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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
Nature Medicine: doi:10.1038/nm.4093
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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
Nature Medicine: doi:10.1038/nm.4093
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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).
Nature Medicine: doi:10.1038/nm.4093
a
c
b RXF media (cachec-c) SKR media
Supplementary Figure 1
d
0
0.2
0.4
0.6
0.8
1
1.2
SKR RXF
Total cell volum
e (fo
ld)
SKR
RXF
*
0
10
20
30
40
50
500 1,000 2,000 8,000 20,000
Rlea-ve freq
uency (%
)
AU
RXF SKR
P < 0.001
Nature Medicine: doi:10.1038/nm.4093
a SKR condi-oned media RXF condi-oned media
Pro-‐CX +DMSO Pro-‐CX +Etomoxir
RXF media +DMSO RXF media +Etomoxir
A
Figure S6
0 200 400 600 800 1000 1200 1400
Concen
tra-
on (p
g/ml) Ac-vin A *
* * *
0
10000
20000
30000
40000
50000
Concen
tra-
on (p
g/ml) Myosta-n
n.s.
10
100
1000
10000
100000
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)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Vehicle Etomoxir
Cell de
ath
(fold change in ethidium fluo
rescen
ce)
SKR RXF *
n.s.
Supplementary Figure 2
b
Nature Medicine: doi:10.1038/nm.4093
1
1.5
2
2.5
3
Vehicle Etomoxir
Adi
pose
wei
ght
(% o
f Bod
y W
eigh
t)
*
G361 LLC
p-‐p38
p-‐AKT (S473)
AKT
p38
Etomoxir Vehicle
GAPDH
IκBα
MHC
Etomoxir Vehicle
Supplementary Figure 3
Nature Medicine: doi:10.1038/nm.4093
5
6
7
8
9
Vehicle Etomoxir SB202190
Limbs’ m
uscle mass
(% bod
y mass)
* *
*
0 50
100 150 200 250 300 350 400
8 10 12 14 16 18 20 Time (d)
RXF Vehicle
Etomoxir
SB202190
n.s.
c
5
6
7
8
9
Limbs’ m
uscle mass
(% bod
y mass)
G361 (n = 5)
*
*
Etomoxir Vehicle
Figure S8
Etomoxir Vehicle
LLC
0
200
400
600
800
1000
7 9 11 13 15 17 19 21 23 25
G361 Vehicle
Etomoxir
SB202190
n.s.
Tumor Volum
e (m
m3 )
e
Time (d)
0
100
200
300
400
500
600
0 5 10 15
LLC Vehicle
Etomoxir
Time (d)
n.s.
f
a b
d
g
RXF (n = 5)
Supplementary Figure 4 Nature Medicine: doi:10.1038/nm.4093
0% 50% 100% 150% 200% 250%
Fold Change
Vehicle Etomoxir
0%
50%
100%
150%
200%
Fold change
Vehicle Etomoxir
b
* *
**
**
* n.s.
a
0%
50%
100%
Decano
yl-‐
carni-ne
Stearoyl-‐
carni-ne
Palm
itoyl-‐
carni-ne
Docosape
ntaeno
yl-‐
carni-ne
Fold change
* * * **
a Eto-‐gel
0%
100%
200%
300%
400%
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%
3.60%
3.80%
4.00%
Eto no treatment Vehicle no treatment
RXF hind
limb muscle (%
bod
y mass)
Normal Hindlimb
-‐gel -‐gel
**
c
0% 50%
100% 150% 200% 250%
2-‐Octen
edioate
CMP
Aden
ine
MNA
6-‐Hy
droxy-‐FA
D
Guanine
Methyl-‐ino
sine
UDP
-‐MurNAC
Dimethylglycine
Ascorbic acid
Hypo
xanthine
GSSG
Fold change
Vehicle Etomoxir * * *
* * * * * *
b
Supplementary Figure 5
Supplementary Figure 6
Nature Medicine: doi:10.1038/nm.4093
0
50
100
150
200
0 50 100 150 200 250
8-‐Oxo-‐guanine
r = 0.92
Phospho-‐p38
Phosph
o-‐p3
8
0
50
100
150
200
non-‐CX CX
Phosph
o-‐p3
8 H-‐score
0
50
100
150
200
non-‐CX CX
Cachexia Patients Non-cachexia Patients
8-‐Oxo-‐guanine
8-‐Oxo-‐guanine
H-‐score
a
Cachexia Non-cachexia
Cachexia Non-cachexia
Supplementary Figure 7
b
c
Supplementary Figure 8
*
*
Nature Medicine: doi:10.1038/nm.4093
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
Supplementary Table 1
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
Supplementary Table 2
Nature Medicine: doi:10.1038/nm.4093
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 Table 4
Nature Medicine: doi:10.1038/nm.4093