Metformin: A Novel but Controversial Drug in Cancer Prevention and

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Metformin: A Novel but Controversial Drug in Cancer Prevention and Treatment Xinbing Sui,*,†,‡ Yinghua Xu,† Xian Wang,†,‡ Weidong Han,†,‡ Hongming Pan,*,†,‡ and Mang Xiao*,§ †

Department of Medical Oncology, Sir Run Run Shaw Hospital, Zhejiang University, 310027 Hangzhou, China Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, 310027 Hangzhou, China § Department of Otolaryngology Head and Neck Surgery, Sir Run Run Shaw Hospital, Zhejiang University, 310027 Hangzhou, China ‡

ABSTRACT: Metformin, a biguanide derivative that is widely used for treating type 2 diabetes mellitus, has recently been shown to exert potential anticancer effects. Many retrospective data and laboratory studies suggest the idea that metformin has antineoplastic activity, but some other studies reach conflicting conclusions. Although the precise molecular mechanisms by which metformin affects various cancers have not been fully elucidated, activation of AMPKdependent and AMPK-independent pathways along with energy metabolism aberration, cell cycle arrest and apoptosis or autophagy induction have emerged as crucial regulators in this process. In this Review, we describe the role of metformin in the prevention and treatment of a variety of cancers and summarize the molecular mechanisms that are currently well documented in the ability of metformin as an anticancer agent. In addition, the scientific and clinical hurdles regarding the potential role of metformin in cancer will be discussed. KEYWORDS: metformin, cancer, prevention, therapy, AMPK

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INTRODUCTION Metformin, a biguanide derivative, is currently the first-line drug for the treatment of type 2 diabetes (T2D) due to its ability to inhibit hepatic gluconeogenesis and trigger glucose uptake in skeletal muscle.1 Different from other biguanides, metformin is a relatively safe and well-tolerated drug, with acknowledged pharmacokinetics and manageable toxicities. Besides glucose-lowering effect, there is increasing interest in its anticancer potential. A huge amount of epidemiologic evidence shows that metformin exposure may reduce cancer incidence and improve cancer patients’ prognosis.2,3 Accumulating preclinical and clinical studies also demonstrate that metformin may not only exert anticancer properties in a spectrum of established malignancies but also have effects in preventing tumor initiation.4−6 The mechanisms involved in the antineoplastic effects of metformin are mainly divided into two categories: “indirect effect” resulting from systemic changes in glucose or insulin levels and “direct effect” on tumor cells (Figure 1).7,8 The direct anticancer effects of metformin are mainly explained by activation of adenosine monophosphate-activated protein kinase (AMPK) and a reduction in mammalian target of rapamycin (mTOR) signaling, which inhibits protein synthesis and gluconeogenesis. However, metformin may also exert antineoplastic properties in an AMPK-independent manner.9 Currently, there is a strong need to understand the underlying molecular mechanisms on the role of metformin in cancer prevention and therapy. Here, we review the anticancer activity and molecular mechanisms of metformin and summarize the epidemiological, preclinical, and clinical © XXXX American Chemical Society

evidence supporting metformin as an anticancer agent. In addition, genetic determinants of metformin response and the clinical challenges will be discussed.

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ANTINEOPLASTIC ACTIONS OF METFORMIN Epidemiology. The epidemiological evidence plays a crucial role in generating the hypothesis that metformin has potential in cancer prevention and treatment. Evans et al. was the first to report that metformin use in patients with type 2 diabetes may reduce their risk of cancer.10 Since then, a large amount of epidemiological studies investigated the association between metformin exposure and a beneficial effect on cancer prevention or treatment.11,12 Thakkar et al. demonstrated that metformin, but not other antidiabetic drugs, reduced cancer risk in subjects with type 2 diabetes.13 Moreover, the magnitude of cancer risk reduction and prolonged cancer patients’ survival was demonstrated to be dependent on the dose of metformin. Significantly greater dose-dependent anticancer effects were observed in cancer patients with diabetes.14 A recent metaanalysis showed a 31% reduction in overall cancer incidence and a 34% decline in cancer mortality when metformin was used in the treatment of cancer patients with diabetes; however, the reduction seemed to be of modest magnitude and not affecting all populations equally.15 Received: July 22, 2015 Revised: September 3, 2015 Accepted: September 27, 2015

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Figure 1. Antineoplastic effects of metformin. The anticancer activity of metformin is associated with indirect and direct effects of this drug. The indirect effects of metformin result from systemic changes of blood glucose and insulin levels, which could affect cancer cells’ survival. The direct anticancer effects of metformin are mainly mediated by AMPK-dependent and AMPK-independent mechanisms.

