Pan-Cancer Analysis of the Mediator Complex Transcriptome ... · Christine Sanders5, Isabella...

Biology of Human Tumors

Pan-Cancer Analysis of the Mediator ComplexTranscriptome Identifies CDK19 and CDK8 asTherapeutic Targets in Advanced Prostate CancerJohannes Br€agelmann1,2,3,4, Niklas Kl€umper5, Anne Offermann5,Anne von M€assenhausen1,2,3, Diana B€ohm5, Mario Deng5, Angela Queisser1,2,3,Christine Sanders5, Isabella Syring1,2,3,6, Axel S. Merseburger7,Wenzel Vogel5,Elisabeth Sievers2,3, Ignacija Vlasic5, Jessica Carlsson8, Ove Andr�en8, Peter Brossart3,4,Stefan Duensing9, Maria A. Svensson10, Zaki Shaikhibrahim5, Jutta Kirfel2,3, andSven Perner5

Abstract

Purpose: The Mediator complex is a multiprotein assembly,which serves as a hub for diverse signaling pathways to regulategene expression. Because gene expression is frequently altered incancer, a systematic understanding of the Mediator complex inmalignancies could foster the development of novel targetedtherapeutic approaches.

Experimental Design: We performed a systematic deconvolu-tion of the Mediator subunit expression profiles across 23 cancerentities (n ¼ 8,568) using data from The Cancer Genome Atlas(TCGA). Prostate cancer–specific findings were validated in twopublicly available gene expression cohorts and a large cohort ofprimary and advanced prostate cancer (n ¼ 622) stained byimmunohistochemistry. The role of CDK19 and CDK8 was eval-uated by siRNA-mediated gene knockdown and inhibitor treat-ment in prostate cancer cell lines with functional assays and geneexpression analysis by RNAseq.

Results: Cluster analysis of TCGA expression data segregatedtumor entities, indicating tumor-type–specific Mediator complexcompositions.Onlyprostate cancerwasmarkedbyhighexpressionof CDK19. In primary prostate cancer, CDK19was associated withincreased aggressiveness and shorter disease-free survival. Duringcancer progression, highest levels of CDK19 and of its paralogCDK8werepresent inmetastases. In vitro, inhibitionofCDK19andCDK8 by knockdown or treatment with a selective CDK8/CDK19inhibitor significantly decreased migration and invasion.

Conclusions: Our analysis revealed distinct transcriptionalexpression profiles of the Mediator complex across cancer entitiesindicating differential modes of transcriptional regulation. More-over, it identified CDK19 and CDK8 to be specifically overex-pressed during prostate cancer progression, highlighting theirpotential as novel therapeutic targets in advanced prostate cancer.Clin Cancer Res; 23(7); 1829–40. �2016 AACR.

IntroductionOne of the crucial factors determining differentiation, growth,

and development of cells is the time- and tissue-specific expres-sion of protein-coding genes by RNApolymerase II (Pol II), whichis therefore tightly controlled (1).The multiprotein Mediatorcomplex (MED) has emerged as one of the main Pol II coactiva-tors governing transcriptional regulation (2). By functioning as ahub for input from numerous signaling pathways and transcrip-tion factors, the Mediator complex induces or represses essentialaspects of the transcription cycle such as initiation, elongation,and chromatin architecture (2, 3). In humans, the Mediatorcomplex consists of 33 dynamically assembled protein subunitseach of which is designated to 1 of 4 distinct MED modules"head," "middle," "tail," and "kinase" (Fig. 1A). Generally, thehead and the middle modules directly interact with Pol II andtogether with the tail module form a relatively stable "core"structure but are devoid of enzymatic activity. The kinase moduleincluding subunits cyclin-dependent kinase 8 (CDK8), MED12,MED13, and cyclin C (CCNC) can reversibly associate with thecore structure, which leads to considerable changes of MEDconfirmation and functional activity (3). In addition, in verte-brates, the subunits CDK19, MED12L, and MED13L have beenshown to take part in formation of the kinase module in a

1Section for Prostate Cancer Research, University Hospital of Bonn, Bonn,Germany. 2Institute of Pathology, University Hospital of Bonn, Bonn, Germany.3Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn,Bonn, Germany. 4Department of Hematology, Oncology and Rheumatology,University Hospital of Bonn, Bonn, Germany. 5Pathology of the UniversityMedical Center Schleswig-Holstein, Campus L€ubeck and the Research CenterBorstel, Leibniz Center for Medicine and Biosciences, L€ubeck and Borstel,Germany. 6Clinic for Urology and Pediatric Urology, University Hospital of Bonn,Bonn, Germany. 7Department of Urology, University Hospital Schleswig-Hol-stein, L€ubeck, Germany. 8Department of Urology, Faculty of Medicine and

Health, €Orebro University, €Orebro, Sweden. 9Molecular Uro-oncology, Depart-ment of Urology, University of Heidelberg, Heidelberg, Germany. 10Department

of Research and Education, Faculty of Medicine and Health, €Orebro University,€Orebro, Sweden.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

J. Br€agelmann, N. Kl€umper and A. Offermann contributed equally to this article.

Corresponding Author: Sven Perner, Pathology of the University Hospital ofLuebeck and Leibniz Research Center Borstel, Institute of Pathology, Ratze-burger Allee 160 (Building 50), 23538 Luebeck, Germany, Department ofPathology, Parkallee 1-40, Borstel 23845, Germany. Phone: 49-451-500-2707;Fax: 49-451-500-3328; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0094

�2016 American Association for Cancer Research.

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mutually exclusive manner with their paralogous counterpartsCDK8, MED12, and MED13, respectively, indicating a highflexibility inMEDcomposition and its quaternary structure (3–5).

