Excessive fatty acid oxidation induces muscle atrophy in cancer cachexia

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1 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 <.1 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 <.5 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 1 µm. (c) Quantification of cell death using ethidium dye, after 6d exposure to RXF media. Data are

2 expressed as means ± s.e.m. P <.5 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 2 mg/kg etomoxir (n = 3 each). Supplementary Figure 4 rescues muscle atrophy in mouse models of cachexia. (a) Representative images of RXF-bearing mice quadriceps muscle morphology with and without etomoxir treatment. -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 SB2219 (n = 5 each). (c) Forelimbs and hindlimbs muscle mass (% body mass) of G361-bearing mice after daily injections of DMSO vehicle, 2 mg/kg etomoxir or 5 mg/kg SB2219 (n = 5 each). Etoxomir and SB2219 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. rescued myofiber atrophy. Bar represents 2 µm. (e-g) Tumor growth curves of (e) RXF, (f) G361, and (g) LLC tumors, with daily injections of DMSO vehicle, 2 mg/kg etomoxir or 5 mg/kg of SB2219 (n = 5 each). Data are expressed as means ± s.e.m. P <.5 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 2

3 mice after intramuscular injections of vehicle- (2% oxidized alginate beads, n = 6) or etomoxirgel (1 µg / 5 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 =.7), 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 2 µm. Data are expressed as means ± s.e.m. P <.1 relative to vehicle-gel control, as determined by Student s t-test. Supplementary Figure 6 inhibited fatty acid oxidation, but not PPARα-associated sterol and carbohydrate metabolism. (a) Acyl-carnitine levels in quadriceps muscles after 2 mg/kg etomoxir treatment (n = 4). (b) Fatty acid and cholesterol levels in quadriceps muscles after 2 mg/kg etomoxir treatment (n = 4). (c) Polar metabolites significantly changed in quadriceps muscles after 2 mg/kg etomoxir treatment (n = 4). 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 <.5 and P <.1 relative to DMSO vehicle control, as determined by Student s t-test. Supplementary Figure 7 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 2 mg/kg etomoxir treatment (n = 3). Pdk4, pyruvate dehydrogenase kinase 4. Fabp3, fatty acid binding protein 3. Ldha, lactate 3

4 dehydrogenase A. Pcx, pyruvate carboxylase. Pck1, phosphoenolpyruvate carboxykinase 1. Data are expressed as means ± s.e.m. P <.5 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-oxoguanine and (b) nuclear phospho-p38 in non-cachexia and cachexia subject muscle biopsies (n = 11), and their quantitative H-scores. Bar represents 5µm. (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 <.5 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 RXFbearing mouse quadriceps muscles, relative to non-cachectic SKR-bearing mouse quadriceps muscles (n = 3 each). Supplementary Table 4 Top 1 downregulated and upregulated gene sets in cachectic RXFbearing mouse quadriceps muscles, after daily intraperitoneal injections of 2 mg/kg etoxomir, relative to vehicle control (n = 3 each). 4

5 a b Rlea-ve frequency (%) , 2, 8, 2, SKR media AU RXF SKR P <.1 RXF media (cachec-c) c SKR 1.2 d 1 RXF Total cell volume (fold) SKR RXF Supplementary Figure 1

6 a RXF condi-oned media SKR condi-oned media b RXF media +DMSO RXF media + c Cell death (fold change in ethidium fluorescence) SKR RXF n.s. Supplementary Figure 2

7 G361 LLC p- p38 p38 p- AKT (S473) AKT IκBα MHC GAPDH Supplementary Figure 3

8 a b Limbs muscle mass (% body mass) RXF (n = 5) 5 SB2219 c 9 G361 (n = 5) d Limbs muscle mass (% body mass) LLC 5 e f g Tumor Volume (mm 3 ) RXF SB2219 n.s Time (d) SB2219 G361 n.s Time (d) LLC n.s Time (d) Supplementary Figure 4

9 a RXF hindlimb muscle (% body mass) 4.% 3.8% 3.6% 3.4% 3.2% Normal Hindlimb b Eto- gel 3.% Eto no treatment no treatment - gel - gel Supplementary Figure 5 a b Fold change 1% 5% % Fold change 2% 15% 1% 5% % n.s. c Fold Change 25% 2% 15% 1% 5% % Decanoyl- carni-ne Stearoyl- carni-ne Palmitoyl- carni-ne Docosapent aenoyl- carni-ne Supplementary Figure 6