Figure 2. Molecular mechanisms of metformin on tumors. Metformin can suppress tumor progression by modulating indirectly IGF signaling, which has a stimulatory effect on tumor cell growth. Alternatively, metformin can also act on cancer cells directly, inhibiting tumorigenesis and cancer progression by modulating PI3K-Akt-mTOR or Ras-MAPK pathway-triggered apoptosis/autophagy, inducing cell cycle arrest, suppressing EMT, activating T cells, and inhibiting SASP activation.

Although accumulating evidence suggests that metformin exposure is associated with the decreased incidence and mortality, some recent epidemiologic studies exhibit conflicting conclusions, and the hypothesis remains controversial. In the previous research, we investigated the effect of metformin on colorectal cancer (CRC) cells. As a result, metformin did not inhibit cell proliferation or induce apoptosis for CRC cell lines in vitro and in vivo.16 In agreement with our results, Thompson et al. reported that metformin did not prevent mammary carcinogenesis in nondiabetic rat and mouse models.17 In addition, Bodmer and colleagues found that metformin did not alter the risk of lung cancer and metformin was also not associated with a decreased risk of colorectal cancer.18,19 These observational studies regarding the effects of metformin should be considered in the ongoing clinical trials where metformin are used as an antineoplastic agent. Preclinical Evidence. Indirect Effects of Metformin on Cancer. As mentioned above, metformin may exhibit indirect effects on cancer cells by reducing circulating glucose and insulin levels. The blood glucose and liver glycogen levels was shown to be associated with benzo(a)pyrene-induced experimental lung tumorigenesis.20 Obesity and insulin resistance was directly associated with liver and colon tumorigenesis in the mice model.21,22 High insulin levels could promote tumorigenesis via activation of insulin-like growth factors (IGF) (Figure 2). The circulating concentrations of IGF-I is reported to be positively associated with cancer risk and contribute to tumorigenesis of breast cancer.23 IGF-II is mainly produced by the liver and has a stimulatory effect on tumor cell growth.24 IGF-binding protein-5 (IGFBP-5) functions as a tumor suppressor to suppress the tumorigenesis of head and neck squamous cell carcinoma.25 Taken together, these studies provide credence that the reduction in glucose or insulin levels may lead to the less aggressive behavior of cancer cells. Thus, metformin has indirect antineoplastic potential by reducing hyperglycemia and hyperinsulinemia. Moreover, this indirect effect does not require any accumulation of the drug in

neoplastic tissue. However, it is not clear whether metformininduced changes in glucose or insulin levels are sufficient to impede carcinogenesis. Direct Effects of Metformin on Cancer. The direct anticancer effects of metformin are mainly mediated by AMPK-dependent and AMPK-independent mechanisms, together with apoptosis, autophagy, cell cycle arrest, energy metabolism aberration, epithelial-to-mesenchymal transition (EMT) suppression, senescence, immune activation, cancer stem cells inactivation, and other biological output (Figure 2). Apoptosis. Increasing evidence witnesses that metformin can inhibit cell proliferation and induce apoptosis in a variety of human cancer types. The anticancer action of metformin is mainly explained by activation of AMPK signaling pathway. Major downstream targets of AMPK include TSC2 and Raptor.26,27 AMPK may directly phosphorylate these targets to inhibit mTOR complex 1 (mTORC1) activity, which is hyperactivated in many types of cancer and can promote cell growth and proliferation.27,28 Metformin treatment selectively induced apoptosis in gastric cancer cells through AMPK/ mTOR-mediated decrease of the antiapoptotic survivin protein.29 Metformin also triggered apoptosis through AMPK-dependent inhibition of unfolded protein response (UPR) signaling in acute lymphoblastic leukemia (ALL) lymphoblasts.30 In human hepatocellular carcinoma (HCC), metformin could induce apoptosis and sensitize HCC cells toward chemotherapy via AMPK activation.31 In addition, metformin-dependent inhibition of mTOR signaling partly contributed to the apoptosis in pancreatic cancer.32 Nonetheless, despite activating AMPK, metformin also actually trigger apoptosis independently of AMPK. Metformin activated p53, Bax, and induced apoptosis in breast cancer cells through the ERK signaling pathway.33 The inhibitory effect of B

