SUPPLEMENTARY INFORMATION Excessive fatty acid oxidation ... · 2 expressed as means ± s.e.m. *P <...

SUPPLEMENTARY INFORMATION Excessive fatty acid oxidation induces muscle atrophy in cancer cachexia Tomoya Fukawa, Benjamin Chua Yan-Jiang, Jason Chua Min-Wen, Elwin Tan Jun-Hao, Dan Huang, Chao-Nan Qian, Pauline Ong, Zhimei Li, Shuwen Chen, Shi Ya Mak, Wan Jun Lim, Hiro-omi Kanayama, Rosmin Elsa Mohan, Ruiqi Rachel Wang, Jiunn Herng Lai, Clarinda Chua, Hock Soo Ong, Ker-Kan Tan, Ying Swan Ho, Iain Beehuat Tan, Bin Tean Teh, Ng Shyh-Chang

Supplementary Figure 1 Human RXF393 cancer cells induce muscle atrophy. (a) Relative

frequency distributions of myofiber cross-sectional area in matching quadriceps muscle biopsies

from SKR- and RXF-bearing mice. AU, arbitrary units of pixels. Data are expressed as means.

P < 0.001 relative to SKR control, as determined by Mann-Whitney test. (b) Representative

phase contrast images of early myotubes derived from human muscle stem cells isolated from

patient biopsies, after 6d exposure to cachectic RXF media or non-cachectic SKR media. (c)

Measurements of total cell volume differences in early myotubes from (b), based on quantitative

phase imaging. Data are expressed as means ± s.e.m. *P < 0.05 relative to SKR control, as

determined by Student’s t-test. (d) Western blot of human myotubes after 6 days of exposure to

cachectic RXF or non-cachectic SKR medias, using antibodies against myogenin, α-actinin, fast

myosin heavy chain (MHC), pan-MHC, GAPDH and tubulin.

Supplementary Figure 2 Cachectic RXF media induces mitochondrial oxidative stress in

human myotubes. (a) Representative MitoSox Red fluorescence images of live human

myotubes after 1h exposure to cachectic RXF versus non-cachectic SKR media. (b)

Representative MitoSox Red fluorescence images of live human myotubes after 1h exposure to

cachectic RXF with and without the fatty acid oxidation inhibitor etomoxir 10 µM. (c)

Quantification of cell death using ethidium dye, after 6d exposure to RXF media. Data are

Nature Medicine: doi:10.1038/nm.4093

2

expressed as means ± s.e.m. *P < 0.05 relative to SKR control, as determined by Student’s t-

test.

Supplementary Figure 3 Human G361 and mouse Lewis lung carcinoma (LLC) cells both

cause excessive fatty acid oxidation to induce p38 MAPK signaling in myofibers as well.

Western blot for phospho-p38, phospho-AKT, IκBα, MHC, and GAPDH levels in quadriceps

myofibers of G361- or LLC-bearing mice after daily intraperitoneal injections of DMSO vehicle or

20 mg/kg etomoxir (n = 3 each).

Supplementary Figure 4 Etomoxir rescues muscle atrophy in mouse models of cachexia. (a)

Representative images of RXF-bearing mice’ quadriceps muscle morphology with and without

etomoxir treatment. Etomoxir-treated mice’ quadriceps preserved their muscle mass. (b)

Forelimb and hindlimb muscle mass (% of body mass) of RXF-bearing mice after daily injections

of DMSO vehicle, etomoxir or SB202190 (n = 5 each). (c) Forelimbs’ and hindlimbs’ muscle

mass (% body mass) of G361-bearing mice after daily injections of DMSO vehicle, 20 mg/kg

etomoxir or 5 mg/kg SB202190 (n = 5 each). Etoxomir and SB202190 rescued limb muscle

loss. (d) Representative H&E histology of quadriceps muscles after daily intraperitoneal

injections of DMSO vehicle or etomoxir in LLC-bearing C57BL/6J mice. Etomoxir rescued

myofiber atrophy. Bar represents 200 µm. (e-g) Tumor growth curves of (e) RXF, (f) G361, and

(g) LLC tumors, with daily injections of DMSO vehicle, 20 mg/kg etomoxir or 5 mg/kg of

SB202190 (n = 5 each). Data are expressed as means ± s.e.m. *P < 0.05 relative to DMSO

vehicle control, as determined by Student’s t-test.

