Preclinical Models and Clinical Trials with Metformin in Breast Cancer

Published OnlineFirst March 28, 2014; DOI: 10.1158/1078-0432.CCR-13-0354

Clinical Cancer Molecular Pathways Research

Molecular Pathways: Preclinical Models and Clinical Trials with Metformin in Breast Cancer

Alastair M. Thompson

Abstract Metformin, an oral biguanide widely used to treat diabetes, has considerable potential and is in clinical trials as an experimental preventive or therapeutic agent for a range of cancers. Direct actions targeting cellular pathways, particularly via AMP-activated protein kinase and through inhibiting mitochondrial ATP synthesis, or systemic mechanisms involving insulin and insulin-like growth factors have been much studied in vitro and in preclinical models. Epidemiologic and retrospective studies also provide clinical evidence in support of metformin as an antitumor agent. Preoperative window-of- opportunity trials confirm the safety of metformin in women with primary breast cancer, and demonstrate reduction in tumor cell proliferation and complex pathways of gene suppression or overexpression attributable to metformin. Confirmation of insulin-mediated effects, independent of body mass index, also supports the potential benefit of adjuvant metformin therapy. Neoadjuvant, adjuvant, and advanced disease trials combining metformin with established anticancer agents are under way or proposed. Companion biomarker studies will utilize in vitro and preclinical understanding of the relevant molecular pathways to, in future, refine patient and tumor selection for metformin therapy. Clin Cancer Res; 20(10); 1–8. 2014 AACR.

Background In vitro, in vivo animal model and human studies support Synthetic biguanides, based on the guanidine deriva- several possible modes of action with extrapolation of in vitro tive galegine originally derived from Galega officinalis studies in diabetes, the range of models, nonphy- (French lilac), have been used clinically since the 1950s siologic metformin levels, the circumstantial evidence from with metformin (N0,N0-dimethylbiguanide) prescribed epidemiologic studies, and retrospective reviews creating to more than 120 million type II diabetics globally. complexity. Focused initially on diabetes and metabolism Phenformin, previously used in the treatment of diabe- more generally, both direct (cellular) pathways and indirect tes, was withdrawn from many countries in the 1970s (systemic) actions have been proposed as key mechanisms due to drug-associated lactic acidosis (1). The potential for the action of biguanides in diabetes that may also be of for repurposing metformin to oncology is supported relevance to cancer. by evidence of reduced cancer incidence and increased AMPK-mediated effects time to develop cancer (2), reduced mortality from cancer (3), with lower HER2-positive breast cancer-spe- The master sensor of cellular energy, the Ser/Thr protein cific mortality (4), or reduced risk of distant metastases kinase complex AMP-activated protein kinase (AMPK), in triple-negative breast cancer (5) in diabetic women. remains central to studies of metformin. AMPK activation Additional supportive evidence has come from studies stimulates TSC2, hence indirectly as well as AMPK directly demonstrating that higher serum insulin levels are asso- inhibits mTOR and consequently by downregulating ciated with increased risk of breast cancer (6) and shorter S6kinase leads to reduced protein synthesis, reduced cellu- survival from breast cancer (7) which may be modified lar growth, and lowers proliferation (Fig. 1). A complex by metformin. containing Liver kinase B1 (LKB1), classed as a tumor sup- The relevance of the multiple proposed mechanisms pressor gene, activates heterodimers of AMPK subunits of action of biguanides to cancer remains controversial. through phosphorylation of AMPKa2Thr-172, established as a link between LKB1, AMPK, and cancer (8). Breast cancer cells lacking LKB1 are resistant to the growth inhibitory in vitro in vivo Author's Affiliation: Department of Surgical Oncology, MD Anderson effects of metformin and (9, 10, 11), and as a Cancer Center, Houston, Texas distinct mechanism, epigenetic inactivation of LKB1 has Corresponding Author: Alastair M. Thompson, MD Anderson Cancer been reported in sporadic papillary breast cancer (12). Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713- Furthermore, the intracellular pathways downstream of 745-2792; Fax: 713-745-7462; E-mail: [email protected] HER2 amplification, PIK3CA mutation, and PTEN muta- doi: 10.1158/1078-0432.CCR-13-0354 tion or loss of PTEN expression converge on elevated mTOR 2014 American Association for Cancer Research. signaling and are considered potential mechanisms for

