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Contents lists available at ScienceDirect

Current Problems in Cancer

journal homepage: www.elsevier.com/locate/cpcancer

Metformin in breast cancer: preclinical and

clinical evidence
Anindita De, PhD1,∗, Gowthamarajan Kuppusamy, PhD1
Department of Pharmaceutics, JSS College of Pharmacy, Ooty, India

a b s t r a c t

Metformin, a well-acknowledged biguanide, safety profile and multiaction drug with low cost for manage-
ment of type 2 diabetes, makes a first-class candidate for repurposing. The off-patent drug draws huge
attention for repositioned for anticancer drug delivery recently. Still few unanswered questions are chal-
lenging, among them one leading question; can metformin use as a generic therapy for all breast cancer
subtypes? And is metformin able to get over the problem of drug resistance? The review focused on the
mechanisms of metformin action specifically for breast cancer therapy and overcoming the resistance; also
discusses preclinical and ongoing and completed clinical trials. The existing limitation such as therapeutic
dose specifically for cancer treatment, resistance of metformin in breast cancer and organic cation trans-
porters heterogeneity of the drug opens up a new pathway for improved understanding and successful
application as repurposed effective chemotherapeutics for breast cancer. However, much more additional
research is needed to confirm the accurate efficacy of metformin treatment for prevention of cancer and
its recurrence.
© 2019 Elsevier Inc. All rights reserved.

a r t i c l e i n f o

Keywords: Metformin; Breast cancer; AMPK; Clinical trials; Limitations

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Molecular mechanisms of metformin for management of breast cancer . . . . . . . . . . . . . . . . . 3
Why metformin is special? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Metformin to overcome the drug resistance in breast carcinoma . . . . . . . . . . . . . . . . . . . 5

∗
Corresponding author.
E-mail addresses: aninditanirupa@gmail.com (A. De), gowthamsang@jssuni.edu.in (G. Kuppusamy).
1
These authors contributed equally.

https://doi.org/10.1016/j.currproblcancer.2019.06.003
0147-0272/© 2019 Elsevier Inc. All rights reserved.

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The efficiency of metformin is glucose-dependent . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 6

Combination therapy of metformin for the breast cancer: preclinical evidence . . . ... . . 7
Therapy of metformin for breast cancer: clinical evidences . . . . . . . . . . . . . . . . . . . ... . . 9
The challenges of metformin for management of breast cancer. . . . . . . . . . . . . . . . ... . . 9
Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 9
Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 11
Targeting EMT/CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 11
Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 11
OCT1 heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 13
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 13
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 14

Introduction

Metformin, a well-acknowledged biguanide, is the first line drug for the management of the
type 2 diabetes mellitus (TDM2) prescribed to over 120 million patients worldwide. The oral
glucose-lowering agent very effectively reduces the hepatic gluconeogenesis, improves insulin
sensibility by enhancing peripheral glucose uptake and lowering the basal and postprandial
plasma glucose.1 This century-old drug first approved in UK on 1958 and it is on the World
Health Organization’s list of essential medicines.2 The antidiabetic drug is considered one of the
safest glucose-lowering agents with no hypoglycaemia.3 The most common side effect is less
than 5% of the patients found with diarrhea, nausea, flatulence, indigestion, abdominal pain, fa-
tigue, and headache. Lactic acidosis is reported with few patients but the condition can easily be
overcome by renal function monitoring. Metformin also reported with anti-inflammatory,4 anti-
apoptotic, anti-cancer, hepatoprotective, cardioprotective,5 otoprotective,6 renoprotective,7 radio-
protective, radiosensitizing, and antioxidant activities.8 Well tolerated safe profile and multiac-
tion drug with low cost make metformin a first-class candidate for repurposing. Many preclinical
and clinical investigations already prove the excellence of the drug for the management of al-
most all type of cancers.
Breast cancer is the most commonly occurring cancer in female and the second most usual
cancer overall. There were near about 2 million new cases in 2018.9 Breast cancer is consid-
ered a genetic disorder due to the mutations and genetic dynamic alteration and changes. The
metabolic pathway alteration of breast cancer from that of the normal cells is one of the leading
causes of mutation which provides a large amount of energy for uncontrolled cell growth and
genetic alterations. The metabolic reprogramming of the breast cancer cells broadly explained
by the metabolic shift from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic gly-
colysis first coined by the Nobel laureate Otto Heinrich Warburg in 1924 known as Warburg
effect.10 Since Warburg observed that cancer cells have a high rate of aerobic glucose uptake
and empathized that cancer cells having a distinguishable metabolic phenotype which opens
new therapeutic strategies.11
In the typical Warburg effect, the cells demand oxygen to burn fuel like glucose to gener-
ate energy for rapid proliferation. Wherever the oxygen supply is limited cells adapted to the
new environment and use fermentation for the fuel and grow uncontrollably, avoid cell death,
and causes DNA mutation. Recent research in genetics investigated that alteration of the breast
cancer cell metabolism gives the breast cells immortality. The investigation focused specifically
for cell metabolism on and off and makes the cancer cells hungry. Research specifically focused
on blocking the proteins that pump this key nutrient into the tumor cells for the treatment of
breast carcinoma.
Recently the antidiabetic drug metformin shows a great potential to alter the metabolic
reprogramming and act as a candidate for cancer management. This initially concluded from
a series of the epidemiological research which suggested that metformin inhibits mammalian