Recently, several subunits have been implicated in a growingnumber of humandiseases including developmental disorders (3,6) and cancer (4, 7). Most evidence has been gathered for thekinase module subunit CDK8, which has been linked to colonand pancreatic cancer (8, 9) and was also investigated as apotential target in cancer therapy (10, 11). Other subunits havealso been reported in neoplastic conditions such as MED12 inuterine leiomyomas, fibroepithelial breast tumors, and prostatecancer (12–14), MED15 in prostate cancer and head and neckcancer (15, 16), and cyclin C in acute lymphoblastic leukemia(17). In addition, some subunits have been linked to alteredhormone receptor signaling such as MED1 in breast cancer andprostate cancer (18, 19).

To systematically characterize Mediator complex expressionprofiles across human tumors, we performed a pan-cancer anal-ysis of the Mediator complex transcriptome in 23 cancer entitiesfor more than 8,000 patients from The Cancer Genome Atlas(TCGA), followed by an in-depth investigation of the role of thekinase subunits CDK19 (formerly known as Cdc2L6 or CDK11-like) and its paralog CDK8 in prostate cancer.

Materials and MethodsFirebrowseR

We developed the R-package FirebrowseR to facilitate access todata generated by the Broad Institute's Firehose pipeline andmade it available for public use under MIT license (github.

Translational Relevance

Tight control of gene expression is needed for physiologicalcell homeostasis, but it is frequently deregulated in cancer. Themultiprotein Mediator complex plays a pivotal role in tran-scriptional regulation being a central binding elementbetween transcription factors and RNA polymerase II. Apan-cancer analysis of the Mediator complex transcriptomerevealed distinct expression profiles distinguishing entities byhierarchical clustering.CDK19was identified to be specificallyoverexpressed in the progression of prostate cancer and wasassociated with clinicopathologic parameters of aggres-siveness and decreased survival. Knockdown of CDK19 andits paralog CDK8 or inhibition with senexin A, one of severalsmall-molecule CDK19/CDK8 inhibitors that were recentlydeveloped, led to significantly decreased migration and inva-sion of prostate cancer cells in vitro. The prognostic andfunctional roles of CDK19 in prostate cancer and the ongoingdevelopment of selective inhibitors thereby highlight thepotential of CDK19 and its paralog CDK8 as therapeutictargets in prostate cancer.

Figure 1.

Expression profiling of the Mediator complex across tumor types. A, Schematic representation of the Mediator complex, its subunits, and interaction with Pol II.B, Heatmap representation for expression levels of Mediator complex subunits (columns) in primary tumor samples of patients within TCGA (rows). Patientsand subunits are arranged on the basis of similarities in Mediator complex expression profile as determined by unsupervised hierarchical clustering. Color keys nextto the rows and columns indicate cancer type and Mediator complex module per individual patient or subunit, respectively. Colored branches in the rowdendrogram correspond to relevant subgroups (A–P) identified by the dynamic tree partitioning algorithm. Bold black boxes indicate high CDK19 expression inprostate cancer (PCa). C, Pairwise Spearman correlation coefficient of the Mediator complex subunits in PCa.

Br€agelmann et al.

Clin Cancer Res; 23(7) April 1, 2017 Clinical Cancer Research1830




com/mariodeng/FirebrowseR). With FirebrowseR, TCGA datasetsand results of Firehose analyses runs (ref. 20; includingmutations,copy number, gene expression, and clinical information) candirectly be imported into the R (21) programming environment.All functions, data types, and output formats provided by theFirehose web application programmable interface (API) are fullyimplemented.

Transcriptome data assemblyTCGA transcriptome sequencing data (RNA-Seq v2) was

imported into R using FirebrowseR from the Broad InstituteFirehose Pipeline standard data analysis run 2015-04-02 (20).Log2-transformed RSEM count values per gene were used forexpression analysis (22) and unsupervised hierarchical clustering(Euclidean distance, Ward clustering; ref. 23). Subclasses gener-ated by clustering patients according to MED expression werederived by partitioning the dendrogram with a dynamic treecutting algorithm (24).

Microarray data from a prostate cancer progression cohort(25) were downloaded from Gene Expression Omnibus (GEO,http://www.ncbi.nlm.nih.gov/geo/, GSE6919) for log-trans-formed, prenormalized array signal intensities. Multiple sam-ples per patient were aggregated using the median normalizedintensity per gene. A second dataset (GSE21032) includedprimary prostate carcinoma samples (pPCa; n ¼ 131) andprostate cancer metastases (n ¼ 19) with gene expression z-scores after normalization to normal samples. Clinical datawere obtained from http://cbio.mskcc.org/cancergenomics/prostate/ (26). Biochemical disease recurrence based on pros-tate-specific antigen (PSA) elevation was defined according toTaylor and colleagues (26).

Immunohistochemical analysisTissue microarrays (TMA) from paraffin-embedded pros-

tatic tissue provided from cohorts of the University Hospitalsof €Orebro, Basel, and T€ubingen were assessed as describedpreviously (Supplementary Table S3; refs. 14, 27, 28).Approval for the present study was obtained from the ethicscommittee of the University Hospital of Bonn. Immunohis-tochemical (IHC) staining was conducted using the VentanaDiscovery automated staining system (Ventana Medical Sys-tem). In brief, slides were incubated at room temperaturewith primary antibodies: anti-CDK19 rabbit polyclonal(1:100, HPA007053, Sigma-Aldrich) and anti-CDK8 rabbitpolyclonal (1:200, A302-501A, Bethyl Laboratories). Primaryantibodies were detected with the ultraView Universal DABDetection Kit (Ventana Medical System). IHC staining qual-ity was validated independently by 2 observers. Samples witha lack of tissue or absence of carcinoma in all cores presenton the TMA were excluded.

For quantification, slides were scanned (Panoramic Desk,3DHistech) and tumor tissue in each sample was marked man-ually blinded to patient and type of tissue. These user-specifiedregions of interest were analyzed with the image analysis softwareDefiniens Tissue Studio 2.1 (Definiens Inc.). Average nuclearstaining intensity (SI, corresponding to the mean brown chro-mogen intensity) was quantified as a continuous value (arbitraryunits). Specificity of the CDK19 antibody for IHC staining wasconfirmed by co-incubation with the corresponding CDK19 anti-gen (APREST71438, Sigma-Aldrich).

