver, but seem not to be essential for the liver99,100. The C-subunit seems to

oppose the functions of the “classical” activins by dimerization with a A-subunit or

with itself. Therefor the A-subunit is no longer available for assembly of the other

activins and or the activin C could block the receptors and inhibit the signal

transduction101. But the roles of the C- and E-subunits have to be elucidated further.

Page 28: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

16

The inhibins are composed of an α-subunit and a -subunit. If A-subunit is part of the

heterodimer it is called inhibin A and inhibin B if the B-subunit is part of the molecule

(see Figure 1.11)

Figure 1.11: Composition of the different inhibins and activins Inhibins are composed of one inhibin chain and the common inhibin α chain. The activins are homo- or heterodimers consisting of inhibin chains

The subunits are synthesized as large precursors, which get assembled into dimeric

molecules via disulfide-linkage. Thereby the typical cysteine-knot, consisting of three

conserved disulfide bonds, of the TGF- superfamily forms102. The maturation occurs

through enzymatic or acid hydrolysis of the propeptide103-105. Hydrophobic residues in

the pro domain of the α-subunit and in the pro- and mature domain in the -subunit are

essential for the dimerization106. The assembly and secretion of activins or inhibins

seems to be regulated by the glycosylation of the α-subunit precursors, predominantly.

Enhanced glycosylation of the precursors seems to favor the assembly and secretion

of inhibins, while less or no glycosylation leads to activin secretion107. The expression

of activins and inhibins is stimulated via several pro-inflammatory and

immunoregulatory pathways. Therefore, activin synthesis and secretion can be

stimulated via interleukin-1 (IL-1), lipopolysaccharide (LPS), tumor necrosis factor α

(TNF-α), but also via itself108-110. At the level of the promoter so far only AP-1 elements

has been described. There seem to be cell specific differences in storage and release

of activins which could to some extent explain why there is a difference in mRNA- and

protein-level in some cell types.

Page 29: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

17

1.4.2) Activin/Inhibin signaling pathways

Like other members of the TGF- superfamily activins signal through an oligomeric

complex. It consists of two different receptor types, type I and type II receptors, with

serin/ threonine kinase activity and the ligand111. The type II receptor is the main player

in binding the activin, because it could bind activin without a type I receptor, but not the

other way round112. There are two type II receptors for activin, activin receptor (ACVR)

IIA (also known as II) and IIB. Also two type I receptors are known for activin signaling,

the activin receptor-like kinase (ALK) 4 and 7 (also called ACVR1B or ACVR1C)112-116.

Thus differences in the actions of the activins could be mediate by the distribution of

the individual receptors on different cells and tissues117. Additionally, for the modulation

of the signaling is the affinity of the different ligands for the receptors. For example,

activin A can only form a complex with ALK4, while activin B can form complexes with

both type I receptors. This may lead to a broader range of actions, by activin B, due to

the use of both type I receptors. But the binding of activin B is much weaker to the

receptor complex, compared to the binding of activin A. This could indicate that in the

presence of activin A, the activin B actions get lost. Activin, similar to other members

of the TGF- superfamily, forms a butterfly-shaped molecule. The monomeric subunits

comprise one α-helix and nine -sheets which form a curled “hand” like structure118,119.

Due to co-crystallization of activin A with its type II receptor it could be revealed that

the “knuckle” region is involved in the binding of the ligand to the receptor118. It is

proposed that different regions are involved in the interaction of activin with the type I

and type II receptor, based on the structure of BMP7 and ALK3118,120,121. The contact

between activin and the type I receptor are mediated through residues in the “wrist”

region of activin.

The activins mediate the assembly of the signaling complex by binding to the type II

receptor which then recruits the type I receptor and forms a tight complex. The type II

receptor is a constitutively active kinase, which phosphorylates after oligomerization

the type I receptor114,122,123. The phosphorylation takes place in a glycine- and serin-

rich (GS) segment in the regulatory region of the type I receptor. This GS segment lies

immediately N-terminal of the kinase domain and is conserved through the type I

receptors of the TGF- superfamily. It keeps the receptor in an inactive state. The

phosphorylation in the GS segment leads to a release of the inactive conformation,

which leads to an activation of the type I receptor and further activation of downstream

mediators.

Page 30: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

18

Figure 1.12: Activin production, signaling and sites of regulation Activins are synthesized as propeptides, which dimerize and gets processed to their mature form. The binding of activins to the activin receptor type and the subsequent oligomerization with the activin receptor type I lead to an active signaling complex, which initiates the intracellular signaling via SMADs or the MAP-Kinase pathway. This activates different cellular processes. The receptor activation can be modulated by various mechanisms. activins can bind to other proteins like follistatin, which inhibits the binding to the receptor. Also the binding of inhibitors like inhibin A to the receptor can inhibit the activation due to a blocked receptor. Membrane bound co-receptors can interfere with the oligomerization and activation of the receptor complex. INHBA = inhibin A, TGFBR3 = TGF- receptor 3, BAMBI = BMP and activin membrane bound inhibitor, SMAD = small mothers against decapentaplegic, ACTR = Activin receptor, MAP kinase = mitogen-activated protein kinase (Modified after Hedger et al., Cytokine & Growth Factor Reviews 2013)

The regulation of the receptor activation can be performed by various mechanisms.

There are on the one hand accessory proteins which can inhibit the binding of activin

to the receptor. On the other hand, the antagonist inhibin consisting of an α- and an -

subunit as described previously, can inhibit the binding of activin to the receptor. Inhibin

can bind to betaglycan (also known as TGF- receptor III (TGFBR3)). The binding

facilitates the formation of a complex between inhibin, betaglycan and activin receptor

type II. Therefore, sequestering the type II receptor and inhibit the formation of active

signaling complex124. Another membrane bound protein, which can inhibit activin

signaling, is BMP and activin membrane bound inhibitor (BAMBI). This transmembrane

protein has an extracellular domain similar to that of type I receptors but only a short

Page 31: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

19

cytoplasmatic tail. It can form a complex with the type I receptors, which abrogates the

signaling125. Another inhibitor of activin signaling is the activin-binding protein follistatin

(FST). Through alternative splicing two major isoforms of 288 amino acids (FST288)

or 315 amino acids (FST315) are produced126. They contain an activin-binding site,

which possesses a similar affinity to activin as the activin receptors. The FST288 also

possesses a heparin-binding site which allows the Follistatin to bind to heparan-

sulphate proteoglycans on the cell surface. The extension of the FST315 inhibits its

binding to Heparin. This isoform circulates, but after the binding to activin the Heparin-

binding site becomes exposed and it can also bind to the cell surface. Therefor

follistatin prevents the binding of activin to the receptors by capture the activin and lead

them, via binding to the cell surface, to lysosomal degradation127-129. The nucleophilin

FKBP12 (12 kDa fk506 binding protein) is another accessory protein which plays a role

in the regulation of the activin signaling. It can bind to the inactive type 1 receptor and

therefore inhibit the binding of SMADs to the receptor. This may constrain the basal

activity of the SMAD pathway by inhibiting ligand-independent activation (see Figure

1.12).

The canonical signaling pathway of the TGF- superfamily is the SMAD pathway. It

consists of receptor activated SMADs (R-SMADs), the common SMAD4 and inhibitory

SMADs (I-SMADs). There are five regulatory SMADs 1, 2, 3, 5 and 8. They can be

divided into two groups. The first group consists of the SMADs 2 and 3. They get

activated by TGF- and activin (see Figure 1.12). The second group with the SMADs

1, 5 and 8 gets activated by BMPs. The activated type I receptor recruits the R-SMADs

and phosphorylates them at the c-terminal SXS-motif. Which R-SMADs are recruited

depends on the combination of type II and type I receptor and the stimulus. This

recruitment may be facilitated by auxiliary proteins. One example is the immobilization

of SMAD2 and 3 by SMAD anchor for receptor activation (SARA), near the plasma

membrane and early endosomes130. The receptor mediated phosphorylation of SARA

bound SMADs occurs at the plasma membrane. More efficiently is the phosphorylation

in the early endosomes to which the activated receptor complex is internalized via

clathrin coated pits131,132. The activated R-SMADs, in case of activin, SMAD2 or 3 can

now form complexes. These complexes consist of a homo- or sometimes heterodimer

out of SMAD2 or 3 and one SMAD4 protein. The complex translocates in the nucleus

and activates under assistance of other co-factors the genexpression of target genes.

Page 32: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

20

All R-SMADs, except SMAD2, and the Co-SMAD4 can bind to DNA by a -Hairpin

structure133,134. The minimal SMAD binding element (SBE) only contains 4 base pairs

5´-AGAC-3´, which leads to a very low specificity135,136. Stimulation with members of

the TGF- superfamily leads to activation and repression of several hundred genes,

depending on the cell type and the state of the cell. This indicates that further

recruitment of coactivators or corepressors are needed to orchestrate the appropriate

answer to the stimulation. Members from many different families of DNA binding

proteins (e.g. forkhead, homeobox, Jun/Fos, Runx, cAMP response element binding

protein (CREBP)) have been shown to cooperate with SMADs137-139. Depending on the

composition of the transcriptional complex SMADs can exhibit activating or repressing

activity.

1.4.3) Activin/Inhibin in cellular homeostasis and cancer

Activin and inhibin signaling play a role in variety of different biological processes and

cellular events. In the human development activin A maintains pluripotency and

self-renewal of human embryonic stem cells (hESC)140-143. Together with nodal, activin

A mediates the expression of the homeobox protein nanog, which is involved in the

expression of genes, necessary to maintain pluripotency144. Beside nanog also other

transcription factors (e.g. octamer-binding transcription factor 4 (OCT4), WNT3

(wingless Int-1 3), fibroblast growth factor-2 (FGF-2)) are induced to maintain

pluripotency143. Maintaining pluripotency can achieve a proper development of the

tissue but also initiate and sustain tumorigenesis. In cooperation with other factors,

activin A can also induce differentiation. For example, prolonged treatment of hESCs

with activin A, BMP4 and Wnt induces the differentiation to definitive endoderm145-147.

Despite the action of activin in stem cells it plays a role in differentiated cells. For

instance, activin A can induce growth suppression in different tissues, e.g. breast148,

vascular endothelial149 or hepatocytes150. The growth arrest is probably induced by

induction of the expression of p21CIP1/WAF1 and suppression of cyclin D2151,152. This

leads to the inhibition Cdk4 and subsequent accumulation of hypophosphorylated

retinoblastoma (RB) protein. Hypophosphorylated RB exhibits a higher binding affinity

to the transcription factor E2F. E2F controls the expression of genes required for cell

cycle progression. The binding to RB sequester its activity and therefor inhibit the cell

cycle progression153.

Page 33: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

21

Activin can also induce apoptosis in liver cells150. It seems that the apoptosis is

mediated by caspases and is promoted through the SMAD pathway. In B-cell

hybridoma cells activin induces Bcl-Xs, which inhibits Bcl-2 and Bcl-X154,155. These

both inhibit caspase activity and prevent apoptosis.

Activins can exert either pro- or anti-tumorigenic effects in different types of cancer.

The growth of cancer is determined by the relationship between the rate of cell

proliferation and cell death. Both can be influenced by activins. It has been shown that

activin A primarily mediates protective effects. For instance the treatment of patient

derived prostate cancer cells and the non-invasive prostate cancer cell line LNCaP

with activin A leads to a cell cycle arrest156. Also the breast cancer cell line T47D shows

increased cell cycle arrest and apoptosis after treatment with activin A157. This is most

likely mediated through the influence on RB protein via p21 and cyclin D2, since cell

cycle regulation largely depends on the RB pathway. However, some tumor cells lose

their ability to respond to growth inhibitory effects of activin A. This occurs through

mutations in genes involved in the activin/nodal/TGF signaling pathways, like ACVRI;

TGFBRI/II, SMAD2 and SMAD4158,159. Despite lower response to activin signals,

activin can also act pro-tumorigenic. Activin A is associated with invasive phenotype in

certain cancers. In oral squamous cell, breast or prostate cancer circulating activin A

level are associated with metastases and a poor prognosis160-162. The underlying

mechanisms are not clear so far. Perhaps activin influence the microenvironment of

the tumor, which then promotes the tumor growth and metastasis. A hint for this theory

is that activin contributes to the switch from T-cells to T-regulatory (Treg)-cells. The

Treg-cells can down regulate the actions of T-cells and limits their ability to recognize

and potentially destroy cancer cells163.

Activin and the activin receptors are present in the intestine. In the healthy intestine all

four activin receptors can be detected. The inhibin A mRNA could only be detected

in inflammatory tissue or intestinal cancer specimen164-168. The levels correlated with

the degree of inflammation. This could also be confirmed for activin A on protein

level165-167. It could be shown that enhanced activin A level inhibits the proliferation of

intestinal cell lines but enhances their migratory capacity165,169. This hints for the

ambiguous role of activin A in the intestine. The inhibited proliferation protects against

carcinogenic transformation but also attenuates the wound healing. The abrogation of

the activin A effects, due to mutations in the signaling pathway could promote

carcinogenesis in the intestine.

Page 34: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

22

1.5) Aim of the study

Colorectal cancer is one of the most leading causes of death especially in the western

world. To develop new therapeutical options and treatments it is necessary to get a

better understanding of the development and progression of colorectal cancer.

Therefor insights into the molecular mechanism in the intestine and their change during

pathogenesis are inevitable.

CSN5 plays a crucial role during many physiological processes like protein

degradation, cell cycle control, apoptosis or signal transduction. Also it has been shown

that the expression pattern of CSN5 in tumors is changed which links CSN5 to cellular

changes during tumor development. In this study the role of CSN5 in colorectal

carcinoma should be further elucidated.

To get an idea which influence aberrant CSN5 expression in colorectal cancer could

have, a reduction in protein expression in a colorectal cancer cell line was performed.

To have an unbiased approach, a gene expression profile was obtained via gene chip

array, to monitor changes in the gene expression due to CSN5 knockdown.

As an interesting link for a possible influence of CSN5 on the pathogenesis of colorectal

cancer the activin/inhibin signaling was identified. This is due to observations in cancer

patients, which show changes in the expression of proteins belonging to this signaling

pathway.

Thus the influence of CSN5, on the expression of proteins involved in the activin/inhibin

signaling pathway, was further validated. Also was examined if physiological effects of

aberrant CSN5 expression could be possibly mediated through activin/inhibin

signaling.

Page 35: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

23

2) Material and methods

2.1) Material

2.1.1) Chemicals and reagents

Chemicals and reagents Manufacturer

30 % Acrylamide/ Bis-solution 29:1 BioRad Laboratories GmbH, Munich

Activin A Peprotech, Hamburg

Bovine serum albumin fraction V (BSA) Carl Roth GmbH + Co. KG, Karlsruhe

Ammonium persulfate (APS) Sigma-Aldrich Chemie GmbH,

Taufkirchen

-Mercaptoethanol Sigma-Aldrich Chemie GmbH,

Taufkirchen

Chloroform Sigma-Aldrich Chemie GmbH,

Taufkirchen

DC Protein Assay BioRad Laboratories GmbH, Munich

Di-Sodium Hydrogen Phosphate

(Na2HPO4) Merck Chemicals GmbH, Darmstadt

Dithiothreitol (DTT) Sigma-Aldrich Chemie GmbH,

Taufkirchen

DMEM + GlutaMAX I cell culture

medium

Thermo Fischer Scientific Inc.,

Darmstadt

Dimethyl sulfoxide (DMSO) Sigma-Aldrich Chemie GmbH,

Taufkirchen

Ethanol absolute VWR International GmbH, Langenfeld

Fetal bovine serum (FBS) Thermo Fischer Scientific Inc.,

Darmstadt

First strand cDNA synthesis kit Thermo Fischer Scientific Inc.,

Darmstadt

Follistatin Peprotech, Hamburg

Glycoblue Thermo Fischer Scientific Inc.,

Darmstadt

Page 36: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

24

Chemicals and reagents Manufacturer

IGEPAL CA-630 Sigma-Aldrich Chemie GmbH,

Taufkirchen

Isopropanol (2-Propanol) Carl Roth GmbH + Co. KG, Karlsruhe

McCoy´s 5a + GlutaMAX I cell culture

medium

Thermo Fischer Scientific Inc.,

Darmstadt

Methanol VWR International GmbH, Langenfeld

MG-132 Merck Chemicals GmbH, Darmstadt

MLN-4924 Active Biochem, Bonn

Novex® Sharp Pre-stained protein

standard

Thermo Fischer Scientific Inc.,

Darmstadt

NuPAGE LDS sample buffer (4 x) Thermo Fischer Scientific Inc.,

Darmstadt

NuPAGE transfer buffer (20 x) Thermo Fischer Scientific Inc.,

Darmstadt

Oligofectamine reagent Thermo Fischer Scientific Inc.,

Darmstadt

OptiMEM I serum reduced medium Thermo Fischer Scientific Inc.,

Darmstadt

Phosphate-buffered saline (PBS) Sigma-Aldrich Chemie GmbH,

Taufkirchen

Penicillin/ Streptomycin-solution Thermo Fischer Scientific Inc.,

Darmstadt

PhosphoSTOP EASYPack Roche Pharma AG, Grenzach-Wyhlen

PolyFect Qiagen GmbH, Hilden

Ponceau S-solution Sigma-Aldrich Chemie GmbH,

Taufkirchen

Potassium Chloride (KCl) Merck Chemicals GmbH, Darmstadt

Potassium Dihydrogen Phosphate

(KH2PO4) Merck Chemicals GmbH, Darmstadt

Protease inhibitor cocktail set I Merck Chemicals GmbH, Darmstadt

RPMI 1640 + GlutaMAX I cell culture

medium

Thermo Fischer Scientific Inc.,

Darmstadt

Page 37: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

25

Chemicals and reagents Manufacturer

Sensi Mix SYBR No-ROX Kit Bioline GmbH, Luckenwalde

SuperSignal® West Dura Extended

Duration Substrate

Thermo Fischer Scientific Inc.,

Darmstadt

SuperSignal® West Femto Maximum

Sensitivity Substrate

Thermo Fischer Scientific Inc.,

Darmstadt

Sodium Chloride (NaCl) Carl Roth GmbH + Co. KG, Karlsruhe

Sodium Deoxycholate Sigma-Aldrich Chemie GmbH,

Taufkirchen

Sodium Dodecylsulfate (SDS) Serva Electrophoresis GmbH,

Heidelberg

N,N,N´,N´-Tetramethylethylendiamin

(TEMED) BioRad Laboratories GmbH, Munich

Transforming growth factor 1(TGF- 1) Peprotech, Hamburg

Trichloroacetic acid (TCA) Sigma-Aldrich Chemie GmbH,

Taufkirchen

Tris(hydroxymethyl)-aminomethan

(TRIS) Carl Roth GmbH + Co. KG, Karlsruhe

TRIS-Hydrogen chloride (TRIS-HCl) Carl Roth GmbH + Co. KG, Karlsruhe

TRIzol Thermo Fischer Scientific Inc.,

Darmstadt

Trypan blue solution Sigma-Aldrich Chemie GmbH,

Taufkirchen

TrypLE Express (Stable Trypsin

replacement enzyme)

Thermo Fischer Scientific Inc.,

Darmstadt

Tween 20 Sigma-Aldrich Chemie GmbH,

Taufkirchen

Page 38: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

26

2.1.2) Buffer and solutions

Buffer Composition

PBS, pH 7,2 137 mM NaCl

2.7 mM KCl

1.5 mM KH2PO4

8.1 mM Na2HPO4

in ddH2O

TBS, pH 7,2 20 mM Tris-HCl

150 mM NaCl

in ddH2O

TBS-T 0.05% (v/v) Tween 20

in TBS

RIPA buffer 10 mM Tris pH 7,4

150 mM NaCl

1% (v/v) IGEPAL CA-630

1% (v/v) Sodium Deoxycholate

0.1% (w/v) SDS

in ddH2O

Blotting buffer 10% (v/v) Methanol

5% (v/v) NuPAGE transfer buffer (20 x)

in ddH2O

Stripping buffer 50 mM Tris (pH 6,8)

100 mM -Mercaptoethanol

2% (w/v) SDS

in ddH2O

Page 39: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

27

2.1.3) Cell lines

Description Origin/Phenotype

CaCo2 Human colorectal carcinoma epithelial cells

HCT116 p53KO Human colorectal carcinoma epithelial cells, exhibit no p53

HCT116 p53wt Human colorectal carcinoma epithelial cells, exhibit a wild type

p53

SW480 Human colorectal carcinoma epithelial cells

2.1.4) Equipment and consumables

2.1.4.1) Equipment

Equipment/ software Manufacturer

-152°C Ultra low MDF-1155 cryogenic

freezer

Sanyo Electric Biomedical Co., Osaka

(Japan)

-80°C Ultra low freezer Sanyo Electric Biomedical Co., Osaka

(Japan)

-20°C freezer Economic-super Robert Bosch GmbH,

Gerlingen-Schillerhöhe

-20°C freezer Premium Liebherr-International Deutschland

GmbH, Biberach an der Riß

4°C refrigerator Liebherr-International Deutschland

GmbH, Biberach an der Riß

Adobe Photoshop CS4 software Adobe Systems GmbH, Munich

AF 100 flake ice machine Scotsman Ice Systems, Milan (Italy)

AIDA Image Analyser software Raytest Isotopenmessgeraete GmbH,

Straubenhardt

Analytical scale Adventurer Ohaus Corporation, New Jersey (USA)

Analytical scale Analytical plus Ohaus Corporation, New Jersey (USA)

Automated cell counter TC20 BioRad Laboratories GmbH, Munich

BioRuptor Diagenode s.a., Seraing (Belgium)

Centrifuge 5417R Eppendorf AG, Hamburg

Centrifuge model J2-21 Beckmann Coulter GmbH, Krefeld

Page 40: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

28

Equipment/ software Manufacturer

Centrifuge Z383K Hermle Labortechnik GmbH,

Wehingen

Centrifuge Z400K Hermle Labortechnik GmbH,

Wehingen

CO2-Incubator MCO-19AK(UV) Sanyo Electric Biomedical Co., Osaka

(Japan)

Electrophoresis power supply EPS 601 GE Healthcare GmbH, Solingen

Endnote X4 citation software Thomson Reuters, New York (USA)

Gel electrophoresis apparatus Horizon 58 Biometra GmbH, Göttingen

Glass flasks Schott AG, Mainz

GraphPad Prism 5 GraphPad Software Inc., La Jolla

(USA)

High temperature dry block heater UBD Grant Instruments, Cambridge (UK)

LAS-3000 Image Reader Fujifilm Europe GmbH, Düsseldorf

Magnetic stirrer MSH basic yellowline IKA-Werke GmbH & Co. KG, Staufen

Micro centrifuge Carl Roth GmbH + Co. KG, Karlsruhe

Microscope Olympus CK40 Olympus Deutschland GmbH,

Hamburg

Neubauer cell chamber Paul Marienfeld GmbH & Co. KG,

Lauda-Königshofen

PCR-Thermocycler Primus 96 MWG AG Biotech, Ebersberg

PHM82 Standard pH-meter Radiometer Analytical SAS,

Villeurbanne Cedex (France)

Pipettes Gilson Inc., Middleton (USA)

Pipetboy acu Integra Biosciences Deutschland

GmbH, Biebertal

Rotor-Gene 6000 Qiagen GmbH, Hilden

Rotor-Gene Series Software 1.7 Qiagen GmbH, Hilden

Shaking device Rocking Platform Biometra GmbH, Göttingen

Shaking device Vibrax VXR basic IKA-Werke GmbH & Co. KG, Staufen

SDS-PAGE NuPAGE® Xcell SureLock

Mini Cell

Thermo Fischer Scientific Inc.,

Darmstadt

Page 41: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

29

Equipment/ software Manufacturer

Sonifier Cell Disruptor B15 Branson Ultrasonics, Danburry (USA)

Spectrophotometer NanoDrop ND-1000 VWR International GmbH, Langenfeld

Tissue culture hood Biowizard KOJAIR Tech Oy, Vilppula (Finland)

VIKTOR2 1420 Multilabel Counter PerkinElmer Inc, Waltham (USA)

Vortex MS2 Minishaker IKA-Werke GmbH & Co. KG, Staufen

Water bath Type 1004 GFL Gesellschaft für Labortechnik

GmbH, Burgwedel

Western-Blot NuPAGE® Xcell II Blot

Modul

Thermo Fischer Scientific Inc.,

Darmstadt

2.1.4.2) Consumables

Consumables Manufacturer

Cell culture flasks (250 ml/50 ml,

75 cm2/25 cm2) Greiner Bio-One GmbH, Frickenhausen

Centrifuge tubes (15 ml, 50 ml) BD Biosciences, Heidelberg

Cell counting slides BioRad Laboratories GmbH, Munich

Cryogenic vials Sigma-Aldrich Chemie GmbH,

Taufkirchen

Multiwell cell culture plates Cellstar

(6 well, 12 well, 24 well, 96 well)

Greiner Bio-One GmbH, Frickenhausen

Nitrocellulose transfer membrane Greiner Bio-One GmbH, Frickenhausen

NuPAGE® gel combs Thermo Fischer Scientific Inc.,

Darmstadt

NuPAGE® gel cassettes

(1.0 mm/1.5 mm)

Thermo Fischer Scientific Inc.,

Darmstadt

Reaction tubes (0.5 ml, 1.5 ml, 2.0 ml) Eppendorf AG, Hamburg

Parafilm „M“ Laboratory Film Bemis Co., Oshkosh (USA)

Pipette tips blue/ yellow (1000 µl/ 200 µl) Brand GmbH + Co. KG, Wertheim

Pipette tips white (10 µl) Sarstedt AG & Co., Nümbrecht

Whatman filter paper Greiner Bio-One GmbH, Frickenhausen

Page 42: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

30

2.2) Methods

2.2.1) Cell culture

2.2.1.1) Cultivation of mammalian cells

The cells were cultivated in T-75 cell culture flasks using the appropriate medium (see

Table 2.1) and stored in a CO2 incubator at 37°C and a humidified 5% CO2

atmosphere. When the cells reached 90% confluency, they were split for further

cultivation. For splitting, the supernatant was removed, and the cells were washed with

3.5 ml PBS. Afterwards, the cells were incubated with 3.5 ml TrypLE Express solution

until the cells detached from the cell culture flask. To stop the possible digestion

through TrypLE Express solution, 6.5 ml complete medium was added. The cell-

solution was transferred into a centrifuge tube and then the cells were spun down at

300 x g for 5 min at room temperature. The supernatant was removed and the cell

pellet was resuspended in 10 ml complete medium. After this the cells were counted

and 2 million cells were seeded in a new flasks containing 15 ml complete medium.