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breast cancer development and of resistance to therapy. This Additional, non-AMPK–mediated mechanisms for the makes the potential for metformin to work via targeting the antineoplastic effect of metformin have been proposed mTOR cascade an attractive mechanism of action (13). through NF-kB, a pleiotropic protein complex that controls Other, AMPK and TSC2 independent, mechanisms of DNA transcription. Metformin may inhibit IkB and IKKa/b mTOR inhibition by metformin include suppression of subunits and hence NF-kB and may prevent the transloca- RAG GTPases (14). tion of NF-kB from the cytoplasm to the nucleus (31). Additional, potentially important, AMPK mechanisms Metformin seems to downregulate genes involved in mito- for oncology include phosphorylating and hence inactivat- sis and may act synergistically in this setting with taxanes ing ACC1 and ACC2, consequently blocking fatty acid (32). Metformin may also act via the DNA damage response synthesis but increasing mitochondrial fatty acid uptake mechanisms through ataxia telangiectasia mutated (ATM) and subsequent breakdown by b-oxidation (15). Inactiva- and Chk2 (33), also potentially accounting for the cancer tion of AMPK promotes a metabolic shift to aerobic glycol- prevention effect. ysis through stabilization of hypoxia-inducible factor-1a Systemic effects (HIF-1a), and increases transcription of genes involved in glycolytic pathways, resistance to apoptosis, angiogenesis, Biguanides have long been known to suppress hepatic and metabolic adaptation (16, 17). AMPK may act as a glucose production (34) with metformin uptake via negative regulator of the Warburg effect under nutrient-rich OCT leading to AMKP inhibition of hepatic gluconeo- and growth factor replete conditions in the absence of genesis, increase peripheral insulin sensitivity (and hence evident energy imbalance (16). In contrast, AMPK activa- glucose uptake, e.g., in skeletal muscle), and reduce tion may increase resistance to stress and viability of cancer circulating insulin and insulin-like growth factor (IGF- cells under hypoxic conditions of metabolic stress (18), I) levels, although it is less clear how metformin regulates which may be of particular relevance as tumor cancer cells IGF-I. migrate and metastasize. Certainly, the insulin receptor lies upstream of the PI3K/ AMPK phosphorylates p53 and leads to cell-cycle AKT/mTOR signaling pathways and breast cancer cells may checkpoint activation, at least in mouse embryonic fibro- have greater sensitivity to the effects of insulin such that blasts, allowing cells to survive energy deprivation (19). metformin reducing insulin levels, and hence binding of Indeed, metformin inhibition of complex 1 of the mito- insulin to receptors on breast cancer cells present very chondrial electron transport chain (20, 21, 22) is partic- attractive mechanisms to indirectly inhibit tumor growth ularly effective in p53-deficient colon cancer cells (23) (13). This provides molecular insights into and supports the although the p53 status did not affect metformin sensi- clinical relevance of insulin/IGF-I reduction in relation to tivity in other cancer cell lines (24, 25). However, loss of cancer progression (7, 35, 36). functional AMPK or p53 (as is common through muta- – tion or p53 network dysfunction in most cancers) may Clinical Translational Advances constitute a sufficient energy crisis to result in metformin Phenformin having a cytotoxic synthetic lethality effect (26). Thus, In murine models, phenformin has been associated with there is evidence that metformin may be therapeutically a reduced incidence of 7,12-dimethylbenz(a)anthracene effective acting via p53, particularly where normal p53 (DMBA)-induced rat mammary tumors (37), enhances the function is abrogated, as is the case in most epithelial effects of chemotherapy (38), and elicits antiproliferative cancers. effects, alongside inducing apoptosis, in neuroblastoma (39). Phenformin, like metformin, is associated with Mitochondrial effects enhanced apoptosis and ATP depletion in AMPK-deficient Biguanides accumulate in mitochondria, with uptake cancer cells and may be most effective in cancer cells promoted (for the cationic metformin at least) by organic without a functional LKB1–AMPK pathway, as demonstrat- cation transporter 1 (OCT1) to inhibit complex 1 of the ed in non–small cell lung cancer (40). mitochondrial respiratory chain, inhibit mitochondrial Phenformin has been shown to prevent and retard ATP synthesis, increase ADP:ATP and AMP:ATP ratios, growth of breast cancer cell lines (41) and to protect against and hence activate AMPK indirectly. Phenformin is 50- tumor development in a spontaneous lymphoma murine fold more potent than metformin as an inhibitor of model exhibiting PTEN heterozygous mutations and LKB1 mitochondrial complex 1 and being lipophilic (21) is hypomorphic mutations with greater potency than metfor- less dependent on OCT (27), retaining the dose-depen- min (11). dent antiproliferative effect in ovarian cancer cell lines Mechanistic insights from xenograft work suggest both even in the presence of OCT1 siRNA (28). Given that systemic and direct tumor/stromal effects may be elicited oxidative phosphorylation-dependent production of ATP (41) and have led to speculation that phenformin may have seems to be secondary to mitochondrial synthesis of the capability to block a reverse Warburg effect of onconu- anabolic precursors in proliferating cells (29), it has been trient production by stroma feeding breast cancer cells suggested that metformin exerts the chemopreventive in vivo (42, 43). effects by functioning as a weak mitochondrial poison Phenformin may initially seem most attractive for trials in inhibiting oxidative phosphorylation (30). the advanced breast cancer setting (providing renal and