Cell linesCell lines RWPE (benign prostate) and prostate cancer cell lines

PC-3, DU-145, and LnCaP were obtained from the ATCC andmaintained according to provider's instructions. Cell lines wereauthenticated by Multiplex Human Cell Line Authentication Test(June 2013; Multiplexion). C4-2 and 22Rv1 were a kind gift ofA. Merseburger and M. Cronauer (UKSH L€ubeck).

Quantitative reverse transcription PCRRNA was isolated using RNeasy Mini Kit (Qiagen) and reverse

transcribed using iScript cDNA synthesis kit (Bio-Rad). PCRreactions were performed as duplicates with Power SYBR Greenkit (Thermo Fisher Scientific) using the Light-Cycler 480 II(Roche). Expression levels of CDK8 and CDK19were normalizedto b-actin using 2�DDCT method. Primers for CDK8 (29), CDK19(6), and b-actin (Applied Biosystems):

CDK8-Fwd: 50-CGGGTCGAGGACCTGTTTG-30, CDK8-Rev: 50-TGCCGACATAGAGATCCCAGT-30; CDK19-Fwd: 50-CGGAACC-TATTTTTCACTGTCG-30, CDK19-Rev: 50-TGTGGGATATTCTGG-CATCTT-30; b-Actin-Fwd: 50-CCTGTGGCA TCCACGAAACT-30,b-actin-Rev: 50-CACTGTGTTGGCGTACAGGTCTT-30.

Western blotProtein extraction and Western blotting were performed as

described previously (17); primary antibodies used were: anti-CDK19 rabbit polyclonal (HPA007053, Sigma-Aldrich), anti-CDK8 rabbit polyclonal (A302-501A, Bethyl Laboratories),anti-b-actin (Sigma-Aldrich), anti-vimentin rabbit monoclonal(D21H3, Cell Signaling), and anti-androgen receptor mouse(554225, BD Pharmingen). Development was carried out withhorseradish peroxidase (HRP)-conjugated secondary antibodiesusing ECL Western Blotting Reagent (GE Healthcare).

CDK8 and CDK19 knockdownPools of 3 siRNAs targeting CDK8 or CDK19 (CDK8: sc-29267,

CDK19/Cdc2L6: sc-72844, Santa Cruz) and a nontargeting scram-bled siRNA as control (sc-37007, Santa Cruz) were transfectedwith Screenfect A (Genaxxon Bioscience GmbH). OptimalCDK8/19 siRNA concentrations (10–50 nmol/L) were determined byWestern blotting. For proliferation assays, siRNA transfection wasperformed in 96-well plates and MTT was added 72 hours later.For Western blot analyses and migration/invasion assays, siRNAtransfection was performed in 6-well plates and cells were seededinto migration/invasion chambers 48 hours after transfection.Proteins were extracted 72 hours after transfection.

CDK8/CDK19 inhibitor senexin ASensitivity of cell lines to senexin A (Cat. no. 4875, Tocris

Bioscience) was assessed by dilution series in 96-well platescompared with DMSO control. Viability was measured by MTTassay72hours later. Formigration/invasion, cellswerepre-treatedwith senexin A for 24 hours before plating into migration/inva-sion chambers.

Cell viability assayViability following senexin A treatment or knockdown was

assessed using thiazolyl blue tetrazolium bromide (MTT). Fivethousand PC-3 or 7000 LnCaP cells per well were plated in 96-well plates (Corning), and 500 mg/mL MTT (Sigma-Aldrich)dissolved in PBS (Gibco Life Technologies) was added 72 hoursafter treatment. Four hours later, 100 mL solubilization buffer

CDK19 as a Therapeutic Target in Advanced Prostate Cancer

www.aacrjournals.org Clin Cancer Res; 23(7) April 1, 2017 1831




[40% vol/vol dimethylformamide (Alfa Aesar), 2% glacial acetic(Merck), 16% SDS (Applichem), pH 4.7)] was added and absor-bance at 595 nm was measured the next day. For LnCaP cells, theexperiments were performed in medium containing charcoal-stripped FCS (ThermoFisher Scientific) without (¼androgen dep-rivation) and with supplementation of dihydrotestosterone(DHT).

Migration and invasion assaysCell lines were pretreated with senexin A or subjected to siRNA

knockdownas described above. A total of 5�105 cellswere platedin the upper chamber of migration inserts (VWR) or Matrigel-coated Transwell inserts (VWR) containing 0% FCSmedium. Thelower chamberwasfilledwithmediumcontaining 10%FCS.After24 hours, cells were fixed with 4% paraformaldehyde (Merck),stained with hematoxylin (Waldeck), and washed with water.Membranes were scanned and cells were counted using Definienssoftware (Definiens Inc.).

30-RNA sequencingTotal RNA of DU-145 cells treated for 48 hours with 10 mmol/L

Senexin A or DMSO was extracted and transcriptome sequencingwith the 30 QuantSeq FWD Kit (Lexogen) and data analysis wasperformed as described previously (30) and in SupplementaryMethods. The experiment was performed in triplicates.

Statistical analysesGroup comparisons were done using nonparametric Mann–

Whitney U or Kruskal–Wallis test unless indicated otherwise.Migration/Invasion assays were analyzed by t tests. To assess theclassification of samples as prostate cancer or cancer of anotherentity by CDK19 mRNA expression in male patients, a logisticregression model was applied. A Hosmer–Lemshow Goodness-of-Fit Test, a receiver operator curve (ROC), and its area under thecurve (AUC)were used to evaluatemodel accuracy. Association ofMediator subunits among each other and with androgen receptor(AR) expression was evaluated by Spearman rank correlation. Alltests were done 2-sided and P values were adjusted for multipletesting using Bonferroni–Holm correction as indicated. Survivalwas evaluated by Kaplan–Meier estimator and log-rank test andhigh CDK19 expression of pPCa samples was defined as valuesgreater than 1.5 SDs above the average expression level in normalsamples. Adjustment for co-variables was done using Cox regres-sionmodels (31). All statistical analyseswere donewithMicrosoftExcel and R (21).