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target of rapamycin-dependent translation initiation in breast cancer cells.12 The signal trans-
duction that regulates Warburg effect during tumorigenesis altered by metformin which has
the ability to inhibit glucose metabolism in cancer cells and offer a benefit by limiting energy
resources and cancer cells proliferation and rapid growth. A meta-analysis on the early stage
breast carcinoma patient taking metformin suggested that diabetic patients receiving neoad-
juvant chemotherapy having the response rate greater (24% in the metformin group vs 8% in
the nonmetformin group, P = 0.007) compared with patients on other diabetes drugs.13 Hirsch
et al (2009) shows that metformin had a synergistic activity along with the chemotherapeutic
agents specifically targeting the breast cancer stem cells in in vitro and in vivo model.14 Cut
down circulating insulin might regulate cancer development and stage at diagnosis in a female
with diabetes who have breast cancer. Department of Medicine, University of Toronto did
an observational study between 2007-2012 shown a lower risk of breast cancers in women
with diabetes treated with metformin.15 This safe and economical drug led to a large number
of preclinical and clinical investigations with or without a chemotherapeutic agent for the
alteration of breast cancer metabolism. This review provides evidence-based data to establish
repositioning metformin for breast cancer. The article also highlights the existing challenges for
the drug in the path of repositioning in cancer management.

Molecular mechanisms of metformin for management of breast cancer

The molecular mechanism for metformin for management of breast cancer is well studied
and reviewed in many clinical and preclinical studies. The anticarcinomas activity of the drug
follow both indirect and direct pathway shows in Fig. 1.16 The reprogrammed metabolism con-
firming cancer cell proliferation and survival leaves cells dangerous for therapeutic strategies.
Metformin enhances glucose uptake of the muscle which reduces plasma insulin level and helps
in the reduction of both preneoplastic and neoplastic cells proliferation.12 The indirect path is
the insulin-lowering activity to reduce the carcinoma cell proliferation via insulin/IGF-1 path-
way whereas the direct pathway target tissue respiratory complex I of the electron transport
chain in mitochondria to reduce the energy intake in cells. Both the pathway activates the AMP-
activated protein kinase to suppress the mammalian target of the rapamycin (mTOR) which is
responsible for the carcinomas cell proliferation, inducing apoptosis and cell-cycle arrest.17 The
multiple studies support both the pathways of the metformin mechanism. Goodwin et al,18 De-
partment of Medicine, University of Toronto conducted a clinical trial with the 32 women with
early breast carcinoma to study the consequence of metformin on the reduction of the insulin
level. The study concluded that metformin significantly lowers insulin levels, and it improves
insulin resistance in nondiabetic women with breast cancer. Dowling et al12 explain the indi-
rect pathway of metformin for the treatment of breast cancer by lowering serum insulin level
and other tumor genesis-related components such as growth factors, sex hormones, inflamma-
tory cells, cytokines, and metabolic intermediates. Studies by Zhuang et al19 showed that low
glucose environment facilities the action of the metformin and concluded that defect in glucose
metabolism and mitochondrial malfunction sensitize metformin action for anticancer activity.
The main focus of the antidiabetic drug is to cut down glucose synthesis via the AMPK path-
way to restrict the energy flow to carcinoma cells and make cancer cell hungry to suppress
proliferation and immortality.

