molecular pathways: preclinical models and clinical trials with ... · 28/03/2014 · officinalis...

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

Alastair M. Thompson

Professor of Surgical Oncology

Department of Surgical Oncology,

MD Anderson Cancer Center,

1515 Holcombe Boulevard,

Houston

Texas 77030, USA

[email protected]

Running title: Metformin in Breast Cancer

No conflicts of interest

Funding sources: Breast Cancer Campaign, Breast Cancer Research Scotland, Cancer Research UK,

Breakthrough Breast Cancer

Research. on March 11, 2021. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 28, 2014; DOI: 10.1158/1078-0432.CCR-13-0354

http://clincancerres.aacrjournals.org/

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ABSTRACT

Metformin, an oral biguanide widely used to treat diabetes, has considerable potential and is in

clinical trials as an experimental preventive or therapeutic agent for a range of cancers. Direct

actions targeting cellular pathways, particularly via AMPK and through inhibiting mitochondrial ATP

synthesis, or systemic mechanisms involving insulin and insulin like growth factors have been much

studied in vitro and in preclinical models. Epidemiological and retrospective studies also provide

clinical evidence in support of metformin as an antitumor agent. Pre-operative window of

opportunity trials confirm the safety of metformin in women with primary breast cancer,

demonstrate reduction in tumor cell proliferation and complex pathways of gene suppression or

overexpression attributable to metformin. Confirmation of insulin mediated effects, independent of

body mass index, also supports the potential benefit of adjuvant metformin therapy. Neoadjuvant,

adjuvant and advanced disease trials combining metformin with established anticancer agents are

underway or proposed. Companion biomarker studies will utilise in vitro and preclinical

understanding of the relevant molecular pathways to, in future, refine patient and tumor selection

for metformin therapy.




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BACKGROUND

Synthetic biguanides, based on the guanidine derivative galegine originally derived from Galega

officinalis (French lilac), have been used clinically since the 1950s with metformin (N’,N’-

dimethylbiguanide) prescribed to over 120 million type II diabetics globally. Phenformin, previously

used in the treatment of diabetes, was withdrawn from many countries in the 1970s due to drug

associated lactic acidosis (1). The potential for re-purposing metformin to oncology is supported by

evidence of reduced cancer incidence and increased time to develop cancer (2), reduced mortality

from cancer (3), with lower HER2 positive breast cancer specific mortality (4) or reduced risk of

distant metastases in triple negative breast cancer (5) in diabetic women. Additional supportive

evidence has come from studies demonstrating higher serum insulin levels are associated with

increased risk of breast cancer (6) and shorter survival from breast cancer (7) which may be modified

by metformin.

The relevance of the multiple proposed mechanisms of action of biguanides to cancer remain

controversial. In vitro, in vivo animal model and human studies support several possible modes of

action with extrapolation of studies in diabetes, the range of in vitro models, non-physiological

metformin levels, the circumstantial evidence from epidemiological studies and retrospective

reviews creating complexity. Focussed initially on diabetes and metabolism more generally, both

direct (cellular) pathways and indirect (systemic) actions have been proposed as key mechanisms for

the action of biguanides in diabetes that may also be of relevance to cancer.

AMPK mediated effects.

The master sensor of cellular energy, the Ser/Thr protein kinase complex AMP activated protein

kinase (AMPK) remains central to studies of metformin. AMPK activation stimulates TSC2, hence

indirectly as well as AMPK directly inhibits mTOR and consequently by down regulating S6kinase

leads to reduced protein synthesis, reduced cellular growth and lowers proliferation (Figure 1). A




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complex containing Liver kinase B1 (LKB1), classed as a tumor suppressor gene, activates

heterodimers of AMPK subunits through phosphorylation of AMPKa2Thr-172, established as a link

between LKB1, AMPK and cancer (8).Breast cancer cells lacking LKB1 are resistant to the growth

inhibitory effects of metformin in vitro and in vivo (9,10,11), and as a distinct mechanism, epigenetic

inactivation of LKB1 has been reported in sporadic papillary breast cancer (12). Furthermore, the

intracellular pathways down stream of HER2 amplification, PIK3CA mutation and PTEN mutation or

loss of PTEN expression converge on elevated mTOR signalling and are considered potential

mechanisms for breast cancer development and of resistance to therapy. This makes the potential

for metformin to work via targeting the mTOR cascade an attractive mechanism of action (13).

Other, AMPK and TSC2 independent, mechanisms of mTOR inhibition by metformin, include

suppression of RAG GTPases (14).