Table 2.1: Full media used in cell culture

Cells Basal Medium Supplements

SW480 RPMI Medium 1640

+ Glutamax® I

10% (v/v) FCS

1% (v/v) Pen/Strep

CaCo2 DMEM + Glutamax® I 10% (v/v) FCS

1% (v/v) Pen/Strep

HCT116 p53wt

HCT116 p53KO

McCoy´s 5a

+ Glutamax® I

10% (v/v) FCS

1% (v/v) Pen/Strep

2.2.1.2) Cell counting

Cells were counted using a Neubauer chamber. Therefor the cells were diluted 1:4 in

Trypan blue. Then the cell/ Trypan blue solution was pipetted into the Neubauer

chamber and the cells were counted under a bright field microscope. The concentration

of the cell suspension was calculated using the formula: cells/ml = n x 104 (n=number

of counted cells).

Page 43: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

31

2.2.1.3) Freezing and thawing of mammalian cells

For long term storage of cells, they were grown to 100% confluence. Then cells were

harvested and counted. They were resuspended at a concentration of 106/ ml in

complete Medium containing 5% (v/v) DMSO. After this 1 ml of the cell suspension

was stored in cryovials. The cryovials were placed in a cryocontainer (containing

Isopropanol) and stored over night at -80°C to ensure a slow cooling. On the next day

the cells were transferred into cryoboxes and for long term storage placed at -152°C.

To thaw cells, the cryovials were shortly placed at 37°C in a water bath. Thereafter the

cells were resuspended in 9 ml complete medium. The cell suspension was centrifuged

for 5 min by 300 x g at room temperature. Afterwards, the supernatant was removed

and the cell pellet was resuspended in 15 ml complete medium. For cultivation the cells

were placed in T-75 cell culture flasks and stored in a CO2-incubator at 37°C and a

humidified 5% CO2 atmosphere.

2.2.1.4) Knockdown of protein expression in mammalian cells

The gene silencing was performed using different siRNAs (Table 2.2) and

Oligofectamine following the manufacturer´s instructions. In most experiments a

mixture of two different siRNAs for one gene was used. In short, cells were seeded in

different numbers according to the used plate format. The cells were grown for 24 h at

37°C in a humidified 5 % CO2 atmosphere. After this the knockdown was performed

as described in the manufacturer´s instruction. In experiments to knockdown two

different genes the total amount of siRNAs per well were doubled up, as also the

amount of Oligofectamine was doubled. For detailed information on the used siRNA

see Table 2.2. Then following 72 h incubation at 37°C and 5 % CO2, the cells were

used for the different experiments.

Page 44: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

32

Table 2.2: siRNAs used for gene silencing

Gene siRNA Sequence (5´→3´)

Control scrRNA AACAGUCGCGUUUGCGACUdTdT

CSN5 CSN5 1 GCUCAGAGUAUCGAUGAAAdTdT

CSN5 2/ UR3 AGAAGUACUUUACCUGAAAUUdTdT

CSN1 CSN1 1 AAGUACGCCUCAUGUCUCAAGdTdT

CSN1 2 GAACCUUUAACGUGGACAUdTdT

CSN2 CSN2 GCACUGAAACAAAUGAUUAdTdT

Inhibin A INHBA 1 ACAUGCUGCACUUGAAGAAdTdT

INHBA 2 GUAGUAGACGCUCGGAAGAdTdT

Cullin1 Cullin1 CUAGAUACAAGAUUAUACAUGCGGdTdT

2.2.1.5) Overexpression of proteins in mammalian cells

For overexpression of proteins, again cells were seeded in an appropriate number to

the used plate format. The cells were incubated for 24 h in a CO2-incubator at 37°C in

a humidified 5 % CO2 atmosphere. After 24 h 0.5 µg or 1 µg (depending if 24 or 12 well

plate was used) of DNA was mixed with 25 µl or 50 µl OptiMEM per well and 3 µl/ 6 µl

Polyfect transfection reagent per well was added. This mix was vortexed for 10 s and

then incubated for 10 min at room temperature. After this 150 µl or 300 µl full medium

per well was added. The supernatant of the cells was removed and 350 µl or 700 µl

fresh full medium was added. Following this 150 µl respectively 300 µl of the

transfection suspension was added per well. The cells then were incubated for 72 h at

37°C and 5 % CO2 and afterwards used in different experiments.

2.2.2) Molecular biological methods

2.2.2.1) Ribonucleic acid (RNA)-Isolation

Two different methods were used for isolation of RNA. On the one hand the RNEasy-

Kit (Qiagen) was used, following the manufacturer´s instruction. On the other hand,

RNA was isolated using the TRIzol-Chloroform extraction. Therefor the cells were

lysed in 400 µl TRIzol and incubated for 3 min at room temperature. Then 80 µl of

chloroform was added and the suspension was shaken for 15 s and again incubated

for 3 min at room temperature. After this the cell suspension was centrifuged at

Page 45: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

33

12000 x g for 15 min at 4°C. From now on the following steps were performed on ice.

The aqueous phase was carefully taken (to avoid contamination with the intermediate

or the organic phase) and placed in a fresh reaction tube. Then 200 µl isopropanol and

1 µl Glycoblue were added. After this the suspension was incubated at -80°C for at

least 1 h (this time period can be expanded indefinitely). The frozen samples then were

centrifuged for 20 min using 12000 x g at 4°C. The supernatant was carefully removed

and 500 µl ice cold 75 % ethanol was added to the pellet. The samples were shortly

vortexed and again centrifuged 15 min at 7400 x g and 4°C. Following this the

supernatant was again removed carefully and the pellet was air dried for around 1 h

until remaining supernatant was vaporized. Then the pellet was resuspend in 15 µl

nuclease-free water and the RNA-concentration was measured at the

Spectrophotometer NanoDrop. Finally, the RNA was stored at -80°C and further used

for complementary DNA (cDNA) synthesis.

2.2.2.2) Complementary DNA (cDNA)-synthesis

For cDNA synthesis out of the isolated RNA the first strand cDNA synthesis kit

(Thermo scientific) was used. The synthesis was carried out as described in the

manual. Shortly, 1 µg RNA filled up with nuclease-free water to 10 µl and incubated

with 1 µl Oligo-dT for 10 min at 65°C. Then 9 µl of the synthesis mastermix (see

Table 2.3) was added to the sample and incubated for 1 h at 37°C. To stop the

reaction, the samples were incubated for 5 min at 70°C. After this the cDNA was

stored at -20°C.

Table 2.3: Mastermix for cDNA synthesis

Reagent Volumes (µl) per sample

5 x Reaction buffer 4

RiboLock RNase Inhibitor 0.5

10 mM dNTP 2

M-MuLV Reverse Transcriptase 1

ddH2O 1.5

Total volume: 9

Page 46: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

34

2.2.2.3) Quantitative real-time polymerase chain reaction (RT-qPCR)

RT-qPCR was used to measure the change in gene expression due to different cell

treatments. First step was to dilute the synthesized cDNA to a concentration of

200 ng/µl or 400 ng/µl. Then 8 µl of the RT-qPCR Mastermix (see Table 2.5),

containing the SYBR-Green dye, was provided in reaction tubes. The SYBR-Green

dye has the ability to bind double stranded DNA. Therefor it is possible to detect the

increase in PCR-products directly throughout the reaction. Then 2 µl of the diluted

cDNA was added to the tubes. The RT-qPCR was performed in the Thermocycler

RotorGene 6000 (Qiagen) by using the SensiMix program. To calculate the change in

gene expression the following formula was used:

ΔCt = Ct (target) – Ct (reference)

ΔΔCt = ΔCt (treated cells) – ΔCt (untreated cells)

Relative mRNA expression: 2(-ΔΔCt)

Table 2.4: Primers used in RT-qPCR

Gene Sequence (5´→3´)

CSN5 Forward

CGC AAA TTG CTT GAG CTG TTG TGG

AA

Reverse CCA GCT GGG CTT CTG ACT GC

CSN1 Forward CAG CAG CTC CTT CAA GTT GT

Reverse GAA ATA CTG GAT GAG GGC AC

CSN2 Forward CCT CAT CCA CTG ATT ATG GGA GT

Reverse CAT CAT AAT TCT TGA AGG CTT CAA AA

Inhibin A Forward CCT CGG AGA TCA TCA CGT TTG

Reverse GGC GGA TGG TGA CTT TGG T

Inhibin α Forward ATG TCT CCC AAG CCA TCC TT

Reverse TG GCC GGA ACA TGT ATC TG

Inhibin B Forward CGT TTC CGA AAT CAT CAG CT

Reverse CCT GGA CCA CAA ACA GGT T

GAPDH Forward GCC TCA AGA TCA TCA GC

Reverse ACC ACT GAC ACG TTG GC

Page 47: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

35

Table 2.5: Mastermix for RT-qPCR

Reagent Volumes (µl) per sample

2 x SensiMix Plus SYBR Green 5

PCR-H2O 1

Forward Primer 1

Reverse Primer 1

Total volume: 8

Table 2.6: RT-qPCR program

Step Temperature Duration Cycles

Initial activation 95°C 20 s

Denaturation 95°C 15 s

40 x Annealing 60°C 30 s

Elongation 72°C 15 s

Melting curve 60°C to 95°C Read every 0.5°C, hold 1 s between reads

2.2.2.4) RNA-Isolation and expression analysis using microarray approach

To perform the microarray RNA was isolated out of cells using the RNEasy Kit

(Qiagen). The RNA isolation was carried out according to manufacturer’s protocol.

Shortly, the cells were harvested by treatment using TrypLE Express solution. A third

of the cell solution was used for protein isolation (see 2.2.2.5) the rest was used for

RNA isolation. For the RNA isolation the cells were spun down at 300 x g for 5 min at

room temperature. Then the supernatant was discarded and the cells were lysed in an

appropriate amount of RLT buffer. The lysates were homogenized using QIAshredder

spin columns according to the protocol. One volume of 70 % ethanol was added to the

homogenized lysates and mixed by pipetting up and down. The samples were

transferred to RNEasy spin columns and centrifuged by room temperature for 15 s at

8000 x g. The flow-through was discarded and the RNEasy spin columns were washed

by adding 700 µl RW1 buffer. The columns were again centrifuged for 15 s at 8000 x g

by room temperature (RT) and the flow-through was discarded. In the next step 500 µl

RPE buffer was added to the columns and centrifuged as described for the RW1 buffer.

Page 48: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

36

In a last washing step, the RNEasy columns were loaded with 500 µl RPE buffer again

and centrifuged for 2 min at 8000 x g by RT. The flow-through was discarded again

and the columns were centrifuged for 1 min at 16000 x g and at room temperature.

Then the RNA was eluted from the columns. Therefor 30 µl of RNAse free water was

pipetted on the columns which were placed in fresh collection tubes. After this the

columns were centrifuged by 8000 x g for 1 min at room temperature. The RNA content

in the flow-through was measured by Nanodrop and the RNA was frozen and stored

at -80°C.

The samples which contained a yield of at least 100 ng/µl RNA and showed a strong

knockdown of CSN5 (at least 70 % reduction in protein level) in the Western blot were

handed over to the IZKF (Interdisziplinäres Zentrum für klinische Forschung) chip

facility. They did a quality check of the RNA and three samples for each treatment were

chosen. The chip facility synthesized the cDNA out of the RNA samples and then

performed the microarray using a GeneChip® Human Gene 2.0 ST (Affymetrix) and

did a first analysis of the results.

2.2.2.5) Protein-Isolation out of whole cell lysates and sample preparation

for gelelectrophoresis

For cell lysis the cells were incubated in an appropriate amount of lysis buffer for 10-30

min on ice. After this a mechanical lysis was performed by scratching in the well, using

a pipette tip. The cell suspension was transferred into a new reaction tube and was

further treated with ultrasound to disrupt the DNA. Therefor the suspension was treated

for an overall pulse time of 5 min in 30 s pulses with 30 s pause in between, using

70 % amplitude. Then the samples were centrifuged 10 min at 16000 x g and 4°C and

the supernatant was transferred into a fresh reaction tube. The protein concentration

was measured from the supernatant using the DC Kit according to the microplate assay

protocol of the manufacturer. In short, the lysate was diluted 1:5 in ddH2O. Then 5 µl

of the diluted lysate and of a BSA standard in ddH2O ranging from 0.2 mg/ml to 1.5

mg/ml was transferred into a clear 96 well plate. 25 µl of reagent A and 200 µl of

reagent B were added. The plate was incubated for 15 min at room temperature in the

dark. Then the absorbance at 590 nm was measured in the VIKTOR2 1420 Multilabel

Counter. The samples then were stored at -20°C or used for gel electrophoresis.

Page 49: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

37

Table 2.7: Composition of the lysis buffer

Reagent

RIPA buffer

Protease inhibitor cocktail 1 % (v/v)

PhosphoStop 10 % (v/v)

For the use in gel electrophoresis the lysate was transferred in a fresh reaction tube

and mixed 4:1 with 4 x LDS sample buffer containing 200 mM DTT and incubated for

10 min at 95°C. To achieve equal protein concentration in each lane the lysates was

previously adjusted to the lysate containing the lowest protein concentration. Therefor

the samples containing higher protein concentrations were diluted with lysis buffer to

gain equal protein concentrations. After incubation at 95°C, the samples were again

spun down for 5 min using 16000 x g at 4°C and then used in gel electrophoresis.

2.2.2.6) Protein-Isolation out of supernatants using TCA-precipitation and

sample preparation

The supernatant was collected in a reaction tube. To avoid contaminations by dead

cells, the supernatant was centrifuged for 5 min at 500 x g by RT. Afterwards, the

supernatant was transferred into a fresh reaction tube and 1 volume of TCA-solution

(20%) was. To mix the solution it gets shortly vortexed and then centrifuged for 15 min

by 16000 x g at 4°C. The supernatant was discarded and the pellet was washed by

pipetting 300 µl Acetone onto the pellet and shortly vortexing. Again the sample was

centrifuged at 16000 x g for 15 min by 4°C. The supernatant was discarded again and

the pellet was dried at room temperature to get rid of solvents. The pellets were then

resuspended in 5-10 µl 1 x LDS sample buffer per 100 µl used supernatant. Then the

samples were incubated at 95°C for 5 min and afterwards stored at -20°C. Before use

in gel electrophoresis the samples were centrifuged for 5 min at 16000 x g and 4°C.

Table 2.8: Composition of TCA-solution

Reagent

Trichloroacetic acid (TCA) 20 % (v/v)

ddH2O

Page 50: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

38

Table 2.9: Composition of 1 x LDS sample buffer

Reagent

NuPAGE LDS sample buffer (4 x) 25 % (v/v)

DTT 50 mM

ddH2O

2.2.2.7) SDS-Polyacrylamid (SDS-PAA) gel electrophoresis and Western

blot

To detect the expression or the change in expression of different proteins SDS-PAA gel

electrophoresis and western blot was used. The SDS-PAA gels were made as

described in Table 2.10 and

Table 2.11. The total volume of the resolving gel was 6 ml for 1 mm gel cassette or 8

ml for a 1.5 mm gel cassette. The stacking gel volume was in all cassettes 2 ml.

Table 2.10: Composition of the different resolving gels used

Volume (µl)/ ml resolving gel

Reagent 9% PAA gel 10% PAA gel

ddH2O 440 410

30 % Acrylamide/Bis-solution 300 330

Resolving gel buffer 250 250

10 % SDS 10 10

APS 10 10

TEMED 1 1

Table 2.11: Composition of the stacking gel

Reagent Volume (µl)/ ml stacking gel

ddH2O 600

Bis-/ Acrylamid 130

Stacking gel buffer 250

10 % SDS 10

APS 5

TEMED 1

Page 51: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

39

The samples, prepared as described in 1.2.4, were loaded onto the gel and then

voltage was applied to the SDS-PAA gel. The gel was run in NUPAGE/X-cell SureLock

chamber (Invitrogen) at 100 V for 15 min and 160 V for 65-75 min.

After separation of the proteins via SDS-PAA gel electrophoresis the proteins were

transferred onto nitrocellulose or PVDF membrane using Xcell II blot module

(Invitrogen). Therefor the blot was run at 35 V for 90 min under cold conditions. After

this the membrane was blocked by incubation in 5% BSA in TBS-T for 1 h at room

temperature while gently shaking. This was done to prevent later on unspecific binding

of the antibodies. Then the membrane was washed 3 to 6 times in TBS-T for 5 min at

room temperature. Afterwards, the membrane was incubated with primary antibodies

according to the protein of interest. The primary antibody was diluted in 1% (w/v) or

5% (w/v) BSA in TBS-T corresponding to the manufacturer´s recommendations (Used

antibodies and conditions see Table 2.12). The membrane was incubated in primary

antibody over night at 4°C under gentle agitation. After this the membrane was washed

again as described previously. The secondary antibody was diluted 1:5000 (anti-

mouse HRP) respectively 1:10000 (anti-rabbit HRP) in 1% BSA in TBS-T. Then the

membrane was incubated in secondary antibody for 2 h at room temperature and

gentle shaking. After this a last washing step was performed as described after the

blocking of the membrane. The membrane was developed using SuperSignal West

Dura Extended Duration ECL reagent or SuperSignal West Femto Extended Duration

ECL reagent (Thermo Scientific) depending on the protein of interest and the used

antibodies. The chemiluminescence was detected using the LAS imager and evaluated

using AIDA Image Analyzer software. Due to the fact that some proteins of interest had

the same size, it was required to strip the membrane. For stripping, the blot was

washed again after detection, like described previously. Then the membrane was

incubated for 8 min at 55°C in stripping buffer. After this the membrane was thoroughly

washed for at least half an hour in TBS-T, under gently agitation. During this washing

step, the TBS-T was changed at least 3 times. Following this the membrane could be

blocked again and incubated with new antibodies as described before.

Page 52: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

40

Table 2.12: List of used primary antibodies

Protein Antibody Manufacturer Dilution

CSN1 Rabbit polyclonal Enzo Life Sciences Inc.

(BML-PW8285)

1:10000

CSN5 Mouse monoclonal

(B-7)

Santa Cruz Biotechnology

(sc-13157)

1:500

CSN8 Rabbit polyclonal Enzo Life Sciences Inc.

(BML-PW8290)

1:5000

Cullin1 Rabbit polyclonal

(H-213)

Santa Cruz Biotechnology

(sc-11384)

1:500

Inhibin A Rabbit polyclonal Abcam (ab97705) 1:1000

Phospho-SMAD2

(Ser465/467)

Rabbit monoclonal

[138D4]

Cell Signaling Technology

(#3108)

1:1000

SMAD2 Rabbit monoclonal

[EP784Y]

Abcam (ab40855) 1:10000

Tubulin Mouse monoclonal

[B-5-1-2]

Sigma-Aldrich (T5168) 1:4000

Table 2.13: Used secondary antibodies

Target Antibody Manufacturer Dilution

Anti-mouse Donkey polyclonal

IgG H&L HRP

Abcam 1:5000

Anti-rabbit Donkey HRP GE Healthcare Life

Sciences (Na-934)

1:10000

2.2.3) Cell biological assays

2.2.3.1) Proliferation assay

In this assay the supernatant of differently treated SW480 cells was transferred onto

CaCo2 cells to examine the effects on the proliferation of the CaCo2 cells.

To collect supernatant of SW480 cells, the knockdown was performed as described in

2.2.1.4). 48 h after starting of the knockdown 80000 CaCo2 cells were seeded in

Page 53: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

41

500 µl/well in a 24 well plate. Following 8 h after seeding of CaCo2 cells the

supernatant was removed and 400 µl/well of serum starvation medium was added.

Then the CaCo2 cells were incubated for 16 h at 37°C in a humidified 5% CO2

atmosphere. Then the supernatant of the SW480 cells were collected and pooled

according to the different knockdowns. The SW480 cells were detached. RNA and

protein was isolated to test the knockdown efficiency. The pooled supernatants were

centrifuged for 5 min by 300 x g at room temperature. Then 1.5 ml of supernatant of

the SW480 cells treated with scrRNA was mixed with activin A (final concentration 0.1

µg/ml) or with Follistatin (final concentration 0.5 µg/ml). 1.5 ml of supernatant of the

SW480 cells treated with CSN5 siRNAs were mixed with Follistatin to a final

concentration of 0.5 µg/ml. Afterwards, the supernatant of the CaCo2 cells were

removed, the cells were washed with PBS and 400 µl of the different supernatants

were added to the cells. Then the cells were incubated for 24 h respectively 48 h at

37°C in CO2 incubator with a humidified 5% CO2 atmosphere. Following the incubation,

the supernatant was again removed, the cells were washed with PBS and 150 µl

Trypsin/EDTA solution was added per well. After the cells completely detached, 150 µl

of complete medium was added and the cell suspension was transferred in 1.5 ml

reaction tubes. To count the cells, the cell suspension was mixed 1:1 with Trypan blue.

10 µl of this mixture was pipetted on the cell counting slides for the BioRad automated

cell counter. After this the cells were counted using the BioRad automated cell counter.

2.2.3.2) Stimulation of CaCo2 cells with supernatants and/or recombinant

protein

To get first hints if the supernatant of SW480 cells activates the classical activin

signaling pathways in CaCo2 cells, the phosphorylation of SMAD2 was measured.

Therefor supernatant was collected as described in 2.2.3.1). CaCo2 cells were seeded

in a 24 well plate with a number of 80000 cells per well. The cells were incubated for 8

h in a CO2 incubator at 37°C in humidified 5 % CO2 atmosphere. Following this the

supernatant was removed and 400 µl of serum starvation medium was added to the

cells. The cells were further incubated for 16 h. Then the medium was again removed

and supernatant or full medium containing recombinant protein as stimuli, were added

to the cells. Then they were again incubated for 15 min and after this, cell lysates were

prepared as describe in 2.2.2.5).

Page 54: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

42

2.2.4) Bioinformatical GeneChip Analysis

After RNA Isolation and GeneChip analysis, performed as described in 2.2.2.4), the

obtained data were analyzed using different methods.

In a first analysis the mean change in expression for each gene was calculated.

Therefor the average signal of all hybridization spots on the chip belonging to a specific

transcript was calculated using internal standards. For each transcript the change in

average signal strength between the cells treated with CSN5 siRNA and scrRNA as

control was calculated.

Next a gene set enrichment analysis was performed. For this all differentially regulated

genes were sorted due to their mean change in expression starting from the most up-

regulated to the most down regulated gene. No threshold was chosen in this analysis

so every gene was taken into account for this ranking. Next genes annotated in certain

gene sets where compared with the resulting list. A gene can be associated with more

than one gene set due to possible multiple functions of the product. These gene sets

were obtained from different databases (see

www.broadinstitute.org/cancer/software/gsea/wiki/index.php/MSigDB_collections).

The genes are grouped in the different gene sets by their biological function,

chromosomal location and regulation170. Additionally gene sets, which display the

expression pattern of genes in different diseases or cancers, are used in this

analysis171. The gene sets result due to computational methods or the analysis of

published experiments like other microarrays. If now a gene of a set is up regulated in

the experiment it gets a positive score, based on the position in the ranking. If a gene

is down-regulated it gets a negative score. To calculate the z-score, the scores of all

genes belonging to a certain gene set were added. If the z-score is significantly

different from the mean z-score of all checked gene sets, it can be concluded that this

gene set is differentially regulated after CSN5 knockdown. A positive z-score indicates

an activation of the gene set, while a negative z-score shows an inhibition of the gene

set. A higher respectively lower z-score indicates a stronger accumulation of up- or

down-regulated genes.

Additionally, a gene ontology enrichment analysis was performed. Therefore, the

overlap of all significantly up- or down-regulated genes with the genes annotated to a

physiological process is determined. Only processes including less than 1000 genes

(term size) were considered, because with an increasing gene number it is more likely

that coincidentally a significant overlap exists. Then the proportion of genes which were

Page 55: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

43

up- or down-regulated in the specific physiological process was calculated (called

precision). The other way round the proportion of genes belonging to a specific

physiological process which were up- or down-regulated in comparison to all up- or

down-regulated genes was calculated (called recall). The processes were sorted due

their precision.