Why metformin is special?

Breast cancer is not anymore the disease of the woman alone even the male also suffering
from this deadly disease.20 The resistance of the existing drugs is challenging for scientist and
boost for searching for new approaches. The molecular level study of breast cancer found differ-
ent energy pathways for the mutation of the gene which gave scientist a new thought to targets

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Fig. 1. The mechanism of action of the metformin fallows basically two pathways. In the direct pathway the drug creates
the energy stress by blocking the complex 1 of the mitochondria and activates the AMPK which leads to the catabolic
downstream along with the cancer cells proliferation inhibition. In the indirect pathways, the AMPK activation in the
liver causes the reduction of the circulating insulin and inhibit the IGF1/phosphoinositide 3-kinase (PI3K) axis in tumor
cells resulted in the inhibition of the cancer cell proliferation via mTOR pathways.

cancer cell metabolism. The old drug metformin in the new approach for the management of
breast cancer was first reported in 200521 and till date, the number of article and preclinical and
the clinical studies are in increase direction. The reason, metformin is one of the best choices
for the repositioned anticancer drug because:
1. The drug is off patent science 200222
2. Easy synthesis in an economical manner enhances global acceptance. The synthesis of met-
formin involves the one-pot reaction of dimethylamine hydrochloride and 2-cyanoguanidine
overheat.23
3. Well-tolerated side-affects which actually positively beneficial for cancer treatment.
4. Metformin works at a whole organism level mainly by reducing the insulin levels which
actually benefited for a cancer cell to make it hungry and produce an energy stress.
5. Metformin even directly work on tumor, by altering energy metabolism which is a distin-
guishing factor in cancer from normal cells and is crucial in sustaining their ability to survive
and proliferate.24

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6. The physical property of metformin itself is anti-neoplastic.25 The hydrophilic characteris-

tics of the drug felicitate direct transport through the cell membrane and action via organic
cation transporters (OCT), plasma monoamine transporter, and multidrug and toxin extrusion
protein.
7. The expression of the drug-specific receptor has shown down-regulated in the breast
carcinoma tissue equated with the non-carcinomas tissues of the surrounding tumor.

Metformin uses for management of breast carcinoma basically along with other chemother-
apeutics but recently the monotherapy of the drug is also in research basically for the Triple
Negative Breast Cancer (TNBC).26 , 27 Even few positive feedbacks obtained from the ER+ and
ER-breast cancer cell lines cell cycle arrest of monotherapy.28 The increasing no of clinical tri-
als shows promising and positive direction for breast cancer with metformin along with radi-
ation and other therapeutics. Though metformin proves its importance in the management of
breast cancer still few questions is unknown. One of the leading questions that bother the sci-
entist the most is; can metformin use as a generic therapy for all breast cancer subtypes? And is
metformin able to get over the problem of drug resistance?

Metformin to overcome the drug resistance in breast carcinoma

Cancer recurrence and chemoresistance are the main challenges for the cancer scientists in
recent time for therapy. The carcinoma cells acquire resistance structurally and mechanically for
almost all existing first-line drugs like doxorubicin, paclitaxel, docetaxel, and vinca alkaloids in
certain conditions.29 Studies also suggested that high glucose concentration is key factors for
rapid cell proliferation due to abandoning nutrition for the cells.30 It is a known fact that un-
controlled cell growth leads to carcinoma.31 Hyperglycemia per se has a direct impact on car-
cinomas cell proliferation, apoptosis, and metastasis.32 Patients with coexpression of IGF1R and
multidrug resistance-associated protein 1 (MRP1) have shown a poorer response with anticancer
agents.33 HER2-negative carcinoma reduces the expression of IGF1R, was correlated with a better
response to the therapy.34-36 Although the function of IGF1R in resistance been confirmed, the
exact mechanism of metformin for overcoming resistance is not very clear till date. Metformin
inactivates IGF signaling pathway by reducing the blood insulin to show antitumor effect. This
hypothesis was supported by a number of preclinical studies.37-39 Goodwin et al18 presented
that, in breast cancer patients without DM, treated with metformin may significantly cut down
insulin levels and improve resistance by activating AMPK via both direct and indirect way.40
Again the molecular level study suggested that overexpression of drug transporters on
surface of cancer cells is another cause of drug resistance.41,42 Among all receptors, ATP binding
cassette (ABC) family members are responsible for drug efflux of almost all chemotherapeutics
and intercellular drug accumulation.43 The research already has proven that the drug-resistant
cancer cells exhibit higher ATP demand and in this point, the biguanides act to disrupt the
supply of energy to suppers the function of the drug pump which facilitates the treatment of
chemoresistant tumors.44 The mechanism of metformin is to inhibit respiratory chain complex I
to diminish the energy render from mitochondria which leads to depletion of ATP and activates
AMPK to inhibit mTOR, which limits the synthesis of biomolecules like protein or nuclear acid,
which basically responsible for cell growth.45 Most of the chemotherapeutics induced resistance
by overexpressing the ABCB1 drug transporter.46 ABCB1 encoded P-gp is up-regulated in many
drug-resistive tumors and responsible for efflux of different drugs. Suppression of P-gp expres-
sion is viable to reverse multidrug resistance. Various clinical and preclinical studies proved that
metformin impairs P-gp function by reducing ATP production. The function leads to increased
intercellular accumulation of chemotherapeutic drugs to produce cytotoxicity.47 Research also
suggested that metformin inhibit ABCB1 expression on mRNA and protein levels facilities
anticancer drug to act synergistically.48 Tamoxifen, one of the leading molecules for treatment
of breast cancer, the resistance overcome by the combination with metformin, is already
shown a positive direction.49,50 In another preclinical research by Qu et al,51 the researcher