ResultsPan-cancer transcriptome profiling of the Mediator complex

To comprehensively analyze the transcriptomic profile of themultiprotein Mediator complex across a large spectrum of themost common tumor entities from thousands of patients, TCGApresents an invaluable resource. To facilitate retrieval of large-scale data sets provided by TCGA and to avoid complicated anderror-prone manual editing of data files, we developed an R-package called FirebrowseR. FirebrowseR enables users to queryservers at the Broad Institute from within the statistical program-ming environment R and directly imports desired cohorts anddata types for further processing. Using FirebrowseR, RNA-Seq–based expression levels of all 33 Mediator complex subunitsacross all 23 tumor entities publicly available at TCGA at the

time of analysis for all 8,137 patients was obtained (see Supple-mentary Tables S1 and S2). A heatmap representation of mRNAlevels shows that the majority of Mediator complex subunits areexpressed at varying degrees across patient samples at medium tohigh levels (Fig. 1B). Anotable exception isMED12L, which showsvery low mRNA expression in almost all samples except for asubpopulation of patients with acute myeloid leukemia (LAML,cluster P, Fig. 1B). In an exploratory approach, unsupervisedhierarchical clustering of patient samples on the basis of theirsimilarity of Mediator complex subunit expression profiles wasperformed and patients (rows) of the heatmap were reorderedaccording to these groupings. Relatedness of clusters is indicatedby a dendrogram (Fig. 1B). A dynamic partitioning algorithmwasused to derive relevant subclasses in these clusters (24). Thisresulted in identification of 16 subclasses (labeled A-P). Uponinspection, it was evident that whereas some of these subclassescontain a heterogeneousmixture of samples fromdifferent tumorentities (e.g., clusters A, D, F, and M), others were primarilycomposed of samples from a single tumor entity (Fig. 1B).Examples of tumor entities that separated well on the basis oftheir Mediator complex expression profile were low-grade glio-mas (LGG) and glioblastomas (GBM, cluster N), thyroid cancers(THCA, cluster J), hepatocellular carcinoma (LIHC, cluster G),and LAML (cluster P). Cluster L contained the majority of pPCaand no samples of other tumor entities. This cluster was markedby the high expression of CDK19, which is part of the kinasemodule and has been implicated in prostate cancer before (32).High CDK19 expression was also present in the second group ofpPCa samples (black boxes, Fig. 1B). Overall, prostate cancersamples showed highest CDK19 expression levels (P ¼ 1 �10�224, Supplementary Fig. S1A). It is known that either CDK19or its paralogCDK8but not both canbe amember of theMediatorcomplex to form a functioning kinase (5). In contrast to CDK19,highest levels of CDK8 are observed in rectal cancer (READ) andcolon cancer (COAD) samples (P ¼ 1 � 10�152, SupplementaryFig. S1B). This is in line with findings indicating a tumor-pro-moting role of CDK8 in colorectal cancer (9) and correspondswith the group of COAD and READ samples in cluster C markedby high CDK8 expression levels (Fig. 1B). In contrast to CDK19,CDK8 levels were not increased in pPCa compared with otherentities (Supplementary Fig. S1B). Within pPCa samples, CDK19has the highest expression of all subunits, whereas CDK8 issignificantly lower (P ¼ 1 � 10�163, Supplementary Fig. S1C).To further unravel the Mediator complex profile in pPCa, allpairwise correlation coefficients of expression levels of Mediatorcomplex subunits were calculated from the TCGA prostate cancercohort. Hierarchical clustering on the basis of these correlationcoefficients revealed 2major clusters (Fig. 1C). All kinasemodulesexhibit strong correlation with each other and were grouped incluster 1. Interestingly, a finer subdivision of the dendrogramrevealed that almost all kinase modules fell into cluster 1.1 withthe exception of CDK19 and CCNC (Fig. 1C), potentially indi-cating differences in expression regulation. BecauseAR signaling isone of the main determinants in prostate cell regulation, weadditionally performed a correlation analysis of ARmRNA levelswith the expression of Mediator complex subunits. In pPCa,tumors revealed a strong positive correlation of AR expressionwith several subunits pre-eminently from the kinasemodule suchas CDK8 (r ¼ 0.60, P < 1 � 10�16, Supplementary Fig. S1D).Interestingly, the highest correlation was observed with MED1(r¼ 0.81, P < 1� 10�16), which has been shown to be involved in






AR signaling and in prostate cancer before (19). In contrast, othersubunits showed a strong negative correlation, whereas CDK19and cyclin C showed only aweak associationwithAR expression (r¼ 0.19, P¼ 1� 10�6, Supplementary Fig. S1D). Thismay indicatea distinct regulation of the kinase module and its constituents inprostate cancer compared with other entities even though direc-tionality and causality of interaction cannot be inferred fromcorrelation.

To further assess the distinct role of CDK19 in pPCa, a logisticregression model was used to predict the probability of a tumorsample from a male patient (n ¼ 3,617) being a prostate canceror any other entity based on CDK19 mRNA expression alone.Interestingly, the odds for a sample being a prostate cancer were9.1-fold increased per one-unit increase of (log-transformed)CDK19 expression [P ¼ 2 � 10�11; OR, 9.1, 95% confidenceinterval (CI), 8.3–11.2]. In addition, the Goodness-of-Fit test wasnonsignificant (P ¼ 0.08), thereby not indicating a lack of fit andthe ROC had an AUC of 0.93 (Supplementary Fig. S1E and S1F).Overall this provides evidence that CDK19 expression as a singlemarker can successfully discriminate tumor samples from males

as prostate versus non–prostate cancer and emphasizes thatCDK19 plays a specific role in prostate cancer.