10 Supplementary Figure 7 a Non-cachexia Patients Cachexia Patients 2 8- Oxo- guanine 8- Oxo- guanine H- score b non- CX Noncachexia Cachexia CX Phospho- p38 c 2 Phospho- p38 H- score non- CX Noncachexia Cachexia CX 8- Oxo- guanine r = Supplementary Figure 8 Phospho- p38

11 Supplementary Table 1 Gene sets Up in cachec=c Mouse myotubes GALINDO_IMMUNE_RESPONSE_TO_ENTEROTOXIN ICHIBA_GRAFT_VERSUS_HOST_DISEASE_D7_UP BAKKER_FOXO3_TARGETS_UP MIZUSHIMA_AUTOPHAGOSOME_FORMATION Gene sets Down in cachec=c Mouse myotubes MANALO_HYPOXIA_DN REACTOME_MRNA_PROCESSING PENG_RAPAMYCIN_RESPONSE_DN KARLSSON_TGFB1_TARGETS_UP 5.23E E E E- 2.E-.E-.E- 3.E- 3 Supplementary Table 2 Gene sets Up in cachec=c Human myotubes DACOSTA_UV_RESPONSE_VIA_ERCC3_DN DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_DN GARGALOVIC_RESPONSE_TO_OXIDIZED_PHOSPHOLIPIDS_UP BUYTAERT_PHOTODYNAMIC_THERAPY_STRESS_UP Gene sets Down in cachec=c Human myotubes ELVIDGE_HIF1A_AND_HIF2A_TARGETS_DN QI_HYPOXIA WINTER_HYPOXIA_UP ELVIDGE_HYPOXIA_BY_DMOG_UP 2.72E E E E- 1.E-.E- 5.45E E- 3 Supplementary Table 3 Top Gene sets Up in cachec=c mouse muscles BUYTAERT_PHOTODYNAMIC_THERAPY_STRESS_UP. REACTOME_ACTIVATION_OF_NF_KAPPAB_IN_B_CELLS.3 DAUER_STAT3_TARGETS_UP.3 KEGG_PROTEASOME.6 Top Gene sets Down in cachec=c mouse muscles MOOTHA_VOXPHOS. REACTOME_RESPIRATORY_ELECTRON_TRANSPORT. REACTOME_GLYCOLYSIS_GLUCONEOGENESIS.3 EBAUER_MYOGENIC_TARGETS_OF_PAX3_FOXO1_FUSION.4

12 Supplementary Table 4 Top Gene sets Down in cachec=c mouse muscles aver KEGG_PROTEASOME. REACTOME_ACTIVATION_OF_NF_KAPPAB_IN_B_CELLS. REACTOME_REGULATION_OF_MRNA_STABILITY_BY_PROTEINS_THAT_BIND_AU_RICH_ELEMENTS. BIOCARTA_PROTEASOME_PATHWAY. REACTOME_P53_DEPENDENT_G1_DNA_DAMAGE_RESPONSE. REACTOME_REGULATION_OF_APOPTOSIS. REACTOME_HOST_INTERACTIONS_OF_HIV_FACTORS. DAUER_STAT3_TARGETS_UP. RASHI_RESPONSE_TO_IONIZING_RADIATION_1. GARGALOVIC_RESPONSE_TO_OXIDIZED_PHOSPHOLIPIDS_BLUE_UP.1 Top Gene sets Up in cachec=c mouse muscles aver CHEMELLO_SOLEUS_VS_EDL_MYOFIBERS_UP. REACTOME_MUSCLE_CONTRACTION. EBAUER_MYOGENIC_TARGETS_OF_PAX3_FOXO1_FUSION. REACTOME_RESPIRATORY_ELECTRON_TRANSPORT_ATP_SYNTHESIS_BY_CHEMIOSMOTIC_COUPLING_A ND_HEAT_PRODUCTION_BY_UNCOUPLING_PROTEINS. REACTOME_RESPIRATORY_ELECTRON_TRANSPORT. REACTOME_TCA_CYCLE_AND_RESPIRATORY_ELECTRON_TRANSPORT. KUNINGER_IGF1_VS_PDGFB_TARGETS_UP. KEGG_CARDIAC_MUSCLE_CONTRACTION. REACTOME_GLUCOSE_METABOLISM.1 DELASERNA_MYOD_TARGETS_UP.2