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Fig. 2. Metformin to overcome the drug resistance. Basically, the inhibition of respiratory chain complex I and suppres-
sion of the ATP play the most significant role in overcoming the resistance. Apart from that reducing blood insulin and
reverse epithelial-mesenchymal transition other significant characteristics of the drug.

proved that the combination of 5-FU along with metformin induce apoptosis; reverse epithelial-
mesenchymal transition (EMT) which enhanced the capacity of cells for invasion, metastasis,
and chemoresistance. Again in the same year Cioce et al52 studied resensitizing property of
metformin for overexpressing aldehyde dehydrogenase (ALDH), an enzyme responsible for
chemoresistance in breast cancer cells also feature an EMT phenotype. Li et al53 studied the ef-
fect of metformin along with doxorubicin in MCF7 and ADR cell lines. The finding indicates that
metformin was very much capable to alter the acquired multidrug resistance during prolonged
doxorubicin treatment. The huge no of the preclinical and clinical evidence proves that even
the drug selectively eliminates cancer stem cells (CSCs), responsible for intrinsic drug resistance
and protracts absolution in multiple cancer cell types. The finding of the research suggests that
metformin is able to resensitize and reversed drug resistance for better management of breast
cancer. The drug resistance overcome mechanism of metformin is summarized in Fig. 2.

The efficiency of metformin is glucose-dependent

High glucose levels induce tumor growth and are linked with resistance to chemother-
apy and metformin play a very significant role in overcoming drug resistance issue. But
recent studies suggested that pharmacological activity of the drug highly depends on the
glucose concentration on site of action. Multiple studies already show that antiprolifera-
tive and apoptosis action of metformin is glucose concentration dependent. Menendez et al
had described that metformin was “synthetically lethal” in the absence of glucose in breast
carcinoma. They pinpointed that glucose deprivation is a classifiable feature of the tumor mi-
croenvironment and contend that, in combination, hypoglycemia and metformin will accelerate
apoptosis.54 Menendez et al54 suggested that glucose was a critical factor for cancer cell apop-
tosis. Under high-glucose circumstances, metformin causes only cell proliferation arrest, but in
glucose withdrawal stress, metformin eluded the ability of oncogenes to protect carcinoma cells
from glucose-deprivation apoptosis. The results indicated that significantly the cell models of
breast carcinoma go through about 90% massive apoptosis glucose-starved cell cultures in the
presence of metformin. Wahdan-Alaswad et al (2013) did a cell line based study and found
that supraphysiologic glucose cuts down the potency of metformin in different molecular sub-
types of carcinoma cells, but may be getting over by using high doses of metformin. Instead of,