Additional, potentially important, AMPK mechanisms for oncology include phosphorylating and

hence inactivating ACC1 and ACC2, consequently blocking fatty acid synthesis but increasing

mitochondrial fatty acid uptake and subsequent breakdown by B-oxidation (15). Inactivation of

AMPK promotes a metabolic shift to aerobic glycolysis through stabilisation of HIF1a, increases

transcription of genes involved in glycolytic pathways, resistance to apoptosis, angiogenesis and

metabolic adaptation (16,17). AMPK may act as a negative regulator of the Warburg effect under

nutrient rich and growth factor replete conditions in the absence of evident energy imbalance (16).

In contrast, AMPK activation may increase resistance to stress and viability of cancer cells under

hypoxic conditions of metabolic stress (18) which may be of particular relevance as tumor cancer

cells migrate and metastasise.

AMPK phosphorylates p53 and leads to cell cycle checkpoint activation, at least in mouse embryonic

fibroblasts, allowing cells to survive energy deprivation (19). Indeed, metformin inhibition of

complex 1 of the mitochondrial electron transport chain (20,21,22) is particularly effective in p53-

deficient colon cancer cells (23) although the p53 status did not affect metformin sensitivity in other




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cancer cell lines (24,25). However, loss of functional AMPK or p53 (as is common through mutation

or p53 network dysfunction in most cancers) may constitute a sufficient energy crisis to result in

metformin having a cytotoxic synthetic lethality effect (26). Thus, there is evidence that metformin

may be therapeutically effective acting via p53 particularly where normal p53 function is abrogated,

as is the case in most epithelial cancers.

Mitochondrial effects

Biguanides accumulate in mitochondria, with uptake promoted (for the cationic metformin at least)

by organic cation transporter 1 (OCT1) to inhibit Complex I of the mitochondrial respiratory chain,

inhibit mitochondrial ATP synthesis, increase ADP:ATP and AMP:ATP ratios and hence activate AMPK

indirectly. Phenformin is 50 fold more potent than metformin as an inhibitor of mitochondrial

complex 1 and being lipophilic (21) is less dependent on OCT (27) retaining the dose-dependent

antiproliferative effect in ovarian cancer cell lines even in the presence of OCT1 siRNA (28). Given

that oxidative phosphorylation-dependent production of ATP appears to be secondary to

mitochondrial synthesis of anabolic precursors in proliferating cells (29), it has been suggested that

metformin exerts the chemopreventive effects by functioning as a weak mitochondrial poison

inhibiting oxidative phosphorylation (30) .

Additional, non-AMPK mediated mechanisms for the antineoplastic effect of metformin have been

proposed through NF-kB, a pleiotropic protein complex that controls DNA transcription. Metformin

may inhibit IkB and IKKa/b subunits and hence NF-kB and prevent the translocation of NF-kB from

the cytoplasm to the nucleus (31). Metformin appears to down regulate genes involved in mitosis

and may act synergistically in this setting with taxanes (32). Metformin may also act via the DNA

damage response mechanisms through ataxia telangiectasia mutated (ATM) and Chk2 (33), also

potentially accounting for the cancer prevention effect.

Systemic effects




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Biguanides have long been known to suppress hepatic glucose production (34) with metformin

uptake via OCT leading to AMKP inhibition of hepatic gluconeogenesis, increase peripheral insulin

sensitivity (and hence glucose uptake for example in skeletal muscle) and reduce circulating insulin

and IGF-1 levels, although it is less clear how metformin regulates IGF-1.

Certainly, the insulin receptor lies upstream of the PI3K/AKT/mTOR signalling pathways and breast

cancer cells may have greater sensitivity to the effects of insulin such that metformin reducing

insulin levels and hence binding of insulin to receptors on breast cancer cells present very attractive

mechanisms to indirectly inhibit tumour growth (13). This provides molecular insights into and

supports the clinical relevance of insulin/IGF-1 reduction in relation to cancer progression (7,35,36).

CLINICAL-TRANSLATIONAL ADVANCES

Phenformin

In murine models, phenformin has been associated with a reduced incidence of DMBA induced rat

mammary tumors (37), enhances the effects of chemotherapy (38) and elicits antiproliferative

effects, alongside inducing apoptosis, in neuroblastoma (39). Phenformin, like metformin, is

associated with enhanced apoptosis and ATP depletion in AMPK deficient cancer cells and may be

most effective in cancer cells without a functional LKB1-AMPK pathway, as demonstrated in non-

small cell lung cancer (40).