Page 56: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

44

3) Results

3.1) siRNA-knockdown of CSN5 changes the gene expression

pattern in SW480 cells

CSN5 and its involvement in the CSN are important for the homeostasis of cells. CSN5

protein levels are deregulated in many cancer entities, including colon carcinoma.

However, the role of CSN5 in the homeostasis and cancerogenesis of the intestinal

epithelium poorly understood. That is why our laboratory was interested to get further

insights in the role of CSN5 in the physiology and patophysiology of the intestinal

epithelium, especially in the large intestine. Dr. Anke Schütz showed in her PhD-thesis

(A. Schütz, doctoral thesis, RWTH Aachen University, 2012) that the induced

homozygous knockout of Csn5 in the intestinal epithelium of mice is lethal172. In first

follow up experiments, we also obtained first hints that the knockout of Csn5 in the

intestinal epithelium of the colon seems to change the differentiation pattern of

intestinal epithelial cells. This leads to changes in the distribution of the different cell

types in the intestinal epithelium. It seemed that the number of absorptive cells in the

epithelium was markedly reduced, while most of the cells obtained a secretive

phenotype (unpublished data). The differentiation of cells is principally organized by

coordinated expression sequences of various genes. Thus, an influence of CSN5 on

gene expression patterns in colon epithelial cells was one of the underlying hypotheses

of this PhD thesis.

Figure 3.1: Strong reduction of the CSN5 protein in the human colorectal carcinoma cell line SW480, after siRNA mediated knockdown Representative Western blot and immunodetection of CSN5 and tubulin after knockdown of CSN5 in SW480 cells. This result is representative of all three experiments used in the microarray (Figure 3.2., Figure 3.3, Figure 3.4 and Table 3.2, Table 3.3, Table 3.4)

Page 57: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

45

In my experiments, the colorectal cancer cell line SW480 was chosen as a surrogate

model for cells of the colon epithelium. To get an unbiased readout of the effects of

CSN5 on the intestinal epithelium, a gene expression analysis was performed, using

microarray technology.

Figure 3.2: Two-fold differentially regulated genes in SW480 cells after CSN5 knockdown Knockdown of CSN5 was performed via siRNA approach. After 72 h of knockdown, RNA was isolated and transcribed into cDNA. For the expression analysis a GeneChip® Human Gene 2.0 ST (Affymetrix) was used. Diagram shows genes which were at least twofold differentially regulated in SW480 cells subjected to a CSN5 knockdown in comparison to SW480 treated with scrRNA. Shown are the means of three independent experiments.

The aim was to get first hints through which mechanisms or genes CSN5 may influence

the differentiation of intestinal epithelial cells. To investigate the influence of CSN5 on

the intestinal epithelium a knockdown of CSN5 was performed using a siRNA approach

as described in 2.2.7.1. After 72 h of knockdown the RNA was isolated and analyzed

by GeneChip microarray. To check for the knockdown efficiency western blot analysis

was performed (see Figure 3.1).

Three experiments, exhibiting substantial reduction of CSN5 protein levels by >60%,

were chosen for the microarray analysis. Quality of the RNA was tested, RNA was

ARL14

EPL

KRTA

P3-1

LCE1F

ACTB

L2

INHBA

GRIK

1-AS2,

BACH1

ANGPT2

SERPIN

B2,

SER

PINB10

PRIC

KLE2,

PRIC

KLE2-

AS1,

LOC10

0

DIR

AS2

ESM1

MIR

548H

3

RNU2-

7P

FABP6

LOC73

0755

, KRTA

P2-4

DUSP19

EREG

BTG

3

HER

C5CR2

TIPRL

PSD2

ACAP1

IL2R

B

CPXM

1

KU-M

EL-3

MUC21

RAB42

KANK4

RHOU, D

USP5P

KRT71

SLC16

A2

KIA

A11

99

DLX3

TREM

2

PPP1R14

C

C9o

rf84

C9o

rf41

RNASE6

SESN3

DKK4

PRR9

C1o

rf18

7

GM

FG

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

Fol

d C

hang

e (li

near

)

Page 58: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

46

transcribed into cDNA and a microarray analysis was performed using a GeneChip®

Human Gene 2.0 ST chip from Affymetrix.

In a first analysis, the change in expression for each gene in the three experiments

was calculated and mean values out of the three experiments were calculated. The

threshold for differentially regulated genes was set to at least two-fold up- or

down-regulation in expression. In this set-up, only 21 genes were up-regulated and 23

genes were down-regulated (see Figure 3.2). If the threshold was raised to at least

three-fold change in gene expression only six genes (5 genes upregulated/ 1 gene

downregulated) were found to be differentially expressed (see Figure 3.3).

Figure 3.3: Only six genes were at least three-fold differentially regulated in SW480 cells after CSN5 knockdown Knockdown of CSN5 was performed via siRNA. After 72 h of knockdown, RNA was isolated and transcribed into cDNA. For the expression analysis a GeneChip® Human Gene 2.0 ST was used. Diagram shows genes which were at least three-fold differentially regulated in SW480 cells subjected to a CSN5 knockdown in comparison to SW480 treated with scrRNA. Shown are the means of three independent experiments. (ARL14EPL = ADP-ribosylation factor-like 14 effector protein-like; KRTAP3-1 = Keratin associated protein 3-1; LCE1F = late cornified envelope 1F; ACTBL2 = actin -like 2; INHBA = inhibin A; GMFG = glia maturation factor )

This unusually short list of highly (at least two-fold) differentially regulated genes

suggests that CSN5 influences the cell behavior probably in other fashions beside

regulating the gene expression. Due to the knockdown time of 72 h, also indirect effects

of CSN5, had to be taken in to account. To check if CSN5 possibly influences the cell

behavior by widely regulate genes involved in certain cellular processes, additional

computational analysis, of the expression profiles, was performed.

ARL14

EPL

KRTA

P3-1

LCE1F

ACTB

L2

INHBA

GM

FG

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

Fo

ld C

han

ge (

linear)

Page 59: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

47

Table 3.1: Table of differentially regulated genes in SW480 cells after CSN5 knockdown List of at least twofold differentially regulated genes in SW480 cells after 72 h of CSN5 knockdown using a siRNA approach. Shown are the gene symbol, the corresponding description of the gene, the linear fold change of gene expression and the ANOVA p-value of the change. The Fold changes are the means of three independent experiments.

Gene symbol Description

Fo

ld C

ha

ng

e

(lin

ea

r)

(CS

N5

vs

. scr)

AN

OV

A p

-valu

e

(CS

N5

vs

. scr)

ARL14EPL ADP-ribosylation factor-like 14 effector protein-like 7,09 0,0063 KRTAP3-1 keratin associated protein 3-1 5,85 0,0000 LCE1F late cornified envelope 1F 3,98 0,0115 ACTBL2 actin, beta-like 2 3,23 0,0031 INHBA inhibin, beta A 3,18 0,0009 GRIK1-AS2, BACH1

GRIK1 antisense RNA 2 (non-protein coding), BTB and CNC homology 1, basic leucine zipper transcription factor 1

2,98 0,0002

ANGPT2 angiopoietin 2 2,85 0,0019 SERPINB2, SERPINB10

serpin peptidase inhibitor, clade B (ovalbumin), member 2, serpin peptidase inhibitor, clade B (ovalbumin), member 10

2,84 0,0002

PRICKLE2, PRICKLE2-AS1, LOC100653177

prickle homolog 2 (Drosophila), PRICKLE2 antisense RNA 1 (non-protein coding), uncharacterized LOC100653177

2,66 0,0037

DIRAS2 DIRAS family, GTP-binding RAS-like 2 2,36 0,0049 ESM1 endothelial cell-specific molecule 1 2,32 0,0213 MIR548H3 microRNA 548h-3 2,27 0,0137 RNU2-7P RNA, U2 small nuclear 7, pseudogene 2,25 0,0140 FABP6 fatty acid binding protein 6, ileal 2,23 0,0172 LOC730755, KRTAP2-4

keratin associated protein 2-4-like, keratin associated protein 2-4

2,19 0,0005

DUSP19 dual specificity phosphatase 19 2,12 0,0437 EREG epiregulin 2,12 0,0059 BTG3 BTG family, member 3 2,11 0,0001 CR2 complement component (3d/Epstein Barr virus)

receptor 2 2,10 0,0206

TIPRL TIP41, TOR signaling pathway regulator-like (S. cerevisiae)

2,01 0,0023

PSD2 pleckstrin and Sec7 domain containing 2 -2,01 0,0238 ACAP1 ArfGAP with coiled-coil, ankyrin repeat and PH

domains 1 -2,02 0,0453

IL2RB interleukin 2 receptor, beta -2,02 0,0368 CPXM1 carboxypeptidase X (M14 family), member 1 -2,03 0,0028 KU-MEL-3 KU-MEL-3 -2,03 0,0339 MUC21 mucin 21, cell surface associated -2,04 0,0291 RAB42 RAB42, member RAS oncogene family -2,07 0,0091

Page 60: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

48

Gene symbol Description

Fo

ld C

ha

ng

e

(lin

ea

r)

(CS

N5

vs

. s

cr)

AN

OV

A p

-valu

e

(CS

N5

vs

. s

cr)

KANK4 KN motif and ankyrin repeat domains 4 -2,09 0,0046 RHOU, DUSP5P

ras homolog family member U, dual specificity phosphatase 5 pseudogene

-2,16 0,0017

KRT71 keratin 71 -2,18 0,0102 SLC16A2 solute carrier family 16, member 2 (thyroid hormone

transporter) -2,18 0,0258

KIAA1199 KIAA1199 -2,24 0,0014 DLX3 distal-less homeobox 3 -2,24 0,0028 TREM2 triggering receptor expressed on myeloid cells 2 -2,26 0,0473 PPP1R14C protein phosphatase 1, regulatory (inhibitor) subunit

14C -2,28 0,0026

C9orf84 chromosome 9 open reading frame 84 -2,30 0,0297 C9orf41 chromosome 9 open reading frame 41 -2,32 0,0011 RNASE6 ribonuclease, RNase A family, k6 -2,37 0,0025 SESN3 sestrin 3 -2,46 0,0080 DKK4 dickkopf homolog 4 (Xenopus laevis) -2,65 0,0038 PRR9 proline rich 9 -2,69 0,0266 C1orf187 chromosome 1 open reading frame 187 -2,88 0,0023 GMFG glia maturation factor, gamma -3,44 0,0148

A possible mechanism how CSN5 may influence cell state could be that genes having

small changes in their expression, after CSN5 reduction, are accumulated in a certain

biological processes/pathways. These small changes can add up and then influence

the whole process. Because of this possibility, a gene set enrichment analysis and a

gene ontology analysis was performed. For the gene set enrichment analysis all

differentially regulated genes were sorted due to their mean change in expression

starting from the most up-regulated to the most down regulated gene. No threshold

was chosen in this analysis so every gene was taken into account for this ranking. Next

genes annotated in certain gene sets where compared with the resulting list. A gene

can be associated with more than one gene set due to possible multiple functions of

the product. These gene sets were obtained from different databases (see

www.broadinstitute.org/cancer/software/gsea/wiki/index.php/MSigDB_collections).

Page 61: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

49

Figure 3.4: Gene set enrichment analysis of the differentially expressed genes in SW480 cells, following siRNA mediated CSN5 knockdown Differentially regulated genes in SW480 cells, following siRNA mediated CSN5 knockdown, were detected by gene array expression analysis. The gene expression profile was used for gene set enrichment analysis. This computational analysis identifies if there is an accumulation of differentially regulated genes in particular gene sets. The z-score describes if there is an accumulation of down- (negative z-score) or up regulated (positive z-score) genes. In total 1342 gene sets were identified to be influenced in SW480 cells by CSN5 knockdown. In this figure 23 gene sets, which seem to be interesting candidates, are depicted. Gene sets belonging to signaling pathways which are speculated or known to be involved in colorectal cancer development, or gene sets belonging to cell cycle processes were chosen. Also, gene sets which are observed in colorectal cancer specimen are depicted.

The gene set enrichment analysis showed that 1342 gene sets were significantly

influenced by CSN5 knockdown in SW480 cells. To pick gene sets for further

analysis, gene sets of interest have to be reviewed by looking at the genes belonging

to the sets and further research in the literature about the genes and gene sets. In

Figure 3.4, gene sets are displayed belonging to processes which were accumulated

in the results or could be associated to colorectal cancer. For example, different

signaling pathways, such as Wnt, TGF- or Notch, seemed to be influenced by CSN5

knockdown in SW480 colorectal cancer cells. Additionally, c-myc-related genes

seemed to be negatively influenced by the knockdown. In Table 3.2 the influenced

gene sets are further described and their corresponding z-score and significance are

shown.

-5 -4 -3 -2 -1 0 1 2 3 4 5

KEGG_NOTCH_SIGNALING_PATHWAY

KEGG_TGF_BETA_SIGNALING_PATHWAY

HALLMARK_WNT_BETA_CATENIN_SIGNALING

HALLMARK_NOTCH_SIGNALING

HALLMARK_TGF_BETA_SIGNALING

REACTOME_SIGNALING_BY_NOTCH

KEGG_WNT_SIGNALING_PATHWAY

SANSOM_WNT_PATHWAY_REQUIRE_MYC

PID_TGFBR_PATHWAY

PID_NOTCH_PATHWAY

POSITIVE_REGULATION_OF_CELL_PROLIFERATION

JECHLINGER_EPITHELIAL_TO_MESENCHYMAL_TRANSITION_UP

KEGG_MAPK_SIGNALING_PATHWAY

PID_WNT_SIGNALING_PATHWAY

WNT_SIGNALING

LAIHO_COLORECTAL CANCER SERRATED UP

GRADE_COLON_CANCER_UP

PID_MYC_ACTIV_PATHWAY

DANG_MYC_TARGETS_UP

MENSSEN_MYC_TARGETS

REN_MIF_TARGETS_DN

REACTOME_CELL_CYCLE

CELL_CYCLE_PROCESS

z score

Page 62: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

50

Also, I aimed to know if the knockdown of CSN5 in SW480 cells influences specific

physiological processes. For this purpose, a gene ontology enrichment analysis was

performed. Therefore, the overlap of all significantly up- or down-regulated genes with

the genes annotated to a physiological process is determined. Only processes

including less than 1000 genes (term size) were considered, because with an

increasing gene number, it is more likely that coincidentally a significant overlap exists.

All identified significant overlaps are depicted Table 3.3. The list is sorted by the

proportion of the overlap due to the size of up- or down-regulated genes (precision).

A higher precision means greater overlap of the up- or down-regulated genes with the

physiological process. It is remarkable that only one process (‘generic transcription

pathway’) seems to be up-regulated after CSN5 knockdown in SW480 cells. This

suggests that CSN5 seems to be involved in the positive regulation of several genes

through its influence on transcriptional regulation. In contrast, there are 24 negatively

influenced processes. It is to mention that many processes belong to the

cardiovascular system or to developmental processes. The influence on

developmental processes may underline the importance of CSN5 in the differentiation

of cells. This could support the finding in the intestinal epithelium of mice, where Csn5

knockdown seems to change the differentiation pattern and is subsequently lethal for

the mice.

Table 3.2: List of the selected 23 candidate gene sets differentially regulated in SW480 cells, following siRNA mediated CSN5 knockdown, and identified by gene set enrichment analysis. List of the 23 candidate gene sets depicted in Figure 3.4. Gene sets were identified by gene set enrichment analysis of the gene expression profile in the colorectal cancer cell line SW480 following siRNA mediated CSN5 knockdown. Gene sets belonging to signaling pathways which are speculated or known to be involved in colorectal cancer development, or gene sets belonging to cell cycle processes were chosen. Also, gene sets which are observed in colorectal cancer specimen were selected. Gene sets are shown with an additional description, the corresponding z-score and the adjusted p value of the z score.

Pathways Description z score adj. p. value

CELL_CYCLE_PROCESS A cellular process that is involved in the progression of biochemical and morphological phases and events that occur in a cell during successive cell replication or nuclear replication events

4,1300 0,0006

REACTOME_CELL_CYCLE Genes involved in Cell Cycle 3,8116 0,0018 REN_MIF_TARGETS_DN Genes down-regulated in SK-N-DZ cells

(neuroblastoma) after knockdown of MIF[GeneID=4282] by antisense RNA

3,2647 0,0078

Page 63: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

51

Pathways Description z score adj. p. value

MENSSEN_MYC_TARGETS Genes up-regulated by adenoviral expression of c-MYC [GeneID=4609] in HUVEC cells

3,2225 0,0086

DANG_MYC_TARGETS_UP Genes up-regulated by MYC [GeneID=4609] and whose promoters are bound by MYC, according to MYC Target Gene Database

2,4903 0,0408

PID_MYC_ACTIV_PATHWAY Validated targets of C-MYC transcriptional activation

2,4887 0,0409

GRADE_COLON_CANCER_ UP

Up-regulated genes in colon carcinoma tumors compared to the matched normal mucosa samples

2,6330 0,0313

LAIHO_ COLORECTAL_CANCER SERRATED_UP

Genes up-regulated in serrated vs conventional colorectal carcinoma (CRC) samples

-2,6032 0,0331

WNT_SIGNALING Genes related to Wnt-mediated signal transduction

-2,6869 0,0280

PID_WNT_SIGNALING_ PATHWAY

Wnt signaling network -2,7204 0,0264

KEGG_MAPK_SIGNALING_ PATHWAY

MAPK signaling pathway -2,8852 0,0190

PID_NOTCH_PATHWAY Notch signaling pathway -3,0281 0,0139 PID_TGFBR_PATHWAY TGF-beta receptor signaling -3,0912 0,0119 SANSOM_WNT_PATHWAY_ REQUIRE_MYC

Wnt target genes up-regulated after Cre-lox knockout of APC [GeneID=324] in the small intestine that require functional MYC

-3,2052 0,0090

KEGG_WNT_SIGNALING_ PATHWAY

Wnt signaling pathway -3,2066 0,0090

HALLMARK_TGF_BETA_ SIGNALING

Genes up-regulated in response to TGFB1

-3,7028 0,0023

HALLMARK_NOTCH_ SIGNALING

Genes up-regulated by activation of Notch signaling

-3,7247 0,0022

HALLMARK_WNT_ BETA_CATENIN SIGNALING

Genes up-regulated by activation of WNT signaling through accumulation of beta catenin CTNNB1

-3,7640 0,0020

KEGG_TGF_BETA_ SIGNALING_ PATHWAY

TGF-beta signaling pathway -4,2332 0,0005

KEGG_NOTCH_SIGNALING _PATHWAY

Notch signaling pathway -4,6255 0,0001

Page 64: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

52

Table 3.3: Gene ontology enrichment analysis of the differentially regulated genes in SW480 cells after CSN5 knockdown by siRNA treatment This analysis shows the accumulation of down- or up regulated genes, identified by gene expression analysis using GeneChip microarray as described in ?, in different physiological processes. Only processes with an assigned total gene number (term size) smaller than 1100 genes were considered. The list is sorted by the relative frequency of up or down regulated genes in the term size (precision =

� � � �� � � ). Query size = total number of up or down regulated genes in the array. Overlap size = number of up or down regulated genes identical with the genes belonging to the physiological process. Recall =

� � � �� � � .

Re

gu

lati

on

term.name

term

.siz

e

qu

ery

.siz

e

ove

rla

p.

siz

e

rec

all

pre

cis

ion

p.v

alu

e

Up Generic Transcription Pathway 491 562 25 0,044 0,051 0,0296

Dow

n

cardiac muscle cell proliferation 40 766 9 0,012 0,225 0,0157

cardiac muscle tissue growth 54 766 10 0,013 0,185 0,0307

heart growth 62 766 11 0,014 0,177 0,0174

O-linked glycosylation 104 764 13 0,017 0,125 0,0388

ear development 206 766 21 0,027 0,102 0,0203

regulation of actin cytoskeleton organization

267 766 26 0,034 0,097 0,0042

ion channel complex 264 766 25 0,033 0,095 0,0109

transporter complex 302 766 27 0,035 0,089 0,0134

transmembrane transporter complex 296 766 26 0,034 0,088 0,0274

regulation of actin filament-based process

313 766 27 0,035 0,086 0,0259

embryonic organ development 428 766 35 0,046 0,082 0,0045

skeletal system development 493 766 36 0,047 0,073 0,0406

cell surface 690 766 50 0,065 0,072 0,0012

blood vessel development 560 766 40 0,052 0,071 0,0233

vasculature development 580 766 41 0,054 0,071 0,0231

epithelial cell differentiation 575 766 40 0,052 0,070 0,0430

embryonic morphogenesis 585 766 41 0,054 0,070 0,0283

organ morphogenesis 934 766 64 0,084 0,069 0,0002

regulation of anatomical structure morphogenesis

800 766 54 0,070 0,068 0,0036

epithelium development 1073 766 68 0,089 0,063 0,0015

MI:hsa-miR-18a 661 766 41 0,054 0,062 0,0316

circulatory system development 884 766 55 0,072 0,062 0,0317

cardiovascular system development 884 766 55 0,072 0,062 0,0317

embryo development 1005 766 62 0,081 0,062 0,0114

Page 65: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

53

3.2) CSN5 influences the expression of activin/inhibin subunits

After knockdown of CSN5 in SW480 cells only a few genes exhibit a high change in

expression, of at least twofold. The gene set enrichment analysis indicated that CSN5

seems to influence many signaling pathways including Wnt, Notch or TGF- . In the

gene ontology analysis processes belonging to epithelial development and

differentiation showed up to be influenced by CSN5.

The inhibin A (INHBA) gene was one of only five genes that was upregulated by more

than threefold following CSN5 knockdown. It is part of the TGF- superfamily, which I

also identified by the gene enrichment analysis to be influenced by the CSN5

knockdown. I decided to take a closer look at the activin/inhibin signaling pathway,

because there is emerging evidence in the literature, that this signaling pathway plays

an important role in the intestine164,165,167-169. Moreover, it is known that the

activin/inhibin signaling pathway plays a fundamental role in the differentiation of stem

cells. In the intestinal epithelium, cryptic stem cells play an important role in the

development and regeneration. From the stem cells located at the bottom of the crypts,

the various cell types of the intestinal epithelium develop. This happens by continuous

division of the stem cells and subsequent differentiation of one of the daughter cells.

This further indicates, that the activin/inhibin signaling pathway might play a role in the

observed change of differentiation after Csn5 knockdown in the intestinal epithelium of

mice.

Table 3.4: Changes in gene expression of inhibin subunits after CSN5 knockdown Changes in gene expression of the different inhibin subunits, as indicated by the GeneChip microarray analysis.

To confirm the results of the microarray, regarding the expression of the activin/inhibin

signaling subunits, which were significantly changed according to microarray data,

RT-qPCR was performed. A knockdown of CSN5 in SW480 cells, using a mixture of

two different siRNAs was performed, as described in 2.2.1.4). The mRNA of the

Page 66: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

54

isolated total RNA was transcribed into cDNA. Then, RT-qPCR was performed using

specific primers for the target genes of the activin/inhibin signaling pathway.

Figure 3.5: The knockdown of CSN5 leads to a significant change in the expression of inhibin. RT-qPCR analysis of mRNA expression in SW480 cells after 72 h of CSN5 knockdown using siRNA. (A) Knockdown of CSN5 leads to increased mRNA expression of inhibin A (B) Decreased inhibin α and inhibin B expression after CSN5 knockdown. (C)(D) The knockdown of CSN5 was efficient and led to a significant decrease of CSN5 mRNA expression and CSN5 protein level. (E) Representative western blot and immunodetection of CSN5 protein level after CSN5 knockdown in SW480 cells. Data represent means ± SEM of independent experiments performed in duplicates (A: n=10; B: n=5-9; C: n=10; D: n=7) (* = p ≤ 0.05, *** = p ≤ 0.005; two-tailed unpaired t-test).

In the microarray two-fold changes was only seen for inhibin A expression after

siRNA mediated CSN5 knockdown (see Table 3.1). In the RT-qPCR analysis, the

Page 67: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

55

expression of the two other prominent subunits inhibin α (INHA) and inhibin B

(INHBB)was checked.

The results of the microarray could be confirmed via RT-qPCR. The knockdown of

CSN5 was efficient and decreased the CSN5 mRNA expression significantly about

80% and subsequently decreased the CSN5 protein level by 50% (see Figure 3.5 C-E).

Like in the microarray the expression of inhibin A mRNA increases significantly about

3.5-fold, after CSN5 knockdown (see Figure 3.5 A). In contrast to the microarray,

additionally a significant change in the mRNA expression of inhibin α and inhibin B

could be detected. CSN5 knockdown leads to a significant decrease in inhibin α mRNA

of about 60%. For inhibin B mRNA expression a reduction of 50% was detected (see

Figure 3.5 B). The other inhibin subunits were not chosen for further investigation, due

to their relatively unknown role and because they did not show a change in expression

in the microarray.

In conclusion it could be shown that the knockdown of CSN5 increases the expression

of inhibin A and decreases the expression of inhibin α and inhibin B. These results

suggest that CSN5 influence the activin/inhibin signaling pathway by directly or

indirectly modulating the expression of its subunits.

3.3) Knockdown of CSN5 do not alter the protein level of inhibin βA

subunits

In a next step I tried to confirm, if the enhanced expression of inhibin A mRNA due to

CSN5 knockdown was accompanied by an increased protein level of inhibin A.

