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Originally published as JCO Early Release 10.1200/JCO.2009.24.5456 on October 26 2009 © 2009 American Society of Clinical Oncology.
If Mammalian Target of Metformin Indirectly Is Mammalian Target of Rapamycin, Then the Insulin-Like Growth Factor-1 Receptor Axis Will Audit the Efficacy of Metformin in Cancer Clinical TrialsCatalan Institute of Oncology-Health Services Division of Catalonia; Girona Biomedical Research Institute, Dr. Josep Trueta University Hospital of Girona, Girona, Catalonia, Spain To the Editor: We read with great interest the editorial recently published by Goodwin et al,1 entitled "Metformin in Breast Cancer: Time for Action," in a recent issue of Journal of Clinical Oncology (JCO). Based on the first evidence of a potential effect of metformin in human breast cancer reported by Jiralerspong et al2 in the same issue of JCO, and considering the ever-growing preclinical studies revealing that metformin can induce strong dose-dependent inhibition of cell proliferation on a series of cultured cancer epithelial cells, Goodwin et al1 illustrate a clinical-molecular interface that strongly supports forthcoming metformin-based clinical trials in the adjuvant breast cancer setting. Although metformin should be considered a dirty drug that targets multiple protein kinases at once in cancer cells (eg, adenosine monophosphate–activated protein kinase [AMPK], S6K1, HER1, HER2, Src),3–8 most researchers in the field have adopted a simplified signal model in which metformin works as a general inhibitor of cancer cell growth by activating AMPK, inactivating the mammalian target of rapamycin (mTOR) and lastly decreasing activity of the mTOR effector S6K1.9,10 In this bench-to-clinic scenario, it is reasonable to suggest that because metformin may have direct (ie, insulin independent) anticancer effects through its inhibition of the AMPK/mTOR/S6K1 pathway, metformin use in the clinical setting might lead to even greater therapeutic benefit than is suggested by metformin-induced insulin lowering alone.10,11 However, the use of rapamycin and its analogs in the clinic has revealed that mTOR pathway is embedded in a network of signaling cross-talks and feedbacks that significantly reduce their effectiveness in cancer.12 Thus, if we assume that mammalian target of metformin (mTOM) indirectly is mTOR,13 then we should acknowledge an important note of caution regarding use of metformin in the adjuvant breast cancer setting. (The expression mTOM has been modified from an original sentence provided by Demidenko and Blagosklonny13: "Figuratively speaking, Target of Resveratrol is "indirectly TOR" (Target of Rapamycin)). Once activated by mTOR (ie, mTORC1), its downstream effector S6K1 mediates phosphorylation of insulin receptor substrate-1 (IRS-1) inhibitory serine sites which lead to IRS-1 degradation.14 Accordingly, suppression of S6K1 activity by rapamycin or rapalogues has been shown to prevent inhibitory IRS-1 phosphorylation, thereby stabilizing IRS-1—the principal insulin-like growth factor-1 receptor (IGF-1R)–docking molecule—and increasing IGF-IR/PI3K signaling to Akt.15 IRS-1 can be also downregulated through transcriptional repression and phosphorylated by other members of the mTOR pathway. mTOR, when inhibited, activates the MAPK pathway throughout S6K1 signaling as well.12 Indeed, this undesired relieving of the negative feedback loops stemming from mTOR/S6K1 inhibition largely accounts for resistance of tumors to killing by mTOR inhibitors such as rapamycin and the rapalogue everolimus (RAD001).12,15 Therefore, any molecular model of metformin action through the mTOR/S6K1 pathway must account for all these phenomena. We have speculated that, if metformin molecularly behaves as an indirect inhibitor of mTOR, cancer cells may rapidly acquire autoresistance to metformin-induced tumoricidal effects by relieving the negative feedback loop between mTORC1/S6K1 and IGF-1R/IRS-1 (Fig 1). Importantly, this process can be incompletely understood by performing experiments that last less than 24 to 48 hours, which is the case for most published studies on metformin-induced killing of cancer cells in vitro. To test this hypothesis, and in order to simulate the clinic where patients will receive metformin on a daily chronic basis, in our laboratory we have begun to develop models of acquired resistance to metformin by chronically exposing epithelial cancer cells to metformin for longer than 4 months before starting any experimental procedure. We have now isolated metformin-resistant (MetR) pool of clones (POOLs) from a series of human carcinoma cell lines that are capable of growing in the continuous presence of 10 mmol/L metformin without any significant effect on cell viability (as confirmed by performing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] –based metabolic assays). We recently performed low-scale semiquantitative phosphoproteomic approaches (ie, Human Phospho-Array Proteome Profiler; R&D Systems, Minneapolis, MN) to simultaneously profile the activation status of 42 different receptor tyrosine kinases in parental (ie, metformin naive) and in resistant (ie, metformin refractory) cancer cells. When analyzing a phosphoproteomic signature distinctively correlating with the acquisition of cancer autoresistance to metformin, and unlike in the metformin-naive parental cancer cells, a dramatic hyperphosphorylation of IGF-1R was likewise observed in metformin-refractory MetR POOLs (Fig 1; Figure shows results obtained in A431 epidermoid carcinoma cells. Equivalent results in terms of metformin-induced IGF-1R hyperactivation have been observed in a series of human carcinoma cell lines including MCF-7 and JIMT-1 breast carcinoma cell lines). Parallel immunofluorescence analyses showing significantly increased levels of tyrosine 1161-phosphorylated IGF-1R protein at the cell membrane (apparently incorporated in focal adhesion sites) of MetR cells strongly suggested that increased IGF-1R signaling underlies, at least in part, the development of acquired autoresistance observed in human cancer epithelial cells chronically exposed to metformin (Fig 1). Intriguingly, vascular endothelial growth factor receptor 3 (also known as Fms-like tyrosine kinase 4) was also found significantly overactivated in MetR cells, supporting the notion that metformin treatment likely promotes a senescence-associated secretory phenotype involving increased gene expression and secretion of vascular endothelial growth factor and other cytokines.16