Supplementary Figure 5 Intramuscular controlled-release formulation of etomoxir only rescues

treated hindlimbs’ muscle mass. (a) Hindlimb muscle mass (% of body mass) of RXF-bearing


3

mice after intramuscular injections of vehicle- (2% oxidized alginate beads, n = 6) or etomoxir-

gel (100 µg / 50 µL, n = 7) into hindlimb thigh muscles every 7 days. Mice were sacrificed when

the mice lost 15% weight. Untreated and vehicle-gel-treated hindlimbs atrophied to 3.2% body

mass, whereas the etomoxir-gel-treated hindlimbs showed a significant rescue from atrophy

(3.8%, P = 0.007), similar to normal non-cachectic hindlimbs (3.6%). (b) Representative images

of etomoxir-gel-treated left quadriceps muscle morphology and H&E histology, relative to the

untreated right quadriceps muscles. Bar represents 200 µm. Data are expressed as means ±

s.e.m. **P < 0.01 relative to vehicle-gel control, as determined by Student’s t-test.

Supplementary Figure 6 Etomoxir inhibited fatty acid oxidation, but not PPARα-associated

sterol and carbohydrate metabolism. (a) Acyl-carnitine levels in quadriceps muscles after 20

mg/kg etomoxir treatment (n = 4). (b) Fatty acid and cholesterol levels in quadriceps muscles

after 20 mg/kg etomoxir treatment (n = 4). (c) Polar metabolites significantly changed in

quadriceps muscles after 20 mg/kg etomoxir treatment (n = 4). Etomoxir altered only 12 of 3743

polar metabolites in quadriceps muscles, none of which were PPARα-associated carbohydrates.

Instead only a polar fatty acid, several nucleotide-related metabolites, and redox-related

metabolites were affected by etomoxir, supporting a fatty acid oxidation-specific mechanism that

regulated the redox state and myocellular growth. CMP, cytidine monophosphate. FAD, flavin

adenine dinucleotide. GSSG, glutathione disulfide. Data are expressed as means ± s.e.m. *P <

0.05 and **P < 0.01 relative to DMSO vehicle control, as determined by Student’s t-test.

Supplementary Figure 7 Etomoxir did not affect PPARα target genes in quadriceps muscles.

Raw microarray expression values for PPARα target genes and PPARα itself, relative to Pax3

and Pax7, which are at the background level, after 20 mg/kg etomoxir treatment (n = 3). Pdk4,

pyruvate dehydrogenase kinase 4. Fabp3, fatty acid binding protein 3. Ldha, lactate


4

dehydrogenase A. Pcx, pyruvate carboxylase. Pck1, phosphoenolpyruvate carboxykinase 1.

Data are expressed as means ± s.e.m. *P < 0.05 relative to DMSO vehicle control, as

determined by Student’s t-test.

Supplementary Figure 8 Oxidative damage is correlated with p38 activation in cachexia

patients’ muscle biopsies. (a-b) Representative immunohistochemical staining for (a) 8-oxo-

guanine and (b) nuclear phospho-p38 in non-cachexia and cachexia subject muscle biopsies (n

= 11), and their quantitative H-scores. Bar represents 50µm. (c) Correlation plot for the H-

scores of 8-oxo-guanine vs. phospho-p38 immunohistochemical staining in non-cachectic

(black) and cachectic (red) subjects’ rectus abdominus muscles (n = 11). Data are expressed as

means ± s.e.m. *P < 0.05 relative to non-cachectic control, as determined by Student’s t-test.

Supplementary Table 1 Top 4 upregulated and downregulated gene sets in mouse myotubes

after 6h exposure to cachectic RXF conditioned media, relative to SKR media (n = 3 each).