Phenformin has been shown to prevent and retard growth of breast cancer cell lines(41) and

toprotect against tumour development in a spontaneous lymphoma murine model exhibiting PTEN

heterozygous mutations and LKB1 hypomorphic mutations with greater potency than metformin

(11).

Mechanistic insights from xenograft work suggest both systemic and direct, tumor/stromal effects

may be elicited (41)and has led to speculation that phenformin may have the capability to block a




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reverse Warburg effect of onconutrient production by stroma feeding breast cancer cells in vivo

(42,43).

Phenformin may initially seem most attractive for trials in the advanced breast cancer setting

(providing renal and hepatic function are adequate) with the potential to avoid lactic acidosis (1) and

potentially enhance cancer cell killby concommitant administration of 2 deoxyglucose with

phenformin (if confirmed beyond colorectal cancer cell lines) (44).

Metformin

In vitro models suggest metformin may be active against breast cancer cell lines regardless of the

estrogen receptor(ER), progesterone receptor (PR), HER2 status or p53 status and subtype of the

cells (24,25).

For ER positive breast cancer, dependent on aromatase conversion of precursors to estrogens in

postmenopausal women, metformin may inhibit aromatase enzyme expression in breast adipose

tissue via AMPK (45). Similar effects on other clinically relevant genes have yet to be explored. Given

that aromatase inhibitors (AI) are now the mainstay of neoadjuvant, adjuvant and extended

treatment in postmenopausal women with breast cancer, the potential for synergism between an AI

and metformin is under exploration in a Korean neoadjuvant trial and is planned for an international

extended adjuvant trial. For premenopausal women, where tamoxifen remains the principal

adjuvant therapy for ER positive breast cancer, the additive effects of metformin to tamoxifen, at

least in terms of reducing cell proliferation (46), merits subgroup analysis within the ongoing MA32

adjuvant metformin trial (47).

For HER2, breast cancer lines expressing HER2 have increased sensitivity to metformin (48) mediated

via inhibition of S6K; in trastuzumab resistant models, metformin disrupted ERBB2/IGF1R complexes

and significantly reduced cell proliferation (49). Metformin, used at comparable clinical doses,

delayed the onset, reduced the growth and increased the lifespan of MMTV-her2/neu mice (50)




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accompanied by reductions in circulating insulin levels. As HER2 targeted therapy extends to other

tumor types (such as gastric and oesophageal cancer) similar metformin therapy approaches may

merit consideration.

For triple negative breast cancer, metformin was effective in vitro (and in an animal model) inducing

AMPK and suppressing EGFR, MAPK and Src phosphorylation and lowering Cyclins D1 and Cyclin E

(51). The effect of metformin on triple negative cell lines may, in part, be attributable to STAT3

inhibition (52).

More generally, reduced immunohistochemical staining for pAMPK was described in 318/349 (91%)

breast cancers, with all subtypes of breast cancer represented (53). Whereas AMPK appears to

protect against initial tumour development, in established breast cancers lack of functional AMPK

dependent pathways may render breast cancer susceptible to metformin therapy.

Additional support for metformin as a therapeutic agent comes from in vivo models where

metformin appears to selectively inhibit CD44+/CD24low cancer stem cell like populations (54),

repress genes associated with epithelial mesenchymal transition such as ZEB1 TWIST, SNAi1 (55) and

work synergistically with chemotherapy agents (54). Additionally, inhibition of glutamine

metabolism, at least in prostate models, may synergise with metformin (56). Injected into tumours,

albeit at potentially non-physiological doses, metformin may be effective alongside paclitaxel and

carboplatinum as part of a combination chemotherapy strategy for the treatment of breast cancer

(57).

However, some have questioned the relevance of selected preclinical data given the concentrations,

doses and delivery of metformin employed (58). Nevertheless, studies have demonstrated cell

inhibition using physiologically possible concentrations of metformin in vitro (51) and in mouse

models (41,50,59). Even so, the multiplicity of mechanisms deduced in vitro may differ significantly




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in vivo while animal models (usually in immune compromised mice) may be different again from the

cancer prevention and cancer therapy settings in humans.

Metformin and breast cancer trials

Given the preclinical data and that metformin has known pharmacokinetics and manageable

toxicities, with extensive use worldwide in diabetes, re-purposing metformin within clinical trials to

seek efficacy against breast cancer is very attractive. Evidence to date suggests metformin could be

clinically useful in the prevention, preoperative, adjuvant, extended adjuvant and advanced disease

settings, although the detail of dose, duration and drug combinations will require elucidation.

