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• W2891855400 abstract "•A pharmacodynamic window study of metformin in patients with breast cancer•Metformin increases 18-FDG flux in primary breast cancer•Two distinct metabolic responses to metformin are identified in tumors•Tumors that upregulate OXPHOS genes show an increase in proliferation score Late-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect. Late-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect. Metformin can reduce proliferation of cancer cell lines in vitro and in vivo, and this effect has been ascribed to inhibition of mitochondrial complex 1 (Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. et al.Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.Elife. 2014; 3: e02242Crossref PubMed Scopus (683) Google Scholar). However, the doses of metformin used have typically been 10- to 1,000-fold greater than peak plasma level in humans (Dowling et al., 2012Dowling R.J. Niraula S. Stambolic V. Goodwin P.J. Metformin in cancer: translational challenges.J. Mol. Endocrinol. 2012; 48: R31-R43Crossref PubMed Scopus (268) Google Scholar). Hence controversy remains as to whether metformin's effects on tumor metabolism at clinical doses are determined by its direct effects on mitochondria or through its action on systemic metabolism via AMPK-dependent inhibition of hepatic gluconeogenesis and subsequent reduced circulating glucose and insulin levels. Several window trials have used immunohistochemistry to investigate metformin's clinical effects in breast, endometrial, and prostate cancer. A number have shown that metformin can reduce the proliferation marker Ki67, but no singular mechanism has been clearly demonstrated. Activation of AMPK suggestive of an energy stress has been observed, while other studies have demonstrated reduced pAKT consistent with decreased insulin signaling (Dowling et al., 2015Dowling R.J. Niraula S. Chang M.C. Done S.J. Ennis M. McCready D.R. Leong W.L. Escallon J.M. Reedijk M. Goodwin P.J. et al.Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study.Breast Cancer Res. 2015; 17: 32Crossref PubMed Scopus (83) Google Scholar, Hadad et al., 2011Hadad S. Iwamoto T. Jordan L. Purdie C. Bray S. Baker L. Jellema G. Deharo S. Hardie D.G. Pusztai L. et al.Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial.Breast Cancer Res. Treat. 2011; 128: 783-794Crossref PubMed Scopus (230) Google Scholar, Schuler et al., 2015Schuler K.M. Rambally B.S. DiFurio M.J. Sampey B.P. Gehrig P.A. Makowski L. Bae-Jump V.L. Antiproliferative and metabolic effects of metformin in a preoperative window clinical trial for endometrial cancer.Cancer Med. 2015; 4: 161-173Crossref PubMed Scopus (109) Google Scholar). Recently published work by Liu et al. comparing the metabolite profile of ten ovarian tumor samples from patients on metformin versus ten control samples (patients not on metformin) demonstrated decreases in the levels of some TCA cycle intermediates and short-chain acyl-carnitines. In addition, the response to metformin seen in the human metabolite profiles could be recapitulated in a mouse model and in vitro when nutrient concentrations were limited (Liu et al., 2016Liu X. Romero I.L. Litchfield L.M. Lengyel E. Locasale J.W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers.Cell Metab. 2016; 24: 728-739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To date, this is the most convincing clinical evidence that metformin has significant and measurable mitochondrial effects at standard therapeutic doses. Here, we present the results of a clinical study that integrates tumor metabolomic profiling with dynamic imaging, transcriptomics, and systemic metabolic markers to further dissect the effects of metformin on systemic and breast tumor metabolism. We recruited 40 female patients with treatment-naive primary breast cancer to the study. Before and after a 13- to 21-day course of metformin, patients underwent a dynamic fluoro-deoxy-D-glucose positron emission tomography-computed tomography (FDG-PET-CT) scan, breast core biopsies from the primary tumor under ultrasound guidance, and blood samples to assay host metabolic markers of the insulin axis (Figure 1A). See Table S1 for details of study entry criteria and Table S2 for tumor features. See Supplemental Information for further detail. Pre-clinical data have shown that inhibition of oxidative phosphorylation (OXPHOS) by metformin increases dependence on glycolysis (Ben Sahra et al., 2010Ben Sahra I. Laurent K. Giuliano S. Larbret F. Ponzio G. Gounon P. Le Marchand-Brustel Y. Giorgetti-Peraldi S. Cormont M. Bertolotto C. et al.Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells.Cancer Res. 2010; 70: 2465-2475Crossref PubMed Scopus (431) Google Scholar, Birsoy et al., 2014Birsoy K. Possemato R. Lorbeer F.K. Bayraktar E.C. Thiru P. Yucel B. Wang T. Chen W.W. Clish C.B. Sabatini D.M. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides.Nature. 2014; 508: 108-112Crossref PubMed Scopus (484) Google Scholar, Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. et al.Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.Elife. 2014; 3: e02242Crossref PubMed Scopus (683) Google Scholar). The FDG radio-tracer is a marker of tissue glucose utilization. Kinetics analysis of FDG uptake time courses obtained from dynamic PET images potentially provides more consistent measures of tumor tracer uptake, adjusted for variations in tracer inflow to the tumor, than standard static FDG-PET-CT (Dunnwald et al., 2011Dunnwald L.K. Doot R.K. Specht J.M. Gralow J.R. Ellis G.K. Livingston R.B. Linden H.M. Gadi V.K. Kurland B.F. Schubert E.K. et al.PET tumor metabolism in locally advanced breast cancer patients undergoing neoadjuvant chemotherapy: value of static versus kinetic measures of fluorodeoxyglucose uptake.Clin. Cancer Res. 2011; 17: 2400-2409Crossref PubMed Scopus (80) Google Scholar). Using an irreversible two-tissue compartment model describing rates of FDG transport and phosphorylation (STAR Methods), we observed an increase in FDG flux (KFDG) into the primary breast cancer following metformin (Figure 1B) but no change in the static uptake measures SULmax and SULmean for tumor (standardized uptake values normalized for lean body mass) (Figures S1A and S1B; Table S3). There was no change in nodal SULmax for patients with FDG avidity within ipsilateral axillary lymph nodes (Figure S1C). There was a significant correlation between change in KFDG in the primary tumor and change in SULmax in the axillary nodes (Figure S1D). The above findings infer that metformin treatment leads to increased glucose uptake by breast tumors and this would be consistent with a switch to glycolytic metabolism. In addition, the analysis emphasizes the sensitivity of dynamic FDG-PET over static scanning in identifying subtle pharmacodynamic changes in glucose metabolism. If normal tissues such as liver absorbed more FDG in response to metformin, FDG activity concentrations in the blood would fall, potentially reducing FDG uptake by the tumor. However, the compartment model/flux constant approach describes tumor FDG uptake after allowing for differences across the whole time course of the dynamic scan in levels of blood-borne tracer flowing into the tumor, determined from imaged activity concentrations in the descending aorta. It is possible that it is precisely because this model controls for the flow of tracer into the tumor that we see a significant change in the flux constant and not standardized uptake values on static PET scanning. We did not observe changes in the levels of the TCA cycle intermediates citrate, succinate, fumarate, and malate in contrast to Liu et al., 2016Liu X. Romero I.L. Litchfield L.M. Lengyel E. Locasale J.W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers.Cell Metab. 