Evaluation of CDK19 and CDK8 in prostate cancer progressionWhen comparing the normal prostate samples available from

TCGA (n¼ 52)with TCGAprimary tumors (n¼ 496),CDK19wassignificantly increased in both an unpaired analysis (P ¼ 2.1 �10�6; Fig. 2A) and in an analysis of matched tumor–normal pairs(P ¼ 9.9 � 10�5, Supplementary Fig. S2A). However, analysis ofTCGA data regarding expression of CDK19 and its paralog CDK8in prostate cancer is limited by the restriction to primary tumors.We therefore additionally analyzed publicly available microarraygene expression data (GSE 6919) including normal prostate tissue(n¼ 81), pPCa (n¼ 65), and also somemetastatic samples (n¼ 4;ref. 25).CDK19 expression increased significantly during prostatecancer progression (P ¼ 5.4 � 10�5; Supplementary Fig. S2B),whereas CDK8 remained at similar levels between normaland primary tissue and was even decreased in metastases (Sup-plementary Fig. S2C). To increase sample size, we extended ouranalysis with a second publicly availablemicroarray data set (GSE

Expression of CDK19, TCGA

Expression of CDK19, GSE 21032

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Low CDK19 (n = 114)High CDK19 (n = 17)

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Figure 2.

Association of CDK19 expression withclinical prostate cancer (PCa) diseasestage. CDK19 mRNA levels areincreased in primary PCa comparedwith normal controls (TCGA geneexpression;A) and during progressionfrom pPCa to metastatic tissue (GSE21032; B). C, Association of CDK19expression with Gleason score. D,Disease-free survival of patients withpPCa with low versus high CDK19expression (�1.5 SDs above theaverage expression in normalprostate tissue).






21032; ref. 26) with clinically annotated pPCa (n ¼ 131) andmetastases (n ¼ 19). This validated a significantly higherCDK19 expression in metastatic samples than in pPCa (P ¼0.002, Fig. 2B). In pPCa, increased CDK19 expression wassignificantly associated with higher pathologic T stage (T2 vs.�T3; P ¼ 0.02, Supplementary Fig. S2D), extracapsular exten-sion (present vs. absent; P ¼ 0.03), presence of an ERG rear-rangement (P ¼ 4 � 10�4, Supplementary Fig. S2E), and alsowith a higher Gleason score (<7 vs. �7; P ¼ 0.001, Fig. 2C).CDK19 overexpression in pPCa specimens, defined as 1.5 SDsabove the average expression in normal tissues, was moreoverassociated with a significantly decreased disease-free survival(log-rank: P ¼ 0.002, Fig. 2D) and a 3.4 times increased hazardfor biochemical disease recurrence (HR, 3.4, 95% CI; 1.5–7.8;P ¼ 0.004). This increased hazard remained significant evenafter adjustment for Gleason score and age (CDK19: HR, 2.7;95% CI; 1.13–6.3; P ¼ 0.02) resulting in 5-year disease-freesurvival rates of 51.3% and 82.6% for high and low CDK19-expressing patients, respectively.

To also assess the expression CDK19 and CDK8 on the proteinlevel and to validate their implication in prostate cancer progres-sion, we subsequently analyzed a large TMA including 622patients with a substantial number of metastatic and castra-tion-resistant samples (Supplementary Table S3) by IHC. TheMediator complex is predominantly interacting with transcrip-tion factors and DNA in the nucleus (3, 19), and following co-incubation of the CDK19 antibody with the recombinant CDK19antigen, only nuclear expression was considered specific (Sup-plementary Fig. S2G). Therefore, analysis of specimens wasrestricted to measurement of average nuclear CDK19 and CDK8staining intensity and was considered for further analyses (Fig. 3Aand B). Comparably to the mRNA data, CDK19 protein expres-sion was low or absent in normal prostate glands but increasedsignificantly frombenign tissue to primary tumors (P¼ 9� 10�4)and from primary tumors to metastases/castration-resistant pros-tate cancer (CRPC; P¼ 4� 10�10, Fig. 3C). CDK8 expression wascomparable in benign and primary tissues (P ¼ 0.28) but wassignificantly increased in metastases/CRPC (P ¼ 2 � 10�8, Fig.3D). Subsets of the current cohort had been analyzed for thepresence of a ERG rearrangement (deletion or translocation) byFISH and ERG/Ki-67 protein expression by IHC in previousstudies (Supplementary Table S3; refs. 27, 28, 33). A combinedanalysis of the datasets showed that presence of an ERG rear-rangement was significantly associated with higher CDK19 levelsin primary tumors (adj. P ¼ 3� 10�7), but high CDK19 levels inmore advanced stages were independent of ERG rearrangementstatus (Fig. 3E). No association of CDK8 expression level and ERGrearrangement was present (Supplementary Fig. S2F). Similarly,the detection of ERG protein expression correlated with signifi-cantly increased CDK19 levels in primary tumors (adj. P ¼ 5 �10�9) but not in increasedCDK19 levels inmetastases/CRPC (adj.P¼ 0.51) or in altered CDK8 levels (Supplementary Fig. S2H andS2I). Ki-67, an indicator of high cell division rate and associatedwithmore aggressive phenotype, was also significantly associatedwith increased CDK19 levels in primary tumors (adj. P ¼0.012, Fig. 3F). For lymph node metastases, a trend towardincreased CDK19 expression with higher Ki-67 index was presentbut remained nonsignificant, potentially due to low samplenumbers (adj. P¼ 0.14), whereas no effect was evident in distantmetastatic CRPC (Fig. 3F). Overall, these results indicate thatCDK19 is elevated already in early prostate cancer progression and

that CDK8 protein levels are upregulated in metastatic tumorsonly. Moreover, CDK19 is significantly associated with ERGrearrangements, ERG protein expression, and with Ki-67 in pri-mary tumors only, but CDK19 levels become independent of ERGin advanced stages.