Therefore, a knockdown of CSN5 in SW480 cells was performed as described before.

After 72 h of knockdown whole cell lysates were made. To detect the protein level of

inhibin A, SDS-PAGE gel electrophoresis and western blot was performed.

immunodetection and densitometry was used to detect and quantify the protein levels.

Western blot analysis of whole cell lysates of SW480 cells exhibiting CSN5 knockdown

showed no significant change in the intensity of the protein bands for inhibin A. (see

Figure 3.6). In comparison, the expression of mRNA of inhibin A increases about 3.5

fold after CSN5 knockdown. Thus it seems that through different mechanisms the cell

may compensate and reduce the increase in inhibin A protein level compared to

inhibin A mRNA expression.

Page 68: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

56

Figure 3.6: CSN5 knockdown does not alter inhibin βA protein levels in SW480 cells. (A) Representative western blot using whole cell lysates of SW480 cells after CSN5 knockdown. immunodetection of inhibin A, CSN5 and tubulin was performed. (B) Quantification of whole cell lysates of SW480 cells after CSN5 knockdown using western blot analysis and AIDA software. Shown is the relative inhibin A protein level normalized to tubulin. Data represents means ± SEM of 30 independent experiments in duplicates.

3.4) Overexpression of CSN5 does not affect the expression of

activin/inhibin subunits

A question that arises from these results is whether the influence on the inhibin

expression is mediated through monomeric CSN5 or through CSN5 as part of the

COP9-signalosome complex. To get a first hint, an overexpression of CSN5 in SW480

cells was performed. To overexpress CSN5, SW480 cells were transfected with a

plasmid coding for flag tagged CSN5 and incubated for 72 h. Following this, RNA was

isolated and the mRNA was transcribed into cDNA. After this the expression of the

inhibin genes of interest were analyzed by RT-qPCR.

The overexpression of CSN5 leads to a strong increase in total CSN5 mRNA

(endogens CSN5 + CSN5-flag) expression around 2000 fold. On protein level the

increase of CSN5 was not such as strong. For the expression of activin/inhibin subunit

mRNA no significant change could be detected (see Figure 3.7). Perhaps a tendency

towards an opposite effect in comparison to CSN5 knockdown is visible for inhibin A

respectively inhibin α and inhibin B mRNA expression.

Page 69: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

57

Figure 3.7: Overexpression of CSN5 in SW480 does not influence the expression of inhibin subunits RT-qPCR analysis of mRNA expression in SW480 cells after 72 h of CSN5 overexpression. (A) Expression of activin/inhibin subunits mRNAs were not influenced, due to CSN5 overexpression. (B) The overexpression of CSN5 led to a significant increase in CSN5 mRNA expression. (C) Representative western blot of whole cell lysates after CSN5 overexpression. Immunodetection of CSN1, CSN5 and tubulin was performed. Data represent means ± SEM of independent experiments performed in duplicates (A: n=5-6; B: n=6) (** = p ≤ 0.01; two-tailed unpaired t-test)

3.5) The knockdown of CSN1 or CSN2 does not alter the expression

of inhibin A

The missing significant influence of CSN5 overexpression on activin/inhibin subunit

expression could indicate that the effects of CSN5 are mediated through the COP9-

signalosome complex rather than the monomeric CSN5. To study this hypothesis

knockdown of other COP9-signalosome subunits was performed. The two largest

subunits CSN1 and CSN2 were chosen for the knockdown, because it was assumed

that this should lead to a breakdown of the COP9-signalosome172,173. The knockdown

of CSN1 and CSN2 was performed as described for CSN5 (see 2.2.7.1), with the

exception that for the CSN2 knockdown only a single siRNA was used.

Page 70: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

58

Figure 3.8: Knockdown of CSN1 in SW480 cells decreases inhibin A and inhibin α expression RT-qPCR analysis of mRNA expression in SW480 cells after 72 h of CSN1 knockdown. (A) CSN1 knockdown decreases significantly the mRNA expression of inhibin A and inhibin α subunits. But no influence on the expression of inhibin B could be observed. (B)(C) Knockdown of CSN1 leads to a significant decrease in CSN1 mRNA expression and CSN1 protein level (D) Representative western blot and immunodetection of CSN1 protein level after CSN1 knockdown in SW480 cells. Data represent means ± SEM of independent experiments performed in duplicates (A: n=4-6; B: n=5; C: n=6) (* = p ≤ 0.05, ** = p ≤ 0.01; two-tailed unpaired t-test).

The knockdown of CSN1 was successful and led to a decrease of about 40 to 60% in

CSN1 mRNA expression. On protein level a decrease between 50 and 60% could be

observed (see Figure 3.8 B-D). CSN1 knockdown significantly decreases the

expression of inhibin A and inhibin α mRNA. For inhibin B mRNA expression no

significant effect in was detectable (see Figure 3.8 A). For the inhibin α expression the

effect is comparable to the effects of the CSN5 knockdown. Interestingly, for the CSN1

knockdown the opposite effect on inhibin A expression, in comparison to the CSN5

knockdown, could be observed.

Another subunit of the CSN which was knocked down via siRNA was CSN2. The

knockdown of CSN2 was highly effective and led to a strong decrease in CSN2 mRNA

Page 71: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

59

amount of around 90%. Similar to the CSN1 knockdown the knockdown of CSN2 led

as well to a significant reduction in inhibin α mRNA expression of about 40%, whereas

the expression of inhibin A mRNA was not influenced by the CSN2 knockdown (see

Figure 3.9).

Figure 3.9: Knockdown of CSN2 in SW480 cells do not alter the inhibin A expression but decreases the inhibin α expression. RT-qPCR analysis of mRNA expression in SW480 cells after 72 h of CSN2 knockdown. (A) The inhibin α mRNA expression is significantly decreased after CSN2 knockdown. No change in inhibin A mRNA expression could be detected. (B) Knockdown of CSN2 decreases the CSN2 mRNA expression. Data represent means ± SEM of 5 independent experiments performed in duplicates (* = p ≤ 0.05; *** = p ≤ 0.005; two-tailed unpaired t-test).

In summary the knockdown of CSN1 and CSN2 led to a significant reduction of

inhibin α, as also seen in the CSN5 knockdown experiments. This would suggest a

functional role of the whole COP9-signalosome in the regulation of the inhibin α-

expression. For the regulation of inhibin A or inhibin B expression the results are not

so clear. Inhibin A expression under the influence of different CSN subunit-

knockdowns could indicate for different influences of the monomeric CSN5 and the

COP9-signalosome complex. While monomeric CSN5 seems to inhibit the expression,

the COP9-signalosome may to some extent promote the expression. For inhibin B it

seems that only the monomeric CSN5 influences the expression.

Page 72: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

60

3.6) Knockdown of different CSN subunits influences the protein

stability of the CSN

Figure 3.10: Knockdown of CSN subunits influences the protein stability of other CSN subunits Western Blot analysis of CSN1/5/8 protein levels after 72 h of knockdown of CSN1/2/5 in SW480 cells. (A) Representative western blot and immunodetection of CSN1/5/8 and tubulin protein level after CSN1/2/5 knockdown in SW480 cells. (B)(C)(D) Quantification of the CSN1/5/8 protein levels after knockdown of CSN1/2/5 using AIDA software. Data represents means ± SEM of independent experiments performed in duplicates (CSN1 and CSN5 protein level n=5; CSN8 protein level n=3), (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.005; two-tailed unpaired t-test)

To more directly investigate how the knockdowns of the different CSN subunits may

influence the COP9-signalosome it was tested how the protein levels of different CSN

subunits are influenced by the knockdowns. Thus it was tested how the knockdowns

of the different CSN subunits influence the protein level of other CSN subunits and

subsequently the COP9-signalosome stability. Therefore, again knockdowns, of the

CSN subunits 1/2/5, was performed using siRNA. The knockdown was done in SW480

cells for 72 h. Following the knockdown, protein levels of CSN1 and CSN8 were

analyzed. These are the largest and the smallest of the PCI-domain subunits of the

Page 73: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

61

COP9-signalosome. Changes in their occurrence could hint towards the occurrence of

the COP9-signalosome complex. Of course, the expression of CSN5, the catalytic

subunit of the COP9-signalosome and the subunit of interest in this thesis, was

analyzed.

As seen in Figure 3.10, the knockdown of CSN1 led to a significant reduction of the

protein levels of the CSN subunits 1/5/8. The knockdown of CSN2 only led to a

significant reduction of CSN5 and CSN8 protein level. Interestingly led the knockdown

of CSN5 to no significant reduction in the protein level of CSN1 and CSN8. By looking

at the CSN5 protein level it seems like the effect is the other way round. The usage of

CSN5 siRNA leads to a strong and significant decrease in CSN5 protein level. This

could also be observed for the CSN1 and CSN2 siRNA treatment. But the knockdown

of CSN1 led to a significant lower reduction of CSN5 protein, in comparison to CSN5

knockdown. The knockdown of CSN2 may also show a tendency to a lower reduction

of CSN5 protein compared to the CSN5 siRNA treatment

This could hint that the knockdown of CSN1 and maybe CSN2 led to a decrease in the

amount of COP9-signalosome but did not alter the amount of monomeric CSN5. In

contrast the CSN5 knockdown seems to reduce the amount of monomeric CSN5.

Additionally, it may reduce the amount of COP9-signalosome.

3.7) Cullin1 seems to compensate the effect of CSN5 on inhibin A

but not inhibin α mRNA expression

The results so far could not clarify whether the effect of CSN5 on inhibin mRNA

expression is mediated through the monomeric form of CSN5 or as part of the CSN-

complex. The missing effects of CSN5 overexpression on activin/inhibin subunit mRNA

expression suggest a COP9-signalosome dependent effect. In contrast the knockdown

of CSN1 and CSN2 rather support a monomeric CSN5 effect.

One of the main functions of the COP9 signalosome is the regulation of cullin E3 RING-

ligases. Because of this, it was tested if the effect on the expression of activin/inhibin

is mediated through cullin1 E3-RING-ligases. For this purpose, a knockdown of cullin1

(CUL1) was performed. The knockdown was performed as described in 2.2.7.1 by

using a single siRNA for cullin1. Afterwards RNA and proteins were isolated from whole

cell lysates. The mRNA was analyzed by using RT-qPCR while the protein lysates

were used for SDS-PAGE gelelectrophoresis.

Page 74: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

62

Figure 3.11: Effects of CSN5 and/ or cullin 1 knockdown in SW480 cells, on the expression of activin/inhibin subunits RT-qPCR analysis of mRNA expression in SW480 cells after knockdown of CSN5 and/or cullin1 for 72 h. (A) The knockdown of cullin1 has no influence on the expression of inhibin A but it can reverse the effect of the CSN5 knockdown. (B) The knockdown of cullin1 exhibit the same effect on inhibin α expression like the CSN5 knockdown and seems to increase the effect together with a CSN5 knockdown. (C) Representative western blot and immunodetection showing cullin1, CSN5 and tubulin protein levels, after CSN5 and/or cullin1 knockdown for 72 h in SW480 cells. Data represents means ± SEM of 5 independent experiments performed in duplicates (* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.005, two-tailed unpaired t-test)

Western blot analysis verified efficient knockdown of cullin1 (see Figure 3.11). The

knockdown of CSN5 again reached 70-80% as seen before. Also, as expected, the

knockdown of CSN5 led to an enhanced NEDDylation of cullin1, as evident by an

increased band density of the higher molecular weight band in the cullin1 blot. CSN5

is the catalytic active subunit of the COP9-signalosome. Therefore, a knockdown of

CSN5 leads to less catalytically active CSN and thus reduced deNEDDylation of

cullins. The knockdown of cullin1 as well as the combined knockdown of cullin1 and

CSN5 was successful (see Figure 3.11 C).

Page 75: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

63

The knockdown of cullin1 per se had no effect on the expression of inhibin A mRNA

but decreased the expression of inhibin α mRNA. By combining the knockdown of

CSN5 and cullin1 in SW480 cells, the increase of the inhibin A expression due to

CSN5 knockdown could be reversed (see Figure 3.11 A). For the expression of

inhibin α in SW480 cells, the combined knockdown of CSN5 and cullin1 seemed to

increase the reduction in comparison to the single knockdowns (see Figure 3.11 B).

These results indicate the involvement of cullin1 RING E3 ubiquitin ligases in the

CSN5-modulated activin/inhibin expression. The results support the hypothesis that

CSN5 mediates its influence on inhibin α expression through its role in the COP9-

signalosome. But for inhibin A the regulation seems far more complicated. The results

of the knockdowns of the different CSN subunits suggest a role of the monomeric

CSN5, while the cullin1 knockdown would suggest the importance of cullin1 RING E3

ubiquitin-ligases and its regulation via the COP9-signalosome.

3.8) The influence of CSN5 on activin/inhibin subunits seems to be

mediated by transcriptional regulation

The results in 3.7 indicate that cullin1 RING E3 ubiquitin-ligases (CRLs) seem to play

a role in the regulation of activin/inhbin subunit expression. To get further hints that

CSN5 influences the activin/inhibin signaling through a cullin1 RING E3 Ubiquitin

ligase, the inhibitor MLN4924 was used. MLN4924 is an inhibitor of the

NEDD8-activating enzyme E1, which is important for the activation of CRLs174,175. The

use of MLN4924 results in a loss of CRL NEDDylation and a subsequent loss of

activity. This should to some extent mimic a cullin1 knockdown. In this experimental

setup a knockdown of CSN5 was again achieved via siRNA treatment and after 56 h

of knockdown the cells were treated for 16 h using MLN4924. Then RNA was isolated

and whole cell lysates were made and analyzed.

The deNEDDylation of cullin RING E3 ubiquitin-ligases via the inhibitor MLN4924

shows different effects for inhibin A expression but not for inhibin α expression in

comparison to the cullin1 knockdown. The treatment using MLN4924 shows no

influence on the expression of inhibin A with or without CSN5 knockdown (see Figure

3.12 A). This is contrary to the results of the cullin1 knockdown. For the expression of

inhibin α mRNA the MLN treatment showed the same effects as the knockdown of

cullin1 (see Figure 3.12 B). This further supports the assumption that cullin RING E3

ubiqutin-ligases play a role in the transcriptional regulation of inhibin α.

Page 76: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

64

Figure 3.12: DeNEDDylated cullin RING E3 ubiquitin-ligases seem to influence inhibin α but not inhibin A expression RT-qPCR analysis of mRNA expression in SW480 cells after knockdown of CSN5 for 56 h and following treatment using MLN4924 or DMSO for 16 h. (A) The treatment of SW480 cells using MLN4924 has no influence on the expression of inhibin A in SW480 cells with or without CSN5 knockdown. (B) In contrast the deNEDDDylation of cullin RING E3 ubiquitin-ligases after treatment using MLN4924 exhibit the same effect on inhibin α expression like the CSN5 or/and cullin1 knockdown and seems to increase the effect together with a CSN5 knockdown. Data represents means ± SEM of 5 independent experiments performed in duplicates (n.s. = not significant, * = p ≤ 0.05; ** = p*** = ≤ 0.01, p ≤ 0.005, two-tailed unpaired t-test)

To check if the inhibitor MLN4924 leds to the deNEDDylation of cullins the expression

of cullin1 was analyzed by using SDS-PAGE gelelektrophoresis and western blot

analysis. The treatment of SW480 cells using MLN4924 nearly abolishes the

NEDDylation of cullin1 as shown by western blot analysis (see Figure 3.13). This

indicates that the inhibition of cullin1 NEDDylation by MLN4924, seem to be effective

in SW480 cells.

The MLN treatment of SW480 cells led to a significant decrease of the inhibin A

protein level. On the other hand, for the inhibin A mRNA expression, no reduction

could be observed. This finding may suggest a posttranscriptional role of CRLs in the

regulation of inhibin A.

Page 77: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

65

Figure 3.13: MLN4924 treatment of SW480 cells leds to a reduction in cullin1 NEDDylation and decreases inhibin βA protein level (A) Representative western blot and immunodetection of cullin1, inhibin A and tubulin. SW480 cells were treated for 16 h with MLN4924 or DMSO as control. (B) Quantification of whole cell lysates of SW480 cells after treatment with MLN4924 or DMSO as control, using western blot analysis. Depicted are the relative inhibin A protein level normalized to tubulin. Data represents means ± SEM of 6 independent experiments performed in duplicates (* = p ≤ 0.05; two-tailed unpaired t-test)

3.9) Inhibition of proteasomal degradation reduces inhibin βA

protein level

To further investigate the involvement of cullin RING E3 ubiquitin-ligases (CRLs) in the

regulation of activin/inhibin expression, MG132 as an inhibitor of the proteasomal

degradation was used176. An important function of CRLs is the regulation of

proteasomal degradation via ubiquitination of target proteins to mark them for

degradation177.

The treatment of SW480 cells with MG132 showed no significant influence on

inhibin A protein levels (see Figure 3.14).

This result would hint that the assumed posttranscriptional regulation by CRLs is not

mediated by supporting the degradation of inhibin A through the proteasomal

pathway.

Page 78: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

66

Figure 3.14: MG132 treatment of SW480 cells seems not to influence inhibin βA protein level (A) Representative western blot and immunodetection of inhibin A and tubulin. Different lanes of the same blot (same gel and exposure) were combined as indicated by the dotted line. The SW480 cells were treated for 16 h with MG132 or DMSO as control. (B) Quantification of whole cell lysates of SW480 cells treatment with MG132 or DMSO as control, using western blot analysis. Depicted are the relative inhibin A protein level normalized to tubulin. Data represents means ± SEM of 3 independent experiments perfomed in duplicates (* = p ≤ 0.05; two-tailed unpaired t-test)

3.10) Increased expression of inhibin βA seems to increase the

secretion of activins/inhibins

In Figure 3.3 I could not detect that the enhanced expression of inhibin A mRNA

subsequently leads to enhanced inhibin A protein level in the cell. Therefore, I

assumed that an enhanced protein synthesis rate of inhibin A was also associated

with an enhanced secretion rate of activin A.

Figure 3.15: Knockdown of CSN5 leads to an enhanced secretion of inhibin βA Representative blot of inhibin A protein in SW480 supernatants after precipitation using TCA. CSN5 knockdown was performed in SW480 cells for 56 h. Afterwards the media was changed to low serum medium. After an additional incubation of 16 h the supernatant was collected and the proteins were precipitated using TCA. (n=5)

To test this assumption, the occurrence of inhibin A in supernatants of SW480 cells

was tested. Therefore, SW480 cells were transfected to knock CSN5 down. After 56 h

of knockdown the growth medium was exchanged against medium containing low

Page 79: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

67

serum levels. Following this the cells were again incubated for 16 h and afterwards the

supernatants were collected and analyzed.

Figure 3.16: Inhibition of secretion enhances the increase of inhibin βA intracellular protein level after CSN5 knockdown in SW480 cells (A) Representative western blot and immunodetection of inhibin A, CSN5 and tubulin. Knockdown of CSN5 was performed for 56 h following 16 h of treatment with Monensin or ddH2O as control. (B) Quantification of whole cell lysates of SW480 cells after CSN5 knockdown and treatment with or without Monensin, using western blot analysis and AIDA software. Depicted is the relative inhibin A protein level normalized to tubulin. Data represents means ± SEM of 5 independent experiments in duplicates (* = p ≤ 0.05; One way ANOVA and Turkey post test).

It appears an enrichment of inhibin A protein in the supernatants of CSN5 knockdown

SW480 cells could be observed. This supports the assumption that the enhanced

expression of inhibin A may lead to an enhanced protein synthesis rate. The cells

may compensate the intracellular accumulation of inhibin A due to the enhanced

protein synthesis rate by an enhanced secretion rate. Therefore, no intracellular

accumulation of inhibin A seems to takes place.

To further test the hypothesis, I used the inhibitor Monensin. Monensin is an inhibitor

of the classical secretion pathway via ER and Golgi. A knockdown of CSN5 was again

Page 80: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

68

achieved via siRNA treatment and after 56 h of knockdown the cells were treated for

16 h with Monensin.

The blocked secretion led to enhanced intracellular protein level of inhibin A after

CSN5 knockdown in SW480 cells. In Monensin treated cells, bearing a CSN5

knockdown, the increase in inhibin A protein level reached up to 3 fold. In untreated

conditions the increase was about 1.3 fold (see Figure 3.16).

These results suggest that the enhanced expression of inhibin A mRNA and the

probable subsequent enhanced protein synthesis rate leads to an enhanced secretion

of inhibin A most likely as activin A. This may explain minor effects of CSN5

knockdown on the intracellular protein level of inhibin A.

3.11) The influence of CSN5 on CRC cell proliferation seems to be at

least partly mediated via activin/inhibin signaling

So far I could show that knockdown of CSN5 leads to increased expression of

inhibin A and decreased expression of inhibin α. Thus, I conclude that the synthesis

of activin A is enhanced and more activin A gets secreted. This hypothesis is supported

by the observation that the inhibition of secretion leads to a stronger intracellular

accumulation of inhibin A after CSN5 knockdown. activin A is known to exhibit growth

suppression in colorectal cancer cells. This effect is mediated via the activation of the

SMAD signaling pathway178. The knockdown of CSN5 also suppresses the

proliferation of colorectal cancer cells85. It could be possible that the growth inhibitory

effect of the CSN5 knockdown is mediated, at least to some extent, by enhanced

activin A levels. To test if the enhanced expression and secretion of activin A after

CSN5 knockdown mediate growth suppression, an in vitro proliferation assay was

performed.

Therefore, a knockdown of CSN5, inhibin A or both was performed in SW480 cells as

described in 2.2.7.1. This cell line was chosen due to the established knockdown

protocol and the high knockdown efficiencies. After 72 h of knockdown the

supernatants were collected and pooled. Some of the supernatant of cells with CSN5

knockdown or scrRNA treated cells were preincubated for 15 min with follistatin.

Follistatin is an inhibitor of activin signaling by binding to activin and thereby prevents

the binding to its receptor.

Page 81: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

69

Figure 3.17: Scheme of the experimental setup of the proliferation assay Knockdown of CSN5, inhibin A or both in SW480 cells were performed for 72 h. The supernatant was transferred onto CaCo2 cells. CaCo2 cells were grown for 24 h in the supernatant. Afterwards CaCo2 cells were counted using Trypan blue staining to measure the proliferation.

Afterwards, CaCo2 cells were incubated with the supernatants for 24 h. This cell line

was chosen because of its functional activin-SMAD signaling pathway. In most

colorectal cell lines this pathway is mutated and more or less not functional. To

measure the proliferation, the cells were counted using Trypan blue method (see

Figure 3.17).

The supernatant of SW480 cells, treated with CSN5 siRNA leads, to a reduction of

about 20% in proliferation of CaCo2 cells compared with CaCo2 cells treated with

supernatant of SW480 treated with scrRNA. The preincubation of the CSN5

knockdown supernatant using follistatin could reverse the effect on the proliferation of

CaCo2 cells (see Figure 3.18). The proliferation of CaCo2 cells, incubated with

supernatant of SW480 cells following a CSN5 and inhibin A knockdown, was

comparable to the proliferation of CaCo2 cells treated with the supernatant of SW480

without a knockdown.

However, the increase in comparison to the CaCo2 cells treated with supernatant of

CSN5 knockdown cells was not significant (see Figure 3.18). The supernatant of

SW480 cell with a knockdown of inhibin A had no influence on the proliferation of

CaCo2 cells. The treatment of CaCo2 cells with supernatant of scrRNA treated SW480

cells preincubated with follistatin showed a tendency towards an increase in

Page 82: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

70

proliferation in comparison to the CaCo2 cells treated with the supernatant of SW480

cells treated with scrRNA only (see Figure 3.18).

Figure 3.18: Supernatant of CSN5 knockdown cells leads to growth suppression in CaCo2 cells, which can be reduced by follistatin or inhibin A knockdown. Treatment of CaCo2 cells with supernatant of SW480 cells exhibiting a CSN5 knockdown decreased the proliferation. This effect could be reversed by 15 min preincubation of the supernatant with follistatin. The double knockdown of CSN5 and inhibin A in SW480 cells and the transfer of the supernatant on CaCo2 cells leads to no significant reversal of the effect but also to no significant reduction in the proliferation. The knockdown of inhibin A has no effect on the proliferation. The treatment with follistatin also showed no significant effect on the proliferation but perhaps a tendency towards an increased proliferation. Shown are means ±SEM of experiments performed in triplicates (scrRNA, CSN5 siRNA, CSN5+INHBA siRNA, INHBA siRNA; n=4), (CSN5 siRNA+follistatin; n=3), (scrRNA+follistatin; n=1), (* = p ≤ 0.05, two-tailed unpaired t-test).

It could be shown that the supernatant of SW480 cells following CSN5 knockdown

inhibit the proliferation of CaCo2 cells by 15-20%. This effect could be reversed by

treatment of these supernatant with follistatin. The additional knockdown of inhibin A

in SW480 cells also seem to prevent the growth suppressive effect mediated by the

CSN5 knockdown. This results indicates that the anti-proliferative effect of CSN5

knockdown, as seen in different cell lines, may be to some extent mediated through

activin signaling.