Supplementary Table 2 Top 4 upregulated and downregulated gene sets in human myotubes

after 6h exposure to cachectic RXF conditioned media, relative to SKR media (n = 3 each).

Supplementary Table 3 Top 4 upregulated and downregulated gene sets in cachectic RXF-

bearing mouse quadriceps muscles, relative to non-cachectic SKR-bearing mouse quadriceps

muscles (n = 3 each).

Supplementary Table 4 Top 10 downregulated and upregulated gene sets in cachectic RXF-

bearing mouse quadriceps muscles, after daily intraperitoneal injections of 20 mg/kg etoxomir,

relative to vehicle control (n = 3 each).


a

c

b RXF media (cachec-c) SKR media

Supplementary Figure 1

d

0

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a SKR condi-oned media RXF condi-oned media

Pro-‐CX +DMSO Pro-‐CX +Etomoxir

RXF media +DMSO RXF media +Etomoxir

A

Figure S6

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Pdk4 Fabp3 Ldha Pcx Pck1 Pax3 Pax7 Ppara

Raw expression values

Vehicle Etomoxir

n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s.

c

SKR condi=oned media (Non-‐CX) RXF condi=oned media (Pro-‐CX)

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G361 LLC

p-‐p38

p-‐AKT (S473)

AKT

p38

Etomoxir Vehicle

GAPDH

IκBα

MHC

Etomoxir Vehicle



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Vehicle Etomoxir SB202190

Limbs’ m

uscle mass

(% bod

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SB202190

n.s.

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uscle mass

(% bod

y mass)

G361 (n = 5)

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Figure S8

Etomoxir Vehicle

LLC

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a b

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RXF (n = 5)

Supplementary Figure 4 Nature Medicine: doi:10.1038/nm.4093

0% 50% 100% 150% 200% 250%

Fold Change

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* n.s.

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0%

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Decano

yl-‐

carni-ne

Stearoyl-‐

carni-ne

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itoyl-‐

carni-ne

Docosape

ntaeno

yl-‐

carni-ne

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* * * **

a Eto-‐gel

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Cer(d1

8:0/20

:0)

Cer(d1

8:1/22

:1(1

3Z))

Glucosylceramide

(d18

:1/24:1(15

Z))

Cer(d1

8:1/24

:1(1

5Z))

% Change

Vehicle Etomoxir

3.00%

3.20%

3.40%

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Eto no treatment Vehicle no treatment

RXF hind

limb muscle (%

bod

y mass)

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-‐gel -‐gel

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c

0% 50%

100% 150% 200% 250%

2-‐Octen

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UDP

-‐MurNAC

Dimethylglycine

Ascorbic acid

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xanthine

GSSG

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* * * * * *

b




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8-‐Oxo-‐guanine

r = 0.92

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Phosph

o-‐p3

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o-‐p3

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Cachexia Patients Non-cachexia Patients

8-‐Oxo-‐guanine

8-‐Oxo-‐guanine

H-‐score

a

Cachexia Non-cachexia

Cachexia Non-cachexia


b

c


*

*


Supplementary Table 3

Top Gene sets Up in cachec=c mouse muscles FDR q-‐val BUYTAERT_PHOTODYNAMIC_THERAPY_STRESS_UP 0.000

REACTOME_ACTIVATION_OF_NF_KAPPAB_IN_B_CELLS 0.003 DAUER_STAT3_TARGETS_UP 0.003

KEGG_PROTEASOME 0.006 Top Gene sets Down in cachec=c mouse muscles FDR q-‐val

MOOTHA_VOXPHOS 0.000 REACTOME_RESPIRATORY_ELECTRON_TRANSPORT 0.000

REACTOME_GLYCOLYSIS_GLUCONEOGENESIS 0.003 EBAUER_MYOGENIC_TARGETS_OF_PAX3_FOXO1_FUSION 0.004

Non-‐CX Pro-‐CX mMuscles


Gene sets Up in cachec=c Human myotubes FDR q-‐val DACOSTA_UV_RESPONSE_VIA_ERCC3_DN 2.72E-‐02

DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_DN 6.01E-‐02 GARGALOVIC_RESPONSE_TO_OXIDIZED_PHOSPHOLIPIDS_UP 6.19E-‐02

BUYTAERT_PHOTODYNAMIC_THERAPY_STRESS_UP 1.64E-‐01 Gene sets Down in cachec=c Human myotubes FDR q-‐val ELVIDGE_HIF1A_AND_HIF2A_TARGETS_DN 0.00E-‐00

QI_HYPOXIA 0.00E-‐00 WINTER_HYPOXIA_UP 5.45E-‐04

ELVIDGE_HYPOXIA_BY_DMOG_UP 2.57E-‐03

Gene sets Up in cachec=c Mouse myotubes FDR q-‐val GALINDO_IMMUNE_RESPONSE_TO_ENTEROTOXIN 5.23E-‐03 ICHIBA_GRAFT_VERSUS_HOST_DISEASE_D7_UP 5.76E-‐03

BAKKER_FOXO3_TARGETS_UP 7.21E-‐03 MIZUSHIMA_AUTOPHAGOSOME_FORMATION 2.03E-‐02

a SKR RXF (cachec-c) mMyotubes

Gene sets Down in cachec=c Mouse myotubes FDR q-‐val MANALO_HYPOXIA_DN 0.00E-‐00

REACTOME_MRNA_PROCESSING 0.00E-‐00 PENG_RAPAMYCIN_RESPONSE_DN 0.00E-‐00 KARLSSON_TGFB1_TARGETS_UP 3.00E-‐03



Top Gene sets Down in cachec=c mouse muscles aVer Etomoxir FDR q-‐val KEGG_PROTEASOME 0.000 REACTOME_ACTIVATION_OF_NF_KAPPAB_IN_B_CELLS 0.000 REACTOME_REGULATION_OF_MRNA_STABILITY_BY_PROTEINS_THAT_BIND_AU_RICH_ELEMENTS 0.000 BIOCARTA_PROTEASOME_PATHWAY 0.000 REACTOME_P53_DEPENDENT_G1_DNA_DAMAGE_RESPONSE 0.000 REACTOME_REGULATION_OF_APOPTOSIS 0.000 REACTOME_HOST_INTERACTIONS_OF_HIV_FACTORS 0.000 DAUER_STAT3_TARGETS_UP 0.000 RASHI_RESPONSE_TO_IONIZING_RADIATION_1 0.000 GARGALOVIC_RESPONSE_TO_OXIDIZED_PHOSPHOLIPIDS_BLUE_UP 0.001

Top Gene sets Up in cachec=c mouse muscles aVer Etomoxir FDR q-‐val CHEMELLO_SOLEUS_VS_EDL_MYOFIBERS_UP 0.000 REACTOME_MUSCLE_CONTRACTION 0.000 EBAUER_MYOGENIC_TARGETS_OF_PAX3_FOXO1_FUSION 0.000 REACTOME_RESPIRATORY_ELECTRON_TRANSPORT_ATP_SYNTHESIS_BY_CHEMIOSMOTIC_COUPLING_AND_HEAT_PRODUCTION_BY_UNCOUPLING_PROTEINS 0.000

REACTOME_RESPIRATORY_ELECTRON_TRANSPORT 0.000 REACTOME_TCA_CYCLE_AND_RESPIRATORY_ELECTRON_TRANSPORT 0.000 KUNINGER_IGF1_VS_PDGFB_TARGETS_UP 0.000 KEGG_CARDIAC_MUSCLE_CONTRACTION 0.000 REACTOME_GLUCOSE_METABOLISM 0.001 DELASERNA_MYOD_TARGETS_UP 0.002



SUPPLEMENTARY INFORMATION Excessive fatty acid oxidation ... · 2 expressed as means ± s.e.m. *P <...

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