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overcome cisplatin chemoresistance by suppressing STAT3 activity independently of the liver kinase B1 (LKB1)−AMPK pathway.55 The pancreatic cancer cell line KLM1 can gain chemoresistance following gemcitabine treatment, but metformin may reverse this resistance and enhance the cytotoxicity of gemcitabine.56 Vujic et al. investigated the effect of metformin on NRAS mutant melanoma cell lines with acquired resistance to trametinib. As a result, metformin could abolish the development of resistance and decreased cell viability.57 In addition, metformin can reverse multidrug resistance in human hepatocellular carcinoma Bel-7402/5-fluorouracil cells.58 These data indicate that metformin may potentiate the resensitization of therapeutic-resistant cancer cells to the anticancer drugs. Cell Cycle Arrest. The cell cycle governs the transition from quiescence (G0) through cell growth to proliferation and plays a crucial role in the majority of cancers.59,60 Metformin inhibited the growth of HCC in vitro and in vivo, possibly by inducing G0/G1 cell cycle arrest.61,62 In breast cancer cells, metformin was reported to induce cell cycle arrest in G0/G1 phase which was mediated by oxidative stress as well as AMPK activation.63 Independent of AMPK, metformin induced cell cycle arrest through activating REDD1, a negative regulator of mTOR.64 Metformin also exhibited anticancer effects via G1 and G2/M cell cycle arrest in endometrial and prostate cancer cells.46,65 These studies suggest that metformin may contribute to the suppression of tumor growth by inducing cell cycle arrest in several types of cancers. Energy Metabolism Aberration. Induction of energy metabolism aberration is another potential antineoplastic mechanism of metformin. In the context of available acetylCoA and cholesterol, metformin could limit fatty acid synthesis in pancreatic tumor cells with mutated K-ras.66 Metformin treatment at least partially impaired glucose metabolism and tumor growth in triple negative breast cancer (TNBC) via directly inhibits the enzymatic function of hexokinase (HK).67 In prostate cancer cells, metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism which contributed to its antiproliferative effect.68 Intriguingly, the anticancer effect of metformin is recently found to be highly dependent on glucose concentration. Under physiological concentrations of glucose, metformin inhibited mTORC1 activation, DNA synthesis, and proliferation of pancreatic ductal adenocarcinoma (PDAC) cells.69 However, metformin did not inhibit the growth of TNBC cells cultured in hyperglycemic conditions.70 In the further study, metformin supplementation mostly caused cell cycle arrest without signs of apoptotic cell death under standard high-glucose conditions. Nevertheless, in response to glucose withdrawal stress, metformin exposed synthetically lethal activity in cancer cells through an increased dependency on Warburg like aerobic glycolysis.71 Taken together, these studies have raised the mechanism that metformin displays additional inhibitory effects on energy metabolism. Epithelial-to-Mesenchymal Transition (EMT) Suppression. Epithelial-mesenchymal transition (EMT) is a process that allows epithelial cells to acquire the highly invasive and metastatic properties of mesenchymal cells and, thus, has been demonstrated to play an important role in promoting cancer progression and metastasis.72,73 Metformin can weaken the ability of TGFβ signaling to fully induce EMT or reverse EMT phenotype in breast cancer cells due to activating AMPK signal pathway.74,75 Metformin also inhibited IL-6-induced EMT in lung adenocarcinoma by blocking STAT3 phosphorylation.76