Fortunately, the research community is actively engaged to address many of these roles and through

companion biomarker studies ascertain the tumor and patient characteristics most amenable to

treatment and whether direct or systemic effects are both of clinical importance.

Trials are underway examining these issues in the setting of breast, brain, colorectal endometrium,

head and neck, lung, lymphoma, melanoma, ovary, pancreas, prostate, renal and thyroid

malignancy. Single centre, phase II, biomarker studies and/or with clinical endpoints predominate;

many trials use gradual increases in dose upto 1500-2250mg metformin per day to mitigate the self-

limiting initial gastrointestinal side effects of nausea, diarrhoea and bloating. Caution over the

potential but rare metformin associated lactic acidosis (1/100,000 years of use) generally restricts

the clinical trials populations to women under 75 years with good renal and hepatic function.

A proposed study of metformin versus placebo to prevent the progression of atypical ductal

hyperplasia, ductal or lobular in situ neoplasias appears attractive given evidence that even low dose

metformin (250mg/day) is sufficient to reduce proliferation and aberrant crypt foci in rectal

epithelium (60). The progression from DCIS to invasive breast cancer may be mechanistically linked




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to glycolytic cancer associated fibroblast activity (exporting lactate to the tumour cells as evidenced

by a fall in caveolin and rise in MCT4 expression) with the potential for blockade by metformin (61).

The first randomised clinical trial using metformin (1000mg bd) in breast cancer was reported in

2011 and used the preoperative window of opportunity setting (62). This novel trial randomised

patients to receive metformin for two weeks or watchful waiting for two weeks between diagnostic

biopsy and definitive surgery. Core needle tumour biopsies were taken at the time of diagnosis and

on the operating table prior to resection of the operable breast cancer to allow direct comparison of

tumour proliferation (Ki67), tumour messenger RNA expression alongside serum insulin. The trial

demonstrated a significant reduction in Ki 67 with metformin but not in the control arm, reduced

gene expression (including in the PI3 kinase pathway) and increased gene expression (for example of

TNFR1), with a flattening of the serum insulin levels in response to surgery for the patients on

metformin. Thus, both in vivo anti-tumour effects and systemic effects were demonstrated for the

first time in women with primary breast cancer receiving metformin. The insulin effect, although

women were not fasting at the time of diagnostic biopsy, and the lack of association between

metformin effects and body mass index confirms the potentially beneficial systemic effect of

metformin as an insulin suppressor in women with breast cancer (7,63).

A second, single centre, preoperative window of opportunity placebo controlled trial of similar size

confirmed the reduction in Ki 67 with metformin use over two weeks, again not associated with BMI,

and provided enhanced evidence for the systemic insulin mediated effects (64). The need for

meticulous trial design was flagged by a subsequent trial (65) where failure to continue metformin

up to the time of surgery and using core samples at diagnosis versus resection specimens (66) make

data interpretation difficult.

Overall, these prospective, preoperative window studies in breast cancer (62,64,65), despite

reservations regarding trial design and methodology, demonstrate metformin is safe for women

with primary breast cancer and confirms the attraction of metformin as a therapeutic agent.




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Given the proposed mechanisms of action of metformin and the preclinical data, synergism of the

biguanide with chemotherapy has been explored. In a retrospective case note review of breast

cancer patients receiving neoadjuvant chemotherapy (67) demonstrated a remarkable 24%

complete pathological response(cPR) in diabetic patients incidentally taking metformin and receiving

neoadjuvant chemotherapy compared with 16% cPR in nondiabetics and 8% cPR in diabetics not

receiving metformin. A similar, also historical, series of epithelial ovarian cancer has demonstrated

clinical benefit with increased progression free survival and overall survival in diabetics taking

metformin receiving taxane/ carboplatin chemotherapy compared with nondiabetics or diabetics

not on metformin (68). However, incidental metformin use has not demonstrated survival benefit in

two case series of diabetics with breast cancer (5,67). This concept of combining metformin with

neoadjuvant chemotherapy is in prospective trials of metformin along side paclitaxel and FAC;

docetaxel, epirubicin and cyclophosphamide +/- metformin; and in ISPY2 (ganitumab + metformin).

An innovative trial being conducted in Oxford combines the window of opportunity and neoadjuvant

concepts. The Oxford trial sequences a two-week run in with metformin, then neoadjuvant

chemotherapy (+/- metformin) is administered up to the time of surgery. Non-invasive PET scanning

and lipid metabolism studies are being used alongside tissue biopsies to examine the effects of

metformin on breast cancer metabolism and lipid synthesis.