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maintenance of glucose homeostasis may better metformin affectivity in some patients. The re-
search supported that metformin brings on a broad spectrum of biological and molecular effects
against breast carcinoma cells and may decrease cellular response to critical factors of tumori-
genesis, including insulin, interleukin 6 (IL-6), and epidermal growth factor (EGF) via downregu-
lation of IGF-1R, (p-Signal transducer and activator of transcription 3) p-Stat-3, and EGFR.55 On
the note of finding the study suggested that the failure to hold the glucose homeostasis may
encourage more aggressive breast carcinoma phenotype. Again Zordoky et al56 analyzed that an
excess amount of glucose in triple negative MDA-MD-231 cell line prevent cell death by met-
formin. The researcher hypnotized that the mortality of the cancer cells was bypass due to the
excess glucose which able to produce enough energy for cell proliferation through aerobic gly-
colysis. Recently in 2018, Wahdan-Alaswad et al57 reported that glucose promotes phenotypic
aggression of carcinoma cells and reduces metformin efficacy. They also demonstrated that met-
formin block several key enzymes necessity to glucose metabolism in TNBC but the efficacy
highly depends on the concentration of glucose on site of action. From the preclinical research
data, it is very clear that glucose monitoring for breast cancer patients is the crucial factor and
metformin pharmacological activity highly depends on it for overcoming the drug resistance and
better therapeutics.

Combination therapy of metformin for the breast cancer: preclinical evidence

Researchers investigated that combination therapy with chemotherapeutics; metformin en-

hances their efficacy as well as reduces adverse effect with a reduction of resistance and
dose.14,27,58 Jiralerspong et al13 first reported the case-control clinical trial of 923 T2DM pa-
tients in the UK and found a 23% reduction in the risk of growing cancer after using metformin.
Science then, the combination therapy is changing the treatment strategy of cancer. In another
meta-analysis using 18 experimental studies and 561,836 patients disclosed that metformin used
along with chemotherapeutics related with overall 27% reduction in the risk of arising from
breast cancer. Rocha et al59 observed that the combination with metformin and paclitaxel sig-
nificantly inhibited cell viability and increased cells arrested in the G2-M phase of the cell cycle.
The study also reveals that AMPK signaling was involved in this process in MCF-7 and A549
cell lines and responsible to overcome paclitaxel resistance. In a very recent study in xenograft
mouse models of breast, lung and prostate cancer, metformin had a synergistic effect when com-
bined with paclitaxel, carboplatin or doxorubicin. Not only chemotherapeutic Sahra et al60 also
showed that metformin combines with 2-deoxyglucose (2-DG) effectively inhibit mitochondrial
respiration and glycolysis through tumor p53-dependent apoptosis via the AMPK pathway. Rico
et al61 combine the antidiabetic drug, metformin, plus propranolol for the treatment of TNBC
in an effective manner with no symptoms of toxicity. Again in the year of 2017 Rabah Rashad
Falah62 combined metformin and curcumin to treat breast cancer in mice model by targeting
angiogenesis inhibition, immune system modulation and induction of p53 independent apop-
tosis showed a promising effect. Xintaropoulou et al63 studied metformin efficacy combination
with of 5 glycolysis pathway molecules inhibitors. This preclinical study very clearly indicated
the therapeutic efficacy of metformin in the combination therapy which basically based on the
glycolytic pathway and increased the sensitivity of inhibitors under normoxic conditions. The in
vitro and in vivo evolution of metformin study in the monotherapy or with combination ther-
apy showed that it inhibits the growth of the breast cancer cells and exhibits a synergistic ac-
tion with vascular endothelial growth factor receptor inhibition as well as EGFR inhibitor to
reduce the cell proliferation in xenografted cell line models.64 The preclinical study also showed
that the radiosensitivity65 of the chemotherapeutic increases a lot when given combined with
metformin. The recent trend of immunotherapy66 proposed that metformin have the immune
modulatory characteristics by inhibiting the immune exhaustion of CD8+ tumor-induced lym-
phocytes (TIL) and enhancing T cell-mediated immune response to the tumor. In a recent study,
the researcher introduces the metformin after the vaccination found an elevated CD8+ mem-
ory T cells which conferred protective immunity upon subsequent tumor challenge.67 Table 1

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Table 1
Combination therapy of metformin for management of breast cancer: preclinical evidence