Functional evaluation of CDK19 and CDK8 in prostate cancercell lines

To evaluate the functional role of the paralogs CDK19 andCDK8 in prostate cancer, first the benign cell line RWPE and the2 malignant cell lines LnCaP (androgen-sensitive) and PC-3(androgen-independent) were investigated. Similar to the IHCand mRNA results in patient samples, mRNA and proteinexpression of CDK19 were higher in the malignant cell lines(Fig. 4A and B). Interestingly, CDK19 and CDK8 increased inLnCaP cells upon androgen deprivation (Fig. 4C). siRNA-medi-ated knockdown of CDK19 and CDK8 did not impact prolif-eration of either LnCaP or PC-3 cells (Fig. 4D and E). Toinvestigate involvement of CDK8 and CDK19 in metastaticspread, Boyden chamber assays were used to assess invasionand migration. This was done with PC-3 cells, as RWPE andLnCaP cells showed no migratory potential. Following a tran-sient CDK19 knockdown, both migration and invasion of PC-3cells were significantly reduced to 44% (P¼ 6� 10�4) and 58%(P ¼ 0.01) compared with control cells, respectively (Fig. 4F).The effect of CDK8 knockdown led to similar but less pro-nounced effects (migration: 61%, P ¼ 0.02; invasion: 68%, P ¼0.06). To further assess CDK8 and CDK19 as potential thera-peutic targets in prostate cancer, we used the selective CDK8/CDK19 inhibitor senexin A (11) and extended analyses in alarger panel of prostate cancer cell lines (LnCaP, C4-2, 22RV1,PC-3, DU-145; refs. 14, 34). All cell lines expressed CDK8/CDK19 (Supplementary Fig. S3A). In accordance with theknockdown results, senexin A treatment showed only modestimpact on proliferation (Supplementary Fig. S3B) with slightlyhigher effects in LnCaPs after androgen deprivation (Supple-mentary Fig. S3C). However, pretreatment of cells with senexinA significantly reduced migration and invasion in all assessablecell lines except for C4-2 (Fig. 5A) with dose-dependent effectsin PC-3 cells (P ¼ 0.01, Supplementary Fig. S3D). Effects weremost pronounced in DU-145 (migration: 21% of control, P <0.001; invasion: 18%, P < 0.001; Fig. 5A). Accordingly, treat-ment of DU-145 with senexin A, knockdown of CDK19 and to alesser extent CDK8 led to reduced vimentin expression (Fig.5B), which has been described as a relevant determinant ofprostate cancer migratory capacity (35). To systematicallyinvestigate the mechanism of action, senexin A–treated DU-145 cells were subjected to RNA sequencing. Among the genesmost strongly upregulated, several have previously been impli-cated in reduced migration of cancer cells or as being repressedduring prostate cancer progression such as such as TAGLN (36,37), CDH1 (38, 39), CDH3 (40), TPM1 (41), orMYL-9 (refs. 39,42; Fig. 5C). A systematic evaluation of transcriptional changesusing an enrichment analysis of Gene Ontology (GO) termsand KEGG pathways revealed that upregulated genes wereenriched for processes governing cytoskeletal structure, mod-ulation of cell motility and focal adhesion, whereas down-regulated processes were primarily involved in transcriptionand translation (Fig. 5D). This is in line with the observedphenotypic changes and the role of the Mediator complex intranscriptional control and provides an explanation how






Normal

CD

K19

CD

K8

CD

K8

CD

K19

pPCa– pPCa+ LNPC CRPC

Normal pPCa LNPC CRPC– CRPC+

Expression of CDK19

Expression of CDK19 by ERG Expression of CDK19 by Ki67 and tumor stage

Expression of CDK8

Nuc

lear

CD

K19

exp

ress

ion

Nuc

lear

CD

K19

exp

ress

ion

Nuc

lear

CD

K19

exp

ress

ion

Nuc

lear

CD

K8

expr

essi

on

Normal pPCA LNPC CRPC

Normal pPCA LNPC CRPCpPCA LNPC CRPC

Normal pPCA LNPC CRPC

P = 9×10–4 P = 0.28P = 4×10–10

P = 2×10–8

P = 3×10–7 P = 0.81 P = 0.58 P = 0.012 P = 0.14 P = 0.81

0.75

0.50

0.25

0.75

0.50

0.25

0.75

0.50

0.25

0.8

0.6

0.4

0.2

A

B

C

E F

D

ERG StatusKi-67

ERG wild-typeERG Rearrangement

0%1–10%>10%

Figure 3.

IHC staining analysis of CDK19 and CDK8 expression in a large cohort of clinical specimens. A and B, Representative images of the CDK19 and CDK8 IHC staining forbenign prostate, pPCa, lymph node metastases (LNPC), and distant metastases of CRPC. �, samples with weak or absent nuclear staining in comparisonto samples with strong nuclear expression (þ). 20� (top) and 40� (bottom) objective magnification. C,Average nuclear CDK19 staining intensity (mean brown) oftissue samples increases significantly throughout disease stages. D, CDK8 nuclear staining intensity is in contrast only elevated in metastatic CRPC samples.E, In primary tumors, CDK19 protein expression is significantly associated with the presence of an ERG rearrangement (gene fusion or deletion, determined by FISH).This association is lost in more advanced stages. F, In primary cancers, CDK19 staining is significantly increased with higher Ki-67 index, a marker of cellproliferation, aggressive behavior, andworse outcome.Of note, benign prostate samples all had aKi-67 index of 0%,whereas distantmetastases/CRPCs had allmorethan 0% Ki-67–positive cells.






interference with the kinase module of the Mediator complexmay impact prostate cancer cell migration.