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Skeletal Colon muscle Liver

Increased Gluconeogenesis M glucose uptake M AMPK M OCT Glucose

Insulin IGFs Aromatase enzymes Cell-cycle amount IR Glycolytic pathways

P p53 PI3K Stabilize HIF-1α

LKB1 AKT

OCT M TSC2 AMPK HER2 amplification

PIK3CA mutation mTOR PTEN mutation Fatty acid P uptake + ACC1 PTEN-reduced β-oxidation P expression ACC2 S6K ADP AMP OCT1 ATP Complex Protein synthesis 1 Growth Proliferation

Mitochondrion Fatty acid synthesis

Cell membrane

Figure 1. Overview of proposed metformin actions in vivo. Metformin (M), absorbed from the gut, acts via systemic and intracellular molecular pathways. Systemic actions of metformin via metformin uptake through OCT in the liver, AMPK inhibition of gluconeogenesis, and consequent reduction in insulin reduces insulin receptor (IR) stimulation and downstream intracellular effects (including via PI3K, akt, and HIF-1a) and leads to increased glucose uptake in tissues such as skeletal muscle. (Continued on the following page.) www.aacrjournals.org Clin Cancer Res; 20(10) May 15, 2014 OF3

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hepatic functions are adequate) with the potential to avoid tumor development, in established breast cancers, lack of lactic acidosis (1) and potentially enhance cancer cell kill by functional AMPK-dependent pathways may render breast concomitant administration of 2 deoxyglucose with phen- cancer susceptible to metformin therapy. formin (if confirmed beyond colorectal cancer cell lines; Additional support for metformin as a therapeutic ref. 44). agent comes from in vivo models where metformin seems þ to selectively inhibit CD44 /CD24 low cancer stem cell- Metformin like populations (54), repress genes associated with epi- In vitro models suggest that metformin may be active thelial to mesenchymal transition such as ZEB1 TWIST, against breast cancer cell lines regardless of the estrogen SNAi1 (55), and work synergistically with chemotherapy receptor (ER), progesterone receptor (PR), HER2 status or agents (54). In addition, inhibition of glutamine metab- p53 status, and subtype of the cells (24, 25). olism, at least in prostate models, may synergize with For ER-positive breast cancer, dependent on aromatase metformin (56). Injected into tumors, albeit at potential- conversion of precursors to estrogens in postmenopausal ly nonphysiologic doses, metformin may be effective women, metformin may inhibit aromatase enzyme alongside paclitaxel and carboplatinum as part of a com- expression in breast adipose tissue via AMPK (45). Similar bination chemotherapy strategy for the treatment of effects on other clinically relevant genes have yet to be breast cancer (57). explored. Given that aromatase inhibitors are now the However, some have questioned the relevance of selected mainstay of neoadjuvant, adjuvant, and extended treat- preclinical data given the concentrations, doses, and deliv- ment in postmenopausal womenwithbreastcancer,the ery of metformin used (58). Nevertheless, studies have potential for synergism between an aromatase inhibitor demonstrated cell inhibition using physiologically possible and metformin is under exploration in a Korean neoad- concentrations of metformin in vitro (51) and in mouse juvant trial and is planned for an international extended models (41, 50, 59). Even so, the multiplicity of mechan- adjuvant trial. For premenopausal women, where tamox- isms deduced in vitro may differ significantly in vivo