In addition to the potentially synergistic effects of metformin with chemotherapy against breast

cancer, murine models suggest a cardio protective effect of metformin which appears to abrogate

the effects of doxorubicin on LDH, CKMB and glutathione (69), although evidence for such effects in

humans is, as yet, lacking.

In the adjuvant setting, the NCIC CTG MA .32 trial (47) is examining the efficacy of adjuvant

metformin for five years after complete resection of invasive breast cancer and following completion

of chemotherapy and/or radiotherapy but concomitant with endocrine therapy or anti-HER2

therapy. Early data suggest beneficial effects of metformin compared with placebo on patient




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weight and metabolic factors which, if sustained, could significantly improve survival from breast

cancer (7). Further clinical efficacy, companion biomarker and mammographic density studies are

eagerly awaited as the trial matures. There are also plans underway to trial metformin in the

extended adjuvant setting, beyond five years, for ER positive patients at risk of recurrence. Further

survivorship trials employing metformin along with exercise in breast and colon cancer are

proposed.

In contrast to most clinical drug development, where molecular pathways are usually explored in

advanced disease, this setting has only recently been reported for metformin. For ER+ advanced

disease, there is no disadvantage to taking metformin concomitant with exemestane on exemestane

levels (70) providing reassurance for the neoadjuvant and adjuvant trails using concomitant

metformin and endocrine therapy underway. Interactions of metformin with a range of agents are

proposed, based on an appreciation of the intracellular pathways outlined above, including trials of

metformin with everolimus and exemestane in ER+ disease, metformin with erlotinib in triple

negative breast cancer and combination chemotherapy +/- metformin in HER2 negative advanced

disease.

Biomarker studies built in to all these trials will be essential to identify the breast cancers with

characteristics (such as LKB1-AMPK or p53 defective pathways) most likely to be susceptible to the

effects of metformin. Cancer heterogeneity of OCT1, and hence for cancer cell uptake of metformin,

as observed in ovarian cancer (28), may merit measurement before considering metformin therapy

in the clinical setting given, for example, the low expression of OCT1 in normal breast tissues. The

potential effects of polymorphisms of LKB1, OCT 1,2,3 and ATM on resistance to metformin (70) and

interactions between proton pump inhibitors and metformin via OCT 1,2,3 (70) will clearly need to

be considered when metformin translates into routine clinical practice.




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Biguanides, and particularly metformin, present an attractive proposition for repurposing as low

toxicity anti-tumor agents, thus demonstrating how understanding the molecular pathways of

cancer combined with clinical data will lead to further advances in oncology.

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Figure 1

Overview of proposed metformin actions in vivo.




17

Metformin (M), absorbed from the gut, acts via systemic and intracellular molecular pathways.

Systemic actions of metformin via metformin uptake through OCT in the liver, AMPK inhibition of

gluconeogenesis and consequent reduction in insulin reduces insulin receptor (IR) stimulation and

downstream intracellular effects (including via PI3kinase, akt and HIF1a) and leads to increased

glucose uptake in tissues such as skeletal muscle. Cell level molecular pathways include reductions in

mitochondrial respiratory function, activation by metformin of AMPK with some of the consequent

intracellular events converging with the systemically driven insulin receptor pathway. AMPK

stimulates phosphorylation of p53, ACC1 and ACC2, inhibits aromatase enzymes, directly and

indirectly inhibits mTOR itself stimulated by key drivers for cancer (HER2, PIK3CA, PTEN) and hence

curtails S6kinase activity and its consequences. Additional potentially important effects, for example

on the stroma and the complexities of subcellular networks are not illustrated.




© 2014 American Association for Cancer Research

ColonSkeletalmuscle

OCT

OCT1 ATP

ADPAMP

Complex1

OCT

AMPK

Gluconeogenesis

Liver

Increasedglucoseuptake

Aromataseenzymes

StabilizeHIFIα

äGlycolyticpathways

Cell cycleamount

LKB1

AMPK

p53

AKT

TSC2HER2

amplification

mTOR

PTEN reducedexpression

Protein synthesisGrowth

Proliferation

Fatty acidsynthesis

Fatty aciduptake +

β-oxidation

Cell membrane

Mitochondrion

ACC1

ACC2 S6K

PTEN mutation

PIK3CA mutation

PI3K

IR

InsulinIGFs

GlucoseM

M

MM

Figure 1:

P

P

P




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