Glutamine activates STAT3 to control cancer cell proliferation. independently of glutamine metabolism

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1 Glutamine activates STAT3 to control cancer cell proliferation S1 independently of glutamine metabolism Andrea Cacace, Martina Sboarina, Thibaut Vazeille, and Pierre Sonveaux Supplementary tables: Table S1. Antibodies used for western blotting Name Species Company Catalog Concentration number Primary antibodies AMPK rabbit Cell signaling /1 000 P-T172--AMPK rabbit Cell signaling /1 000 ASCT2 rabbit Millipore ABN73 1/1 000 β-actin mouse Sigma A5441 1/5 000 Caspase 3 rabbit Cell signaling /1 000 c-myc mouse BD Biosciences /2 000 EGFR rabbit Cell signaling /1 000 P-Y1068-EGFR rabbit Cell signaling /1 000 ERKs mouse BD Biosciences /5 000 Phospho-ERKs mouse Santa Cruz sc /1 000 GDH goat Novus NBP /500 Biological HIF-1α mouse BD Biosciences /1 000 Hsp90 mouse BD Biosciences /5 000 Jak2 rabbit Cell signaling /1 000 Phospho-T1007/1008-Jak2 rabbit Cell signaling /1 000 LAT1 rabbit Cell signaling /1 000 MCT1 rabbit Millipore AB3538P 1/1 000 MCT4 rabbit Reference 1-1/1 000 mtor rabbit Cell signaling /1 000 P-S2448-mTOR rabbit Cell signaling /1 000 p38 rabbit Cell signaling /1 000 Phospho-T180/Y182-p38 rabbit Cell signaling /1 000 p70 S6K rabbit Cell signaling /1 000 Phospho-T389-p70 S6K rabbit Cell signaling /1 000 PARP rabbit Cell signaling /1 000 Src rabbit Cell signaling /1 000 P-Y416-Src rabbit Cell signaling /1 000 STAT3 mouse Cell signaling /1 000 P-Y705-STAT3 rabbit Cell signaling /1 000 P-S727-STAT3 mouse Cell signaling /1 000 Secondary antibodies goat anti-rabbit goat Jackson /5 000 goat anti-mouse goat Jackson /5 000 rabbit anti-goat rabbit Jackson /5 000

2 S2 Table S2. Primers used for qrt-pcr Target Forward sequence Reverse sequence hβ-actin 5 -CCCGCGAGCACAGAGC-3 5 -TCATCATCCATGGTGAGCTGG-3 hhif-1α 5 -TCATCCATGTGACCATGAGG-3 5 -TTCTTCCTCGGCTAGTTAGGG-3 hmct1 5 -GTGGCTCAGCTCCGTATTGT-3 5 -GAGCCGACCTAAAAGTG-GTG-3 hmct4 5 -CAGTTCGAGGTGCTCATGG-3 5 -ATGTAG-ACGTGGGTCGCATC-3 hrpl19 5 -CAAGCGGATTCTCATGGAACA-3 5 -TGGTCAGCCAGGAGCTTCTT-3

3 Supplementary Figures: S3 Figure S1. Glutamine deprivation for 48 h decreases intracellular glutamine levels but does not cause the apoptosis of cancer cells. (a-b) MDA-MB-231, HeLa and SiHa cancer cells were cultured in assay medium containing 2 mm of L-glutamine (+Q) or not (-Q). (a) Measurement of intracellular glutamine content after 48 h of culture in the presence of 10% FBS or not (data shown as means ± SEM; ***P < compared with media +Q +FBS; ns = not significant; n = 6). (b) Western blots are representative of n = 3 and show PARP, caspase-3 and β-actin expression.