while ifen remains the principal adjuvant therapy for ER-pos- animal models (usually in immune compromised mice) itive breast cancer, the additive effects of metformin to may be different again from the cancer prevention and tamoxifen, at least in terms of reducing cell proliferation cancer therapy settings in humans. (46), merits subgroup analysis within the ongoing MA32 adjuvant metformin trial (47). Metformin and breast cancer trials For HER2, breast cancer lines expressing HER2 have Given the preclinical data and that metformin has known increased sensitivity to metformin (48) mediated via pharmacokinetics and manageable toxicities, with extensive inhibition of S6K; in trastuzumab-resistant models, met- use worldwide in diabetes, repurposing metformin within formin disrupted ERBB2/IGF1R complexes and signifi- clinical trials to seek efficacy against breast cancer is very cantly reduced cell proliferation (49). Metformin, used at attractive. Evidence to date suggests that metformin could comparable clinical doses, delayed the onset, reduced be clinically useful in the prevention, preoperative, adju- the growth, and increased the lifespan of MMTV-her2/ vant, extended adjuvant, and advanced disease settings, neu mice (50) accompanied by reductions in circulating although the detail of dose, duration, and drug combina- insulin levels. As HER2-targeted therapy extends to tions will require elucidation. Fortunately, the research other tumor types (such as gastric and esophageal can- community is actively engaged to address many of these cer), similar metformin therapy approaches may merit roles and through companion biomarker studies ascertain consideration. the tumor and patient characteristics most amenable to For triple-negative breast cancer, metformin was effec- treatment and whether direct or systemic effects are both tive in vitro (and in an animal model) inducing AMPK and of clinical importance. suppressing EGF receptor, mitogen-activated protein Trials are under way examining these issues in the kinase, and Src phosphorylation and lowering cyclin D1 setting of breast, brain, colorectal, endometrium, head and cyclin E (51). The effect of metformin on triple- andneck,lung,lymphoma,melanoma,ovary,pancreas, negative cell lines may, in part, be attributable to STAT3 prostate, renal, and thyroid malignancy. Single-center, inhibition (52). phase II biomarker studies and/or with clinical endpoints More generally, reduced immunohistochemical stain- predominate; many trials use gradual increases in dose up ing for pAMPK was described in 318 of 349 (91%) breast to 1,500 to 2,250 mg metformin per day to mitigate the cancers, with all subtypes of breast cancer represented self-limiting initial gastrointestinal side effects of nausea, (53). Although AMPK seems to protect against initial diarrhoea, and bloating. Caution over the potential but

(Continued.) Cell level molecular pathways include reductions in mitochondrial respiratory function and activation by metformin of AMPK with some of the consequent intracellular events converging with the systemically driven insulin receptor pathway. AMPK stimulates phosphorylation of p53, ACC1, and ACC2, inhibits aromatase enzymes, directly and indirectly inhibits mTOR itself stimulated by key drivers for cancer (HER2, PIK3CA, and PTEN), and hence curtails S6kinase activity and its consequences. Additional potentially important effects, for example, on the stroma, and the complexities of subcellular networks are not illustrated.