2016; 24: 728-739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, or aspartate, a key marker of electron transport chain integrity (Figure 1C). Ornithine is condensed with carbamoyl phosphate to produce citrulline in the only intra-mitochondrial reaction of the urea cycle and citrulline levels decreased (mean log2FC = −0.53; p = 0.007). Some investigators have observed an increase in the ADP/ATP and AMP/ATP ratios typically under in vitro nutrient-deprived conditions but there was no significant increase in intratumoral ADP/ATP or AMP/ATP ratios post-metformin (Figure S1E), and this is consistent with metabolomic data from ovarian tumors published in Liu et al., 2016Liu X. Romero I.L. Litchfield L.M. Lengyel E. Locasale J.W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers.Cell Metab. 2016; 24: 728-739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar. The discordance in findings with metabolomic profiling from pre-clinical studies may reflect the heterogeneity inherent in a study analyzing clinical samples and the difficulty of making very precise measurements when there may be only small changes in the levels of these metabolites. Mitochondrial dysfunction under the tissue culture conditions described in the literature cited above is likely to be greater than in our study. Indeed, Gaude et al., 2018Gaude E. Schmidt C. Gammage P.A. Dugourd A. Blacker T. Chew S.P. Saez-Rodriguez J. O'Neill J.S. Szabadkai G. Minczuk M. et al.NADH shuttling couples cytosolic reductive carboxylation of glutamine with glycolysis in cells with mitochondrial dysfunction.Mol. Cell. 2018; 69: 581-593.e7Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar showed that, at lower levels of mitochondrial dysfunction, there was little or no decrease in TCA cycle metabolites and aspartate. Uptake from the stroma in an in vivo system may help maintain aspartate levels (for example, Birsoy et al., 2015Birsoy K. Wang T. Chen W.W. Freinkman E. Abu-Remaileh M. Sabatini D.M. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis.Cell. 2015; 162: 540-551Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar used a cell line lacking in the transporter SLC1A3, which was expressed at the mRNA level in our clinical samples). In contrast to findings in some other studies (Dowling et al., 2015Dowling R.J. Niraula S. Chang M.C. Done S.J. Ennis M. McCready D.R. Leong W.L. Escallon J.M. Reedijk M. Goodwin P.J. et al.Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study.Breast Cancer Res. 2015; 17: 32Crossref PubMed Scopus (83) Google Scholar, Hadad et al., 2011Hadad S. Iwamoto T. Jordan L. Purdie C. Bray S. Baker L. Jellema G. Deharo S. Hardie D.G. Pusztai L. et al.Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial.Breast Cancer Res. Treat. 2011; 128: 783-794Crossref PubMed Scopus (230) Google Scholar) tumor immunohistochemistry demonstrated no change in AMPK phosphorylation following metformin (paired t test, p = 0.801) (Figure S1F). There was no correlation between change in pAMPK and change in KFDG (Figure S1G). Whole-transcriptome RNA sequencing pre- and post-metformin revealed significant upregulation of several pathways linked to metabolism (Figure 2A) and more specifically to mitochondrial pathways and disease (Table S4). This included four KEGG pathways that we predicted would be targeted by metformin based on extensive pre-clinical data (Birsoy et al., 2014Birsoy K. Possemato R. Lorbeer F.K. Bayraktar E.C. Thiru P. Yucel B. Wang T. Chen W.W. Clish C.B. Sabatini D.M. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides.Nature. 2014; 508: 108-112Crossref PubMed Scopus (484) Google Scholar, Fendt et al., 2013Fendt S.M. Bell E.L. Keibler M.A. Davidson S.M. Wirth G.J. Fiske B. Mayers J.R. Schwab M. Bellinger G. Csibi A. et al.Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism.Cancer Res. 2013; 73: 4429-4438Crossref PubMed Scopus (160) Google Scholar, Liu et al., 2016Liu X. Romero I.L. Litchfield L.M. Lengyel E. Locasale J.W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers.Cell Metab. 2016; 24: 728-739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, Mullen et al., 2011Mullen A.R. Wheaton W.W. Jin E.S. Chen P.H. Sullivan L.B. Cheng T. Yang Y. Linehan W.M. Chandel N.S. DeBerardinis R.J. Reductive carboxylation supports growth in tumour cells with defective mitochondria.Nature. 