DiscussionRecently, an increasing number of Mediator complex subunits

have been identified as relevant components in a range of humandiseases including different cancer entities (3, 4, 7). To foster theknowledge of Mediator complex biology in human cancers and toevaluate its complex and potentially entity-specific subunit com-position, we performed a pan-cancer analysis ofMediator complextranscriptome profiles. To facilitate entity-wide analyses usingTCGA data, we developed the R-package FirebrowseR, which wemade available to the public. In an exploratory approach, RNA-Seqexpression data of all 33 MED subunits for more than 8,000patients across 23 cancer entities available from TCGA were sub-jected to anunsupervisedhierarchical clustering and closely relatedsubgroups were determined by a dynamic partitioning algorithm(24). Using mRNA expression data, other modes of changes in theMediator complex such asmutations and copy number alterationscannot be evaluated and the correspondence of mRNA levels toprotein levels and to MED composition can be inferred onlyindirectly. Nevertheless, this analysis resulted in several subclustersthat were almost entirely composed of only one tumor entity,whereas others showed a more heterogeneous mixture of samples

from different entities. This segregation of tumor entities solely byMED expression indicates that theMediator complex plays distinctroles in different cancer types and allows generation of severalnovel hypotheses regarding MED biology and potential therapeu-tic targets. The LAML samples (cluster P) are for example furthersubdivided into 2 groups on the basis of differences in MED12Lexpression, which presents a valuable topic for further research.Very highCDK8 expressionwas observed in the group of colorectalcancers (cluster C, Fig. 1B, Supplementary Fig. S1B), which is inaccordance with prior data (9, 43) and presents validation for ourapproach. One of the entities resulting in a very homogenousgroup was pPCa (cluster L, Fig. 1B). Compared with all otherentities, pPCa samples were marked by exceptionally high CDK19expression and an increase from normal prostate tissue to pPCa,which indicates a distinct role of this kinase subunit in prostatecancer and a potential candidate for targeted cancer-specific ther-apy. High CDK19 expression in prostate cancer has also beenobserved by studies not primarily investigating the Mediatorcomplex (32, 44) andwas linked tomigratory potential in prostatecancer cells (45). However, an in-depth evaluation of CDK19 hadnot been performed yet.

For confirmation of the TCGA results, additional cohorts includ-ing normal tissue, primary tumors, and metastatic cancers ofpublicly available mRNA expression data and an IHC cohort ofmore than 600 clinical prostate specimens were used. Because of

Figure 4.

In vitro analysis of CDK19 and CDK8 in prostate cancer (PCa) cell lines. A and B, CDK19 mRNA (meanþ SD) and protein levels as measured by reverse transcriptaseqPCR normalized to b-actin using the 2�DCT method and by Western blotting were increased in malignant cell lines LnCaP (androgen-sensitive) and PC-3(androgen-independent) comparedwith the benign cell line RWPE (n¼ 3). CDK8 levelswere similar in all cell lines.C,CDK19, CDK8, andAR expression in LnCaP cellsafter androgen deprivation (AD) or after treatment with DHT at indicated concentrations. D, Transient knockdown of CDK19 or CDK8 by siRNA was establishedat the respective optimal concentrations and verified by Western blotting for LnCaP and PC-3 cells. E, No effect of CDK19 or CDK8 knockdown on cellviability after 72 hours as measured by MTT assay in comparison to scrambled (scr) RNA control (n ¼ 3; mean þ SD). F, Migration and invasion were significantlydecreased in PC-3 cells after CDK19 and CDK8 knockdown compared with control (n ¼ 4, mean þ SD).






the predominant localization and function of the Mediator com-plex in the nucleus (3, 19), nuclear protein expression was eval-uated in the IHC.We could thereby validate the findings regardingCDK19 and CDK8 in pPCa and also established a further increaseof CDK19 in metastatic and CRPC (Figs. 2 and 3). CDK8 was incontrast strongly elevated in distant metastases only on a proteinlevel but showed slightly decreased mRNA expression. Very inter-estingly, in the 2 cohorts with information on ERG rearrangement,

CDK19 was significantly associated with ERG rearrangements andERG expression in primary tumors but not in more advancedstages. This is in agreement with the observation that rearrange-ments and expression of ERG, a member of the ETS transcriptionfactor family, are early events in prostate cancer carcinogenesis butmost likely lose relevance inmore advanced stages (46). Similar toour results, a recent study reported CDK19 as 1 of only 2 candidategenes associated with invasion and aggressiveness from in vivo and

Figure 5.

Inhibition of CDK19 and CDK8 in prostate cancer (PCa) cell lines with senexin A. A, Migration and invasion of PCa cell lines in Transwell Boyden chamberassays following 24-hour senexin A (10 mmol/L) pretreatment compared with DMSO control (n ¼ 3, mean þ SD). 22Rv1 cells did not invade under control ortreatment conditions. n.s., nonsignificant; � , P < 0.05; �� , P < 0.01; �� , P < 0.001. B, Treatment of DU-145 cells with senexin A (10 mmol/L) or siRNA knockdown ofCDK19/CDK8 led to reduced expression of vimentin. C, Volcano plot of RNAseq results for senexin A versus DMSO-treated DU-145 cells (n ¼ 3) indicatinglog2 fold change (x-axis) and false discovery rate (FDR)-adjusted significance (y-axis). The 15 most significantly up- or downregulated genes are indicated by name.D, Top five most significantly enriched GO processes and KEGG pathways inferred from genes significantly (adj. P < 0.01) up- (Up) or downregulated (Down).Dashed line indicates adjusted GO enrichment: P ¼ 0.05; �� , adjusted P < 0.001.






in vitro gene expression studies of prostate tumors with rearrange-ment of another gene from the ETS transcription factor familycalled ETV-1 (47). In addition, in primary tumors, a correlation ofCDK19 levels with Ki-67 protein expression was observed, whichhas been linked to aggressive prostate cancer and was shown to bean independent prognostic biomarker (48). Given the low impactof CDK19 and CDK8 knockdown and inhibition on prostatecancer cell viability in vitro suggests that CDK19 and CDK8 donot causally influence proliferation but are instead concordantlyupregulated in more aggressive tumors.

Because of the limited availability of patient data and follow-upinformation for most of the datasets, evaluation of clinical asso-ciations andoutcome could unfortunately only be done for one ofthe cohorts. In this cohort, CDK19 expression was significantlyassociated with clinical parameters of malignancy such asadvanced stage and higher pathologic Gleason score. Moreover,high CDK19 expression was significantly correlated to worseoutcome for patients with pPCa with a 30% absolute differenceof freedom from disease at 5 years. It has to be noted, however,that due to the low incidence of fatal events, biochemical recur-rence was used as a surrogate endpoint and that the cohortconsisted exclusively of patients who had undergone surgicalprostatectomy (26). Extrapolation of conclusions drawn fromthese findings to other populations and different primary treat-ment strategies such as "watchful waiting" is therefore limited.Instead, these association and potential causal links with CDK19should be investigated in subsequent molecular biological andepidemiologic studies, ideally in a prospective manner.