Page 83: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

71

3.12) Transfer of supernatants of SW480 cells on CaCo2 cells seems

to lead to an increased SMAD2 phosphorylation

Figure 3.19: Relative SMAD2 phosphorylation increases in CaCo2 cells after treatment using supernatants of CSN5 knockdown SW480 cells CaCo2 cells were incubated for 15 min with supernatant of SW480 cells treated with scrRNA or CSN5 siRNA. Follistatin treated supernatants were preincubated for 15 min prior to the transfer onto the CaCo2 cells. (A) Western Blot and immunodetection of phosphorylated SMAD2, total SMAD2 and tubulin in CaCo2 cells treated with supernatant of wildtype or CSN5 knockdown SW480 cells, and supernatants treated with follistatin. (B) Quantification using AIDA software of the phosphorylated SMAD2 normalized to total SMAD2. The data represents the mean of one experiment performed in duplicates.

As described before is the suppressive effect of activin A mediated in colorectal cancer

cells by SMAD signaling178. Because of this we wanted to elucidate if the treatment of

CaCo2 cells with supernatant of SW480 cells exhibiting a CSN5 knockdown possibly

induce the activation of SMAD signaling.

Therefore (like in 3.11) we transferred the supernatant of SW480 cells exhibiting a

CSN5 knockdown or were treated with scrRNA on CaCo2 cells. In addition, we

incubated the supernatant of SW480 cells treated with scrRNA or CSN5 siRNA with

follistatin for 15 minutes. Follistatin should bind to activins and thereby inhibit the signal

transduction via the activin receptor. The CaCo2 cells were incubated for 15 minutes

Page 84: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

72

with the supernatants followed by lysis of the cells and protein analysis using SDS-

PAGE gelelectrophoresis, western blot and immunodetection.

The treatment of CaCo2 cells with supernatant of CSN5 knockdown SW480 cells,

leads to 2.5-fold increase in phosphorylation of SMAD2 in contrast to the cells treated

with scrRNA (see Figure 3.19). Preincubation of the supernatant of SW480 cells

exhibiting a CSN5 knockdown with follistatin led to a decrease in phosphorylation of

SMAD2 in comparison to the CaCo2 cells treated with supernatant of CSN5

knockdown SW480 cells. But treatment of CaCo2 cells with supernatant of scrRNA

treated SW480 cells incubated with follistatin also increased the phosphorylation of

SMAD2 (around 1.7 fold). This effect is not as strong as the effect of the CSN5

knockdown supernatant. This result suggests that the knockdown of CSN5 leads to an

enhanced secretion of activin A which subsequently increases the phosphorylation of

SMAD2. This may to some extent inhibit the proliferation of CaCo2 cells. Because this

was a first experiment it needs to be repeated to validate the obtained data.

3.13) Effects of CSN5 on inhibin A mRNA expression seem to

depend on the p53 status in HCT116 cells

A second colorectal cancer cell line was used to confirm the results obtained in the

SW480 cells. Thereby, it should be proved that the effects of CSN5 on activin/inhibin

subunit expression are not cell line dependent. In these experiments the two different

subclones of the colorectal cancer cell line HCT116 were used. One clone possessed

a functional wild type p53 and the other one had a p53 knockout. The knockdown of

CSN5 was performed as described for the SW480 cells.

The knockdown of CSN5 showed contradictory results in the two used HCT116

subclones. In the clones having a wild type p53, the CSN5 knockdown led to a

decreased inhibin A mRNA expression. This result is contrary to the results obtained

in SW480 cells. But using HCT116 cells possessing a p53 knockout, we get an

increase in inhibin A mRNA expression comparable to the increase detected in

SW480 cells (see Figure 3.20). These results support the notion that CSN5 can

modulate the expression of activin/inhibin subunits. They also hint that p53 and

possible mutations in the protein may also influence the expression of inhibin A.

Page 85: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

73

Figure 3.20: Inhibin A expression in HCT116 cell lines after CSN5 knockdown depends on the p53 status RT-qPCR analysis of inhibin A mRNA expression in HCT116 p53wt/p53KO cells after knockdown of CSN5 for 72 h. (A) Significant decrease in inhibin A mRNA expression in HCT116 p53wt after CSN5 knockdown (B) The knockdown of CSN5 increased the inhibin A mRNA expression in HCT116 p53KO cells. Data represents means ± SEM of 5 independent experiments in duplicates (* = p ≤ 0.05; *** = p ≤ 0.005; two-tailed unpaired t-test).

Page 86: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

74

4) Discussion

4.1) CSN5 influences the gene expression in SW480 cells

The constitutive photomorphogenesis 9 (COP9) signalosome is a multiprotein

complex, which is evolutionary conserved between different species. It was first

described as a regulator of light induced growth in plants30. Until now, many different

cellular processes are described to be influenced by the CSN. A pivotal role is the

regulation of protein degradation by cullin RING E3 ligases54. The CSN consist of 8

subunits, which are numbered according to their weight37. The subunits can form the

CSN holocomplex, but also smaller subcomplexes consisting only of a few subunits

are described38. The fifth subunit (CSN5) has been discovered independently from the

CSN. It was found to stabilizes the transcription factor AP-144, consisting of a protein

of the Jun and Fos family. Thus it was first named c-jun activation domain binding

protein (Jab1). CSN5 is also known to be involved in many different cellular processes

such as protein degradation, regulation of signal transduction or cell cycle control179-

182. Thus aberrations in the cellular CSN5-level may consequently influence the

homeostasis of cells. This could lead to malignant transformation of cells and

subsequently cancerogenesis26,183. Anke Schütz showed in her PhD-thesis, that the

deletion of CSN5 in the intestinal epithelium causes severe changes in the cellular

composition of the intestinal epithelium which is eventually lethal172. This finding hints

towards the importance of CSN5 in the differentiation of cells in the intestinal

epithelium. The differentiation of cells is a complicated process which is regulated by

switching on and off genetic programs during the differentiation84,184,185. Therefore, I

set out wanted to investigate the influence of CSN5 on the expression of genes in the

intestinal epithelium. This might hint towards how CSN5 leads to this drastic phenotype

in mouse due to the knockout in the intestinal epithelium. As a surrogate for the

intestinal epithelium, I used the human colorectal adenocarcinoma cell line SW480 and

knocked CSN5 down by using siRNAs.

Surprisingly, despite the diverse function of the CSN and CSN5, in different cellular

processes. The knockdown of CSN5 leads only to a small number of strongly

differentially regulated genes. Only 44 genes showed a change in expression of about

two-fold (see figure 3.1 and table 3.1). If the threshold was raised to changes of at least

three fold in expression, only 6 genes were left. This is a rather small number of genes

Page 87: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

75

which expression seemed to be strongly influenced by CSN5. A reason for this could

be that the knockdown of CSN5 does not lead to a total loss of CSN5. The siRNA

mediated knockdown reduced the CSN5 expression around 80% and the protein level

around 70%. Thus the remaining CSN5 may be sufficient for the execution of some or

the critical functions of the protein in the cells. Therefore, the effects are likely not as

strong as in knockout cells. Indeed, it has been shown that the homozygous CSN5

knockout in mice is lethal. But, already the heterozygous knockout in mice and mouse

embryonic fibroblasts, shows a reduced growth and changes in cell cycle progression

(Kato, JBC 2004). On the other hand, SW480 are a colorectal carcinoma cell line.

These cells inherit mutations in genes of different proteins and subsequently signal

transduction and regulatory pathways. This can possibly influence the effects of CSN5

on the regulation of the transcription and may be an explanation for the observed

effects. The six genes which were threefold differentially regulated are the genes for

ADP-ribosylation factor-like 14 effector protein-like (ARL14EPL), keratin associated

protein 3-1 (KRTAP3-1), late cornified envelope protein 1F (LCE1F), -actin-like

protein 2 (ACTBL2), inhibin A (INHBA) and glia maturation factor (GMFG) (see

figure 3.2).

ADP-ribosylation factor-like 14 effector protein-like is so far only confirmed in mice.

Not much is known about the protein. It belongs to the ADP ribosylation factor family,

which are GTPases, acting as molecular switches by converting guanosine

triphosphate (GTP) into guanosine diphosphate (GDP)186,187.

Keratin associated protein 3-1 belongs to a protein family, which is important for the

interfilamentous matrix in the hair cortex. It leads to a rigid and resistant hair shaft.

Beside this, the expression in other tissues like breast or adipose tissue is known,

where it perhaps plays a role in the intracellular matrix188,189.

Late cornified envelope protein 1F is a precursor protein of the cornified envelope

in the stratum corneum. It belongs to a gene cluster of the epidermal differentiation

complex, which can be subdivided. So far only the expression in the skin was verified

for two groups (1 (where also LCE1F belongs to) and 2). But the isoforms of the third

group seem also to be expressed in other tissues190. New results indicate that group 1

is expressed in the intestinal epithelium and is regulated via p53191.

Actin -like protein 2 is a protein, which shares similarities with actins. It seems to be

a part of the central proteome192. Its function is unknown, but due to the similarity it is

Page 88: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

76

proposed to have functions in the cytoskeleton and is expressed ubiquitous in

eukaryotic cells.

Glia maturation factor is a barely described protein. The gene was identified and it

has been discovered as a part of the so called central proteome192. It seems to possess

kinase activity which can regulate the activation of other kinases and plays a role as

growth factor and in the cytoskeleton modeling193.

Inhibin A is part of the activin/inhibin signaling pathway. This protein family belongs

to the TGF- superfamily. Inhibin A can be a part of the activins which are homo- or

heterodimeric molecules. activins are involved in a variety of physiological processes

like proliferation, differentiation and apoptosis. Thus they play important roles in acute

and chronic inflammation, as also in carcinogenesis194-196. To date connections

between activin signaling and colorectal cancer are described. For example, the

inhibin A mRNA could be detected in intestinal cancer specimen but not in the

corresponding normal tissue167.

Despite huge changes in expression of a distinct gene, also the accumulation of small

changes in expression of genes belonging to a certain cellular process can be sufficient

to influence the homeostasis of cells. To investigate this option, the obtained data was

used for gene set enrichment analysis and gene ontology analysis. These types of

analysis are used to get further insights in the accumulation of differentially regulated

genes in certain processes after CSN5 knockdown. The gene set enrichment analysis

showed that the CSN5 knockdown influences different signaling pathways in colorectal

cancer cells (see figure 3.3 and table 3.2). A prominent pathway is the Wnt/ -catenin

pathway. The Wnt/ -catenin signaling is important for intestinal homeostasis and

renewal. Because of its function in the pluripotent intestinal stem cells, its dysregulation

is an important mechanism in the carcinogenesis of colorectal cancer197. About 90%

of colorectal cancers develop mutations in this pathway198. Mostly mutations occur in

the APC gene, which codes for a protein of the -catenin destruction complex199. But

also mutations in other components of the signaling pathway like -catenin itself

occur200. The mutations lead to an inhibited phosphorylation and following an inhibited

degradation of -catenin. This subsequently leads to a constitutively activation of the

-catenin signaling pathway201. CSN5 can influence the Wnt/ -catenin signaling

pathway by regulating the degradation of -catenin by cullin RING E3 Ubiquitin-

Ligases85,86. But also -catenin can influence CSN5 by transcriptional regulation of the

expression202,203. Recently an alternative degradation pathway of -catenin without the

Page 89: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

77

need of -catenin phosphorylation has been discovered204,205. This pathway also

seems to be influenced by CSN587. Because of these facts it is likely that changes in

CSN5 protein level influences this pathway.

Notch signaling is another important pathway in the control of differentiation in the

intestine206. Together with Wnt/ -catenin signaling it orchestrate the differentiation of

the epithelial cells in the intestine during the renewal of the epithelium207. In colon

cancer an upregulation of Notch1 could be detected208,209. But so far no connection

between CSN5 and Notch has been described. It could be shown that Notch1 interacts

with p53 and subsequently inhibit the transcription of p53 target genes210. This could

be a link between CSN5 and Notch signaling because CSN5 is known to regulate the

degradation of p5346,56,57. This could influence the transactivation abilities of Notch by

increasing the accessibility for other transcriptional activators, like p300 which can be

inhibited by p53211. Another possibility could be an influence of CSN5 or the COP9-

signalosome on the ubiquitination and degradation in the Notch pathway. The stability

of the intracellular domain of Notch1 is regulated by SCF E3 Ubiquitin-Ligase due to

interaction by Fbw7212. The stability of Fbw7 depends on the COP9-signalosome and

CSN5. Thus a knockdown of CSN5 leads to reduced Fbw7 levels213. This could lead

to an accumulation of intracellular domains of Notch1 and an enhanced activation of

target genes. Therefor CSN5 could influence the Notch signaling at different stages.

Another monitored signaling pathway which seems to be influenced by the CSN5

knockdown belongs to the TGF- superfamily. The TGF- superfamily plays an

important role in maintenance of pluripotency in stem cells and in the differentiation

beside other functions214. Because of its role in stem cells it seems obvious that

aberrant TGF- superfamily signaling is important in diseases and

carcinogenesis215,216. Key molecules in the signal transduction in the TGF-

superfamily are the SMADs. Upon activation by ligands of the TGF- superfamily

SMADs form a heterotrimeric complex which translocate in to the nucleus and enforces

gene transcription138,217. An inhibitory element in this pathway is SMAD7 which

antagonize the signaling by blocking the receptor complex218. CSN5 can regulate the

TGF- signaling by binding SMAD7 and promoting the degradation50. Another

important SMAD is the so-called Co-SMAD4 which is part of all heterotrimeric

complexes219. SMAD4 gets ubiqutinated and subsequently degraded by the Ubiqutin-

Ligase SCF -TrCP1220. CSN5 can directly interact with SMAD4 and promote the

ubiquitination and degradation via SCF -TrCP149,220. Furthermore, could the

Page 90: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

78

transcription factor AP-1 be a link between TGF- superfamily signaling and CSN5. It

is known that CSN5 can stabilize the transcription factor AP-148. Also described are

interactions between the SMAD complex and other transcription factors for example

AP-1221. Thus CSN5 may influence the transcriptional activities of TGF- superfamily

signaling by influencing the stability of AP-1. As seen for the signaling pathways before,

CSN5 seems to influence the TGF- superfamily signaling at different levels. The

analysis of the gene expression profile already showed a strong influence from CSN5

on inhibin A. Inhibin A is part of the activin/inhibin signaling pathway, which belongs

to the signaling pathways of the TGF- superfamily. This indicates, that the TGF-

superfamily, especially the activins/inhibins possibly play an important role in the

effects of the CSN5 knockdown.

Interestingly, there is an accumulation of up-regulated gene sets, which involves the

transcription factor c-myc. C-myc is a pleiotropic transcription factor and known as

oncogene. It is involved in diverse cellular processes like cell cycle control, apoptosis

or cell growth222,223. Therefore it also play an essential role in the tumorigenesis224. In

the intestine it has been shown that c-myc together with Wnt-signaling plays a role in

the homeostasis of the intestine and in the tumorigenesis225. Furthermore, c-myc

seems to be a target gene of Wnt-signaling226. C-myc is important for the proliferation

of intestinal cells and a loss of it leads to a reduced proliferation227,228. In human

colorectal cancer samples an overexpression of c-myc is commonly observed229.

C-myc is a target of a cullin RING Ubiquitin E3 ligase and the depletion of CSN5 leads

to an increase in c-myc level213. This could indicate a link between CSN5 and intestinal

tumorigenesis by up-regulation of c-myc and subsequently the up-regulation of c-myc

dependent gene sets.

Also, gene sets which comprise genes involved in cell cycle regulation are up-regulated

due to CSN5 knockdown. As described earlier CSN5 is known to have influence on

the cell cycle control for example by influencing p27KIP1, p53 or c-myc182. It is likely that

the deregulation of genes respectively proteins which contribute to the cell cycle control

leads to tumorigenesis230. Consequently, an influence of CSN5 on the cell cycle control

could be expected and also be a link between tumorigenesis and CSN5.

In the gene ontology analysis, it is compared, if up- or down-regulated genes are

accumulated in specific physiological processes. Here, it is noticeable that an

accumulation of up-regulated genes takes place only, in the generic transcription

pathway. This aligns with the findings that many signal transduction pathways, like

Page 91: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

79

Wnt/ -catenin, Notch or TGF- as discussed earlier, seem to be influenced by CSN5

in colorectal cancer cells. In 24 processes there is an accumulation of down-regulated

genes. Many of these processes belong to the cardiovascular system. Of interest could

be the processes located in the epithelium (epithelial cell differentiation, epithelium

development), because most colorectal cancer develop from the intestinal

epithelium231. These findings could support the important role of CSN5 in the

homeostasis of the intestinal epithelium.

For the whole analysis of the results it has to be kept in mind that this experiment was

performed in a colorectal cancer cell line. Therefore, the significance of the influence

of CSN5 on gene expression respectively changes in gene sets may be to some extent

due to the malignant transformation of the cells. Additionally, the extent of changes

after CSN5 knockdown in gene expression or the influence of gene sets could be

influenced by the malignant state of the cells. Thus it would be helpful to verify some

results in non-transformed primary intestinal cells or cell lines which do not originate

from tumors.

Nevertheless, the role of different signaling pathways in the development and

homeostasis of the intestinal epithelium has been discussed so far. A starting point for

the project, was the result that the knockout of CSN5 in the intestinal epithelium of mice

leads to changes in the cellular composition of the epithelium. Because of the role of

signaling pathways in the differentiation of the intestinal epithelium we decided to take

a closer look at the different signaling pathways84. The activin/inhibin signaling pathway

belongs to the TGF- superfamily. In our results inhibin A, one of the subunits of the

activin/inhibin signaling molecules, was strongly influenced in expression by CSN5.

Because so far no connection between CSN5 und activin/inhibin signaling was made,

we started to investigate the influence of CSN5 on this signaling pathway.

4.2) CSN5 and the COP9-signalosome modulate the expression of

inhibin βA in SW480 cells

The knockdown of CSN5 showed only a low number of strongly differentially regulated

genes. Among them was inhibin A which codes for a protein of the activin/inhibin

signaling pathway that belongs to the TGF- superfamily. In the gene enrichment

analysis, the TGF- signaling was identified as significantly influenced by the CSN5

knockdown. This encouraged me to take a closer look at this signaling pathway. In a

first step I could verify that the CSN5 knockdown influences the expression of the best-

Page 92: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

80

characterized inhibin subunits. The RT-qPCR could validate the enhancing effect of

the CSN5 knockdown on inhibin A mRNA expression, which was detected in the

microarray. Nevertheless, it could be shown also that the inhibin α mRNA as also the

inhibin B expression is significantly decreased after CSN5 knockdown.

So far not much is known about the regulation of the expression of activins and

inhibins. For inhibin A an enhancer region called DR-1 and a promoter region called

PR-2 are described, to be important for the expression232. The inhibin A enhancer

contains a core region with two identified binding sites. One of the sites is a AP-1 like

sites, TRE (12-O-tetradecanoylphorbol-13-acetate response element) and the other a

CREBP/ATF (cAMP response element) site232. Beside this the binding of two specific

protein complexes to this region could be shown. These protein complexes seem to

contain AP-1 transcription factors, but they seem not necessarily required for

transcription. Because deletion of the AP-1 site only moderately decreases the

expression, while deletion of the CREBP/ATF site abolishes the expression232. These,

AP-1 like sites, could be one possibility where the regulation via CSN5 takes place. It

is known that CSN5 can stabilize the transcriptional coactivator AP-144,47,48 but also

other transcription factors like HAND2 or HIF-1α51,60. But the enhancing effect of the

CSN5 knockdown on the inhibin A expression is contrary to a possible AP-1

stabilizing and subsequent expected enhancing effect of CSN5. The deletion of the

intermediate region between these two binding sites leads to a moderate induction of

the transcription. In this region a putative binding site for the drosophila protein dorsal

could be identified, which belongs to the transcription factors of the Rel protein

family232. These transcription factors normally repress the transcription. This could be

a possible site of influence for CSN5.

To get further evidences, CSN5 overexpression was performed. The overexpression

of CSN5 did not influence the protein levels of CSN1 but increases the CSN5 protein

level. Because of this we suppose that most probably the overexpression of CSN5

leads to an enrichment of monomeric CSN5, but barely changes the amount of COP9-

signalosomes. The overexpression did not significantly influence the expression of

inhibin mRNAs (see figure 3.7). This could rather indicate a function of the COP9-

signalosome in the regulation of the expression than an effect of the monomeric CSN5.

But it has to be kept in mind that we were working with a colorectal cancer cell line.

Thus the CSN5 amount could be very high in the cells, from the beginning. A further

increase in CSN5 might have no effect because a saturation of CSN5 already exists.

Page 93: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

81

The catalytic activity of CSN5 in the COP9-signalosome is one of the best described

functions of CSN5. There are transcription factors, as also transcriptional pathways,

which are influenced through regulation of degradation by the CSN. For example IRF5

associates with the CSN and upon stimulation this association gets lost and IRF5 gets

degraded via the proteasom233. Additionally, there are first hints that CSN itself can

regulate transcription through association with transcriptional elements on chromatin75.

Therefore, different mechanisms are possible for CSN5 to regulate the transcription as

part of the COP9-signalosome. To proof this possibility a knockdown of other subunits

of the COP9-signlosome was performed. The subunit CSN1 was chosen because it

has been shown that CSN1 can only be found in the COP9-signalosome and not as

monomer or in subcomplexes27. As a second subunit CSN2 was chosen.

It has been shown that the reduction of CSN5 leads to a reduction of other subunits of

the COP9-signalosome172,173. This could be verified in our hands. The knockdown of

CSN5 leads to less reduction of other CSN subunits than of CSN5 itself. Interestingly,

the knockdown of other CSN subunits, in our case CSN1 and CSN2, reduced the

CSN1 and CSN8 protein level strongly (about 70-80%) but did not as much influence

the CSN5 level (about 30-50%). That may hint towards a stronger effect of the CSN5

knockdown on the monomeric CSN5 than on the COP9-signalosome, where the

knockdowns of other CSN subunits strongly reduce the amount of the COP9-

signalosome but the monomeric CSN5 is not affected to such an extent. The

knockdown of CSN1 reduced the inhibin A mRNA expression significantly. In contrast

the knockdown of CSN2 has no influence on the inhibin A mRNA expression. The

result of the CSN1 knockdown could indicate a role of the COP9-signalosome in the

regulation of inhibin A expression, while the knockdown of CSNβ does not.

Surprisingly is the effect of the CSN1 knockdown contrary to the effect of the CSN5

knockdown.

To further evaluate a possible role of the COP9-signalosome in the regulation of

inhibin A expression, I took a closer look at a main target of the catalytic activity of

the COP9-signalosome. Cullin RING E3-Ubiquitin ligases are regulated in their

specificity and activity by DeNEDDylation through the COP9-signalosome54,67,213,234-

236. Therefor a knockdown of CSN5, cullin1 and both together was performed. The

cullin1 knockdown shows no influence on the expression of inhibin A mRNA. In

contrast, the knockdown of cullin1 and CSN5 decreased the inhibin A mRNA

expression to a level comparable with the expression in scrRNA treated control cells.

Page 94: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

82

This result hints towards a transcriptional regulator, which gets degraded by cullin1

RING Ubiquitin E3 ligases. Because the knockdown of cullin1 has no effect on the

expression of inhibin A, it seems that the degradation is not constitutive. May be

CSN5 prevents the ubiquitination, and subsequent degradation of the repressor

through cullin1 based CRLs. Despite this it could also be that the remaining cullin1

RING E3 Ubiquitin-Ligases are sufficient for the basal degradation of the repressor.

Thus no effect on the inhibin A expression after cullin1 knockdown can be observed.

But the rescue effect by knockdown of cullin1 additionally to CSN5 may be an

indication that CSN5 inhibits the degradation of the transcriptional regulator. Thus a

knockdown of CSN5 may render a regulator accessible for cullin1 RING E3-Ubiquitin

ligase dependent ubiquitination and subsequent degradation.

Figure 4.1: Proposed model for the influence of CSN5 on inhibin A expression CSN5 stabilize a repressor or repressor complex (X) perhaps by reducing the turnover rate of the repressor at the transcriptional site. This leads to a reduction of the transcription. The reduction of CSN5 enhances the turnover rate of the repressor from the transcriptional site. This possibly enhances the cullin1 RING Ubiquitin E3 ligase mediated ubiquitination and subsequent degradation.

To further investigate this hypothesis, I used the NEDDylation inhibitor MLN4924174,175.

MLN leads to deNEDDylated cullins which should lead to inactivation of CRLs and

subsequent degradation. In contrast to the cullin1 knockdown did the MLN treatment

additionally to the CSN5 knockdown not influence the inhibin A expression. A possible

explanation for this observation could be the different durations of the treatments. The

knockdowns last for about 72 h, while the MLN treatment only takes place in the last

16 h of the knockdown. Therefore, the degradation of the biggest amount of

Page 95: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

83

transcriptional regulator may occur before the MLN treatment starts. Thus the MLN

treatment cannot influence the degradation due to the absence of the regulator.

Perhaps treatment of the cells using MLN in the first hours of the knockdown may lead

to effects comparable with the cullin1 knockdown.

Due to the results gained in the RT-qPCRs the following model for the regulation of

inhibin A expression could be proposed. An unknown repressor or a repressor

complex (X) gets stabilized by or in the presence of CSN5. This could be done, for

example by enhancing the DNA binding. It has been shown for the transcription factor

AP-148 or HIF1-α237 that CSN5 can stabilize transcriptional elements. Also could the

shuttling between nucleus and cytoplasm and therefor the turnover rate of the

repressor at the promoter be influenced. That CSN5 can facilitate the subcellular

localization is already known for other proteins like p27KIP1, p53 or RUNX355,56,238.