2011; 481: 385-388Crossref PubMed Scopus (898) Google Scholar, Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. et al.Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.Elife. 2014; 3: e02242Crossref PubMed Scopus (683) Google Scholar): oxidative phosphorylation (KEGG:00190); TCA cycle (KEGG:00020); glycolysis and gluconeogenesis (KEGG:00010); and alanine, aspartate, and glutamate metabolism (KEGG:00250). Taking all genes that were significantly up- or downregulated from these pathways we observed that for one hierarchical cluster of patients fold change in expression was strikingly increased for this set of genes (OXPHOS responders or OTR [OXPHOS transcriptional response]). All patients in the OTR group were estrogen receptor-positive (Figure 2B). Coherent with this observation, unsupervised hierarchical clustering of the expressed nuclear whole transcriptome showed that patients in the OTR group also clustered together in this analysis (Figure S2A). Notably, clustering of the OTR group also occurred for expressed genes of the mitochondrial transcriptome (Figure S2B). For patients with limited OTR there was evidence of increased glucose uptake defined by an increase in KFDG (FDG responders or FR) in contrast to the OTR group. Consistent with mitochondrial targeting it has recently been shown that metformin treatment leads to a decrease in the levels of short-chain acyl-carnitines in ovarian cancer (Liu et al., 2016Liu X. Romero I.L. Litchfield L.M. Lengyel E. Locasale J.W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers.Cell Metab. 2016; 24: 728-739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Metabolomic profiling of paired pre- and post-metformin samples showed that acetyl- and propionylcarnitine levels decrease (mean log2FC = −1.32, p = 0.046 and log2FC = −1.01, p = 0.039, respectively). Acetylcarnitine is a short-chain acyl-carnitine derived from glucose carbons (Schooneman et al., 2013Schooneman M.G. Vaz F.M. Houten S.M. Soeters M.R. Acylcarnitines: reflecting or inflicting insulin resistance?.Diabetes. 2013; 62: 1-8Crossref PubMed Scopus (402) Google Scholar) and, in contrast to the OTR group, their FR counterparts were able to maintain acetylcarnitine levels (Figure 2C). There was a strong correlation between change in KFDG and change in acetylcarnitine levels (Figure 2D). Figure S2C shows the interquartile range and median fold change for metabolites in the OTR and FR groups. It is unclear why intratumoral acetylcarnitine levels dropped, and this finding is at odds with Chen et al., 2016Chen W.W. Freinkman E. Wang T. Birsoy K. Sabatini D.M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism.Cell. 2016; 166: 1324-1337.e11Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, who showed that complex 1 inhibition in a cell line model resulted in a severalfold increase in acetylcarnitine levels within whole cells and mitochondria. However, this may be due to the discordance between the very different environmental conditions and strength of mitochondrial inhibition in our clinical study compared with cell line models. In addition, Chen et al. only assayed the mitochondrial matrix, and used a different complex 1 inhibitor in a non-breast cancer model. Notably, carnitine o-acetyltransferase, which catalyzes the bidirectional conversion of acetylcarnitine to acetyl-coenzyme A (CoA) within both mitochondria and peroxisomes, was differentially upregulated in the OTR group (all Figure 3A). Hence, we speculate that altered flux in this pathway may be a consequence of metformin treatment. The positive correlation between change in FDG flux and intratumoral acetylcarnitine levels possibly reflects increased flux of glucose carbons toward acetyl-CoA. To fully understand the effects of metformin and mitochondrial defects on acyl-carnitine metabolism will require further work in pre-clinical models. Maintaining aspartate levels has been shown to be a key resistance mechanism to electron transport chain inhibition and biguanides (Birsoy et al., 2015Birsoy K. Wang T. Chen W.W. Freinkman E. Abu-Remaileh M. Sabatini D.M. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis.Cell. 2015; 162: 540-551Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, Cardaci et al., 2015Cardaci S. Zheng L. MacKay G. van den Broek N.J. MacKenzie E.D. Nixon C. Stevenson D. Tumanov S. Bulusu V. Kamphorst J.J. et al.Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis.Nat. Cell Biol. 2015; 17: 1317-1326Crossref PubMed Scopus (177) Google Scholar, Sullivan et al., 2015Sullivan L.B. Gui D.Y. Hosios A.M. Bush L.N. Freinkman E. Vander Heiden M.G. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells.Cell. 2015; 162: 552-563Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). There was no difference in aspartate metabolite levels between the FR and OTR groups (Figures 1C and S3A). However, several genes involved in aspartate metabolism were significantly upregulated and it was striking that the increase in expression of three of the five genes that encode for units of the malate-aspartate shuttle (GOT2, MDH1, and MDH2) was significantly greater in the OTR group compared with the FR group (Figure 3A). Dependency on glutamine as a source of citrate for either lipid or aspartate biosynthesis has been shown to be a key resistance mechanism to metformin and other mitochondrial insults (Birsoy et al., 2015Birsoy K. Wang T. Chen W.W. Freinkman E. Abu-Remaileh M. Sabatini D.M. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis.Cell. 2015; 162: 540-551Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, Fendt et al., 2013Fendt S.M. Bell E.L. Keibler M.A. Davidson S.M. Wirth G.J. Fiske B. Mayers J.R. Schwab M. Bellinger G. Csibi A. et al.Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism.Cancer Res. 2013; 73: 4429-4438Crossref PubMed Scopus (160) Google Scholar, Mullen et al., 2011Mullen A.R. Wheaton W.W. Jin E.S. Chen P.H. Sullivan L.B. Cheng T. Yang Y. Linehan W.M. Chandel N.S. DeBerardinis R.J. Reductive carboxylation supports growth in tumour cells with defective mitochondria.Nature. 2011; 481: 385-388Crossref PubMed Scopus (898) Google Scholar) and we observed increased expression of multiple genes that regulate glutamine metabolism. Two key checkpoints in this process were differentially upregulated in the OTR group, mitochondrial isocitrate dehydrogenase (IDH2) and the citrate transporter, SLC25A1, which delivers glutamine-derived citrate to the cytosol where it is cleaved by ATP citrate lyase to oxaloacetate and acetyl-CoA for aspartate and lipid synthesis, respectively (Figure 3A). Previous work has also shown that both isoforms of isocitrate dehydrogenase, IDH1 and IDH2, support growth in cells that use glutamine-dependent reductive carboxylation. Hence, tumors harboring IDH mutations may be more susceptible to biguanide therapy. Metformin has been shown to modulate a number of systemic metabolic and inflammatory markers in diabetic populations. In our study metformin lowered circulating levels of serum glucose, insulin, c-peptide, and an insulin resistance score (homeostatic model assessment or HOMA), but not leptin, adiponectin, C-reactive protein, tumor necrosis factor α, or interleukin-6 (Figures 3B and S3B; Table S5). However, there were no significant differences between the OTR and FR groups in pre-/post-metformin changes in levels of any of these circulating metabolic markers (Figure S3C). There was a marked overlap in genes whose change in expression correlated with change in KFDG and change in acetylcarnitine (hypergeometric test, p < 0.00001), but little corresponding overlap with genes related to change in c-peptide, glucose, insulin, or HOMA (Figures 3C and S3D). Eighteen of the genes correlating with change in KFDG and acetylcarnitine were KEGG-annotated metabolism genes most notably associated with oxidative phosphorylation, carbohydrate, amino acid, and nucleotide metabolism pathways (Table S6). There was an increase in pAKT expression on tumor immunohistochemistry (paired t test, p = 0.026), but no correlation between change in pAKT expression and change in c-peptide, glucose, insulin, or HOMA, and no significant difference between the FR and OTR groups (Figures S3E–S3G). There was also no difference in pAMPK expression between the FR and OTR groups (Figure S3G). The increase in tumor pAKT expression was unexpected and not consistent with a decrease in insulin receptor signaling or findings in prior studies. AKT activation increases ATP levels in cells and has been identified in a number of studies as being a key player in the regulation of both glycolysis and oxidative phosphorylation (Robey and Hay, 2009Robey R.B. Hay N. Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis.Semin. Cancer Biol. 2009; 19: 25-31Crossref PubMed Scopus (425) Google Scholar). Recent work has shown that mitochondrial AKT activation occurs in the context of tumor energy and hypoxic stress, switching metabolism toward glycolysis (Chae et al., 2016Chae Y.C. Vaira V. Caino M.C. Tang H.Y. Seo J.H. Kossenkov A.V. Ottobrini L. Martelli C. Lucignani G. Bertolini I. et al.Mitochondrial Akt regulation of hypoxic tumor reprogramming.Cancer Cell. 2016; 30: 257-272Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, we cannot exclude metformin's systemic effects on host metabolism being a significant factor in modulating tumor metabolism and proliferation, and indeed we would expect a decrease in insulin levels to have some effect on tumor intracellular signaling. Our study only recruited patients with normal systemic glucose levels, and for patients with diabetes or glucose intolerance any effect on insulin signaling via the hypoglycemic activity of metformin is likely to be greater. We then investigated the relationship between tumor metformin levels and metabolic response. Although serum and tumor levels were significantly correlated with each other (Figure 3D) they did not differ between the OTR and FR groups (Figure S4A). Previously published pre-clinical data suggested that expression of the organic cation transporter, OCT1 (encoded by gene SLC22A1), is required for tumor uptake of metformin and metabolic response (Chandel et al., 2016Chandel N.S. Avizonis D. Reczek C.R. Weinberg S.E. Menz S. Neuhaus R. Christian S. Haegebarth A. Algire C. Pollak M. Are metformin doses used in murine cancer models clinically relevant?.Cell Metab. 2016; 23: 569-570Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, Dowling et al., 2016Dowling R.J. Lam S. Bassi C. Mouaaz S. Aman A. Kiyota T. Al-Awar R. Goodwin P.J. Stambolic V. Metformin pharmacokinetics in mouse tumors: implications for human therapy.Cell Metab. 2016; 23: 567-568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). There was no significant correlation between baseline OCT1 gene expression and tumor metformin levels but notably the patient with highest tumor metformin levels also had the greatest expression of tumor OCT1 (Figure S4B). Furthermore, there was no difference in baseline OCT1 expression between the OTR and FR groups (Figure S4C). Baseline OCT1 expression did correlate with change in KFDG, although the relevance of this finding is unclear given that there was no such relationship with tumor metformin levels (Figure S4D). Glucose transporter gene expression may determine the sensitivity of cell lines to biguanides (Birsoy et al., 2014Birsoy K. Possemato R. Lorbeer F.K. Bayraktar E.C. Thiru P. Yucel B. Wang T. Chen W.W. Clish C.B. Sabatini D.M. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides.Nature. 2014; 508: 108-112Crossref PubMed Scopus (484) Google Scholar). Expression of the glucose transporter, GLUT1 (encoded by gene SLC2A1), has previously been shown to correlate with uptake of FDG on PET-CT (Bos et al., 2002Bos R. van Der Hoeven J.J. van Der Wall E. van Der Groep P. van Diest P.J. Comans E.F. Joshi U. Semenza G.L. Hoekstra O.S. Lammertsma A.A. et al.Biologic correlates of (18)fluorodeoxyglucose uptake in human breast cancer measured by positron emission tomography.J. Clin. Oncol. 