metformin in prostate cancer was reported to be dependent on disruption of the midline-1 translational regulator complex and androgen receptor downregulation but not AMPK activation.34,35 AKT phosphorylation also was demonstrated to contribute to the apoptotic response to metformin in human leukemic cells.36 In addition, pro-apoptosis activities of metformin are partly due to downregulation of specificity protein (Sp) transcription factors and Sp-regulated genes.37 Autophagy. Autophagy, an important homeostatic cellular recycling mechanism, is now emerging as a crucial regulator in cancer development and response to therapy.38,39 Current evidence supports that autophagy can have a pro-survival or pro-death role in response to metabolic and therapeutic stresses, which contributes to the anticancer efficacy of these drugs as well as drug resistance.26 On the one hand, metformin may induce pro-survival autophagy. Systemic treatment with metformin activated pro-survival autophagy in p53(+/+) cells but not p53(−/−) cells, indicating that metformin was selectively toxic to p53-deficient cells.40 Metformin treatment induced interdependent activation between autophagy and PERK/eIF2a pathway, which protected ovarian cancer cells against metformin-induced apoptosis.41 Metformin also triggered autophagy in esophageal squamous cell carcinoma (ESCC) by signal transducer and activator of transcription 3 (STAT3)-Bcl-2 pathway and pharmacological or genetic suppression of autophagy sensitized ESCC cells to metformin-induced apoptotic cell death.42 On the other hand, metformin can activate pro-death autophagy. Metformin potentially blocked lymphoma and melanoma cell growth via selectively induction of autophagy through AMPK activation.43,44 Metformin inhibited the proliferation of the retinoblastoma cells in vitro, which was associated with activation of autophagy.45 Metformin was also proved to suppress endometrial cancer cell survival via the activation of autophagy and apoptosis.46 In addition, metformin could induce both autophagy and apoptosis in cervical cancer cells when LKB1 is expressed, resulting in tumor growth inhibition.47 Recently, pharmacologic screens revealed that metformin suppressed GRP78-dependent autophagy to enhance the antimyeloma effect of bortezomib.48 These findings indicate that the antiproliferative effects of metformin are partially or completely dependent on autophagy. Potentiating Anticancer Drug Efficacy. Metformin can sensitize cancer cells to the effects to conventional chemotherapeutic agents and other therapeutic strategies. Metformin synergistically enhanced antitumor activity of histone deacetylase inhibitor trichostatin A in osteosarcoma cell line.49 Metformin treatment could sensitize human bladder cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through mTOR/S6K1-mediated downregulation of c-FLIP.50 Moreover, metformin was also demonstrated to enhance gefitinib chemosensitivity in human lung adenocarcinoma cell line.51 Metformin also may reverse anticancer drug resistance. Currently, the EGF receptor tyrosine kinase inhibitors (EGFRTKI) have become a standard therapy for advanced non-smallcell lung cancer (NSCLC) patients with EGFR mutation.52,53 However, acquired resistance to EGFR-TKIs limits their clinical efficacy. Fortunately, metformin in combination with TKIs may reverse TKI resistance in the patients with NSCLC harboring EGFR mutations.54 Cisplatin is another drug for treating advanced NSCLC; however, the activation of STAT3-mediated drug resistance restricts its anticancer efficacy. Metformin can C

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Molecular Pharmaceutics Table 1. Active Clinical Trials with Metformin in Cancer Therapy identifier

cancer type

NCT02285855 NCT 01529593

NSCLC advanced cancers

metformin metformin + temsirolimus

II I

NCT 01666730 NCT 01310231

pancreatic cancer breast cancer

metformin + modified FOLFOX 6 metformin + chemotherapy

II II

NCT 01980823

breast cancer

metformin + atorvastatin

0

NCT 02028221

breast cancer

metformin

II

NCT 02176161 NCT 02065687

metformin metformin + paclitaxel and carboplatin metformin + platinum

I/II II III

NCT 02019979

prostate cancer endometrial cancer NS-NSCLC

NCT 01793948

breast cancer

metformin

0

NCT 01620593

metformin + castration

II

metformin

II

metformin

III

NCT 01528046 NCT 02278965

advanced prostate cancer neuroendocrine tumors endometrial cancer solid tumors breast cancer

metformin metformin + omega-3 fatty acids

I 0

NCT 01796028

prostate cancer

metformin + docetaxel

II

NCT 02005419

pancreatic cancer

metformin + gemcitabine

II

NCT 01650506

metformin

I

metformin

0

Pilot Study of Metformin in Head and Neck Squamous Cell Cancer and Its Effects on Stromal-Epithelial Metabolic Uncoupling

metformin

I

Dose-Finding Study of Metformin with Chemoradiation in Locally Advanced Head and Neck Squamous Cell Carcinoma