Posttranslational modifications, which may be influenced by CSN5, could also play a

role for stabilizing the repressor, as well. They could influence the binding to the DNA,

the shuttling between nucleus and cytoplasm. The mechanism could be comparable

to the effects of CSN5 on CDK2. The loss of CSN5 results in an increased

phosphorylation of CDK2 and cytoplasmic accumulation239. Due to binding of the

repressor to the DNA or the nuclear localization, the repressor might be less accessible

for the degradation. This subsequently leads to a low transcription rate. A reduction or

loss of CSN5 destabilizes the repressor/ repressor complex and thus makes him

accessible for degradation, perhaps in the cytoplasm. This reduction may be mediated

through cullin1 RING E3 Ubiquitin-Ligases and Proteasom dependent degradation.

The loss of the repressor leads to an enhanced expression of inhibin A.

The next step could be the identification of possible transcriptional elements in the

promoter region of inhibin A, which may interact with CSN5. The identification of the

possible repressor or repressor complex could be done by an electrophoretic mobility

shift assay (EMSA) using different sequences of the promoter region. In this assay the

whole cell lysates or nuclear protein fraction of CSN5 knockdown cells could be

compared to scrRNA treated cells as control. By using in silico methods possible

repressor binding sites could be identified. Using mass spectrometry perhaps possible

repressor proteins could be identified. Also a possible involvement of CSN5 in this

repressor complex could be investigated. Via chromatin immunoprecipitation (ChIP)

and immunoprecipitation (IP) it could be further examined if CSN5 is directly involved

in the repressor DNA interaction.

Page 96: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

84

4.3) CSN5 does not seem to alter inhibin βA protein levels

The enhanced expression of Inhibin A mRNA and the decreased expression of

inhibin α mRNA would suggest an enhanced assembly of activin A which consist of

two inhibin A subunits.

It has been shown that activin A in the context of the intestine seems to inhibit the

proliferation and induces apoptosis165,168,178. The knockdown of CSN5 also shows an

inhibition of proliferation in different colorectal cancer cell lines85. Thus it could be that

anti-proliferative effect of CSN5 may be to some extend mediated via an increased

activin A synthesis and secretion. The analysis of whole cell lysates of SW480 cells

exhibiting a CSN5 knockdown showed no changes in the inhibin A protein level

compared to scrRNA treated cells. Thus the question arises if the enhanced inhibin A

mRNA level really leads to enhanced Inhibin A protein synthesis. It has to be

mentioned that the used inhibin A antibody detects only the pro A peptide with a

weight of around 46 kDa240. But for activin A and other TGF- superfamily ligands, it

has been shown that the pro-peptides are important for the assembly and secretion of

the mature dimeric proteins103,106. This hints that an increased synthesis of mature

activin A should be reflected in enhanced levels of inhibin A pro-peptides.

Nevertheless, it would be important to detect the mature activin A in whole cell lysates

using an appropriate antibody. Unfortunately, so far no antibody against mature

activin A for western blot analysis could be found in the literature.

An explanation for the obtained results could be posttranslational regulation. This could

be, for example, enhanced secretion which may prevent an accumulation of protein in

the cells. To test whether enhanced secretion may play a role, Monensin as an inhibitor

for classical protein secretion via the Golgi-apparatus was used241. Indeed, we could

monitor an increase of around 3 fold in intracellular inhibin A protein level, after CSN5

knockdown in SW480 cells using Monensin. This increase was comparable to the

increase in inhibin A mRNA expression after CSN5 knockdown. This indicates that

the secretion of activins or inhibin seems to be increased after CSN5 knockdown. So

far not much is known about the regulation of activin A secretion in intestinal epithelial

cells. It seems that the expression and secretion of activin A depends on the

differentiation state of cell in the intestinal epithelium. In the colorectal cancer cell line

CaCo2 could be observed that the differentiation of this cell line to absorptive like cells

leads to a decrease in inhibin A expression168. Beside this, the fact that activin A

Page 97: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

85

inhibits proliferation and induces apoptosis it seems likely that the expression occurs

in differentiated cells which are mainly at the upper areas of the villi in the intestine.

The possible enhanced secretion should lead to rise in activin A levels in the

supernatant. Unfortunately, we could not detect reliably activin A in the supernatants

via ELISA measurement (data not shown) neither in the scrRNA treated cells nor in

the CSN5 siRNA treated cells. Thus we decided to precipitate the proteins out of the

supernatants and tried to detect the inhibin A pro-peptide which should be secreted

together with activin A103,106. Using western blot analysis, we could detect an increase

in inhibin A pro-peptide in the supernatants. This hints that there could be an increase

in secreted activin A. The fact that an increase in the amount of pro-peptide could be

detected while not in mature activin A could be due to different reasons. It could be

that the activin A is hidden by the bound pro-peptides. Thus the activin A cannot bind

to the capture antibody of the ELISA or the detection antibody cannot recognize the

bound activin A. But the fact that we used a commercial available ELISA which is used

in other publications makes it rather unlikely that this is happening. Also could it be that

the secreted activin A is rapidly bound in auto- and paracrine fashion to the cells and

subsequently gets internalized while the pro-peptide has a longer half life time in the

supernatant. Therefore, activin A cannot be detected by ELISA while the pro-peptide

can be precipitated out of the supernatant.

Also enhanced degradation, due to enhanced protein levels, might compensate the

increase in inhibin A expression after CSN5 knockdown. Therefore, we used in our

experiments the inhibitor MG132 as an inhibitor of the proteasomal degradation or

MLN4924 as an inhibitor of the cullin RING E3 ubiquitin-ligase dependent

ubiquitination174-176.

A little bit puzzling are the results that the inhibition of proteasomal degradation at

different stages (ubiquitination respectively degradation) leads to reduced inhibin A

protein level. A reduction of protein level after inhibition of degradation may hint

towards an imbalance of loss and new synthesis of protein. Because no accumulation

of inhibin A takes place after blockage of the proteasomal degradation the loss may

be due to secretion of inhibin A as activin A or inhibin A. The blockage of the

proteasomal degradation may prevent the degradation of a transcriptional repressor.

Thus the transcription may be decreased and lower as the secretion.

But the results of the RT-qPCR after MLN treatment as also after cullin1 knockdown

are contrary to this hypothesis. The treatments did not influence the expression of

Page 98: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

86

inhibin A mRNA. These findings suggest that there are no influences of CRLs in the

transcriptional regulation of inhibin A expression.

It remains so far unclear why the inhibition of proteasomal degradation leads to a

decrease in inhibin A protein level. To test if the reduction of inhibin A is due to an

imbalance between secretion and new synthesis the inhibitor Monensin could be used.

Monensin should inhibit the secretion of inhibin A respectively activin A as shown

before. If the use of Monensin additionally to MLN or MG132 would reduce the effects

on inhibin A protein level, it would be a hint that enhanced secretion and less new

synthesis may lead to the observed results. Also should be checked if the cullin1

knockdown exhibits the same influences on inhibin A protein level as the MG132 or

MLN treatment.

4.4) Enhanced activin A secretion after CSN5 knockdown can inhibit

the proliferation of colorectal cancer cells

The knockdown of CSN5 in SW480 increases the expression of inhibin A mRNA and

decreases the expression of inhibin α mRNA and inhibin B mRNA. But on protein

level we could not confirm an increase of inhibin A. Also an increase of Inhibin A

protein after CSN5 knockdown could be obtained by blocking the classical secretion.

Therefore, we suggested an enhanced activin A assembly, which gets subsequently

secreted. In the literature the activin A signaling is linked to proliferation, as well as

CSN5. Schütz et al. could show that CSN5 knockdown lead to growth suppression in

colorectal cancer cell lines and Bauer et al. could show that activin A acts as a growth

suppressor in colorectal cancer cell lines85,178. Because of these findings we wanted to

investigate if proliferative effects of CSN5 may be, to some extent, mediated through

activin/inhibin signaling. To test this hypothesis a knockdown of CSN5 was performed

in SW480 cells, using a siRNA approach and the supernatant was transferred onto

CaCo2 cells. This was done because SW480 cells have an impaired SMAD signaling

pathway. It was shown that the growth suppressive effects of activin A are mediated

through SMAD signaling178. Thus we did not expect any activin A mediated growth

suppressive effects in SW480 cells which was also showed by Bauer et al178.

An additional reason why we performed the CSN5 knockdown in SW480 cells is the

knockdown efficiency. The SW480 cells exhibit a higher knockdown efficiency and this

is supposed to lead to higher secretion of activin A. Also we were not able to detect

any inhibin A expression in CaCo2 cells. Thus we could not be sure that CaCo2 cells

Page 99: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

87

are able to secret activin A and stimulate themselves in an auto- or paracrine fashion.

Furthermore, this setting excludes any adaption of the cells to a permanent stimulation

via activin A which may lead to a decrease in the proliferative effect due to possible

desensibilization.

The transfer of supernatant, of SW480 cells exhibiting a CSN5 knockdown, lead to a

significant decrease in proliferation in CaCo2 cells, compared to CaCo2 cells treated

with supernatant of the scrRNA treated SW480 control cells. To confirm if the effects

may be mediated via secreted activin A, supernatant of CSN5 knockdown SW480 cells

were preincubated with Follistatin. Afterwards CaCo2 cells were stimulated with these

supernatants. Follistatin is an inhibitory protein for activin A, which binds to it and

prevents the assembly of a functional signaling complex consisting of activin A and the

two activin receptors127,242. Indeed, did Follistatin restore the proliferation rate of

CaCo2 cells treated with CSN5 knockdown supernatant compared to the scrRNA

supernatant treated cells. As a second hint a knockdown of CSN5 or/and inhibin A in

SW480 cells were performed. Then CaCo2 cells were stimulated using these

supernatants. The aim was to compensate the enhanced expression of inhibin A by

knocking it down. This approach leads to no significant reduction in proliferation in

comparison to the control cells. But also did not significantly enhance the proliferation

in comparison to the CaCo2 cells treated with supernatant of CSN5 knockdown SW480

cells. These inconsistent results could be due to the fact that a knockdown, using a

siRNA approach, barely leads to a total termination of the expression. It could be that

the knockdown of inhibin A could not totally compensate the enhanced inhibin A

expression after CSN5 knockdown. Thus some protein synthesis remains which is

followed by secretion of activin A into the supernatant. This could subsequently induce

small growth inhibitory effects. Beside this the reduction of proliferation after treatment

using supernatant, of CSN5 knockdown cells, is not very high. Thus to get significant

effects the results must be very consistent. Because of different knockdown

efficiencies, of CSN5 and inhibin A, in each experiment, the amount of secreted

activin A may also varies strongly between experiments. If the number of experiments

would be increased, perhaps it could be possible to reach some significance for this

approach.

It has been shown that the growth inhibition through activin A is mediated via the SMAD

signaling pathway157,178,243. Bauer et al. also showed that the growth inhibitory effect of

activin A in colorectal cancer cells, is mediated via the SMAD signaling pathway178.

Page 100: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

88

This is the reason, why we checked the phosphorylation of SMAD2 in CaCo2 cells

after stimulation with supernatant of SW480 cells. In CaCo2 cells treated with

supernatant of SW480 cells exhibiting a CSN5 knockdown we could detect an

increased phosphorylation in comparison to the control cells. This phosphorylation

could be decreased by preincubation of the supernatants with Follistatin. These results

support the hypothesis that the knockdown of CSN5 lead to enhanced activin A

synthesis and secretion, which mediates a growth suppressive effect via SMAD

signaling. This experiment was so far only performed once and need to be repeated to

validate the results.

In future experiments the setup could be used to validate the results in other colorectal

cancer cell lines. Also it should be checked if the observed growth suppressive effect

could be mediated auto- or paracrine. Therefore, a knockdown of CSN5 in another

colorectal cancer cell line, with a functional SMAD signaling pathway and activin A

secretion should be performed. Then it should be checked if possible growth

suppressive effects are mediated via activin A by using for example Follistatin or a

knockdown of inhibin A.

4.5) The p53 status in the cell influences the expression of

inhibin A

The protein p53 is often called the gatekeeper of the cell. Usually it gets rapidly

degraded via MDM2244. Different stress stimuli, like DNA damage, lead to a

stabilization of p53 and subsequent transcriptional activity245,246. It has been shown

that CSN5 can influence the stability of p53 via MDM246,57. In most tumors occur

mutations of p53, which lead to changes up to loss of functionality247. In our

experiments we could show that the p53 status, of a cell seem to influence the

expression of inhibin A. In HCT116 p53KO cells we could see the same effects on

inhibin A transcription after CSN5 knockdown as in SW480 cells. SW480 cells

possess a truncated p53, which may lose some functions248. In contrast, in HCT116

p53wt cells we could observe an opposite effect. The CSN5 knockdown decreases the

inhibin A expression.

P53 might repress the expression of inhibin A through the p53-p21 –DREAM

or -RB/E2F complex249. This effect might be stronger than the possible influence of the

proposed transcriptional repressor. Therefore, an enhanced expression due to loss of

the repressor might be compensated by the effect of the stabilized p53. CSN5

Page 101: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

89

stabilizes MDM2 and therefore antagonize the stability of p5357. Thus a knockdown of

CSN5 should lead to stabilization of p53. In p53 knockdown cells the CSN5 knockdown

cannot lead to enhanced p53 levels and subsequent not mediate the transcriptional

repression. In SW480 cells p53 is also truncated. The fact that the knockdown of CSN5

in SW480 cells mimics the effect in HCT116 p53KO cells may be a hint that the

truncated p53 has lost its repressive ability. To test if p53 has an inhibitory function on

inhibin A expression, a reconstitution of wildtype p53 in SW480 or HCT116 p53KO

cells during a CSN5 knockdown should be performed. If the reconstitution of p53 in

these cell lines oppose the effect of the CSN5 knockdown, this would be a hint that

p53 has a suppressive effect on inhibin A expression. Also it could be done the other

way round and a knockdown of p53 and CSN5 in HCT116 p53wt cells could be

performed. If this would enhance the inhibin A expression, it would also hint towards

an inhibitory effect of p53.

4.6) CSN5 seems to influence inhibin α expression via cullin1 RING

E3 Ubiquitin-Ligases

In rats it has been discovered that different regulatory mechanism seem to be

responsible for the transcription of inhibin α and inhibin B250. The results so far

suggest that there is a different regulatory mechanism for the transcriptional regulation

of inhibin α then for inhibin A.

A transcriptional repressor or repressor complex (Y) seems to be constitutively

degraded via cullin1 RING E3 Ubiquitin-Ligase. The knockdown of CSN subunits,

leads to a reduced degradation of the repressor complex due to decreased

deNEDDylation of the CRLs. A sustained NEDDylation of CRLs leads to auto-

ubiquitination of the CRLs and subsequent degradation67,213. Furthermore, the

impaired amount of CRLs may lead to an accumulation of the repressor. This could

enhance the binding of the repressor to the promoter region of the inhibin α gene and

decrease the inhibin α expression. In line with the hypothesis that the CSN influences

the expression of inhibin α via CRLs, the knockdown of cullin1 as well decreases the

inhibin α expression. Also the inhibition of NEDDylation of CRLs and subsequent

deactivation of CRLs via the inhibitor MLN4924 leads to a decreased inhibin α

expression. These facts further support the theory that CRLs play an important role in

the transcriptional regulation of inhibin α.

Page 102: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

90

Figure 4.2: Proposed model for the influence of CSN5 on inhibin α expression A transcriptional repressor (Y) gets probably, constitutively ubiquitinated by cullin1 RING Ubiquitin E3 ligases and subsequently degraded. The reduction of CSN5, or the loss of CRL functionality, leads to an accumulation of the repressor and decreases the expression of inhibin α.

Some things are known of the inhibin α promoter region and possible transcriptional

regulation but not in intestinal cells. The promoter region seem to contain no TATA

boxes and also no GC rich sequences250. These promoters are usually not

constitutively active but regulated during differentiation or development251. A potential

transcriptional element, which was identified, is a binding site for cAMP- and

phorbolester-responsive element (CRE)250,252. This site is also highly homologous to

the transcription factor AP-1 consensus binding site250. But in cytotrophoblasts it

could be shown that AP-1 seems not to be involved in inhibin α expression253. In

contrast it could be shown that cAMP via CRE-binding protein (CREB) in cooperation

with AP-2 transcription factor influences the expression of inhibin α in rat ovarian

granulosa cells respectively in human trophoblasts254,255. Another transcription factor

family which is described to be involved in the regulation of Inhibin α are the

transcription factors of the GATA family256,257. As a repressor for inhibin α expression

the CCAAT/enhancer binding protein- (C/EBP- ) could be identified in ovarian

granulosa cells258. So far no implications of CSN5 or the COP9-signalosome on one

of these transcription factors are described. For CSN5 it is only known that its

promoter also exhibits C/EBP- binding sites and that the expression is also inhibited

by binding of C/EBP- binding259. Thus further efforts have to be made to identify the

way how the COP9-signalosome influences the inhibin α expression.

The next step like for inhibin A could be the identification of possible transcriptional

elements in the promoter region of Inhibin α, which may interact with CSN5. As

Page 103: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

91

described for inhibin A the identification of the possible repressor or repressor

complex could be done by an electrophoretic mobility shift assay (EMSA), mass

spectrometry (MS) or via chromatin immunoprecipitation (ChIP) and

immunoprecipitation (IP).

Page 104: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

92

5) Summary

CSN5 is a subunit of the constitutive photomorphogenesis (COP9) signalosome

(CSN). This multifunctional 600 kDa protein complex functions as a deNEDDylase,

regulating SCF E3 ubiquitin ligase mediated ubiquitin/26S proteasome

(UPS)-dependent protein degradation and signaling platform to control pivotal cellular

processes such as protein degradation, signal transduction or the cell cycle. CSN5

harbors the deNEDDylase activity of the CSN, but also has several activities

independent of its integration into the CSN complex. For example, it can act as a

transcriptional coactivator for various transcription factors (e.g. AP-1, NF-κB, 53BP1 or

HAND2). CSN5 also influences the nuclear export and degradation of proteins like p27,

p53 or SMAD7. CSN5 also has been implicated in tumorigenesis in several cancer

entities. At the same time CSN5 drives cell proliferation and is critical for cell

homeostasis. Moreover, prior work in our laboratory demonstrated, that the conditional

homozygous deletion of Csn5 in the intestinal epithelium is lethal for mice. It also

causes severe changes in the cellular composition of the intestinal epithelium (Schütz,

Bernhagen et al., unpublished data and Schütz A. PhD thesis, RTWH Aachen

University, 2011). This finding hints towards the importance of CSN5 in the

differentiation of cells in the intestinal epithelium. The differentiation of cells is a

complex process which is regulated by the switching-on and -off of central genetic

programs. The overall aim of my PhD thesis was to investigate the influence of CSN5

on the expression of genes in the intestinal epithelium. As a surrogate for the intestinal

epithelium I used the human colorectal adenocarcinoma cell line SW480. Csn5 gene

deletion was mimicked by siRNA-mediated gene silencing of CSN5.

I showed that the knockdown of CSN5 led to a change in the gene expression profile

of SW480 cells. Surprisingly the overall number of strongly regulated genes was

unusually low. Only 44 genes showed a significant twofold change in expression while

only 6 of them were differentially regulated by an at least three-fold range. These genes

were ADP-ribosylation factor-like 14 effector protein-like (ARL14EPL),

keratin-associated protein 3-1 (KRTAP3-1), late cornified envelope protein 1F

(LCE1F), -actin-like protein 2 (ACTBL2), inhibin A (INHBA) and glia maturation

factor (GMFG). In further analysis I demonstrated by gene set enrichment analysis

that the knockdown of CSN5 leads to the accumulation of differentially regulated genes

Page 105: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

93

in certain cellular processes e.g. KEGG_NOTCH_SIGNALING_PATHWAY,

KEGG_TGF_BETA_SIGNALING_ PATHWAY, HALLMARK_WNT_BETA_CATENIN

SIGNALING, CELL_CYCLE_PROCESS or REACTOME_CELL_CYCLE. Notably

genes of different signaling pathways were negatively influenced by the CSN5

knockdown. These pathways (e.g. Notch, Wnt or TGF- ) have been described to be

involved in the orchestration of the differentiation process in the intestinal epithelium

and may constitue a link between CSN5 and the differentiation process of the intestinal

epithelium. Furthermore, genes assigned to cell cycle processes were found to be

influenced by the CSN5 knockdown. Gene onthology analysis revealed that CSN5

negatively influences genes in different processes regarding development or

differentiation of a variety of tissues (e.g. epithelium, heart or skeletal system). This

may further support the hypothesis that CSN5 plays a role in the regulation of

differentiation.

Based on these results and on the identification of inhibin A in the knockdown

experiment, I investigated the role of CSN5 in the activin/inhibin signaling pathway.

This pathway belongs to the TGF- superfamily of signaling pathways. There is also

emerging evidence in the literature, supporting the hypothesis that this signaling

pathway plays a role in the intestinal epithelium. I showed that the CSN5 knockdown

influences the expression of different members of the activin/inhibin signaling pathway.

Using RT-qPCR I could validate that the knockdown of CSN5 led to an increase in

inhibin A expression and a decrease in inhibin α and inhibin B expression. Despite

this the overexpression of CSN5 did not influence the expression of any of the inhibin

subunits. The knockdown of the CSN subunits 1 and 2 also led to a decreased inhibin α

expression. The inhibin A expression was also reduced by CSN1 knockdown in

contrast to the CSN5 knockdown. Applying Western blot experiments, I could show

that the knockdown of CSN5 led to a reduction of all studied CSN subunits (CSN1, 5

and 8). But the knockdown of CSN1 and 2 led to a significantly stronger reduction of

CSN1 and 8 protein levels in comparison to CSN5. Thus, I hypothesized that CSN5 as

part of the COP9-signalosome influences the inhibin α expression while inhibin A

expression may additionally be influenced by the CSN5 monomer.

One of the best-described functions of the CSN is the regulation of cullin RING E3

ubiquitin ligases. These ligases mark proteins for proteasomal degradation. By

knocking down cullin1 additionally to CSN5 I could restore inhibin A expression

almost back to normal. While the cullin1 knockdown alone did not influence the

Page 106: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

94

inhibin A expression as measured by RT-qPCR. Inhibin α expression was also

decreased by cullin1 knockdown, which even seemed to enhance the effects of CSN5

knockdown. The inhibition of cullin RING E3 ubiquitin ligases by MLN4924 treatment

showed the same effect as the cullin1 or CSN5 knockdown for inhibin α. For inhibin A

inhibition by MLN4924 did not alter the effect of the CSN5 knockdown. On the other

hand, MLN treatment alone led to a decrease in inhibin A protein level.

Another factor that may influence activin/inhibin subunit expression by CSN5 is the

tumor suppressor protein p53. In the colon adenocarcinoma cell line HCT116

harboring a p53 knockout, I could show that CSN5 knockdown led to comparable

results regarding inhibin A expression, as in SW480 cells. It should be noted, that

SW480 cells harbor a truncated p53. By contrast, wildtype HCT116 cells expressing

normal levels of wildtype p53 showed contrary results. This indicates that p53 may be

important for the influence of CSN5 on activin/inhibin subunit expression.

On protein level I could not detect any changes in inhibin A after CSN5 knockdown.

Pilot experiments suggest that the possible enhanced protein synthesis may be

compensated by enhanced secretion of inhibin A. This may prevent an intracellular

accumulation of inhibin A.

On functional level I could show that the supernatant transfer of CSN5 knockdown cells

onto untreated cells results in a decrease of proliferation in the recipient cells. This

inhibitory effect seems to be mediated by activins, as it was revertable by follistatin, an

inhibitory protein of activins.

This thesis provided evidence that CSN5 plays a role in the differentiation of the colon

epithelium. This influence seems to be, at least in part, mediated by transcriptional

effects of CSN5, as differential gene expression profiles were obtained in colorectal

cancer cells upon CSN5 knockdown. In focusing on one of these regulated factors, my

thesis showed that CSN5 influences activin/inhibin signaling pathway. This pathway

may be a specific link between CSN5 and the homeostasis of the intestinal and

especially the colonic epithelium. My results might be a start point, to take a closer look

at the role of CSN5 or the COP9-signalosome in intestinal differentiation and

homeostasis, as also the cancerogenesis of the intestine.

Page 107: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

95

6) Zusammenfassung

CSN5 ist die fünfte Untereinheit des COP9-Signalosoms (CSN), eines 600 kDa

schweren Multi-Proteinkomplexes mit vielen Funktionen in der Zelle. Eine der

wichtigsten ist die Regulation der Proteinlevel in der Zelle. Dies geschieht durch

deNEDDYlierung von Ubiquitin-Ligasen, welche wiederum im NEDDyliertem Zustand

Ubiquitin an Proteine binden. Diese werden dadurch für den Abbau durch das 26S

Proteasom markiert. Daneben erfüllt es aber noch weitere Funktionen, unter anderem

bei der Signaltransduktion und hat dadurch Einfluss auf viele zelluläre Prozesse (z. B.

den Zellzyklus). Die wichtige deNEDDylase-Aktivität des COP9-Signalosoms, wird

durch CSN5 vermittelt. Daneben konnten aber auch viele andere Funktionen von

CSN5, auch außerhalb des COP9-Signalosoms, bereits identifiziert werden. CSN5

kann als Cofaktor für viele Transkriptionsfaktoren (z. B. AP-1, NF-κB, 5γBP1 oder

HAND2) wirken. Desweiteren, wurde die Beteiligung von CSN5 am Export von

verschiedenen Proteinen (z.B. p27, p53 oder SMAD7) aus dem Zellkern beschrieben.

CSN5 hat Einfluss auf das Zellwachstum und spielt eine wichtige Rolle in der

Homöostase von Zellen. In verschiedenen Tumorarten konnte bereits eine Rolle für

CSN5 nachgewiesen werden. In unserem Labor konnten wir bereits zeigen, dass der

homozygote Knockout von Csn5 im Darmepithel von Mäusen tödlich verläuft. Der

Knockout führt zu schwerwiegenden Veränderungen in der zellulären

Zusammensetzung des Darmepithels. Dies war ein Hinweis, dass CSN5 scheinbar

eine Rolle in der Differenzierung von Zellen im Darmepithel spielt. Die Differenzierung

von Zellen ist ein komplexer Prozess, der durch die genaue Regulation von

Genexpression und Proteinlevel koordiniert wird.

Das Ziel meiner Doktorarbeit, war die Untersuchung möglicher Einflüsse von CSN5

auf die Genexpression im Darmepithel. Als Modelle für das Darmepithel wurden

verschiedene Kolorektalkrebs-Zelllinien benutzt, vor allem die Zelllinie SW480. Der

knockout von Csn5 im Darmepithel der Mäuse wurde durch siRNA vermittelten

Knockdown in der Zelllinie simuliert.

In meiner Arbeit konnte ich zeigen, dass der knockdown von CSN5 zu einer

Veränderung der Genexpression führt. Erstaunlicherweise war die Zahl der Gene,

Page 108: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

96

welche stark in ihrer Expression beeinflusst wurden, gering. Nur 44 Gene zeigten eine

mehr als zweifache Veränderung der Expression, wobei sogar nur 6 Gene eine

dreifache Veränderung, in ihrer Expression, zeigten. Diese 6 Gene waren: ADP-

ribosylation factor-like 14 effector protein-like (ARL14EPL), Keratin associated protein

3-1 (KRTAP3-1), Late cornified envelope protein 1F (LCE1F), -actin-like protein 2

(ACTBL2), Inhibin A (INHBA) and Glia maturation factor (GMFG). Durch eine

sogenannte gene set enrichment Analyse konnte ich zeigen, dass es in bestimmten

zellulären Prozessen (z.B. KEGG_NOTCH_SIGNALING_PATHWAY,

KEGG_TGF_BETA_SIGNALING_ PATHWAY,

HALLMARK_WNT_BETA_CATENIN_SIGNALING, CELL_CYCLE _PROCESS oder

REACTOME_CELL_CYCLE) zu einer Akkumulation von Genen kommt, welche durch

den knockdown von CSN5 in ihrer Expression beeinflusst werden. Besonders fiel auf,

dass es zu einer Akkumulation von negativ regulierten Genen in Signalwegen (vor

allem Notch, Wnt/ -Catenin und TGF- ) kam, für welche schon bekannt ist, dass sie

eine Rolle in der Differenzierung des intestinalen Epithels spielen. Auch konnte gezeigt

werden, dass Gene, welche eine Rolle im Zellzyklus spielen, durch den Knockdown

von Csn5 beeinflusst werden. Durch eine Gen Onthologie Analyse konnte gezeigt

werden, dass CSN5, Gene negativ beeinflusst, welche eine Funktion in der

Entwicklung und Differenzierung der unterschiedlichsten Gewebe (z.B. Epithel, Herz,

Skelet) haben. Alle diese Hinweise unterstützen die Hypothese, dass CSN5 eine Rolle

in der Differenzierung von Zellen und Geweben spielt.

Aufgrund dieser Ergebnisse, untersuchte ich weitergehend den Einfluss von CSN5 auf

den Activin/Inhibin-Signalweg. Dieser Signalweg, gehört zu der „Superfamilie“ der

TGF- -Signalwege. In der Literatur gibt es ebenfalls Hinweise, dass dieser Signalweg

eine Rolle in der Differenzierung des intestinalen Epithels spielt. Ich konnte in meiner

Arbeit zeigen, dass der Knockdown von CSN5 die Expression der verschiedenen

Untereinheiten der Activin/Inhibin-Signalweg-Liganden beeinflusst. Der Knockdown

von CSN5 führt zu einer verstärkten Expression von Inhibin A und einer Verringerung

der Expression von Inhibin α und Inhibin B. Die Überexpression von CSN5 zeigte

aber keinen Einfluss auf die Expression der Untereinheiten. Der Knockdown weiterer

Untereinheiten des COP9-Signalosoms (hier CSN1 und CSN2) führte ebenfalls zu

einer Verringerung der Inhibin α Expression. Im Gegensatz zum CSN5-Knockdown

führte der Knockdown von CSN1 aber zu einer verringerten Inhibin A Expression.

Daher vermute ich, dass der Einfluss von CSN5 auf die Expression von Inhibin α durch

Page 109: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

97

das COP9-Signalsom vermittelt wird, wohingegen CSN5 auch als Monomer einen

Einfluss auf die Expression von Inhibin A haben kann.

Eine der am besten untersuchten Funktionen des COP9-Signalsoms ist, wie oben

beschrieben, die Regulation der Cullin RING E3 Ubiquitin Ligasen. Diese Ligasen

markieren Proteine mit Ubiquitin für den proteasomalen Abbbau. Der zusätzliche

Knockdown von Cullin1 konnte den Effekt des Knockdowns von CSN5 auf die

Expression von Inhibin A wieder aufheben. Der Knockdown von Cullin1 alleine zeigte

jedoch keine Auswirkung auf die Expression von Inhibin A. Die Inhibin α Expression

konnte durch Knockdown von Cullin1 verringert werden. Wurde der Cullin1-

Knockdown zusätzlich zu dem Knockdown von CSN5 durchgeführt, schien sich der

Effekt noch zu verstärken. Der Effekt des CSN5-Knockdowns auf die Inhibin A

Expression konnte, im Gegensatz zum Cullin1-Knockdown, durch die Inhibierung von

Cullin RING E3 Ubiquitin Ligasen mittels MLN4924 nicht beeinflusst werden. Die

Inhibierung durch MLN4924 alleine, führte jedoch zu einer Verringerung der Inhibin

A. Für Inhibin α konnte der Effekt des Cullin1-Knockdowns durch die Inhibierung der

Cullin RING E3 Ubiquitin Ligasen mittels MLN4924 bestätigt werden.

Ein weiterer Faktor, welcher die Expression der Activin/Inhibin-Untereinheiten

beeinflussen kann, ist das Tumorsuppressor-Protein p53. In der Kolorektalkrebs-

Zelllinie HCT116, welche einen p53 Knockout besitzen, führte der Knockdown von

CSN5 ebenfalls zu einer Verringerung der Inhibin A Expression. Diese war

vergleichbar mit dem Effekt, wie in der SW480-Zelllinie, welche ein verkürztes

p53-Protein besitzt. Im Gegensatz dazu führt der Knockdown von CSN5 in

HCT116-Zellen, welche ein Wildtyp p53-Protein besitzen, zu einer verstärkten

Expression von Inhibin A. Dies zeigt, dass p5γ wahrscheinlich eine Rolle beim

Einfluss von CSN5 auf die Expression der Activin/Inhibin-Untereinheiten hat.

Auf Proteinebene, konnte ich keine Veränderungen des Inhibin A-Levels nach

CSN5-Knockdown nachweisen. Erste Pilotexperimente deuten aber darauf hin, dass

vielleicht eine erhöhte Sekretion von Activin, die erhöhte Synthese von Inhibin A

kompensiert. Dies könnte unter anderem die Erhöhung der intrazellulären Inhibin A

Proteinlevel ausgleichen.

Auf funktionaler Ebene konnte ich zeigen, dass der Transfer des Überstandes von

Zellen, in denen ein Knockdown von CSN5 durchgeführt wurde, in den

Empfängerzellen zu einer Verringerung des Wachstums führte. Dieser Effekt, konnte

durch die Zugabe von Follistatin, einem natürlichen Activin-Inhibitor, zum Teil

Page 110: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

98

aufgehoben werden. Dies deutet darauf hin, dass dieser Effekt unter anderem durch

Activin beeinflusst zu sein scheint.

Durch meine These liefere ich Hinweise, dass CSN5 wahrscheinlich einen Einfluss auf

die Differenzierung des Kolon-Epithels hat. Dieser Einfluss scheint, zum Teil, durch die

Effekte von CSN5 auf die Transkription vermittelt zu werden. Darauf deuten die

veränderten Genexpressions-Profile nach CSN5-Knockdown in Kolorektalkrebs-

Zelllinien hin. Im weiteren Verlauf, habe ich mich näher mit dem Einfluss von CSN5

auf den Activin/Inhibin-Signalweg beschäftigt. Ich konnte zeigen, dass CSN5 einen

Einfluss auf die Expression der verschiedenen Activin/Inhibin-Untereinheiten hat.

Dieser Signalweg könnte somit eine Verbindung zwischen CSN5 und der Homöostase

und Differenzierung von intestinalen Zellen, vor allem im Epithel des Kolons, sein.

Meine These könnte der Startpunkt für die weitere Untersuchung der Rolle von CSN5

und des COP9-Signalosoms in der Differenzierung und Homöostase des intestinalen

Epithels, sowie der kolorektalen Krebsentstehung sein.

Page 111: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

99

References

1. Möricke B, Mergenthaler. Biologie des Menschen. Vol. 15. Auflage: Nikol-Verlag; 2007.

2. Faller A. SM. Der Körper des Menschen. Vol. 15. Auflage. Stuttgart: Thieme Verlag; 2008.

3. Noah TK, Donahue B, Shroyer NF. Intestinal development and differentiation. Exp Cell Res. 2011;317(19):2702-2710.

4. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet. 2006;7(5):349-359.

5. Medema JP, Vermeulen L. Microenvironmental regulation of stem cells in intestinal homeostasis and cancer. Nature. 2011;474(7351):318-326.

6. Arvelo F, Sojo F, Cotte C. Biology of colorectal cancer. Ecancermedicalscience. 2015;9:520.

7. Coyle YM. Lifestyle, genes, and cancer. Methods Mol Biol. 2009;472:25-56.

8. Slattery ML. Physical activity and colorectal cancer. Sports Med. 2004;34(4):239-252.

9. Larsson SC, Rutegard J, Bergkvist L, Wolk A. Physical activity, obesity, and risk of colon and rectal cancer in a cohort of Swedish men. Eur J Cancer. 2006;42(15):2590-2597.

10. Chao A, Thun MJ, Connell CJ, et al. Meat consumption and risk of colorectal cancer. JAMA. 2005;293(2):172-182.

11. Slattery ML, Curtin K, Anderson K, et al. Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors. J Natl Cancer Inst. 2000;92(22):1831-1836.

12. Cho E, Smith-Warner SA, Ritz J, et al. Alcohol intake and colorectal cancer: a pooled analysis of 8 cohort studies. Ann Intern Med. 2004;140(8):603-613.

13. Triantafillidis JK, Nasioulas G, Kosmidis PA. Colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies. Anticancer Res. 2009;29(7):2727-2737.

14. Tierney RP, Ballantyne GH, Modlin IM. The adenoma to carcinoma sequence. Surg Gynecol Obstet. 1990;171(1):81-94.

15. Hamilton SR. The adenoma-adenocarcinoma sequence in the large bowel: variations on a theme. J Cell Biochem Suppl. 1992;16G:41-46.

16. Miyaki M, Konishi M, Kikuchi-Yanoshita R, et al. Characteristics of somatic mutation of the adenomatous polyposis coli gene in colorectal tumors. Cancer Res. 1994;54(11):3011-3020.

17. Takayama T, Ohi M, Hayashi T, et al. Analysis of K-ras, APC, and beta-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis. Gastroenterology. 2001;121(3):599-611.

Page 112: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

100

18. Naccarati A, Polakova V, Pardini B, et al. Mutations and polymorphisms in TP53 gene--an overview on the role in colorectal cancer. Mutagenesis. 2012;27(2):211-218.

19. Nagothu KK, Jaszewski R, Moragoda L, et al. Folic acid mediated attenuation of loss of heterozygosity of DCC tumor suppressor gene in the colonic mucosa of patients with colorectal adenomas. Cancer Detect Prev. 2003;27(4):297-304.

20. Pruitt K, Der CJ. Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Lett. 2001;171(1):1-10.

21. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000;408(6810):307-310.

22. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88(3):323-331.

23. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010;138(6):2073-2087 e2073.

24. Leach FS, Nicolaides NC, Papadopoulos N, et al. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell. 1993;75(6):1215-1225.

25. Bronner CE, Baker SM, Morrison PT, et al. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature. 1994;368(6468):258-261.

26. Lee MH, Zhao R, Phan L, Yeung SC. Roles of COP9 signalosome in cancer. Cell Cycle. 2011;10(18):3057-3066.

27. Adler AS, Littlepage LE, Lin M, et al. CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression. Cancer Res. 2008;68(2):506-515.

28. Shintani S, Li C, Mihara M, Hino S, Nakashiro K, Hamakawa H. Skp2 and Jab1 expression are associated with inverse expression of p27(KIP1) and poor prognosis in oral squamous cell carcinomas. Oncology. 2003;65(4):355-362.

29. Sui L, Dong Y, Ohno M, et al. Jab1 expression is associated with inverse expression of p27(kip1) and poor prognosis in epithelial ovarian tumors. Clin Cancer Res. 2001;7(12):4130-4135.

30. Chamovitz DA, Wei N, Osterlund MT, et al. The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch. Cell. 1996;86(1):115-121.

31. Seeger M, Kraft R, Ferrell K, et al. A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits. FASEB J. 1998;12(6):469-478.

32. Wei N, Tsuge T, Serino G, et al. The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr Biol. 1998;8(16):919-922.

33. Wee S, Hetfeld B, Dubiel W, Wolf DA. Conservation of the COP9/signalosome in budding yeast. BMC Genet. 2002;3:15.

34. Maytal-Kivity V, Pick E, Piran R, Hofmann K, Glickman MH. The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes. Int J Biochem Cell Biol. 2003;35(5):706-715.

Page 113: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

101

35. Luke-Glaser S, Roy M, Larsen B, et al. CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo. Mol Cell Biol. 2007;27(12):4526-4540.

36. Freilich S, Oron E, Kapp Y, et al. The COP9 signalosome is essential for development of Drosophila melanogaster. Curr Biol. 1999;9(20):1187-1190.

37. Deng XW, Dubiel W, Wei N, Hofmann K, Mundt K. Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet. 2000;16(7):289.

38. Kato JY, Yoneda-Kato N. Mammalian COP9 signalosome. Genes Cells. 2009;14(11):1209-1225.

39. Kapelari B, Bech-Otschir D, Hegerl R, Schade R, Dumdey R, Dubiel W. Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome. J Mol Biol. 2000;300(5):1169-1178.

40. Henke W, Ferrell K, Bech-Otschir D, et al. Comparison of human COP9 signalsome and 26S proteasome lid'. Mol Biol Rep. 1999;26(1-2):29-34.

41. Scheel H, Hofmann K. Prediction of a common structural scaffold for proteasome lid, COP9-signalosome and eIF3 complexes. BMC Bioinformatics. 2005;6:71.

42. Wei Z, Zhang P, Zhou Z, Cheng Z, Wan M, Gong W. Crystal structure of human eIF3k, the first structure of eIF3 subunits. J Biol Chem. 2004;279(33):34983-34990.

43. Lingaraju GM, Bunker RD, Cavadini S, et al. Crystal structure of the human COP9 signalosome. Nature. 2014;512(7513):161-165.

44. Claret FX, Hibi M, Dhut S, Toda T, Karin M. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature. 1996;383(6599):453-457.

45. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature. 1999;398(6723):160-165.

46. Bech-Otschir D, Kraft R, Huang X, et al. COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J. 2001;20(7):1630-1639.

47. Bianchi E, Denti S, Granata A, et al. Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity. Nature. 2000;404(6778):617-621.

48. Kleemann R, Hausser A, Geiger G, et al. Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1. Nature. 2000;408(6809):211-216.

49. Wan M, Cao X, Wu Y, et al. Jab1 antagonizes TGF-beta signaling by inducing Smad4 degradation. EMBO Rep. 2002;3(2):171-176.

50. Kim BC, Lee HJ, Park SH, et al. Jab1/CSN5, a component of the COP9 signalosome, regulates transforming growth factor beta signaling by binding to Smad7 and promoting its degradation. Mol Cell Biol. 2004;24(6):2251-2262.

Page 114: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

102

51. Bae MK, Ahn MY, Jeong JW, et al. Jab1 interacts directly with HIF-1alpha and regulates its stability. J Biol Chem. 2002;277(1):9-12.

52. Wei N, Deng XW. Making sense of the COP9 signalosome. A regulatory protein complex conserved from Arabidopsis to human. Trends Genet. 1999;15(3):98-103.

53. Cope GA, Suh GS, Aravind L, et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science. 2002;298(5593):608-611.

54. Cope GA, Deshaies RJ. COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003;114(6):663-671.

55. Tomoda K, Kubota Y, Arata Y, et al. The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex. J Biol Chem. 2002;277(3):2302-2310.

56. Oh W, Lee EW, Sung YH, et al. Jab1 induces the cytoplasmic localization and degradation of p53 in coordination with Hdm2. J Biol Chem. 2006;281(25):17457-17465.

57. Zhang XC, Chen J, Su CH, Yang HY, Lee MH. Roles for CSN5 in control of p53/MDM2 activities. J Cell Biochem. 2008;103(4):1219-1230.

58. Dechend R, Hirano F, Lehmann K, et al. The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators. Oncogene. 1999;18(22):3316-3323.

59. Kwak HJ, Kim SH, Yoo HG, Park SH, Lee CH. Jun activation domain-binding protein 1 is required for mitotic checkpoint activation via its involvement in hyperphosphorylation of 53BP1. J Cancer Res Clin Oncol. 2005;131(12):789-796.

60. Dai YS, Hao J, Bonin C, Morikawa Y, Cserjesi P. JAB1 enhances HAND2 transcriptional activity by regulating HAND2 DNA binding. J Neurosci Res. 2004;76(5):613-622.

61. Chiba T, Tanaka K. Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system. Curr Protein Pept Sci. 2004;5(3):177-184.

62. Bosu DR, Kipreos ET. Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div. 2008;3:7.

63. Read MA, Brownell JE, Gladysheva TB, et al. Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha. Mol Cell Biol. 2000;20(7):2326-2333.

64. Wu K, Chen A, Pan ZQ. Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. J Biol Chem. 2000;275(41):32317-32324.

65. Podust VN, Brownell JE, Gladysheva TB, et al. A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination. Proc Natl Acad Sci U S A. 2000;97(9):4579-4584.

66. Lydeard JR, Schulman BA, Harper JW. Building and remodelling Cullin-RING E3 ubiquitin ligases. EMBO Rep. 2013;14(12):1050-1061.

Page 115: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

103

67. Wee S, Geyer RK, Toda T, Wolf DA. CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability. Nat Cell Biol. 2005;7(4):387-391.

68. Wu JT, Lin HC, Hu YC, Chien CT. Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation. Nat Cell Biol. 2005;7(10):1014-1020.

69. Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA. Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p. Mol Cell. 2003;11(4):927-938.

70. Hetfeld BK, Helfrich A, Kapelari B, et al. The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1. Curr Biol. 2005;15(13):1217-1221.

71. Choi HH, Gully C, Su CH, et al. COP9 signalosome subunit 6 stabilizes COP1, which functions as an E3 ubiquitin ligase for 14-3-3sigma. Oncogene. 2011;30(48):4791-4801.

72. Lykke-Andersen K, Schaefer L, Menon S, Deng XW, Miller JB, Wei N. Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. Mol Cell Biol. 2003;23(19):6790-6797.

73. Yan J, Walz K, Nakamura H, et al. COP9 signalosome subunit 3 is essential for maintenance of cell proliferation in the mouse embryonic epiblast. Mol Cell Biol. 2003;23(19):6798-6808.

74. Tomoda K, Yoneda-Kato N, Fukumoto A, Yamanaka S, Kato JY. Multiple functions of Jab1 are required for early embryonic development and growth potential in mice. J Biol Chem. 2004;279(41):43013-43018.

75. Menon S, Chi H, Zhang H, Deng XW, Flavell RA, Wei N. COP9 signalosome subunit 8 is essential for peripheral T cell homeostasis and antigen receptor-induced entry into the cell cycle from quiescence. Nat Immunol. 2007;8(11):1236-1245.

76. Patil MA, Gutgemann I, Zhang J, et al. Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis. 2005;26(12):2050-2057.

77. Bhansali M, Shemshedini L. COP9 subunits 4 and 5 target soluble guanylyl cyclase alpha1 and p53 in prostate cancer cells. Mol Endocrinol. 2014;28(6):834-845.

78. Zhao R, Yeung SC, Chen J, et al. Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers. J Clin Invest. 2011;121(3):851-865.

79. Adler AS, Lin M, Horlings H, Nuyten DS, van de Vijver MJ, Chang HY. Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet. 2006;38(4):421-430.

80. Luo J, Emanuele MJ, Li D, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell. 2009;137(5):835-848.

Page 116: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

104

81. Tsujimoto I, Yoshida A, Yoneda-Kato N, Kato JY. Depletion of CSN5 inhibits Ras-mediated tumorigenesis by inducing premature senescence in p53-null cells. FEBS Lett. 2012;586(24):4326-4331.

82. Tomoda K, Kato JY, Tatsumi E, Takahashi T, Matsuo Y, Yoneda-Kato N. The Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase activity and facilitates cell-cycle progression. Blood. 2005;105(2):775-783.

83. Nishimoto A, Kugimiya N, Hosoyama T, Enoki T, Li TS, Hamano K. JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cells. Biochem Biophys Res Commun. 2013;438(3):513-518.

84. Sancho E, Batlle E, Clevers H. Signaling pathways in intestinal development and cancer. Annu Rev Cell Dev Biol. 2004;20:695-723.

85. Schutz AK, Hennes T, Jumpertz S, Fuchs S, Bernhagen J. Role of CSN5/JAB1 in Wnt/beta-catenin activation in colorectal cancer cells. FEBS Lett. 2012;586(11):1645-1651.

86. Huang X, Langelotz C, Hetfeld-Pechoc BK, Schwenk W, Dubiel W. The COP9 signalosome mediates beta-catenin degradation by deneddylation and blocks adenomatous polyposis coli destruction via USP15. J Mol Biol. 2009;391(4):691-702.

87. Jumpertz S, Hennes T, Asare Y, Vervoorts J, Bernhagen J, Schutz AK. The beta-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells. Cell Signal. 2014;26(9):2051-2059.

88. McCullagh DR. Dual Endocrine Activity of the Testes. Science. 1932;76(1957):19-20.

89. Vale W, Rivier J, Vaughan J, et al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986;321(6072):776-779.

90. Ling N, Ying SY, Ueno N, et al. A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem Biophys Res Commun. 1986;138(3):1129-1137.

91. Vale W, Rivier C, Hsueh A, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res. 1988;44:1-34.

92. de Kretser DM, Robertson DM. The isolation and physiology of inhibin and related proteins. Biol Reprod. 1989;40(1):33-47.

93. Vliegen MK, Schlatt S, Weinbauer GF, Bergmann M, Groome NP, Nieschlag E. Localization of inhibin/activin subunits in the testis of adult nonhuman primates and men. Cell Tissue Res. 1993;273(2):261-268.

94. Gurusinghe CJ, Healy DL, Jobling T, Mamers P, Burger HG. Inhibin and activin are demonstrable by immunohistochemistry in ovarian tumor tissue. Gynecol Oncol. 1995;57(1):27-32.

95. Spencer SJ, Rabinovici J, Jaffe RB. Human recombinant activin-A inhibits proliferation of human fetal adrenal cells in vitro. J Clin Endocrinol Metab. 1990;71(6):1678-1680.

Page 117: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

105

96. Furukawa M, Eto Y, Kojima I. Expression of immunoreactive activin A in fetal rat pancreas. Endocr J. 1995;42(1):63-68.

97. Yasuda H, Mine T, Shibata H, et al. Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J Clin Invest. 1993;92(3):1491-1496.

98. Dolter KE, Palyash JC, Shao LE, Yu J. Analysis of activin A gene expression in human bone marrow stromal cells. J Cell Biochem. 1998;70(1):8-21.

99. Fang J, Wang SQ, Smiley E, Bonadio J. Genes coding for mouse activin beta C and beta E are closely linked and exhibit a liver-specific expression pattern in adult tissues. Biochem Biophys Res Commun. 1997;231(3):655-661.

100. Vejda S, Erlach N, Peter B, et al. Expression of activins C and E induces apoptosis in human and rat hepatoma cells. Carcinogenesis. 2003;24(11):1801-1809.

101. Wada W, Maeshima A, Zhang YQ, Hasegawa Y, Kuwano H, Kojima I. Assessment of the function of the betaC-subunit of activin in cultured hepatocytes. Am J Physiol Endocrinol Metab. 2004;287(2):E247-254.