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rare metformin-associated lactic acidosis (1/100,000 case note review of patients with breast cancer receiving years of use) generally restricts the clinical trials popula- neoadjuvant chemotherapy (67) demonstrated a remark- tions to women under 75 years of age with good renal and able 24% complete pathologic response (cPR) in diabetic hepatic function. patients incidentally taking metformin and receiving A proposed study of metformin versus placebo to neoadjuvant chemotherapy compared with 16% cPR in prevent the progression of atypical ductal hyperplasia, nondiabetics and 8% cPR in diabetics not receiving met- ductal, or lobular in situ neoplasias seems attractive given formin. A similar, also historical, series of epithelial the evidence that even low-dose metformin (250 mg/day) ovarian cancer has demonstrated clinical benefit with is sufficient to reduce proliferation and aberrant crypt foci increased progression-free survival and overall survival in rectal epithelium (60). The progression from ductal in diabetics taking metformin and receiving taxane/car- carcinoma in situ (DCIS) to invasive breast cancer may be boplatin chemotherapy compared with nondiabetics or mechanistically linked to glycolytic cancer-associated diabetics not on metformin (68). However, incidental fibroblast activity (exporting lactate to the tumor cells as metformin use has not demonstrated survival benefit in evidenced by a fall in caveolin and rise in MCT4 expres- two case series of diabetics with breast cancer (5, 67). This sion) with the potential for blockade by metformin (61). concept of combining metformin with neoadjuvant che- The first randomized clinical trial using metformin motherapy is in prospective trials of metformin alongside (1,000 mg bd) in breast cancer was reported in 2011 and paclitaxel and FAC; docetaxel, epirubicin, and cyclophos- used the preoperative window-of-opportunity setting phamide metformin; and in ISPY2 (ganitumab þ (62). This novel trial randomized patients to receive metformin). An innovative trial being conducted in metformin for 2 weeks or watchful waiting for 2 weeks Oxford combines the window-of-opportunity and neoad- between diagnostic biopsy and definitive surgery. Core juvant concepts. The Oxford trial sequences a 2-week run needle tumor biopsies were taken at the time of diagnosis in with metformin and then neoadjuvant chemotherapy and on the operating table before resection of the oper- ( metformin) is administered up to the time of surgery. able breast cancer to allow direct comparison of tumor Noninvasive positron emission tomography scanning proliferation (Ki-67) and tumor messenger RNA expres- and lipid metabolism studies are being used alongside sion alongside serum insulin. The trial demonstrated a tissue biopsies to examine the effects of metformin on significantreductioninKi-67withmetforminbutnotin breast cancer metabolism and lipid synthesis. the control arm, reduced gene expression [including in In addition to the potentially synergistic effects of metfor- the phosphoinositide 3-kinase (PI3K) pathway], and min with chemotherapy against breast cancer, murine mod- increased gene expression (e.g., TNFR1), with a flattening els suggest a cardio-protective effect of metformin which of the serum insulin levels in response to surgery for the seems to abrogate the effects of doxorubicin on lactate patients on metformin. Thus, both in vivo antitumor dehydrogenase, CKMB, and glutathione (69), although evi- effects and systemic effects were demonstrated for the dence for such effects in humans is, as yet, lacking. first time in women with primary breast cancer receiving In the adjuvant setting, the NCIC CTG MA .32 trial (47) metformin. Although women were not fasting at the time is examining the efficacy of adjuvant metformin for 5 of diagnostic biopsy, the insulin effect and the lack of years after complete resection of invasive breast cancer association between metformin effects and body mass and following completion of chemotherapy and/or radio- index (BMI) confirm the potentially beneficial systemic therapy but concomitant with endocrine therapy or anti- effect of metformin as an insulin suppressor in women HER2 therapy. Early data suggest beneficial effects of with breast cancer (7, 63). metformin compared with placebo on patient weight and A second, single-center, preoperative window-of-oppor- metabolic factors which, if sustained, could significantly tunity placebo-controlled trial of similar size confirmed improve survival from breast cancer (7). Further clinical the reduction in Ki-67 with metformin use over 2 weeks, efficacy, companion biomarker, and mammographic again not associated with BMI, and provided enhanced density studies are eagerly awaited as the trial matures. evidence for the systemic insulin-mediated effects (64). There are also plans under way to trial metformin in the The need for meticulous trial design was flagged by a extended adjuvant setting, beyond 5 years, for ER-positive subsequent trial (65) where failure to continue metformin patients at risk of recurrence. Further survivorship trials up to the time of surgery and using core samples at using metformin along with exercise in breast and colon diagnosis versus resection specimens (66) make data inter- cancer are proposed. pretation difficult. In contrast with most clinical drug development, where Overall, these prospective, preoperative window studies molecular pathways are usually explored in advanced in breast cancer (62, 64, 65), despite reservations about trial disease, this setting has only recently been reported for þ design and methodology, demonstrate that metformin is metformin. For ER advanced disease, there is no disad- safe for women with primary breast cancer and confirm the vantage to taking metformin concomitant with exemes- attraction of metformin as a therapeutic agent. tane on exemestane levels (70) providing reassurance for Given the proposed mechanisms of action of metfor- the neoadjuvant and adjuvant trails using concomitant min and the preclinical data, synergism of the biguanide metformin and endocrine therapy under way. Interactions with chemotherapy has been explored. A retrospective of metformin with a range of agents are proposed on the