NCT01717482 NCT 01997775 NCT 01926769

triple negative breast cancer head and neck squamous cell cancer head and neck squamous cell cancer NSCLC lung cancer colorectal cancer

metformin metformin metformin + chemotherapy

II II II

NCT 02048384

pancreatic cancer

metformin + rapamycin

I/II

NCT 01864681 NCT 02143050

NSCLC melanoma

II I/II

NCT 01638676 NCT 01750567

melanoma CLL

metformin + gefitinib metformin + dabrafenib and trametinib metformin + vemurafenib metformin

Metformin as a Chemoprevention Agent in Nonsmall Cell Lung Cancer Metformin in Stage IV Lung Adenocarcinoma A Phase II Study to Determine the Safety and Efficacy of Second-Line Treatment with Metformin and Chemotherapy (FOLFOX6 or FOFIRI) in the Second Line Threatment of Advanced Colorectal Cancer A Study of Metformin With or Without Rapamycin as Maintenance Therapy After Induction Chemotherapy in Subjects With Pancreatic Cancer Combination of Metformin With Gefitinib to Treat NSCLC Study of Dabrafenib, Trametinib and Metformin for Melanoma Patients

NCT 01725490

familial adenomatous polyposis AML

metformin

II

metformin + cytarabine

I

NCT 02279758 NCT 01697566

NCT 02083692 NCT 02325401

NCT 01849276

drugs

phase

II

title Metformin in NSCLC Temsirolimus in Combination With Metformin in Patients With Advanced Cancers Metformin Plus Modified FOLFOX 6 in Metastatic Pancreatic Cancer A Trial of Standard Chemotherapy With Metformin (vs Placebo) in Women With Metastatic Breast Cancer Pre-Surgical Trial of the Combination of Metformin and Atorvastatin in Newly Diagnosed Operable Breast Cancer Phase II Study of Metformin for Reduction of Obesity-Associated Breast Cancer Risk Metformin Prostate Cancer Adjuvant Trial Paclitaxel and Carboplatin With or Without Metformin Hydrochloride in Treating Patients With Stage III, IV, or Recurrent Endometrial Cancer Metformin and Carbohydrate Restriction With Platinum Based Chemotherapy In Stage IIIB/IV Non-Squamous NSCLC (NS-NSCLC) Metformin Hydrochloride vs Placebo in Overweight or Obese Patients at Elevated Risk for Breast Cancer Castration Compared to Castration Plus Metformin as First Line Treatment for Patients with Advanced Prostate Cancer A Pilot Study of Metformin Treatment in Patients with Well-Differentiated Neuroendocrine Tumors An Endometrial Cancer Chemoprevention Study of Metformin Metformin in Children With Relapsed or Refractory Solid Tumors Metformin and Omega-3 Fatty Acids in Woman With a History of Early Stage Breast Cancer Metformin-Docetaxel Association in Metastatic Hormone-Refractory Prostate Cancer Metformin Combined with Gemcitabine as Adjuvant Therapy for Pancreatic Cancer after Curative Resection Study of Erlotinib and Metformin in Triple Negative Breast Cancer

I/II II

A Phase I/II Trial of Vemurafenib and Metformin to Melanoma Patients A Pilot Study of Metformin Therapy in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL) and Untreated CLL The Chemopreventive Effect of Metformin in Patients with Familial Adenomatous Polyposis: Double Blinded Randomized Controlled Study Metformin + Cytarabine for the Treatment of Relapsed/Refractory AML

(SASP) in tissues where senescent cells were not normally cleared.79 Metformin also inhibited SASP by blocking with IKK/NF-κB activation, which suppressed the growth of prostate cancer cells.80 In TNBC cells, metformin induced a senescence-associated gene signature which contributed to metformin’s antiproliferative effect.81 Low concentration of metformin was reported to induce a p53-dependent senescence in HCC cells via activation of the AMPK pathway.82 Nonetheless, metformin also protected endothelial cells from hyperglycemia-induced senescence in mouse microvascular

In addition, metformin was proved to suppress epithelialmesenchymal transition in prostate cancer cells, which may involve upregulation of miR30a and downregulation of SOX4.77 These findings provide an attractive therapeutic strategy for inhibiting cancer metastasis and abrogating chemoresistance. Senescence, Cancer Stem Cells, and Cell Immune. Although less studied than apoptosis, cell senescence has gained increasing attention in tumorigenesis and tumor suppression.78 Metformin can prevent cancer by modulating the activity of the senescence associated secretory phenotype D

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Molecular Pharmaceutics endothelial cells.83 So, the role of metformin in cell senescence remains unresolved. In addition, metformin can target cancer stem cells. Unlike most cancer cells within a tumor, cancer stem cells (CSCs) often resist chemotherapeutic drugs and are key drivers of tumorigenesis and potential recurrence of cancer. Low doses of metformin could selectively kill cancer stem cells and acted together with doxorubicin to reduce tumor mass and prolong remission.84 Coencapsulation of epirubicin and metformin in PEGylated liposomes contributed to the arrest of CD133+ cancer stem-like cells in the G0/G1 phase and inhibited the recurrence of murine sarcoma.85 Metformin in combination with salinomycin exerted a modest growth inhibitory effect on CSCs in NSCLC cell lines, moreover, this effect is irrespective of their EGFR, KRAS, EML4/ALK, and LKB1 status.86 Metformin also exhibited anticancer action against ovarian cancer stem cells through the modulation of energy metabolism.87 Taken together, metformin might exert pivotal roles in the attenuation of CSCs phenotypes and functions. Recently, immune-mediated mechanism was also involved in the antitumor effect of metformin. Oncogene-driven major histocompatibility complex class I (MHC-I) deficiency restricted immunosurveillance and facilitated tumorigenesis and cancer cell survival in most types of human cancers. Metformin could rescue MHC-I deficiency, which represented a previously unrecognized mechanism underlying the cancerpreventive effects of metformin.88 Metformin protected CD8+ tumor-infiltrating lymphocytes (TILs) from apoptosis and the inevitable functional exhaustion in the tumor microenvironment, which eventually resulted in tumor growth inhibition.89 Metformin was also proved to have immunomodulatory actions through the inhibition of Th17 cell differentiation and the upregulation of regulatory T cell differentiation.90 Moreover, metformin may increase CD127+ 132− and CD127+ 132+ cell ̈ T cells and CD127+ 132− population in populations in naive + CD4 T lymphocytes, which might be helpful for the normal functioning of T-cell-dependent immunosurveillance.91 Importantly, metformin treatment could regulate T-cell responses to immune activation, and this effect was mediated by downregulated STAT3 and STAT4 phosphorylation via the AMPKmTORC 1 pathway.92 However, the effects of metformin on T cell immune responses were also shown to have no requirement for expression of AMPK in T cells.93 Therefore, the immune regulatory effects exhibit different anticancer mechanism of metformin. Genetic Determinants of Metformin Response. Although a number of studies have reported that metformin exhibits both chemopreventive and chemotherapeutic actions that may be used to treat specific types of cancer, marked interindividual variability in response attenuates its optimal use. This may be associated with the various genetic backgrounds of the different tumor types. Thus, it will be important to understand why some cancer cells are resistant to the effects of metformin, what are the genetic determinants of metformin response and subsequently identify predictable biomarkers of response to the treatment. LKB1, the major upstream kinase of AMPK, was recently shown that its inactivation confered therapeutic response of cancer cells to the metabolism drug metformin or phenformin.94,95 p53 could be regulated by activation of AMPK. Mutation of p53 might be the important genetic determinant of metformin response. Systemic treatment with metformin selectively impaired p53-deficient tumor cell growth.40 The head and neck squamous cell carcinoma

(HNSCC) cells with mutant TP53 are more sensitive to metformin treatment, compared to wild-type TP53-expressing cells.96 Metformin has antineoplastic potential in preclinical models of endometrial cancer, lung cancer, and pancreatic cancer; however, the greatest response was observed in cells harboring K-Ras mutation.97,98 So, K-Ras status may determine the response of these cancer types to metformin treatment. It is also demonstrated that dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin.99 Taken together, these targets are important for design of future clinical trials evaluating the anticancer potential of metformin. Clinical Studies. As mentioned above, metformin exhibits both indirect and direct actions activity for multiple cancers. Dozens of epidemiological and preclinical studies raise enthusiasm for clinical trials of metformin.100 Currently, more than 30 phase I/II/III cancer clinical trials (http://clinicaltrials. gov/) involving metformin are active around the world (Table ). This research mainly comprises two broad areas. One examines whether metformin has utility for cancer prevention or treatment and safety and dose-finding of the drug. A few phase I clinical trials (NCT02285855, NCT 02176161, NCT 01650506, and NCT 02048384) are investigating the effect and dose of metformin on various cancers, including NSCLC, prostate cancer, breast cancer, and pancreatic cancer. Some small clinical trials have been reported. Metformin suppressed colorectal aberrant crypt foci in a short-term clinical trial, suggesting its promise for the chemoprevention of colorectal cancer.101 Neoadjuvant metformin treatment reduced the cell proliferation of prostate cancer patients.102 The other field assesses the efficacy of combination metformin with conventional chemotherapeutics. However, the information about the anticancer activity of this combination is underwhelming to date. In addition, several neoadjuvant studies are ongoing and planned in the different cancer types. Therefore, few clinical results have been shown, although many clinical trials are ongoing.