In prostate cell lines, increased expression of CDK19 mRNAand protein could be detected in the malignant cell lines com-pared with nonmalignant RWPE cells. Interestingly, proliferationof prostate cancer cell lineswasnot reduced following knockdownof CDK8 or CDK19 and only modestly impacted by treatmentwith the selective CDK8/CDK19 small-molecule inhibitorsenexin A (11) with viability reduction of 50% at concentrationswell above 20 mmol/L. This is in contrast to colon cancer andmelanoma cell lines with high endogenous CDK8 levels in whichit has been shown that CDK8 knockdown leads to reduced cellproliferation (9, 29). Other reports paradoxically suggest thatCDK8 may possess tumor-suppressive properties (17, 49). ForCDK19, no such knowledge has been available. In contrast, strongreductions of migration and invasion, which are essential traits inmetastatic spread and constitute an aggressive phenotype, couldbe achieved in PC-3 cells withCDK19 andCDK8 knockdown (Fig.4E and F) andby pretreatmentwith senexin A in 22Rv1, PC-3, andespecially in DU-145 cells (Fig. 5A). This inhibition of migrationand invasion corresponds well to the high expression of CDK19and CDK8 in metastatic samples and indicates their functionalrole in prostate cancer. Interestingly, the increasing recognition ofthe Mediator complex and its kinase module as a potentialtherapeutic cancer target has recently led to the development ofadditional selective CDK8/CDK19 inhibitors (10), which couldfurther enhance research into novel targeted treatment strategiesfor patients with prostate cancer. A focused assessment of vimen-tin, a major determinant of prostate cancer motility (35), showeddecreased protein expression in DU-145 cells after senexin Atreatment and knockdown of CDK19 or CDK8. Moreover, asystematic investigation via RNA sequencing following senexinA treatment showed differential regulation of several genes andprocesses involved in cancer cell migration, cytoskeleton, andfocal adhesion (Fig. 5C and D; refs. 36–42). These observations

therefore provide a mechanism for decreased motility, whichcorresponds well to the observed phenotype and the expectedrole of the Mediator complex. Interestingly, some genes differen-tially expressed in response to senexin A have been linked toWnt/b-catenin pathway before (50). Nevertheless, it should be kept inmind that RNA interference and small-molecular inhibitors maypossess off-target effects and that interference with a fundamentalcellular component such as the Mediator complex may provokedifferential responses depending on the cell type. Further, eval-uation of the MED kinase module in prostate cancer and otherentities will therefore be needed to gain an improved understand-ing of the Mediator complex as a therapeutic target in cancer.

Taken together, our data for the first time provide evidence thattumor entities can be differentiated by their Mediator complexexpression profile and that CDK19—and to a lesser extent itsparalog CDK8—plays an important and distinct role in prostatecancer, which gains impact throughout disease progression.Thereby, our study yields new insights into the differential reg-ulation of theMediator complex across tumor entities and its rolein human disease, fosters the understanding of prostate cancerbiology, and moreover presents CDK19 and CDK8 as novelpotential therapeutic targets especially in the metastatic setting.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Br€agelmann, N. Kl€umper, I. Syring, P. Brossart,S. PernerDevelopment of methodology: J. Br€agelmann, N. Kl€umper, S. PernerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Br€agelmann, A. von M€assenhausen, A. Queisser,I. Syring O. Andr�en, P. Brossart, M.A. Svensson, J. Kirfel, S. PernerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Br€agelmann, N. Kl€umper, A. Offermann, M. Deng,I. Syring, A.S. Merseburger, J. Carlsson, S. Duensing, J. Kirfel, S. PernerWriting, review, and/or revision of the manuscript: J. Br€agelmann,N. Kl€umper, A. Offermann, I. Syring, A.S. Merseburger, I. Vlasic, J. Carlsson,O. Andr�en, P. Brossart, S. Duensing, M. Svensson, S. PernerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Offermann, D. B€ohm, M. Deng, C. Sanders,I. Syring, W. Vogel, E. Sievers O. Andr�en, P. Brossart, M.A. Svensson, S. PernerStudy supervision: S. PernerOther (review/revision of article): Z. Shaikhibrahim

AcknowledgmentsThe results shown here are in part based upon data generated by the TCGA

Research Network: http://cancergenome.nih.gov/. We thank Anke R€oper, Ker-stin Ludwig and Sugirthan Sivalingam for help with experiments and MarcusCronauer for C4-2 and 22Rv1 cells.

Grant SupportThe study was supported by a grant of the Rudolf Becker-Foundation, a grant

of the Wilhelm Sander Foundation (2011.077.2), and of the German ResearchFoundation (DFG PE1179/9-1 and PE1179/11-1) to S. Perner. I. Syring wassupported by the Ferdinand Eisenberger-Fellowship of the German Society ofUrology (DGU, SYI1/FE-13). J. Br€agelmann was supported by a Gerok-Fellow-ship grant (2014-11-06) of the Medical Faculty of the University of Bonn. A.Offermannwas supported by amedical doctoral fellowship grant (BONFOR) ofthe Medical Faculty of the University of Bonn and C. Sanders by a "Mildred-Scheel medical doctoral programme" grant of the German Cancer Aid.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 15, 2016; revised August 28, 2016; accepted September 15,2016; published OnlineFirst September 27, 2016.






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2017;23:1829-1840. Published OnlineFirst September 27, 2016.Clin Cancer Res Johannes Brägelmann, Niklas Klümper, Anne Offermann, et al. Prostate CancerIdentifies CDK19 and CDK8 as Therapeutic Targets in Advanced Pan-Cancer Analysis of the Mediator Complex Transcriptome

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