Chemotherapeutics Mechanism of Action References

11 , 77 , 78 , 53 , 79 ,
Doxorubicin (DOX) Combination therapy inhibits nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κ B) activity in tumor cells, which further decreased 80 , 27

tumor necrosis factor-α (TNF-α ) and L-6 expressions in tumor tissues

and suppressed tumor cell proliferation and enhance apoptosis. It’s
simultaneously targeted tumors and cancer-related inflammation. The
therapy enhanced nuclear DOX accumulation and overcame drug
resistance by down-regulating Pgp and intracellular ATP content in a
reduced dose
DOX+ 2 DG Combination therapy started strong metabolic stress in low dose through 81

inhibiting glucose uptake, lactate, fatty acid and ATP production. The
therapy reversing Multidrug-resistant (MDR.
DOX+ 2FDG Increase Phospho-AMPK by combination but decrease phospho-Akt and 82

phospho-ERK expressions.
50
Tamoxifen Combination synergistically inhibits proliferation, DNA replication and
trigger apoptosis with reduced dose involving in BAX/BCL-2 apoptotic
pathway and AMPK/mTOR/p70S6 growth pathway.
83
Paclitaxal Co-delivery synergistically induced higher cytotoxicity and apoptosis via
Toll-like receptor (TLR) signaling by activation of TLR-MyD88-ERK which
responsible for tumor growth, progression, metastasis and drug
resistance.
84
5-Fluorouracil, Metformin accelerated glucose consumption and lactate production in
epirubicin, and breast CSCs and severely hampered intracellular ATP. Combination
cyclophosphamide leading to a severe energy crisis induced DNA damage
85 , 62
Curcumin Combination exhibited dose-dependent cytotoxicity and antiproliferative
activity by decline hTERT expression, reduced VEGF expression, induced
Trp53 independent apoptosis and triggered Th2 immune response.
86
Ursolic acid Co-drug significantly inhibited and migration of Transforming growth
factor–β (TGF-β at low concentration and down-regulation of CXCR4,
uPA, vimentin, E-cadherin, N-cadherin, and MMP-2/9 proteins expression
and also regulate AMPK signaling.
2 DG Combination therapy enhanced γ H2A.X foci formation by 50 μ Ci (131I, 65 , 87 , 88 , 89

decrease cell viability and increase apoptosis. Combination increase in

intracellular oxidation, autophagy and completely inhibit colony
formation by activation of AMPK
90 , 91
Everolimus Combination inhibited cell survival, clonogenicity, mTOR signalling activity
and mitochondrial respiration and abrogated S6 and
4EBP1phosphorylation.
64
Erlotinib Combination enhanced reduction of EGFR, AKT, S6 and 4EBP1
phosphorylation, with inhibition of mammosphere outgrowth.
74 , 92
Aspirin Aspirin and metformin enhanced 4T1 cell apoptosis by inducing secretion
of TGF-β 1. The activity of combination partly relied on cyclooxygenase-2
(COX-2) up-regulation, without production of lipoxins.
Aspirin + Atenalol Combination therapy increased angiogenesis in local and metastatic 93

growth of HER2+ and TNBC both in immune-deficient and

immune-competent models. Therapy reduces endothelial cell component
of tumor vessels inducing apoptosis via AMPK
61
Propranolol Combination inhibits glucose metabolism through ADRB2-dependent
posttranscriptional down-regulation of hexokinase-2 and activation of
AMPK along with the anti-oxidation activity.
94 , 95
DCA DCA and metformin synergistic induction CASPASE-dependent apoptosis
via oxidative damage through PDK1 inhibition and diminishes lactate
production. Expression of glycolytic enzymes including HK2, LDHA and
ENO1 down-regulated to induced cell death
96
Topotecan Metformin active AMPK and down-regulates excision repair
cross-complementation group 1 and Topotecan suppress DNA replication
by inhibiting nuclear enzyme topoisomerase I. Combination depolarize
mitochondrial membrane and induced cell cycle arrest.
97
Vitamin D3 The combination activate expression of cleaved CASPASE-3, BAX and AMPK
and inhibit BCL-2, c-Myc, IGF-IR, mTOR, P70S6K and S6.
(continued on next page)

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Table 1 (continued)