4 S4 Figure S2. Glutathione and N-acetyl-glucosamine do not restore the proliferation of glutamine-deprived cancer cells. (a-h) MDA-MB-231, HeLa and SiHa cancer cells were cultured in assay medium containing 2 mm of L-glutamine (+Q) or not (-Q). (a) The cells also received dimethyl-glutamate (DM-glutamate, 7 mm) or dimethyl-2-oxoglutarate (DM-2-oxoglutarate, 7 mm), and intracellular glutamine was measured after 48 h of culture (**P < 0.01, ***P < compared with medium +Q or as indicated; ns not significant; n = 6). (b) as in (a) but measuring intracellular glutamate (**P < 0.01, ***P < compared with medium +Q or as indicated; ns not significant; n = 6) (c) The cells also received dimethyl-glutamate (DM-glutamate, 2 mm) or dimethyl-2-oxoglutarate (DM-2-oxoglutarate, 2 mm). Lactate production and glucose consumption were measured using a CMA600 analyzer (***P < comparing glucose consumption; ### P < comparing lactate production; n = 3). (d) Where indicated, the cells were supplemented or not with DM-2-oxoglutarate (7 mm) or DM-glutamate (7 mm). Cell number was determined at the indicated time points using a SpectraMax i3 multi-mode microplate reader (***P < compared with media +Q; n = 8). (e) Where indicated, the cells also received 2 mm of D-glutamine. After 72 h in culture, cell proliferation was measured using Ki-67 staining (***P < compared with medium +Q; n = 4). (f) As in (e) but the cells received 5 mm of reduced glutathione or not (***P < compared with medium +Q; n = 4). (g) The cells were treated as in (f). Cell number was assessed at indicated time points using a SpectraMax i3 multi-mode microplate reader (***P < compared with medium +Q; ### P < compared with medium -Q; ns not significant; n = 5 for MDA-MB-231; n = 4 for HeLa). (h) As in (g) but supplementing the cells with the indicated doses of N-acetyl-glucosamine (***P < compared with medium +Q; n = 5 for MDA-MB-231 and SiHa; n = 4 for HeLa). (a-h) All quantitative data show means ± SEM.

5 S5 Figure S3. Glutamine restriction increases SLC16A1/MCT1 transcription and dimethyl-2- oxoglutarate activates HIF-1 in cancer cells. (a-d) MDA-MB-231, HeLa and SiHa cancer cells were cultured in complete medium containing 2 mm of L-glutamine (+Q) or not (-Q). (a) qrt-pcr analysis of SLC16A1/MCT1 mrna levels after 24 h of culture. Data were normalized for β-actin mrna levels (**P < 0.01, ***P < 0.005; n = 8). (b) Western blots representative of n = 3 showing HIF-1α and β-actin expression in the cells transfected during 48 h with a control sirna (sictr) or a sirna against HIF-1α (sihif-1α). (c) Cells were also supplemented or not with dimethyl-2- oxoglutarate (DM-2-oxoglutarate, 7 mm). The activity of HIF prolylhydroxylases (PHDs) was measured using an ODD-luciferase reporter in cells grown in the indicated media for 24 h (*P < 0.05, **P < 0.01, ***P < 0.005; ns = not significant; n = 4). (d) Cells were treated as in (c). HIF-1 activity was measured using a HRE-luciferase reporter (**P < 0.01, ***P < 0.005, ns = not significant compared with medium +Q at the same treatment time; # P < 0.05, ## P < 0.01, ### P < compared with medium Q at the same treatment time; n = 4). (a-d) All quantitative data show means ± SEM.

6 S6 Figure S4. Validation of the efficacy of sirnas and pharmacological inhibitors. (a-d) MDA- MB-231, HeLa and SiHa cancer cells were cultured in complete medium containing 2 mm of L- glutamine (+Q) or not (-Q). (a) The cells were also supplemented with mtor inhibitors rapamycin (10 nm) or AZD8055 (15 nm) during 48 h. Western blots are representative n = 3 and show phospho-t389-p70s6k, mtor and β-actin expression. (b) The cells were transfected with a control sirna (sictr) or a sirna against c-myc (sic-myc). Western blots are representative of n = 3 and were acquired 48 h after the transfection. They show c-myc and β-actin expression. (c) The cells were transfected with a control SmartPool sirna (sictr) or SmartPool sirnas against STAT3 (sistat3). Western blots are representative of n = 3 and were acquired 48 h after the transfection. They show phospho-y705-stat3, total STAT3 and β-actin expression. (d) The cells were treated with the indicated amounts of STAT3 inhibitor Stattic inhibitor for 48 h. Western blots are representative of n = 3 and show phospho-y705-stat3, total STAT3 and β-actin expression.