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basis of an appreciation of the intracellular pathways need to be considered when metformin translates into outlined above, including trials of metformin with ever- routine clinical practice. þ olimus and exemestane in ER disease, metformin Biguanides, and particularly metformin, present an with erlotinib in triple-negative breast cancer, and com- attractive proposition for repurposing as low toxicity anti- bination chemotherapy metformininHER2-negative tumor agents, thus demonstrating how understanding the advanced disease. molecular pathways of cancer combined with clinical data Biomarker studies built in to all these trials will be will lead to further advances in oncology. essential to identify the breast cancers with characteristics (such as the LKB1–AMPK or p53-defective pathways) most Disclosure of Potential Conﬂicts of Interest likely to be susceptible to the effects of metformin. Cancer No potential conﬂicts of interest were disclosed. heterogeneity of OCT1, and hence for cancer cell uptake of Grant Support metformin, as observed in ovarian cancer (28), may merit This work was supported by Breast Cancer Campaign, Breast Cancer measurement before considering metformin therapy in the Research Scotland, Cancer Research UK, and Breakthrough Breast Cancer. clinical setting given, for example, the low expression of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked OCT1 in normal breast tissues. The potential effects of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate polymorphisms of LKB1, OCT 1,2,3, and ATM on resistance this fact. to metformin (70) and interactions between proton pump inhibitors and metformin via OCT 1,2,3 (70) will clearly Received September 26, 2013; revised January 14, 2014; accepted February 7, 2014; published OnlineFirst March 28, 2014.

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OF8 Clin Cancer Res; 20(10) May 15, 2014 Clinical Cancer Research

Molecular Pathways: Preclinical Models and Clinical Trials with Metformin in Breast Cancer

Alastair M. Thompson

Clin Cancer Res Published OnlineFirst March 28, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-0354

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