2002; 20: 379-387Crossref PubMed Scopus (487) Google Scholar), and in our study change in KFDG positively correlated with the change in expression of GLUT1 (Figure 3E). However, there was no significant difference in GLUT1 expression between the two groups although there was for another glucose transporter, GLUT4 (encoded by gene SLC2A4) (Figure S4E). Several clinical studies have shown that metformin can reduce breast, prostate, and endometrial cancer cell proliferation (Hadad et al., 2011Hadad S. Iwamoto T. Jordan L. Purdie C. Bray S. Baker L. Jellema G. Deharo S. Hardie D.G. Pusztai L. et al.Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial.Breast Cancer Res. Treat. 2011; 128: 783-794Crossref PubMed Scopus (230) Google Scholar, Joshua et al., 2014Joshua A.M. Zannella V.E. Downes M.R. Bowes B. Hersey K. Koritzinsky M. Schwab M. Hofmann U. Evans A. van der Kwast T. et al.A pilot 'window of opportunity' neoadjuvant study of metformin in localised prostate cancer.Prostate Cancer Prostatic Dis. 2014; 17: 252-258Crossref PubMed Scopus (59) Google Scholar, Laskov et al., 2014Laskov I. Drudi L. Beauchamp M.C. Yasmeen A. Ferenczy A. Pollak M. Gotlieb W.H. Anti-diabetic doses of metformin decrease proliferation markers in tumors of patients with endometrial cancer.Gynecol. Oncol. 2014; 134: 607-614Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, Mitsuhashi et al., 2014Mitsuhashi A. Kiyokawa T. Sato Y. Shozu M. Effects of metformin on endometrial cancer cell growth in vivo: a preoperative prospective trial.Cancer. 2014; 120: 2986-2995Crossref PubMed Scopus (94) Google Scholar, Niraula et al., 2012Niraula S. Dowling R.J. Ennis M. Chang M.C. Done S.J. Hood N. Escallon J. Leong W.L. McCready D.R. Reedijk M. et al.Metformin in early breast cancer: a prospective window of opportunity neoadjuvant study.Breast Cancer Res. Treat. 2012; 135: 821-830Crossref PubMed Scopus (196) Google Scholar, Schuler et al., 2015Schuler K.M. Rambally B.S. DiFurio M.J. Sampey B.P. Gehrig P.A. Makowski L. Bae-Jump V.L. Antiproliferative and metabolic effects of metformin in a preoperative window clinical trial for endometrial cancer.Cancer Med. 2015; 4: 161-173Crossref PubMed Scopus (109) Google Scholar). We explored the effect of metformin on a validated human breast cancer proliferation signature (Desmedt et al., 2008Desmedt C. Haibe-Kains B. Wirapati P. Buyse M. Larsimont D. Bontempi G. Delorenzi M. Piccart M. Sotiriou C. Biological processes associated with breast cancer clinical outcome depend on the molecular subtypes.Clin. Cancer Res. 2008; 14: 5158-5165Crossref PubMed Scopus (651) Google Scholar) and overall observed no significant change following metformin treatment (Figure 4). However, it was striking that an increase in metagene expression occurred in the OTR group, while, in contrast, there was a decrease for several patients in the FR group, the change in metagene expression consequently differing significantly between the two groups (Figure 4). Under in vitro low-glucose conditions the ability for cell lines to upregulate OXPHOS predicts for sensitivity to biguanides (Birsoy et al., 2014Birsoy K. Possemato R. Lorbeer F.K. Bayraktar E.C. Thiru P. Yucel B. Wang T. Chen W.W. Clish C.B. Sabatini D.M. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides.Nature. 2014; 508: 108-112Crossref PubMed Scopus (484) Google Scholar), and our data suggest that a reactive increase in OXPHOS and aspartate synthesis gene transcription may be critical for resistance to metformin. None of the circulating or tumor immunohistochemical markers, metformin levels, KFDG, or significantly altered metabolites correlated with change in expression of the proliferation metagene (Figure S4F). Our work outlines two types of breast cancer metabolic response to metformin and links the effects of metformin on mitochondrial metabolism with its effects on breast cancer proliferation at a transcriptional level. Tumors that were able to upregulate OXPHOS gene transcription in response to metformin showed an increase in their proliferation score suggestive of resistance following metformin treatment." @default.
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