7 S7

8 Figure S5. STAT3 controls cancer cell proliferation and metabolism in a glutamine-dependent context. (a) MDA-MB-231, HeLa and SiHa cancer cells were cultured the indicated amounts of time in complete medium containing 2 mm of L-glutamine (+Q) or not (-Q) and 100 ng/ml of epithelial growth factor (EGF) or not. Representative western blots of n = 3 show phospho-y1068- EGFR, total EGFR, phospho-y705-stat3 and total STAT3. Hsp90 was used as a loading control. (b) The cells were treated as in (a), and also with or without 5 µm of STAT3 inhibitor Stattic. Representative western blots of n = 3 show phospho-y1068-egfr, total EGFR, phospho-y705- STAT3, phospho-s727-stat3 and total STAT3 72 h after the treatments. Hsp90 was used as a loading control. (c-l) MDA-MB-231, HeLa and SiHa cancer cells were cultured for 72 h in assay medium containing 2 mm of L-glutamine (+Q) or not (-Q). (c) The cells were also supplemented or not with 100 ng/ml of EGF. Cell number was assessed over time using a SpectraMax i3 multimode microplate reader (***P < 0.005, ns = not significant compared to +Q; n = 8). (d) The cells were also supplemented or not with 20 ng/ml of interleukin-6 (IL-6) ± 2 µm of Stattic. Representative western blots of n = 3 show phospho-y1007/1008-jak2, total JAK2, phospho- Y705-STAT3 and total STAT3 72 h after the treatments. β-actin was used as a loading control. (e) Cell number was quantified in medium containing dialyzed FBS instead of FBS, ± EGF (100 ng/ml) ± Stattic (2 µm) (***P < compared to medium +Q, n = 8). (f) As in (e) but using IL-6 (20 ng/ml) instead of EGF (***P < compared to medium +Q, n = 8). (g) Cell number was assessed when the cells were also supplemented or not with EGF (100 ng/ml), IL-6 (10 ng/ml) and/or glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES, 10 µm) (***P < compared with medium +Q -BPTES; ns = not significant; n = 8). (h) The cells were transfected with a control sirna (sictr) or SmartPool sirnas against glutamate dehydrogenase (sigdh), and experimental medium was provided 24 h later. Representative western blots of n = 3 show GDH and β-actin expression and were acquired 48 h after transfection. (i) The cells were treated as in (h), after which cell number was assessed at the indicated time points (***P < compared with media +Q; n = 8 for MDA-MB-231 and SiHa; n = 6 for HeLa). (j) The cells were treated ± Stattic (5 µm). The graph shows HIF-1α mrna levels after 24 h of culture. Data are normalized for β-actin mrna levels (***P < 0.005; ns not significant; n = 8). (k) As in (j), but analyzing SLC16A3/MCT4 mrna levels. (***P < 0.005; ns not significant; n = 8). (l) The cells were transfected with a control sirna (sictr) or a sirna against HIF-1α, and experimental medium was delivered 24 h later. Cells were transfected with a control sirna (sictr) or a sirna against HIF-1α (sihif-1α), and experimental medium was provided 24 h later. Cell number was assessed at the indicated time points (***P < 0.005; n = 8 for MDA-MB-231 and SiHa; n = 6 for HeLa. (a-l) All quantitative data show means ± SEM. S8 Supplementary reference: 1. Vegran F, Boidot R, Michiels C, Sonveaux P, Feron O. Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-kappaB/IL-8 pathway that drives tumor angiogenesis. Cancer Res 2011; 71: