REVIEW Metformin in cancer: translational challenges

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Metformin in cancer: translational challenges
Ryan J O Dowling1,2, Saroj Niraula2, Vuk Stambolic1,2 and Pamela J Goodwin3
    Division of Signalling Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9
    Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
    Division of Clinical Epidemiology, Department of Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
     University of Toronto, 1284-600 University Avenue, Toronto, Ontario, Canada M5G 1X5

(Correspondence should be addressed to P J Goodwin; Email:

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin, effective in treating
type 2 diabetes and the insulin resistance syndromes, improves insulin resistance by reducing hepatic gluconeogenesis
and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin
use with decreased cancer incidence and cancer-related mortality. Furthermore, numerous preclinical and clinical
studies have demonstrated anti-cancer effects of metformin, leading to an explosion of interest in evaluating this agent
in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers
associated with hyperinsulinaemia; however, metformin may also directly inhibit tumour growth. In this review, we
describe the mechanism of action of metformin and summarise the epidemiological, clinical and preclinical evidence
supporting a role for metformin in the treatment of cancer. In addition, the challenges associated with translating
preclinical results into therapeutic benefit in the clinical setting will be discussed.
Journal of Molecular Endocrinology (2012) 48, R31–R43

Introduction                                                                                  bypassed the traditional Phase I pharmacokinetic/
                                                                                              toxicity assessment and has moved directly into Phase II
Emerging evidence from several areas of research                                              and Phase III testing, reflecting rapid accumulation of
suggests that metformin, a commonly used anti-diabetic                                        preclinical, clinical and epidemiological evidence that
drug, may be useful in the prevention and treatment of                                        it may have beneficial anti-cancer effects, despite the
cancer. Metformin, a biguanide derivative, has been                                           fact that precise mechanisms of potential anti-cancer
used for half a century to treat non-insulin-requiring                                        action remain unresolved (Goodwin et al. 2011).
diabetes. It is a relatively safe drug, with known                                               Current understanding of potential anti-cancer
pharmacokinetics and manageable toxicities. Its most                                          effects of metformin raises the intriguing possibility
common toxicity is mild-to-moderate gastrointestinal                                          of a duality of action – metformin may act directly on
discomfort, usually self-limited and ameliorated by a                                         the tumour or it may act indirectly on the host by
graduated ramp up in dose. Its most serious toxicity,                                         lowering insulin levels. This, along with the fact that
lactic acidosis, is rare (Bodmer et al. 2008), occurring                                      the key host-related mediator (insulin) is a critical
once per 100 000 years of use. Lactic acidosis is more                                        component of culture medium used in in vitro experi-
common in elderly patients (over age 80 years) and in                                         ments, leads to enhanced challenges in translating
those with impaired renal, cardiac and/or hepatic                                             results of preclinical research into the clinical setting,
function. Although metformin has only recently been                                           underscoring the importance of cross-disciplinary
directly tested in conjunction with anti-cancer agents in                                     research approaches.
the clinical setting, its common use in the treatment of                                         Here, we summarise emerging evidence from research
diabetes has resulted in its co-administration with                                           on humans (epidemiological, clinical and metabolic)
virtually all anti-cancer agents in diabetic cancer                                           and from the preclinical setting (mechanistic, in vitro
patients. No evidence of important interactions of                                            and in vivo research) suggesting that metformin has anti-
metformin with standard cancer therapies has emerged                                          cancer effects (Fig. 1). We then integrate this informa-
through this experience. Because of this, clinical                                            tion to develop recommendations for translational
evaluation of metformin as an anti-cancer agent has                                           evaluation of metformin in the clinical setting.
Journal of Molecular Endocrinology (2012) 48, R31–R43                                                                                                    DOI: 10.1530/JME-12-0007
0952–5041/12/048–R31 q 2012 Society for Endocrinology           Printed in Great Britain                                    Online version via

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                                          Epidemiologic                                     of metformin (Memmott et al. 2010), coupled with
                                      associations in diabetes
                                                                                            emerging clinical evidence that metformin may be
                                                                                            associated with improved lung cancer outcomes
                                                                                            (reviewed below Mazzone et al. (2010) and Tan et al.
                                                                                            (2011)), suggest that other factors, such as nicotine,
       Associations of obesity and                                Evidence of metformin
        insulin with risk/prognosis                              activity in human cancer   may lead to insulin resistance in individuals with
                                            Metformin                                       smoking associated cancers or that non-insulin-mediated
                                                                                            mechanisms of action are important.
                                                                                               Although epidemiological studies have fairly consist-
                                                                       Preclinical          ently reported reduced cancer incidence and/or
                                                                   In vitro evidence of
           In vivo evidence of
           anti-cancer activity
                                                                   anti-cancer activity     mortality in diabetic patients who receive metformin
                                                                                            in standard clinical doses (1500–2250 mg/day in
                                                                                            adults), these studies have important methodological
                                                                                            limitations. Most were conducted retrospectively and
                                                                                            many sampled their cases from hospital or clinical
      Figure 1 Evidence supporting the use of metformin in the                              registries rather than population-based registries,
      treatment of cancer. The potential use of metformin in the
      prevention and treatment of cancer is based on emerging evidence
                                                                                            thereby limiting external validity and introducing
      from a number of research fields. Metabolic factors such as obesity                   potential selection biases. Inclusion criteria varied.
      and hyperinsulinaemia are associated with cancer risk while                           Some studies did not exclude individuals with prior
      epidemiological studies have demonstrated a reduced incidence                         diagnosis of cancer, thus introducing a possible reverse
      of cancer in diabetic patients receiving metformin. Moreover,                         causation bias. Other studies included both invasive
      preclinical studies have provided evidence of the anti-cancer
      effects of metformin and defined its mechanism of action.                             and non-invasive cancers in their analyses (Bodmer et al.
                                                                                            2010). Many studies included patients exposed to a
                                                                                            variety of treatments for diabetes complicating the
      Research on humans                                                                    analysis of metformin associations. Self-reporting of key
                                                                                            variables such as concomitant medication use and
      Epidemiologic                                                                         cancer risk factors such as obesity, tobacco use and
                                                                                            family history may have introduced exposure biases;
      Evans et al. (2005) were the first to report a potential                              imbalances in these factors, as well as others such as age
      association of metformin use with reduced cancer inci-                                and severity of diabetes, may have led to confounding of
      dence. In diabetics receiving metformin (as opposed                                   associations of metformin with cancer incidence or
      to other therapies), overall cancer incidence was                                     mortality. Nonetheless, it remains biologically plausible
      lowered (odds ratio (OR) 0.86 and 95% confidence                                      that metformin use may be causally associated with
      interval (95% CI) 0.73–1.02) and there was evidence of                                lower cancer incidence and mortality. However, caution
      a dose response in relation to total duration of                                      should be exercised in translating these observations to
      use or number of prescriptions dispensed. Since then,                                 non-diabetic patients because their physiological milieu
      at least 17 epidemiological studies examining the                                     and the cancers they develop may differ in important
      association of metformin treatment of diabetes with                                   ways from the situation in diabetic patients. Notably,
      cancer incidence and mortality have reported similar                                  insulin resistance (characterised by high levels of
      findings (see Table 1), and evidence has emerged that                                 circulating insulin and, in later stages, by high glucose
      metformin use may reverse the increased cancer risk                                   levels with or without sustained high insulin levels) may
      associated with administration of insulin or insulin                                  have been present for many years before the clinical
      secretagogues (Li et al. 2009). A recent meta-analysis                                diagnosis of diabetes (Kendall & Bergenstal 2001),
      (Decensi et al. 2010) has identified a 31% reduction in                               leading to the development of insulin- and/or glucose-
      overall cancer incidence or mortality when metformin                                  dependent cancers that are more sensitive to insulin-
      is used in the treatment of diabetes. There is                                        and/or glucose-lowering effects of metformin. Because
      emerging evidence that metformin use is associated                                    of this, it is possible that similar physiological and/or
      with reduced risk of several common cancers, including                                anti-cancer effects may not be present in non-diabetics.
      colorectal, pancreas, hepatocellular, breast and lung,
      although not all studies have identified significant
      effects. The reduced risk of obesity-associated cancers
      such as breast and colorectal is not surprising, given                                Retrospective clinical data suggest that metformin use in
      that metformin reduces insulin levels that appear to                                  diabetics may be associated with the development of
      be important mediators of associations of obesity with                                cancers that differ from those seen in the absence of
      cancer. Preclinical reports suggesting reduced tobacco–                               metformin. Specifically, in breast cancer, metformin
      carcinogen-induced lung cancer burden with the use                                    use has been associated with tumours that are more
      Journal of Molecular Endocrinology (2012) 48, R31–R43                                                                  

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Table 1 Epidemiological studies examining the association of metformin use by diabetics with cancer incidence and mortality

                                         Sample                                                  Results (HR/OR/RR
Study type                   Outcome     size   Comparison                       Site            and 95% CI)                  Reference

Case–control                 Incidence   11 876     Any vs no metformin          Multiple        OR 0.86 (0.73–1.02)          Evans et al. (2005)
Retrospective cohort         Mortality   10 309     Metformin                    Multiple        HR 1.0                       Bowker et al. (2006)
                                                    Insulin                                      HR 1.9 (1.5–2.4)
                                                    Sulphonylureas (SU)                          HR 1.3 (1.1–1.6)
Case–control                 Incidence   390        Any vs no metformin          Multiple        OR 0.28 (0.13–0.57)          Monami et al. (2009)
Retrospective cohort         Incidence   8170       Any vs no metformin          Bowel           HR 0.60 (0.38–0.94)          Libby et al. (2009)
                                                                                 Lung            HR 0.70 (0.43–1.15)
                                                                                 Breast          HR 0.60 (0.32–1.10)
                                                                                 Overall         HR 0.63 (0.49–0.81)
Retrospective cohort         Incidence   62 809     SU                           Breast          HR (relative to              Currie et al. (2009)
                                                                                                   metformin users)
                                                                                                 B 0.98 (0.69–1.41)
                                                                                 Colorectal      C 1.80 (1.29–2.53)
                                                                                 Pancreas        P 4.95 (2.74–8.96)
                                                                                 Prostate        R 1.07 (0.76–1.49)
                                                    MetforminCSU                                 B 0.90 (0.67–1.21)
                                                                                                 C 1.43 (1.05–1.94)
                                                                                                 P 0.38 (0.13–1.12)
                                                                                                 R 1.18 (0.89–1.57)
                                                    Insulin-based                                B 1.07 (0.79–1.44)
                                                                                                 C 1.69 (1.23–2.33)
                                                                                                 P 4.63 (2.64–8.10)
                                                                                                 R 1.10 (0.79–1.52)
Prospective cohort           Mortality   1353       Any vs no metformin          Multiple        HR 1.47 (1.22–1.76)          Landman et al. (2010)
Case–control                 Incidence   973        Insulin                      Pancreas        OR 5.04 (2.38–10.7)          Li et al. (2009)
                                                    Insulin secretagogues                        OR 1.74 (0.80–3.77)
                                                    Thiazolidinediones                           OR 1.65 (0.71–3.87)
                                                    Metformin                                    OR 0.41 (0.19–0.87)
Case–control                 Incidence   1001       Any vs no metformin          Prostate        OR 0.56 (0.32–1.00)          Wright & Stanford
Case–control                 Incidence   1573    Insulin or SU                   Liver           OR 2.99 (1.34–6.65)          Donadon et al. (2009)
                                                 Metformin                                       OR 0.33 (0.1–0.7)
Case–control                 Incidence   1524    Biguanides                      Liver           OR 0.3 (0.2–0.6)             Hassan et al. (2010)
                                                 Thiazolidinediones                              OR 0.3 (0.1–0.7)
                                                 SU                                              OR 7.1 (2.9–16.9)
                                                 Insulin                                         OR 1.9 (0.8–4.6)
Case–control                 Incidence   305     Any vs no metformin             Breast          OR 0.44 (0.24–0.82)          Bodmer et al. (2010)
Prospective cohort           Incidence   480 984 Any vs no metformin             Colorectal      HR 0.36 (0.13–0.98)          Lee et al. (2011)
                                                                                 Liver           HR 0.06 (0.02–0.16)
                                                                                 Pancreas        HR 0.15 (0.03–0.79)
                                                                                 Overall         HR 0.12 (0.08–0.19)
Retrospective cohort         Mortality   595        Any vs no metformin          Colorectal      HR 1.45 (1.08–1.93)          Lee et al. (2012)
Retrospective cohort         Mortality   3685       Metformin                    Multiple        HR 0.56 (0.34–0.94)          Bo et al. (2012)
                                                    Insulin                                      HR 1.56 (1.22–1.99)
Case–control                 Incidence   10 781     Metformin                    Ovarian         OR 0.61 (0.30–1.25)          Bodmer et al. (2011)
                                                    SU                                           OR 1.26 (0.65–2.44)
                                                    Insulin                                      OR 2.29 (1.13–4.65)
Nested case–control          Incidence   8098       Any vs no metformin          Prostate        RR 1.23 (0.99–1.52)          Azoulay et al. (2011)
Case–control                 Incidence   4323       Any vs no metformin          Breast          OR 0.81 (0.63–0.96)          Bosco et al. (2011)
Case–control                 Incidence   1340       Metformin                    Multiple        OR 0.46 (0.25–0.85)          Monami et al. (2011)
                                                    SU                                           OR 0.75 (0.39–1.45)
B, Breast Cancer; C, Colorectal Cancer; P, Pancreatic Cancer; R, Prostate Cancer; HR, Hazard Ratio; RR, Risk Ratio; OR, Odds Ratio.

frequently progesterone receptor (PgR) positive (Berstein                   cancer if they are receiving metformin. Jiralerspong
et al. 2010a) and less frequently triple negative (Meiers                   et al. (2009) studied pathological complete response
et al. 2010), whereas in lung cancer, metformin use has been                (pCR) rates in breast cancer patients receiving
associated with a higher frequency of adenocarcinomas                       neoadjuvant chemotherapy – pCR was significantly
(vs other histologies; Mazzone et al. 2010, Tan et al. 2011).               higher in diabetics receiving metformin than in
    Similar retrospective data suggest that cancer out-                     diabetics not receiving metformin (24 vs 8%), with
comes may be better in diabetics with breast or lung                        intermediate pCR in non-diabetics who were not                                                                      Journal of Molecular Endocrinology (2012) 48, R31–R43

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      exposed to metformin (16%). Although imbalances in            ongoing studies on the metastatic and adjuvant setting
      covariates such as age, obesity, insulin and taxane use       in breast, prostate, pancreatic and endometrial cancer
      were present, differences in pCR were significant in          (, will provide important additional
      adjusted analyses; however, metformin use was not             information on the anti-cancer effects of metformin
      significantly associated with survival. Likewise, metfor-     and help elucidate the clinically important mechanisms
      min use by patients with triple-negative breast cancer        of metformin action in diabetic and non-diabetic
      was not associated with beneficial effects on distant         cancer patients.
      metastasis-free survival, recurrence-free survival or over-
      all survival (Bayraktar et al. 2012). However, patients
      receiving metformin had a slightly lower risk of distant      Metabolic
      metastases (compared with diabetics not receiving             Obesity has been associated with increased overall
      metformin and non-diabetics). In a retrospective review       cancer risk and increased risk of most common solid
      of lung cancers in diabetics, Mazzone et al. (2010)           tumours and haematological malignancies (Calle et al.
      reported more frequent occurrence of metastatic               2003); lung cancer is a notable exception to this
      disease at diagnosis in those receiving insulin or            association. Although many factors have been postu-
      thiazolidinediones (which also lower insulin; 20 vs           lated to mediate effects of obesity on cancer, including
      42% in those not receiving these agents, PZ0.027) and         sex hormones such as oestrogen, adipocytokines such
      a reduced risk of death in those receiving metformin          as leptin and adiponectin, and inflammatory cytokines
      (hazard ratio (HR) 0.56, PZ0.056). Recent evidence            such as interleukins and tumour necrosis factor a
      also suggests that chemotherapy outcomes may be               (TNFa), recent research has focused on insulin (or
      improved by metformin in diabetic non-small cell lung         insulin resistance as part of the metabolic/insulin
      cancer patients (Tan et al. 2011). Because of the             resistance syndrome) as a potentially important
      retrospective design of these studies and the non-            mediator. In a number of meta-analyses (Everhart &
      random allocation of metformin (and thiazolidine-             Wright 1995, Larsson et al. 2005, 2006, 2007, Mitri et al.
      diones), these results should be considered as                2008, Larsson & Wolk 2011), type 2 diabetes (associated
      hypothesis generating. Nonetheless, they suggest that         with a period of hyperinsulinaemia) has been associ-
      clinically important beneficial effects may result from       ated with increased risk of common solid tumours
      metformin use in cancer patients.                             including those of the breast, colon/rectum, pancreas
         Prospective data regarding metformin use in non-           and kidney but not the prostate (Kasper & Giovannucci
      diabetic cancer patients are beginning to emerge. We          2006, Kasper et al. 2009). Furthermore, higher insulin
      have reported a 22% reduction in insulin levels when          or C-peptide (cleaved from proinsulin when insulin is
      metformin is given to non-diabetic breast cancer              released) levels have also been associated with risk of
      survivors in a standard clinical dose of 1500 mg/day          breast, colorectal and other cancers (Pisani 2008).
      (Goodwin et al. 2008). Hadad et al. (2011) have recently      These observations provide credence to an insulin-
      completed a preoperative, randomised, ‘window of              lowering mechanism of metformin action in cancer
      opportunity’ trial examining the effects of metformin         prevention.
      on non-diabetic women with operable invasive breast              In the setting of established cancer, higher levels of
      cancer. Metformin (1000 mg twice daily) was well              insulin or C-peptide have been independently associ-
      tolerated and caused a significant reduction in Ki-67         ated with poor outcomes (increased risk of recurrence
      staining in tumours and altered the expression of             and death) in breast and prostate cancers (Goodwin
      numerous genes including those involved in metab-             et al. 2002, Pasanisi et al. 2006, Ma et al. 2008, Emaus et al.
      olism, inflammation and AMPK (PRKAA) and mTOR                 2010, Duggan et al. 2011, Erickson et al. 2011, Irwin et al.
      signalling. Interim data from additional ongoing              2011, Pritchard et al. 2011). Our group has demon-
      neoadjuvant studies on breast cancer have provided            strated a more than doubled risk of distant recurrence
      evidence that metformin administration (in standard           and tripled risk of death in women with locoregional
      clinical dose) to newly diagnosed breast cancer patients      breast cancer whose insulin levels were in the highest
      is safe and has favourable effects on tumour cell             quartile, even though those levels were within the
      proliferation and apoptosis in the absence of other           normal range (Goodwin et al. 2002). In prostate cancer,
      anti-cancer treatment (Bonanni et al. 2010, Niraula et al.    this association may be limited to men with body mass
      2010). Furthermore, Hosono et al. (2010) have                 index (BMI) O25 kg/m2 (Ma et al. 2008).
      reported a randomised trial showing that a very low              The presence of insulin receptors (IRs) on many
      dose of metformin (250 mg daily) reduces proliferation        cancers provides a biological rationale for the effects
      and aberrant crypt foci formation in the rectal               of insulin on cancer risk and prognosis – recent under-
      epithelium of non-diabetic patients with previous             standing of the nature of these receptors and the
      colorectal polyps when compared with placebo.                 signalling pathways they activate when ligand binding
      Completion of these studies, as well as planned and           occurs is reviewed in the following section.
      Journal of Molecular Endocrinology (2012) 48, R31–R43                                           

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Preclinical evidence                                                                              AMPK (Kahn et al. 2005). The resulting increase in AMP
                                                                                                  activates AMPK by at least three separate mechanisms
Mechanism of action of metformin as an anti-cancer                                                (Long & Zierath 2006): i) allosteric activation,
agent                                                                                             ii) promotion of activation-specific phosphorylation of
                                                                                                  the a catalytic subunit on residue Thr172 by the upstream
The role of metformin in the inhibition of cancer is                                              kinase LKB1 (STK11) and iii) interference with de-
postulated to be associated with both direct and                                                  posphorylation of AMPK Thr172 by protein phosphatases.
indirect effects of the drug, as shown in Fig. 2. The                                             Upon activation, AMPK phosphorylates a number of
indirect effects are linked to the ability of metformin                                           effectors leading to the activation of ATP-generating
to inhibit the transcription of key gluconeogenesis                                               pathways, such as glycolysis and fatty acid oxidation,
genes in the liver and stimulate glucose uptake in                                                and the inhibition of ATP-consuming pathways, such
muscle, thus increasing insulin sensitivity and reducing                                          as fatty acid and protein synthesis (Kahn et al. 2005).
blood glucose and lowering insulin levels. The direct
effects of metformin are believed to be primarily
mediated by activation of AMPK, a serine/threonine                                                Direct (insulin-independent) effects of metformin
protein kinase involved in regulating cellular energy                                             Protein synthesis is one of the most energy-consuming
metabolism, leading to a reduction in mTOR signalling                                             processes in the cell and, as such, is a major target of
and protein synthesis in cancer cells. Metformin                                                  AMPK signalling under conditions of cellular energy
activates AMPK by inhibiting complex I of the mito-                                               stress. Upon activation by metformin, AMPK phosphor-
chondrial respiratory chain, which leads to impaired                                              ylates TSC2, stimulating its GTPase-activating protein
mitochondrial function and conditions that effectively                                            (GAP) activity towards the small GTPase RHEB, which
mimic cellular energy stress (El-Mir et al. 2000, Brunmair                                        causes a reduction in mTOR signalling (Inoki et al.
et al. 2004). Examples of these stresses include glucose                                          2003). mTOR controls protein synthesis by regulating
deprivation, hypoxia, oxidative stress, ischaemia and                                             the phosphorylation of key proteins involved in mRNA
muscle contraction or exercise, all of which lead to                                              translation, such as the 4E-BPs, S6Ks and the initiation
an increase in the ratio of AMP:ATP and activation of                                             factor eIF4G. Inhibition of mTOR signalling leads to a
                                                                                                  reduction in phosphorylation of its major downstream
                                                                                                  effectors, the 4E-BPs and S6Ks, and a net inhibition of
                                                                                                  global protein synthesis (Zakikhani et al. 2006, Dowling
                                                                                                  et al. 2007; Fig. 2). Reduced protein synthesis is thought
                                                                                                  to be an important mechanism of direct metformin
  Circulating insulin levels
                                        Metformin                     IGF1R       IR              action in the inhibition of cancer cell proliferation.
                                                                                                  Indeed, the growth-suppressive effect of metformin
                                                                                  PI3K            in breast cancer cell lines was associated with a
                               OCT                               LKB1
                                                    OCT                                           suppression of mTOR signalling and a general
   Gluconeogenesis             AMPK                            AMPK
                                                                                                  inhibition of protein synthesis (Zakikhani et al. 2006,
                                                                                                  Dowling et al. 2007).
                                                                         TSC2                        Considering the complexity of the molecular cascade
 Liver                                                    Metformin                               mediating the effect of metformin on protein synthesis,
                                                                                         Cancer   the status of several factors can impact metformin
                                                                                                  sensitivity. For instance, in mouse embryonic fibroblasts
                                                                  Protein synthesis,
                                                               cell growth, proliferation         (MEFs), loss of TSC2 leads to resistance to the effects of
                                                                                                  metformin on mTOR signalling, protein synthesis and
           Indirect                                                      Direct
                                                                                                  cell proliferation (Dowling et al. 2007). Nevertheless, a
Figure 2 Mechanism of action of metformin in cancer. The anti-                                    recent report demonstrates that in MEFs, AMPK can
cancer activity of metformin is associated with direct and indirect
effects of the drug. The direct insulin-independent effects of                                    directly suppress mTOR in the absence of TSC2 by
metformin are mediated by activation of AMPK and a reduction in                                   phosphorylating the mTOR-associated adaptor protein
mTOR signalling and protein synthesis in cancer cells. The                                        raptor (Gwinn et al. 2008). The tumour suppressor
tumour suppressors LKB1 and TSC2 are important in mediating                                       LKB1 is also an important determinant of metformin
the effects of metformin on AMPK and mTOR respectively;
however, metformin may also inhibit mTOR independently of                                         sensitivity. As indicated earlier, LKB1 is the immediate
LKB1, AMPK and TSC2. The indirect insulin-dependent effects of                                    AMPK activator in response to cellular energy stress and
metformin are mediated by its ability to activate AMPK and inhibit                                phosphorylates a key AMPK residue (Thr172) obligate
gluconeogenesis in the liver and stimulate glucose uptake in                                      for catalytic activity. Cells lacking LKB1 exhibit
muscle. The resulting reduction in circulating insulin alleviates
activation of PI3K/AKT/mTOR signalling in cancer cells. The cell
                                                                                                  decreased AMPK activation and elevated mTOR signal-
membrane transporter OCT1 is required for metformin uptake in                                     ling even under conditions of cellular energy stress or
tissue and therefore may play a key role in efficacy.                                             treatment with AMPK agonists (Shaw et al. 2004). LKB1                                                                                        Journal of Molecular Endocrinology (2012) 48, R31–R43

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      is also required for metformin-mediated AMPK acti-            (SLC22A2), and -3 (SLC22A3)). OCT1 is primarily
      vation. A variety of cells lacking LKB1, including those      responsible for metformin transport into cells and
      derived from breast and cervical cancers and mouse            polymorphisms in the gene encoding OCT1 that affect
      embryonic stem cells, are resistant to the growth             hepatic uptake of metformin in vitro and have been
      inhibitory effects of metformin in vitro (Zakikhani et al.    associated with decreased efficacy in patients (Shu et al.
      2006, Dowling et al. 2007, Huang et al. 2008). Moreover,      2007, Jin et al. 2009, Tzvetkov et al. 2009, Memmott et al.
      LKB1 is required for liver AMPK activation and the            2010). OCT1 is highly expressed in liver tissue,
      associated reduction in blood glucose in response to          with lower levels present in the kidney and minimal
      metformin treatment (Shaw et al. 2005). Thus, LKB1            levels present in the lung and small intestine (Jin et al.
      and TSC2 represent key factors in determining tumour          2009, Tzvetkov et al. 2009, Memmott et al. 2010). Little is
      sensitivity to the effects of metformin.                      known about the expression of OCT1 in human
         Increased mTOR-dependent protein synthesis and             tumours; however, an assessment of its expression
      cell growth are hallmark features of tumorigenesis            patterns in tumour tissue will be critical to elucidate
      downstream of the activated PI3K/AKT signalling               the mechanism of anti-tumour action of metformin
      pathway, one of the most frequently deregulated               and the identification of patients best suited for
      molecular networks in human breast cancer. Activating         metformin therapy.
      mutations in the PI3K catalytic subunit (PIK3CA) have            Recent reports have raised the possibility that
      been found in 20–35% of primary breast specimens              metformin exhibits additional inhibitory effects on
      (Bachman et al. 2004, Lee et al. 2005), while mutations       gluconeogensis and mTOR signalling independent of
      or loss of the tumour suppressor PTEN, a negative             AMPK and TSC2 (Gwinn et al. 2008, Foretz et al. 2010,
      regulator of the PI3K/AKT cascade, has been found in          Kalender et al. 2010). Metformin inhibited mTOR
      up to 40% of breast tumours (Sansal & Sellers 2004,           activity in cells lacking TSC2 and AMPK by suppressing
      Perez-Tenorio et al. 2007). In addition, over-expression      RAG GTPases, which are involved in mTOR activation
      of HER2 (ERBB2), an upstream activator of PI3K, is            (Kalender et al. 2010). Furthermore, metformin
      observed in 30% of human breast cancers (Yu & Hung            lowered hepatic gluconeogenesis in the absence of
      2000). Notably, PI3K/AKT signalling correlates with           AMPK, or its kinase, LKB1, by reducing hepatic energy
      breast cancer progression and contributes to resistance       levels (Foretz et al. 2010). These observations highlight
      of breast cancer cells to chemotherapy, trastuzumab           the need for additional research focusing on the
      and tamoxifen (Zhou et al. 2004, Morgensztern &               mechanism of action of metformin. Nevertheless, it
      McLeod 2005). A number of physiological stimuli,              appears likely that the ability of metformin to exert
      including a variety of growth factors and hormones            direct effects on mTOR signalling and indirect effects
      such as insulin, also activate PI3K signalling in cancer      on circulating insulin remain integral to its potential
      cells. Considering the prevalence of genetic changes          mechanism of action in the treatment of cancer.
      leading to elevated mTOR signalling, targeting this
      cascade using metformin represents a possibly viable
                                                                    Indirect (insulin-dependent) effects of metformin
      therapeutic option in breast cancer.
         In addition to mTOR, other targets of AMPK may also        As noted earlier, metformin may also exhibit indirect
      be implicated in cancer cell proliferation and survival.      effects on tumour growth by reducing circulating
      For example, AMPK phosphorylates p53 (a tumour                glucose and insulin levels via inhibition of hepatic
      suppressor that is a downstream target of AMPK) upon          gluconeogenesis and stimulation of glucose uptake in
      energy stress in MEFs leading to activation of a cell cycle   muscle (Fig. 2). High insulin levels associated with
      checkpoint, allowing cells to survive periods of energy       obesity, insulin resistance and type 2 diabetes may
      deprivation (Jones et al. 2005). p53 may also impact          promote tumourigenesis via activation of the foetal
      sensitivity to metformin. Metformin inhibited the             isoform of the IR-A, which is highly expressed in human
      growth of p53-deficient human colon carcinoma cells           breast cancer. The IR lies upstream of a number
      in vitro and in tumour xenografts in mice but had no          of growth-promoting pathways including the PI3K/
      effect on p53 wild-type cells (Buzzai et al. 2007). This      AKT/mTOR signalling network (Belfiore & Frasca
      difference in sensitivity was attributed to the ability of    2008, Pollak 2008). The IGF1R, also proposed to be
      p53 wild-type cells to undergo a metabolic adaptation,        involved in breast cancer, is less highly expressed in
      which maintained cell survival during periods of              breast tumours but may hybridise with IR-A to bind
      metformin-mediated AMPK activation. However, p53              insulin and stimulate proliferation of breast cancer cells
      status did not impact metformin sensitivity in other          at concentrations that do not activate IGF1R dimers
      cancer cell lines (Zhuang & Miskimins 2008, Alimova           (Papa & Belfiore 1996, Mulligan et al. 2007). Together,
      et al. 2009). Sensitivity to the anti-tumoral effects of      these observations suggest that, unlike normal adult
      metformin may also be governed by expression levels of        epithelial cells, breast cancer cells may acquire sensi-
      the organic cation transporters (OCT1 (SLC22A1), -2           tivity to the growth and survival effects of insulin.
      Journal of Molecular Endocrinology (2012) 48, R31–R43                                          

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Reduction in insulin levels by metformin may counter          human doses in the latter) reduced tumour growth
act this sensitivity by reducing ligand binding to            when mice were fed a high-energy diet (which induces
IR-A/IGF1R, indirectly inhibiting tumour growth and           weight gain and insulin resistance; Algire et al. 2008,
improving breast cancer outcomes.                             Phoenix et al. 2010). In these models, the high-energy
                                                              diet enhanced tumourigenesis, whereas metformin
                                                              significantly reduced the effect of this diet on tumour
In vivo models of anti-tumour activity of metformin           growth. Metformin improved insulin sensitivity in
The effects of metformin on spontaneous breast                tumour-bearing high-energy diet fed mice and resulted
tumour development have been examined in MMTV–                in AMPK activation and reduced IR-mediated signalling
Her2/Neu transgenic mice that exhibit mammary-                in tumours. Paradoxically, metformin failed to inhibit
specific expression of the HER2/NEU oncoprotein,              the growth of tumours in mice on a control diet, despite
resulting in the almost universal development of focal        activation of AMPK in tumour tissue (Algire et al. 2008).
mammary adenocarcinomas, which metastasise to the             These observations are consistent with an indirect,
lungs. Metformin treatment, in a dose comparable to           insulin-lowering mechanism of metformin action;
that used clinically in humans, significantly delayed the     however, it must be recognised that the tumour cells
appearance of mammary adenocarcinomas, reduced                were dependent on high insulin levels in culture
the size of tumours and prolonged the lifespan of             medium before their injection into mice and
MMTV–Her2/Neu mice (Anisimov et al. 2005). These              mechanisms may differ in the clinical setting in which
effects were accompanied by a significant reduction in        this dependence is not present.
blood glucose and a statistically non-significant dimin-
ution in circulating insulin levels (Anisimov et al. 2005).
                                                              In vitro models of anti-cancer effects of metformin
In mice heterozygous for the tumour suppressor PTEN
(highly prone to tumours in various organs), metfor-          In vitro work focusing on the effects of metformin on
min (administered at a dose of 300mg/kg body weight           breast cancer cells demonstrated that metformin
per day, more than tenfold higher than the standard           inhibited the growth of a variety of breast cancer cells
clinical dose) delayed tumour onset by 25% (Huang             regardless of oestrogen receptor (ER), PR, HER2 or
et al. 2008), an effect that required LKB1-mediated           p53 status (Zhuang & Miskimins 2008, Alimova et al.
AMPK activation. Metformin was also effective in              2009). Recently, it was reported that metformin
reducing the growth of intestinal polyps in mice deficient    induced unique responses in the triple-negative (ER,
for the tumour suppressor APC (Tomimoto et al. 2008).         PR and HER2 negative) breast cancer cell line MDAMB-
   Orally administered metformin (at plasma levels            231, leading to an S phase cell cycle arrest, reduced cell
(2.7–10.3 mM) equivalent to those achieved in patients        proliferation and colony formation, as well as increased
(2 . 8–15 mM)) also reduced tobacco carcinogen-               apoptosis (Liu et al. 2009). In this system, metformin
induced lung tumourigenesis (4-(methylnitrosamino)-           also induced AMPK activation, reduced the phosphoryl-
1-(3-pyrdyl)-1-butanone (NNK)) in mice (tumour                ation of epidermal growth factor receptor (EGFR),
burden reduced by 53%; Memmott et al. 2010).                  MAPK and Src and lowered the levels of cyclins D1
Intraperitoneal administration, resulting in higher           and E (Liu et al. 2009). Growth inhibitory effects were
plasma levels of metformin (24.2 mM), reduced tumour          recapitulated in vivo where metformin significantly
burden by 72%. Although AMPK activation was                   reduced the growth of triple-negative breast cancer
observed in the liver and circulating insulin levels          cell xenografts in nude mice. Some of the anti-
were reduced after i.p. injection, metformin failed to        cancer effects of metformin on triple-negative breast
activate AMPK in lung tumour tissue. A concomitant            cancer cells were also associated with STAT3 inhibition.
reduction in phosphorylation of IGF1R/IR, AKT and             Treatment of triple-negative cells with metformin led to
ribosomal protein S6 in tumours suggests that the             a reduction in STAT3 activation and downstream
indirect (insulin-mediated) effects of metformin were         signalling and metformin acted synergistically with a
responsible for the reduction in lung tumour burden.          STAT3 inhibitor to decrease cell growth and induce
   In addition to spontaneous and carcinogen-induced          apoptosis (Deng et al. 2012). Metformin may also be
tumours, metformin has been found to impact growth            effective against ER-positive breast cancers by
in mouse xenograft and syngeneic tumour models – the          inhibiting aromatase expression in tumour stroma.
tumour cells injected in these models were grown in           In primary human breast adipose stromal cells,
culture media that contained high levels of glucose,          metformin treatment led to an increase in the
insulin and other growth factors. In at least two             phosphorylation of AMPK, which was associated with
syngeneic model systems (Lewis lung carcinoma LLC1            an inhibition of nuclear translocation of CRTC2, a
cells in C57BL/6J mice and triple negative 66c14 cells        CREB co-activator known to increase aromatase
in Balb/c mice), metformin (at doses comparable to            expression (Brown et al. 2010). Furthermore, metfor-
human doses in the former and w40 times higher than           min interacted additively with tamoxifen to reduce                                                   Journal of Molecular Endocrinology (2012) 48, R31–R43

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R38   R J O DOWLING   and others . Metformin and cancer

      breast cancer cell proliferation (Berstein et al. 2010b).      effective in preventing tumour growth and relapse in
      These results indicate that metformin may represent a          mice (Hirsch et al. 2009, Iliopoulos et al. 2011).
      potentially effective therapy in women with ER-positive        Significantly, no CD44C/CD24lo cells could be isolated
      breast tumours.                                                from mice after metformin treatment, raising the
         Emerging evidence indicates that breast cancer cells        possibility that metformin may specifically target
      over-expressing Her2, either through gene amplifi-             tumour-initiating cells. Metformin was also effective in
      cation or ectopic expression, exhibit increased sensi-         preventing tumour relapse in mice when combined
      tivity to the growth inhibitory effects of metformin           with the standard chemotherapeutic agents paclitaxel
      (Vazquez-Martin et al. 2009). The effects of metformin         and carboplatin, highlighting the potential application
      were associated with translational suppression of HER2         of metformin as part of a combinatorial therapeutic
      protein expression mediated by the inhibition of S6K1,         strategy (Iliopoulos et al. 2011). However, in some
      a downstream effector of mTOR (Vazquez-Martin et al.           experiments the regimen of metformin administration
      2009). Of interest, the mTOR inhibitor rapamycin               complicates the interpretation of results (Hirsch et al.
      caused similar translational suppression of erbB3              2009). Large volumes (up to 0.5 ml) of metformin
      (an EGFR, also known as HER3 in humans) in breast              solution were directly injected into tumour xenografts,
      cancer cells and mammary tumours in mice (Liu et al.           likely resulting in exposure of tumour cells to non-
      2005). Taken together, these data indicate that                physiological doses of the drug, as well as necrosis (due
      metformin and other inhibitors of mRNA translation             to direct tumoral injection). It is unclear how a
      act by reducing translation of mRNAs encoding specific         comparable drug delivery can be achieved in patients.
      growth factor receptors. Metformin also exhibited              Nonetheless, these data imply a possible benefit in
      inhibitory effects on trastuzumab-resistant breast cancer      combining metformin with standard chemotherapies
      cells. In trastuzumab-resistant models, metformin              in breast cancer treatment.
      disrupted ERBB2/IGF1R complexes and significantly
      reduced cell proliferation (Liu et al. 2011).
         Metformin has also been reported to exert anti-
      proliferative effects against a number of other cancer         Limitations of preclinical models
      cell types including those derived from prostate,
                                                                     One of the major limitations in interpreting many
      endometrial and brain tumours (Isakovic et al. 2007,
                                                                     preclinical studies is the high concentration of
      Ben Sahra et al. 2008, Cantrell et al. 2010). In prostate
                                                                     metformin used in vitro in relation to the concentration
      cancer cells, metformin treatment caused a G1 cell cycle
                                                                     that can be safely obtained in the clinical setting
      arrest (mediated by a reduction in cyclin D1 levels and
                                                                     (Fig. 3). Most in vitro studies report using doses of
      phosphorylation of Rb), as well as an increase in the
                                                                     metformin between 1 and 40 mM (165–6600 mg/l),
      levels of the cell cycle inhibitor p27kip, resulting in a
                                                                     which is well above the feasible therapeutic plasma
      strong anti-proliferative effect both in vitro and in
                                                                     levels (0.465–2.5 mg/l or 2.8–15 mM) in humans
      tumour xenografts in mice (Ben Sahra et al. 2008). In
                                                                     (Stambolic et al. 2009). Thus, it is possible that
      endometrial cancer cells, metformin induced a
                                                                     metformin caused a degree of energy stress (and resul-
      G1 arrest and at high doses caused apoptosis while
                                                                     ting AMPK activation) in these studies that far exceeds
      suppressing mTOR signalling (Cantrell et al. 2010).
                                                                     effects that would be seen clinically. For example,
      Finally, while causing cell cycle arrest in low-density
                                                                     Phoenix et al. (2009); reported pro-angiogenic effects of
      cultures of rat glioma cells, metformin induced
      apoptosis in confluent cell cultures (Isakovic et al. 2007).                                   Concentration of metformin
         A major obstacle in the effective treatment of breast
      and other cancers is the resistance of tumour cells to
      drug therapy. The existence of tumour-initiating (stem)
      cells, a specific subset of tumour cells that are believed
      to be involved in tumour initiation, progression,
      heterogeneity and recurrence, has been proposed as
      one of the underlying causes of this resistance (Camp-            Clinical/epidemiological               In vivo                     In vitro
                                                                      0·15–1·0 × therapeutic dose    2·0–45 × therapeutic dose    25–>1000 × therapeutic dose
      bell & Polyak 2007, Polyak & Weinberg 2009).                         250–2250 mg/day              750 mg/kg per day                2–50 mM
      Metformin specifically inhibited the growth of
      CD44C/CD24lo (a putative stem cell marker signature;           Figure 3 Concentrations of metformin used in clinical and
      Hirsch et al. 2009) cells in culture and reduced their         preclinical studies. The anti-cancer effects of metformin have
      ability to form tumours when injected into nude mice.          been tested over a wide range of doses. Clinical and epidemio-
                                                                     logical studies have utilised metformin at standard doses of up to
      When metformin was combined with doxorubicin, a                2250 mg/day. Conversely, preclinical studies often involve extre-
      striking reduction in both CD44C/CD24lo and other              mely high, non-physiological concentrations of metformin that are
      cancer cells was observed; this combination was also           in excess of the therapeutic levels achieved in human patients.

      Journal of Molecular Endocrinology (2012) 48, R31–R43                                                        

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Metformin and cancer .        R J O DOWLING    and others       R39

metformin in a mouse xenograft model; however,                 glucose within culture media are required for
metformin concentrations were hundreds of times                physiological modelling of metformin in vitro. It is
higher than those that are deemed safe clinically; it is       anticipated that such models will be more likely to
possible that these extremely high concentrations led to       inform clinical studies.
intracellular metabolic stresses (and responses to those
stresses) beyond those that would be seen clinically,
raising questions as to whether such pro-angiogenic
effects are clinically relevant.                               Translational challenges
   Nonetheless, recent studies indicate that metformin
is effective at reducing cancer cell proliferation in vitro    The anti-cancer effects of metformin are associated with
                                                               both direct (insulin-independent) and indirect
at concentrations as low as 10 mM, well within the
                                                               (insulin-dependent) actions of the drug. This duality
therapeutic range of the drug (Liu et al. 2009).
                                                               of action has increased the complexity of clinical
Moreover, metformin is capable of inhibiting
                                                               evaluation of metformin, including the translation of
tumour growth in mouse models at doses equivalent
                                                               preclinical observations. Existing preclinical, clinical
to those used in humans without causing toxicity
                                                               and epidemiological evidence has incorporated a broad
(Anisimov et al. 2005, Ben Sahra et al. 2008).
                                                               range of insulin, glucose and metformin concen-
Importantly, only slight levels of AMPK activation are
                                                               trations and has often focused on different potential
required to trigger downstream signalling events
                                                               mechanisms of metformin action (Fig. 3). Effects that
(Hawley et al. 2002).
                                                               have been identified in preclinical work, including
   Another concern arises from the growth conditions
                                                               differential effects in different molecular subtypes of
of the cell culture models used to assess the inhibitory
                                                               breast cancer, although plausible, require confirmation
effects of metformin in vitro. The majority of cancer cell
                                                               in the clinical setting. Focused research in the
lines are grown in culture media that contains
                                                               neoadjuvant and metastatic settings, with biopsies pre-
extremely high amounts of growth factors and glucose.
                                                               and post-metformin administration, and concurrent
For example, average tissue culture media contains
                                                               evaluation of physiological effects will help to elucidate
glucose at concentrations between 10 and 25 mM, well
                                                               the clinical relevance of these effects.
above the fasting levels of glucose observed in non-
                                                                  Differentiation of the relative importance of indirect
diabetic patients (!6 mM). Most media is also
                                                               (insulin-mediated) and direct (insulin-independent)
supplemented with 5–10% foetal bovine serum, which             metformin effects is critical for optimal evaluation of
contains high concentrations of growth factors and             this agent. The postulated mechanisms of action give
hormones, including insulin and EGF. Thus, cancer cell         rise to a broad range of therapeutic targets, leading to a
lines are often maintained in non-physiological con-           large number of potential clinical markers of metfor-
ditions that are optimised for maximum growth and              min benefit (Table 2). Understanding the relative
proliferation. The excessive concentrations of insulin,        importance of therapeutic targets and markers of
glucose and growth factors in culture media, combined          benefit is required for appropriate selection of patients
with the presence of oncogenic mutations and variable          who might benefit from metformin. For example, if
expression of the membrane transporter OCT1, may               indirect insulin-mediated effects of metformin predo-
account for the elevated doses of metformin required           minate, patient selection should target individuals with
to elicit cellular responses in vitro.
   A host response is not present in vitro, and as a result,   Table 2 Predictors of metformin benefit in human cancer
the direct (insulin-independent) mechanism(s) of
metformin action have been the focus of this research.                                                Indirect              Direct
Because many of the conditions evaluated in this in vitro                                              effect               effect
                                                                                                      (insulin-            (insulin-
work are outside the boundaries possible in the clinical       Marker                                dependent)         independent)
setting, it is conceivable that many of the direct effects
of metformin reported in in vitro work will not be
relevant in the clinical situation.                             High BMI                                   C
   Thus, although a beneficial anti-cancer effect of            Physical inactivity                        C
metformin at lower concentrations that are encoun-              High fasting insulin                       C
tered clinically remains plausible, mechanistic effects         Insulin resistance                         C
                                                                OCT1/2/3 expression (liver)                C
identified in the preclinical studies may differ from          Tumour
those that are most relevant in the clinical setting.           IR/IGF1R expression                        C
It is therefore necessary to exercise caution in                Increased PI3K/mTOR                        C                   C
further studies of the effects of metformin in vitro.           OCT1/2/3 expression                                            C
Careful dosing, equivalent to therapeutic levels in vivo,       LKB1 expression                                                C
                                                                TSC2 expression                                                C
and strategies for varying the levels of insulin and                                                      Journal of Molecular Endocrinology (2012) 48, R31–R43

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R40   R J O DOWLING   and others . Metformin and cancer

      high insulin levels and/or obesity, germline expression     Declaration of interest
      of OCTs (particularly OCT1) and tumour characteristics
      including IR/IGF1R expression and PI3K pathway              P J G is the principal investigator and V S is the correlative science
                                                                  chair of a Phase III adjuvant metformin trial (NCIC CTG MA.32).
      activation. In contrast, if direct (insulin-independent)    Apotex Canada, a generic drug company, supplies drug and placebo to
      mechanisms of metformin action are key, tumour              NCIC CTG for use in this trial.
      characteristics such as LKB1 and TSC2 expression,
      OCT1 positivity and PI3K/AKT/mTOR activation will
      likely emerge. It is possible that the predominant
      mechanism of metformin action (and key markers of           Funding
      activity) will differ across types of tumours. Once key
      mechanisms of action (and their associated markers)         The authors would like to acknowledge support from the Canadian
      are understood, metformin can be used in a targeted         Breast Cancer Foundation, the Ontario Institute for Cancer Research,
      fashion, incorporating patient and tumour attributes        the Canadian Cancer Society Research Institute, the Susan G Komen
                                                                  for the Cure Foundation and the Breast Cancer Research Foundation.
      consistent with known mechanisms of action.                 R J O D is supported by a Banting Postdoctoral Fellowship from the
         Although evidence of direct (insulin-independent)        Canadian Institutes of Health Research.
      anti-cancer effects of metformin that has arisen from
      preclinical research is strong, leading to a focus on
      these direct effects by many researchers, emerging
      clinical and in vivo data suggest that indirect insulin-    References
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