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FUTURE DIRECTIONS Currently, the role of metformin in cancer is drawing more and more attentions because of its minimal side effects and low cost. However, to maximize the anticancer potential to be applied for more stringent clinical application, many issues need to be further investigated in the future. First, most of these studies are based on retrospective reviews and exclusively involved diabetic patients, so the question is whether the anticancer effect of metformin may be applicable to nondiabetic subjects. If so, what is the appropriate doses and safety of metformin in nondiabetic individuals? This will be a crucial issue in the future. Second, metformin levels in humans are in micromolar levels, but it is not clear whether the normal concentration of metformin in human is sufficient to impede tumor biology. So, a crucial knowledge gap that needs to be addressed is adequate drug concentration of metformin in neoplastic tissue. Third, the antineoplastic actions of metformin are associated with both direct and indirect effects of the drug, which increases the complexity of clinical evaluation of metformin. Differentiation of the indirect or direct effects is critical for optimal benefit of this agent. Thus, the potential clinical markers of metformin benefit need to be identified in the future, which is important for appropriate selection of patients who might benefit from metformin. E

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(5) Pernicova, I.; Korbonits, M. Metformin–mode of action and clinical implications for diabetes and cancer. Nat. Rev. Endocrinol. 2014, 10 (3), 143−156. (6) Greenhill, C. Gastric cancer: Metformin improves survival and recurrence rate in patients with diabetes and gastric cancer. Nat. Rev. Gastroenterol. Hepatol. 2015, 12 (3), 124. (7) Pollak, M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nat. Rev. Cancer 2012, 12 (3), 159− 169. (8) Martin-Castillo, B.; Vazquez-Martin, A.; Oliveras-Ferraros, C.; Menendez, J. A. Metformin and cancer: doses, mechanisms and the dandelion and hormetic phenomena. Cell Cycle 2010, 9 (6), 1057− 1064. (9) Luengo, A.; Sullivan, L. B.; Heiden, M. G. Understanding the complex-I-ty of metformin action: limiting mitochondrial respiration to improve cancer therapy. BMC Biol. 2014, 12, 82. (10) Evans, J. M.; Donnelly, L. A.; Emslie-Smith, A. M.; Alessi, D. R.; Morris, A. D. Metformin and reduced risk of cancer in diabetic patients. BMJ 2005, 330 (7503), 1304−1305. (11) Morales, D. R.; Morris, A. D. Metformin in cancer treatment and prevention. Annu. Rev. Med. 2015, 66, 17−29. (12) Tseng, C. H. Metformin significantly reduces incident prostate cancer risk in Taiwanese men with type 2 diabetes mellitus. Eur. J. Cancer 2014, 50 (16), 2831−2837. (13) Thakkar, B.; Aronis, K. N.; Vamvini, M. T.; Shields, K.; Mantzoros, C. S. Metformin and sulfonylureas in relation to cancer risk in type II diabetes patients: a meta-analysis using primary data of published studies. Metab., Clin. Exp. 2013, 62 (7), 922−934. (14) Lin, H. C.; Kachingwe, B. H.; Lin, H. L.; Cheng, H. W.; Uang, Y. S.; Wang, L. H. Effects of metformin dose on cancer risk reduction in patients with type 2 diabetes mellitus: a 6-year follow-up study. Pharmacotherapy 2014, 34 (1), 36−45. (15) Gandini, S.; Puntoni, M.; Heckman-Stoddard, B. M.; Dunn, B. K.; Ford, L.; DeCensi, A.; Szabo, E. Metformin and cancer risk and mortality: a systematic review and meta-analysis taking into account biases and confounders. Cancer Prev. Res. 2014, 7 (9), 867−885. (16) Sui, X.; Xu, Y.; Yang, J.; Fang, Y.; Lou, H.; Han, W.; Zhang, M.; Chen, W.; Wang, K.; Li, D.; Jin, W.; Lou, F.; Zheng, Y.; Hu, H.; Gong, L.; Zhou, X.; Pan, Q.; Pan, H.; Wang, X.; He, C. Use