We here present evidence that disruption of the AMPK/mTOR/S6K1 axis on chronic exposure to metformin efficiently relieves negative feedback suppression on the IGFR-1/IRS-1 axis, leading to elevation of cell survival signals and thus counteracting the antitumor activity of metformin. These findings largely recapitulate those observed with rapamycin and rapalogues and might have important therapeutic implications because part of the poor anticancer activity of mTOR inhibitors in clinical trials is due to this negative feedback.12,14,15 Therefore, we would like to suggest that: strategies that prevent the activation of the IGFR-1/IRS-1 axis by metformin (eg, anti-IGF-1R antibodies or small molecule IGF-1R compounds) are likely to have synergistic activity on tumor kill while preventing or delaying the appearance of auto-resistance phenomena in metformin-treated epithelial cancer cells; there is a need to incorporate the activation status of the IGF-1R/IRS-1 axis (and that of its downstream Akt and MAPK prosurvival signaling cascades) as a pivotal biomarker in metformin-based cancer clinical trials. Our division of clinical trials at the Catalan Institute of Oncology (ICO) is currently involved in developing a phase II, randomized, open label, multicentric clinical trial of neoadjuvant chemotherapy and trastuzumab with or without metformin in women diagnosed with HER2-positive primary breast cancer (METTEN-01). In exploratory work, a secondary end point of the study will be to test the differences in the percentage change induction of IGF-1R before and after metformin treatment and to compare the data with placebo group. Only then could we confirm that upstream activation of the IGF-1R/IRS-1 axis is auditing the effectiveness of metformin in cancer clinical trials. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest. ACKNOWLEDGMENT A.V.-M. is the recipient of a Sara Borrell post-doctoral contract (CD08/00283, Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria, Spain); J.A.M. is the recipient of a Basic, Clinical and Translational Research Award (BCTR0600894) from the Susan G. Komen Breast Cancer Foundation (TX) and was also supported by a grant from the Fundación Científica de la Asociación Española Contra el Cáncer (Spain); this work was supported in part by Grants No. CP05-00090, PI06-0778, and RD06-0020-0028 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria, Spain, to J.A.M.). REFERENCES
1. Goodwin PJ, Ligibel JA, Stambolic V: Metformin in breast cancer: Time for action. J Clin Oncol 27:3271–3273, 2009. 2. Jiralerspong S, Palla SL, Giordano SH, et al: Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J Clin Oncol 27:3297–3302, 2009. 3. Dowling RJ, Zakikhani M, Fantus IG, et al: Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res 67:10804–10812, 2007. 4. Shell SA, Lyass L, Trusk PB, et al: Activation of AMPK is necessary for killing cancer cells and sparing cardiac cells. Cell Cycle 7:1769–1775, 2008.[Medline] 5. Vazquez-Martin A, Oliveras-Ferraros C, del Barco S, et al: The antidiabetic drug metformin: A pharmaceutical AMPK activator to overcome breast cancer resistance to HER2 inhibitors while decreasing risk of cardiomyopathy. Ann Oncol 20:592–595, 2009. 6. Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA: The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR effector p70S6K1 in human breast carcinoma cells. Cell Cycle 8:88–96, 2009.[Medline] 7. Alimova IN, Liu B, Fan Z, et al: Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8:909–915, 2009.[Medline] 8. Liu B, Fan Z, Edgerton SM, et al: Metformin induces unique biological and molecular responses in triple negative breast cancer cells. Cell Cycle 8:2031–2040, 2009.[Medline] 9. Hadad SM, Fleming S, Thompson AM: Targeting AMPK. A new therapeutic opportunity in breast cancer. Crit Rev Oncol Hematol 78:1–7, 2008. 10. Cazzaniga M, Bonanni B, Guerrieri-Gonzaga A, et al: Is it time to test metformin in breast cancer clinical trials? Cancer Epidemiol Biomarkers Prev 18:701–705, 2009. 11. Pollak M: Insulin and insulin-like growth factor signaling in neoplasia. Nat Rev Cancer 8:915–928, 2008.[CrossRef][Medline] 12. Carracedo A, Baselga J, Pandolfi PP: Deconstructing feedback-signaling networks to improve anticancer therapy with mTORC1 inhibitors. Cell Cycle 7:3805–3809, 2008.[Medline] 13. Demidenko ZN, Blagosklonny MV: At concentrations that inhibit mTOR, resveratrol suppresses cellular senescence. Cell Cycle 8:1901–1904, 2009.[Medline] 14. O'Reilly KE, Rojo F, She QB, et al: MTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66:1500–1508, 2006. 15. Easton JB, Kurmasheva RT, Houghton PJ: IRS-1: auditing the effectiveness of mTOR inhibitors. Cancer Cell 9:153–155, 2006.[CrossRef][Medline] 16. Oliveras-Ferraros C, Vazquez-Martin A, Menendez JA: Genome-wide inhibitory impact of the AMPK activator metformin on [kinesins, tubulins, histones, auroras and polo-like kinases] M-phase cell cycle genes in human breast cancer cells. Cell Cycle 8:1633–1636, 2009.[Medline]
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Copyright © 2009 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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