102. Sun PD, Davies DR. The cystine-knot growth-factor superfamily. Annu Rev Biophys Biomol Struct. 1995;24:269-291.

103. Gray AM, Mason AJ. Requirement for activin A and transforming growth factor--beta 1 pro-regions in homodimer assembly. Science. 1990;247(4948):1328-1330.

104. Huylebroeck D, Van Nimmen K, Waheed A, et al. Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily. Mol Endocrinol. 1990;4(8):1153-1165.

105. Antenos M, Zhu J, Jetly NM, Woodruff TK. An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells. J Biol Chem. 2008;283(48):33059-33068.

106. Walton KL, Makanji Y, Wilce MC, Chan KL, Robertson DM, Harrison CA. A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (TGFbeta) ligands. J Biol Chem. 2009;284(14):9311-9320.

107. Antenos M, Stemler M, Boime I, Woodruff TK. N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers. Mol Endocrinol. 2007;21(7):1670-1684.

108. Shao L, Frigon NL, Jr., Sehy DW, et al. Regulation of production of activin A in human marrow stromal cells and monocytes. Exp Hematol. 1992;20(10):1235-1242.

109. Eramaa M, Hurme M, Stenman UH, Ritvos O. Activin A/erythroid differentiation factor is induced during human monocyte activation. J Exp Med. 1992;176(5):1449-1452.

110. Keelan JA, Zhou RL, Evans LW, Groome NP, Mitchell MD. Regulation of activin A, inhibin A, and follistatin production in human amnion and choriodecidual explants by inflammatory mediators. J Soc Gynecol Investig. 2000;7(5):291-296.

Page 118: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

106

111. de Caestecker M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev. 2004;15(1):1-11.

112. Attisano L, Carcamo J, Ventura F, Weis FM, Massague J, Wrana JL. Identification of human activin and TGF beta type I receptors that form heteromeric kinase complexes with type II receptors. Cell. 1993;75(4):671-680.

113. Tsuchida K, Nakatani M, Yamakawa N, Hashimoto O, Hasegawa Y, Sugino H. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Cell Endocrinol. 2004;220(1-2):59-65.

114. Attisano L, Wrana JL, Montalvo E, Massague J. Activation of signalling by the activin receptor complex. Mol Cell Biol. 1996;16(3):1066-1073.

115. Lebrun JJ, Vale WW. Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation. Mol Cell Biol. 1997;17(3):1682-1691.

116. ten Dijke P, Yamashita H, Ichijo H, et al. Characterization of type I receptors for transforming growth factor-beta and activin. Science. 1994;264(5155):101-104.

117. Thompson TB, Cook RW, Chapman SC, Jardetzky TS, Woodruff TK. Beta A versus beta B: is it merely a matter of expression? Mol Cell Endocrinol. 2004;225(1-2):9-17.

118. Thompson TB, Woodruff TK, Jardetzky TS. Structures of an ActRIIB:activin A complex reveal a novel binding mode for TGF-beta ligand:receptor interactions. EMBO J. 2003;22(7):1555-1566.

119. Daopin S, Piez KA, Ogawa Y, Davies DR. Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily. Science. 1992;257(5068):369-373.

120. Sebald W, Mueller TD. The interaction of BMP-7 and ActRII implicates a new mode of receptor assembly. Trends Biochem Sci. 2003;28(10):518-521.

121. Greenwald J, Vega ME, Allendorph GP, Fischer WH, Vale W, Choe S. A flexible activin explains the membrane-dependent cooperative assembly of TGF-beta family receptors. Mol Cell. 2004;15(3):485-489.

122. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-beta receptor. Nature. 1994;370(6488):341-347.

123. Willis SA, Zimmerman CM, Li LI, Mathews LS. Formation and activation by phosphorylation of activin receptor complexes. Mol Endocrinol. 1996;10(4):367-379.

124. Lewis KA, Gray PC, Blount AL, et al. Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature. 2000;404(6776):411-414.

125. Onichtchouk D, Chen YG, Dosch R, et al. Silencing of TGF-beta signalling by the pseudoreceptor BAMBI. Nature. 1999;401(6752):480-485.

126. Shimasaki S, Koga M, Esch F, et al. Porcine follistatin gene structure supports two forms of mature follistatin produced by alternative splicing. Biochem Biophys Res Commun. 1988;152(2):717-723.

127. Schneyer A, Schoen A, Quigg A, Sidis Y. Differential binding and neutralization of activins A and B by follistatin and follistatin like-3 (FSTL-3/FSRP/FLRG). Endocrinology. 2003;144(5):1671-1674.

Page 119: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

107

128. Harrington AE, Morris-Triggs SA, Ruotolo BT, Robinson CV, Ohnuma S, Hyvonen M. Structural basis for the inhibition of activin signalling by follistatin. EMBO J. 2006;25(5):1035-1045.

129. Hashimoto O, Nakamura T, Shoji H, Shimasaki S, Hayashi Y, Sugino H. A novel role of follistatin, an activin-binding protein, in the inhibition of activin action in rat pituitary cells. Endocytotic degradation of activin and its acceleration by follistatin associated with cell-surface heparan sulfate. J Biol Chem. 1997;272(21):13835-13842.

130. Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL. SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell. 1998;95(6):779-791.

131. Lu Z, Murray JT, Luo W, et al. Transforming growth factor beta activates Smad2 in the absence of receptor endocytosis. J Biol Chem. 2002;277(33):29363-29368.

132. Hayes S, Chawla A, Corvera S. TGF beta receptor internalization into EEA1-enriched early endosomes: role in signaling to Smad2. J Cell Biol. 2002;158(7):1239-1249.

133. Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Pavletich NP. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell. 1998;94(5):585-594.

134. Chai J, Wu JW, Yan N, Massague J, Pavletich NP, Shi Y. Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding. J Biol Chem. 2003;278(22):20327-20331.

135. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 1998;17(11):3091-3100.

136. Zawel L, Dai JL, Buckhaults P, et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell. 1998;1(4):611-617.

137. Attisano L, Wrana JL. Smads as transcriptional co-modulators. Curr Opin Cell Biol. 2000;12(2):235-243.

138. Massague J, Wotton D. Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. 2000;19(8):1745-1754.

139. ten Dijke P, Miyazono K, Heldin CH. Signaling inputs converge on nuclear effectors in TGF-beta signaling. Trends Biochem Sci. 2000;25(2):64-70.

140. Vallier L, Alexander M, Pedersen RA. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci. 2005;118(Pt 19):4495-4509.

141. Brown S, Teo A, Pauklin S, et al. Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors. Stem Cells. 2011;29(8):1176-1185.

142. Duggal G, Heindryckx B, Warrier S, et al. Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential. Stem Cells Dev. 2013;22(23):3141-3155.

Page 120: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

108

143. James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 2005;132(6):1273-1282.

144. Xu RH, Sampsell-Barron TL, Gu F, et al. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell. 2008;3(2):196-206.

145. McLean AB, D'Amour KA, Jones KL, et al. Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem Cells. 2007;25(1):29-38.

146. Teo AK, Ali Y, Wong KY, et al. Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells. 2012;30(4):631-642.

147. Toivonen S, Lundin K, Balboa D, et al. Activin A and Wnt-dependent specification of human definitive endoderm cells. Exp Cell Res. 2013;319(17):2535-2544.

148. Liu QY, Niranjan B, Gomes P, et al. Inhibitory effects of activin on the growth and morpholgenesis of primary and transformed mammary epithelial cells. Cancer Res. 1996;56(5):1155-1163.

149. McCarthy SA, Bicknell R. Inhibition of vascular endothelial cell growth by activin-A. J Biol Chem. 1993;268(31):23066-23071.

150. Schwall RH, Robbins K, Jardieu P, Chang L, Lai C, Terrell TG. Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology. 1993;18(2):347-356.

151. Yamato K, Koseki T, Ohguchi M, Kizaki M, Ikeda Y, Nishihara T. Activin A induction of cell-cycle arrest involves modulation of cyclin D2 and p21CIP1/WAF1 in plasmacytic cells. Mol Endocrinol. 1997;11(8):1044-1052.

152. Zauberman A, Oren M, Zipori D. Involvement of p21(WAF1/Cip1), CDK4 and Rb in activin A mediated signaling leading to hepatoma cell growth inhibition. Oncogene. 1997;15(14):1705-1711.

153. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995;81(3):323-330.

154. Koseki T, Yamato K, Krajewski S, Reed JC, Tsujimoto Y, Nishihara T. Activin A-induced apoptosis is suppressed by BCL-2. FEBS Lett. 1995;376(3):247-250.

155. Koseki T, Yamato K, Ishisaki A, Hashimoto O, Sugino H, Nishihara T. Correlation between Bcl-X expression and B-cell hybridoma apoptosis induced by activin A. Cell Signal. 1998;10(7):517-521.

156. Wang Q, Tabatabaei S, Planz B, Lin CW, Sluss PM. Identification of an activin-follistatin growth modulatory system in the human prostate: secretion and biological activity in primary cultures of prostatic epithelial cells. J Urol. 1999;161(4):1378-1384.

157. Burdette JE, Jeruss JS, Kurley SJ, Lee EJ, Woodruff TK. Activin A mediates growth inhibition and cell cycle arrest through Smads in human breast cancer cells. Cancer Res. 2005;65(17):7968-7975.

Page 121: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

109

158. Jeruss JS, Sturgis CD, Rademaker AW, Woodruff TK. Down-regulation of activin, activin receptors, and Smads in high-grade breast cancer. Cancer Res. 2003;63(13):3783-3790.

159. Togashi Y, Sakamoto H, Hayashi H, et al. Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer. Mol Cancer. 2014;13:126.

160. Leto G, Incorvaia L, Badalamenti G, et al. Activin A circulating levels in patients with bone metastasis from breast or prostate cancer. Clin Exp Metastasis. 2006;23(2):117-122.

161. Incorvaia L, Badalamenti G, Rini G, et al. MMP-2, MMP-9 and activin A blood levels in patients with breast cancer or prostate cancer metastatic to the bone. Anticancer Res. 2007;27(3B):1519-1525.

162. Chang KP, Kao HK, Liang Y, et al. Overexpression of activin A in oral squamous cell carcinoma: association with poor prognosis and tumor progression. Ann Surg Oncol. 2010;17(7):1945-1956.

163. Huber S, Stahl FR, Schrader J, et al. Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol. 2009;182(8):4633-4640.

164. Hubner G, Brauchle M, Gregor M, Werner S. Activin A: a novel player and inflammatory marker in inflammatory bowel disease? Lab Invest. 1997;77(4):311-318.

165. Dignass AU, Jung S, Harder-d'Heureuse J, Wiedenmann B. Functional relevance of activin A in the intestinal epithelium. Scand J Gastroenterol. 2002;37(8):936-943.

166. Dohi T, Ejima C, Kato R, et al. Therapeutic potential of follistatin for colonic inflammation in mice. Gastroenterology. 2005;128(2):411-423.

167. Wildi S, Kleeff J, Maruyama H, Maurer CA, Buchler MW, Korc M. Overexpression of activin A in stage IV colorectal cancer. Gut. 2001;49(3):409-417.

168. Sonoyama K, Rutatip S, Kasai T. Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2000;278(1):G89-97.

169. Jung BH, Beck SE, Cabral J, et al. Activin type 2 receptor restoration in MSI-H colon cancer suppresses growth and enhances migration with activin. Gastroenterology. 2007;132(2):633-644.

170. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-15550.

171. Segal E, Friedman N, Koller D, Regev A. A module map showing conditional activity of expression modules in cancer. Nat Genet. 2004;36(10):1090-1098.

172. Schütz A. Funktionelle Rolle von CSN5/JAB1 und seines Interaktionspartners MIF bei Entzündung und Kolorektalkrebs. Aachen; 2011.

Page 122: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

110

173. Peth A, Berndt C, Henke W, Dubiel W. Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability. BMC Biochem. 2007;8:27.

174. Soucy TA, Smith PG, Milhollen MA, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458(7239):732-736.

175. Brownell JE, Sintchak MD, Gavin JM, et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol Cell. 2010;37(1):102-111.

176. Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 1998;8(10):397-403.

177. Hua Z, Vierstra RD. The cullin-RING ubiquitin-protein ligases. Annu Rev Plant Biol. 2011;62:299-334.

178. Bauer J, Sporn JC, Cabral J, Gomez J, Jung B. Effects of activin and TGFbeta on p21 in colon cancer. PLoS One. 2012;7(6):e39381.

179. Bech-Otschir D, Seeger M, Dubiel W. The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis. J Cell Sci. 2002;115(Pt 3):467-473.

180. Chamovitz DA, Segal D. JAB1/CSN5 and the COP9 signalosome. A complex situation. EMBO Rep. 2001;2(2):96-101.

181. Chamovitz DA. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Rep. 2009;10(4):352-358.

182. Shackleford TJ, Claret FX. JAB1/CSN5: a new player in cell cycle control and cancer. Cell Div;5:26.

183. Richardson KS, Zundel W. The emerging role of the COP9 signalosome in cancer. Mol Cancer Res. 2005;3(12):645-653.

184. Dalton S. Linking the Cell Cycle to Cell Fate Decisions. Trends Cell Biol. 2015;25(10):592-600.

185. Bertrand FE, Angus CW, Partis WJ, Sigounas G. Developmental pathways in colon cancer: crosstalk between WNT, BMP, Hedgehog and Notch. Cell Cycle. 2012;11(23):4344-4351.

186. Dias Neto E, Correa RG, Verjovski-Almeida S, et al. Shotgun sequencing of the human transcriptome with ORF expressed sequence tags. Proc Natl Acad Sci U S A. 2000;97(7):3491-3496.

187. Gillingham AK, Munro S. The small G proteins of the Arf family and their regulators. Annu Rev Cell Dev Biol. 2007;23:579-611.

188. Gong H, Zhou H, McKenzie GW, et al. An updated nomenclature for keratin-associated proteins (KAPs). Int J Biol Sci. 2012;8(2):258-264.

189. Fratini A, Powell BC, Rogers GE. Sequence, expression, and evolutionary conservation of a gene encoding a glycine/tyrosine-rich keratin-associated protein of hair. J Biol Chem. 1993;268(6):4511-4518.

190. Jackson B, Tilli CM, Hardman MJ, et al. Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation. J Invest Dermatol. 2005;124(5):1062-1070.

Page 123: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

111

191. Deng Z, Matsuda K, Tanikawa C, et al. Late Cornified Envelope Group I, a novel target of p53, regulates PRMT5 activity. Neoplasia. 2014;16(8):656-664.

192. Burkard TR, Planyavsky M, Kaupe I, et al. Initial characterization of the human central proteome. BMC Syst Biol. 2011;5:17.

193. Zaheer A, Lim R. In vitro inhibition of MAP kinase (ERK1/ERK2) activity by phosphorylated glia maturation factor (GMF). Biochemistry. 1996;35(20):6283-6288.

194. Hedger MP, de Kretser DM. The activins and their binding protein, follistatin-Diagnostic and therapeutic targets in inflammatory disease and fibrosis. Cytokine Growth Factor Rev. 2013;24(3):285-295.

195. Chen YG, Wang Q, Lin SL, Chang CD, Chuang J, Ying SY. Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis. Exp Biol Med (Maywood). 2006;231(5):534-544.

196. Antsiferova M, Werner S. The bright and the dark sides of activin in wound healing and cancer. J Cell Sci. 2012;125(Pt 17):3929-3937.

197. Krausova M, Korinek V. Wnt signaling in adult intestinal stem cells and cancer. Cell Signal. 2014;26(3):570-579.

198. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta. 2003;1653(1):1-24.

199. Hinoi T, Yamamoto H, Kishida M, Takada S, Kishida S, Kikuchi A. Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin. J Biol Chem. 2000;275(44):34399-34406.

200. Samowitz WS, Powers MD, Spirio LN, Nollet F, van Roy F, Slattery ML. Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res. 1999;59(7):1442-1444.

201. Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275(5307):1787-1790.

202. Hsu MC, Chang HC, Hung WC. HER-2/neu transcriptionally activates Jab1 expression via the AKT/beta-catenin pathway in breast cancer cells. Endocr Relat Cancer. 2007;14(3):655-667.

203. Yang KT, Wang MC, Chen JY, Hsu MC, Hung WC. Bcr-Abl oncogene stimulates Jab1 expression via cooperative interaction of beta-catenin and STAT1 in chronic myeloid leukemia cells. J Cell Physiol. 2011;226(11):2849-2856.

204. Liu J, Stevens J, Rote CA, et al. Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol Cell. 2001;7(5):927-936.

205. Matsuzawa SI, Reed JC. Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses. Mol Cell. 2001;7(5):915-926.

206. Noah TK, Shroyer NF. Notch in the intestine: regulation of homeostasis and pathogenesis. Annu Rev Physiol. 2013;75:263-288.

Page 124: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

112

207. Fre S, Pallavi SK, Huyghe M, et al. Notch and Wnt signals cooperatively control cell proliferation and tumorigenesis in the intestine. Proc Natl Acad Sci U S A. 2009;106(15):6309-6314.

208. Reedijk M, Odorcic S, Zhang H, et al. Activation of Notch signaling in human colon adenocarcinoma. Int J Oncol. 2008;33(6):1223-1229.

209. Zhang Y, Li B, Ji ZZ, Zheng PS. Notch1 regulates the growth of human colon cancers. Cancer. 2010;116(22):5207-5218.

210. Kim SB, Chae GW, Lee J, et al. Activated Notch1 interacts with p53 to inhibit its phosphorylation and transactivation. Cell Death Differ. 2007;14(5):982-991.

211. Oswald F, Tauber B, Dobner T, et al. p300 acts as a transcriptional coactivator for mammalian Notch-1. Mol Cell Biol. 2001;21(22):7761-7774.

212. Oberg C, Li J, Pauley A, Wolf E, Gurney M, Lendahl U. The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem. 2001;276(38):35847-35853.

213. Cope GA, Deshaies RJ. Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels. BMC Biochem. 2006;7:1.

214. Watabe T, Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell Res. 2009;19(1):103-115.

215. Akhurst RJ, Hata A. Targeting the TGFbeta signalling pathway in disease. Nat Rev Drug Discov. 2012;11(10):790-811.

216. Wakefield LM, Hill CS. Beyond TGFbeta: roles of other TGFbeta superfamily members in cancer. Nat Rev Cancer. 2013;13(5):328-341.

217. Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells. 2002;7(12):1191-1204.

218. Nakao A, Afrakhte M, Moren A, et al. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature. 1997;389(6651):631-635.

219. Nakao A, Imamura T, Souchelnytskyi S, et al. TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J. 1997;16(17):5353-5362.

220. Wan M, Tang Y, Tytler EM, et al. Smad4 protein stability is regulated by ubiquitin ligase SCF beta-TrCP1. J Biol Chem. 2004;279(15):14484-14487.

221. Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature. 1998;394(6696):909-913.

222. Bretones G, Delgado MD, Leon J. Myc and cell cycle control. Biochim Biophys Acta. 2015;1849(5):506-516.

223. Pelengaris S, Khan M, Evan G. c-MYC: more than just a matter of life and death. Nat Rev Cancer. 2002;2(10):764-776.

224. Dang CV. MYC on the path to cancer. Cell. 2012;149(1):22-35.

225. Myant K, Sansom OJ. Wnt/Myc interactions in intestinal cancer: partners in crime. Exp Cell Res. 2011;317(19):2725-2731.

Page 125: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

113

226. He TC, Sparks AB, Rago C, et al. Identification of c-MYC as a target of the APC pathway. Science. 1998;281(5382):1509-1512.

227. Bettess MD, Dubois N, Murphy MJ, et al. c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium. Mol Cell Biol. 2005;25(17):7868-7878.

228. Muncan V, Sansom OJ, Tertoolen L, et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol. 2006;26(22):8418-8426.

229. Erisman MD, Rothberg PG, Diehl RE, Morse CC, Spandorfer JM, Astrin SM. Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene. Mol Cell Biol. 1985;5(8):1969-1976.

230. Nath S, Ghatak D, Das P, Roychoudhury S. Transcriptional control of mitosis: deregulation and cancer. Front Endocrinol (Lausanne). 2015;6:60.

231. Tanaka T. Colorectal carcinogenesis: Review of human and experimental animal studies. J Carcinog. 2009;8:5.

232. Tanimoto K, Yoshida E, Mita S, Nibu Y, Murakami K, Fukamizu A. Human activin betaA gene. Identification of novel 5' exon, functional promoter, and enhancers. J Biol Chem. 1996;271(51):32760-32769.

233. Korczeniewska J, Barnes BJ. The COP9 signalosome interacts with and regulates interferon regulatory factor 5 protein stability. Mol Cell Biol. 2013;33(6):1124-1138.

234. Wolf DA, Zhou C, Wee S. The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases? Nat Cell Biol. 2003;5(12):1029-1033.

235. von Arnim AG. On again-off again: COP9 signalosome turns the key on protein degradation. Curr Opin Plant Biol. 2003;6(6):520-529.

236. Choo YY, Boh BK, Lou JJ, et al. Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity. Mol Biol Cell. 2011;22(24):4706-4715.

237. Bemis L, Chan DA, Finkielstein CV, et al. Distinct aerobic and hypoxic mechanisms of HIF-alpha regulation by CSN5. Genes Dev. 2004;18(7):739-744.

238. Kim JH, Choi JK, Cinghu S, et al. Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3. J Cell Biochem. 2009;107(3):557-565.

239. Yoshida A, Yoneda-Kato N, Kato JY. CSN5 specifically interacts with CDK2 and controls senescence in a cytoplasmic cyclin E-mediated manner. Sci Rep. 2013;3:1054.

240. Mason AJ, Farnworth PG, Sullivan J. Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A. Mol Endocrinol. 1996;10(9):1055-1065.

241. Mollenhauer HH, Morre DJ, Rowe LD. Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim Biophys Acta. 1990;1031(2):225-246.

Page 126: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

114

242. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. Activin-binding protein from rat ovary is follistatin. Science. 1990;247(4944):836-838.

243. Chen L, Zhang W, Liang HF, et al. Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells. Cell Commun Signal. 2014;12:18.

244. Iwakuma T, Lozano G. MDM2, an introduction. Mol Cancer Res. 2003;1(14):993-1000.

245. Lavin MF, Gueven N. The complexity of p53 stabilization and activation. Cell Death Differ. 2006;13(6):941-950.

246. Pflaum J, Schlosser S, Muller M. p53 Family and Cellular Stress Responses in Cancer. Front Oncol. 2014;4:285.

247. Muller PA, Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell. 2014;25(3):304-317.

248. Liu Y, Bodmer WF. Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines. Proc Natl Acad Sci U S A. 2006;103(4):976-981.

249. Fischer M, Steiner L, Engeland K. The transcription factor p53: not a repressor, solely an activator. Cell Cycle. 2014;13(19):3037-3058.

250. Feng ZM, Li YP, Chen CL. Analysis of the 5'-flanking regions of rat inhibin alpha- and beta-B-subunit genes suggests two different regulatory mechanisms. Mol Endocrinol. 1989;3(12):1914-1925.

251. Smale ST, Baltimore D. The "initiator" as a transcription control element. Cell. 1989;57(1):103-113.

252. Comb M, Birnberg NC, Seasholtz A, Herbert E, Goodman HM. A cyclic AMP- and phorbol ester-inducible DNA element. Nature. 1986;323(6086):353-356.

253. Debieve F, Thomas K. Control of the human inhibin alpha chain promoter in cytotrophoblast cells differentiating into syncytium. Mol Hum Reprod. 2002;8(3):262-270.

254. Depoix CL, Debieve F, Hubinont C. Inhibin alpha gene expression in human trophoblasts is regulated by interactions between TFAP2 and cAMP signaling pathways. Mol Reprod Dev. 2014;81(11):1009-1018.

255. Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE. Regulation of the alpha inhibin gene by cyclic adenosine 3',5'-monophosphate after transfection into rat granulosa cells. Mol Endocrinol. 1991;5(4):521-534.

256. Anttonen M, Parviainen H, Kyronlahti A, et al. GATA-4 is a granulosa cell factor employed in inhibin-alpha activation by the TGF-beta pathway. J Mol Endocrinol. 2006;36(3):557-568.

257. Feng ZM, Wu AZ, Chen CL. Testicular GATA-1 factor up-regulates the promoter activity of rat inhibin alpha-subunit gene in MA-10 Leydig tumor cells. Mol Endocrinol. 1998;12(3):378-390.

258. Burkart AD, Mukherjee A, Sterneck E, Johnson PF, Mayo KE. Repression of the inhibin alpha-subunit gene by the transcription factor CCAAT/enhancer-binding protein-beta. Endocrinology. 2005;146(4):1909-1921.

Page 127: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

115

259. Shackleford TJ, Zhang Q, Tian L, et al. Stat3 and CCAAT/enhancer binding protein beta (C/EBP-beta) regulate Jab1/CSN5 expression in mammary carcinoma cells. Breast Cancer Res. 2011;13(3):R65.

Page 128: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and
Page 129: Gene expression analysis identifies the Activin/Inhibin ...publications.rwth-aachen.de/record/707129/files/707129.pdf · Referat. His ideas and comments were always very helpful and

Erklärung

Ich erkläre eidesstattlich, dass ich diese Dissertation selbstständig verfasst und alle in

Anspruch genommenen Hilfen in der Dissertation angegeben habe.

Aachen,

Thomas Hennes