of metformin alone is not associated with survival outcomes of colorectal cancer cell but AMPK activator AICAR sensitizes anticancer effect of 5fluorouracil through AMPK activation. PLoS One 2014, 9 (5), e97781. (17) Thompson, M. D.; Grubbs, C. J.; Bode, A. M.; Reid, J. M.; McGovern, R.; Bernard, P. S.; Stijleman, I. J.; Green, J. E.; Bennett, C.; Juliana, M. M.; Moeinpour, F.; Steele, V. E.; Lubet, R. A. Lack of effect of metformin on mammary carcinogenesis in nondiabetic rat and mouse models. Cancer Prev. Res. 2015, 8 (3), 231−239. (18) Bodmer, M.; Becker, C.; Jick, S. S.; Meier, C. R. Metformin does not alter the risk of lung cancer: a case-control analysis. Lung cancer 2012, 78 (2), 133−137. (19) Bodmer, M.; Becker, C.; Meier, C.; Jick, S. S.; Meier, C. R. Use of metformin is not associated with a decreased risk of colorectal cancer: a case-control analysis. Cancer Epidemiol., Biomarkers Prev. 2012, 21 (2), 280−286. (20) Anandakumar, P.; Kamaraj, S.; Jagan, S.; Ramakrishnan, G.; Devaki, T. Effect of capsaicin on glucose metabolism studied in experimental lung carcinogenesis. Nat. Prod. Res. 2009, 23 (8), 763− 774. (21) Nakamura, A.; Tajima, K.; Zolzaya, K.; Sato, K.; Inoue, R.; Yoneda, M.; Fujita, K.; Nozaki, Y.; Kubota, K. C.; Haga, H.; Kubota, N.; Nagashima, Y.; Nakajima, A.; Maeda, S.; Kadowaki, T.; Terauchi, Y. Protection from non-alcoholic steatohepatitis and liver tumourigenesis in high fat-fed insulin receptor substrate-1-knockout mice despite insulin resistance. Diabetologia 2012, 55 (12), 3382−3391. (22) Algire, C.; Amrein, L.; Zakikhani, M.; Panasci, L.; Pollak, M. Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase. Endocr.-Relat. Cancer 2010, 17 (2), 351−360.

Fourth, antineoplastic activity of metformin as a single agent is limited. To optimize the anticancer potential of metformin, nanoparticles will hopefully provide a promising therapeutic approach. O-Carboxymethyl chitosan encapsulated metformin nanoparticles were shown to have significant anticancer potential.103 The combination of metformin and boswellic acid nanoparticles decreased colony formation and exhibited high rate of induction of apoptosis in pancreatic cancer cells.104

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CONCLUSIONS Metformin is an extensively used and well-tolerated antidiabetic drug tha tis widely used for treating type 2 diabetes mellitus. During the past decade, increasing epidemiological, preclinical, and clinical reports have revealed that metformin has also an antineoplastic potential in vitro and in vivo. Although a large number of indirect and direct effects have been reported to contribute to the anticancer action of metformin,105 the precise mechanisms that underlie its antineoplastic effects have not been well elucidated.106 The role of metformin in cancer needs to be further investigated in the future. Our increased knowledge regarding the role of metformin in cancer will hopefully provide a promising therapeutic strategy to impede carcinogenesis, inhibit cancer cell survival, and circumvent resistance for cancer patients.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-0571-86436673. E-mail: [email protected]. (H.P.) *Tel.: +86-0571-86606239. E-mail: [email protected]. (M.X.) *Tel.: +86-0571-86006926. E-mail: [email protected]. (X.S.) Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is supported by grants from National Natural Science Foundation of China (grant no. 81301891), Zhejiang Provincial Natural Science Foundation of China (grant no. LQ13H160008, LY13H130002, and LY15H160028), Zhejiang province science and technology project of TCM (grant no. 2015ZB033) and Zhengshu Medical Elite Scholarship Fund.

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