Chemotherapeutics Mechanism of Action References

98
Spautin-1 Autophagy inhibitor and mitochondrial complex I inhibitor effective in
limiting oxidative respiratory capacity, colony formation and tumor
growth.
99
Silibinin (SIL) Combination synergistically down-regulated expression levels of hTERT and
cyclin D1 and enhance therapeutic efficacy
100
Chrysin Combination synergistically reduce cyclin D1 and hTERT gene
101
Flavone Combination decrease protein expression of MDMX, regulate p53
downstream target genes BCL-2 and cleaved CASPASE-3.
102
Melatonin Combination reduces tumor by increased expression of BAX and
CASPASE-3 and inhibit DMBA-induced tumor growth.
66
Denosumab Metformin prevents BRCA1 haploinsufficiency-driven RANKL gene
over-expression and disrupting an auto-regulatory feedback control of
RANKL-addicted CSCs. Synergistic therapy decline breast cancer-initiating
cell population and self-renewal capacity
103
Lily-polysaccharide LP1 down-regulate BCL-2 expression and metformin up-regulated BAX
1 (LP1) enhances anti-proliferation and apoptosis

contains the preclinical evidence of the therapeutic efficacy of metformin in combination

therapy for breast cancer.

Therapy of metformin for breast cancer: clinical evidences

Presently 11 ongoing and 13 completed clinical trials are in different stages fully focusing to
study the efficacy of metformin as monotherapy or in combination with chemotherapy and/or
radiotherapy exclusively for the management of breast cancer. The ongoing (Table 2) and the
completed clinical trials (Table 3) basically focused on the process of establishing the effects of
metformin on markers of cellular proliferation, pathological response rate, progression-free sur-
vival, tolerated, safe dose, and recurrence-free survival for breast cancer. The most ongoing trails
focused to study the combination therapy of the metformin with the other chemotherapeutics
for locally advanced breast cancer or reduction of obesity-related breast cancer along with the
study of different biomarker and toxicity studies. The completed clinical trials finished with
the primary outcome of the safety efficacy of metformin in the combination or individual for
the treatment of breast cancer.

The challenges of metformin for management of breast cancer

Through metformin shows excellent activity against the carcinoma condition still few chal-
lenges irrespective of drug, dose and delivery bother the research scientist for repositioning the
drug in breast cancer management.

Dose
Basically, the antidiabetic drug, act on the cancer cells via direct and indirect way.68 As per
the in vitro studies, the dose for AMPK activation is 5 to 10 mM whereas patients with T2DM
required around 10 μM and can reach as high as 40 μM.69 But research showed that such low
concentrations are not sufficient to cause AMPK activation, although metabolic alteration can be
evoked.1,70 In this scenario, the scientist focuses on the other activator with different mechanics
to put off the limitation of low dose. Other pharmacological agents may be used together with
metformin for enhancing the therapeutic efficacy of the drug71,72 still the effective and safe pro-
file dose for breast cancer is a great challenge.

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Table 2
Therapy of metformin for the management of the breast cancer: ongoing Clinical trial evidence

Phase Study Design Primary Objective Registration no on

ClinicalTrials.gov
(https://clinicaltrials.gov/,
accessed December 6, 2018

Phase II 500 mg/m2 of 5- Fluorouracil, Study combination effect of NCT02506777

Doxorubicin and
Cyclophosphamide in
combination with 850 mg/m2
metformin for 21 days for 32
female patients above the age
18
Phase II 850mg/m2 metformin BID for 4
weeks 150 Premenopausal
obese women 21-54 age
Phase II Combination therapy of
multicenter Toremifene 60 mg/m2 and
randomized study metformin 850 mg/m2 BID 96
women patient above the age
of 18
Early phase 1 Combination therapy of 80 mg
Atorvastatin at bedtime with
Metformin 500 mg in morning
and 10 0 0 mg in night for 2
weeks QD for 40 women with
operable DCIS breast carcinoma
had core needle biopsy
followed by surgical excision
above the age of 21
Phase II 500mg/m2 Metformin and 2.5
mg/m2 Letrozole QD for
22weeks for 208 women
patients with ER+ breast
cancer between 18 -80 years.
Phase II Combination therapy of
Taxotere 75mg/m2, Carboplatin repurposed of metformin for
Herceptin 6mg/kg Pertuzumab
the chemotherapeutics for
locally advanced breast
cancer.
Study effect of metformin for NCT02028221104
reduction of obesity-related
breast cancer and breast
density along with serum
IGF-1 to IGFBP-3 ratio
Study the NCT02506790
pathomorphological and
toxicological effect of
combination therapy
Study Pre-Surgical Trial of NCT01980823
combination therapy for
newly diagnosed operable
breast cancer along the
change in proliferation
marker Ki-67 in breast tissue
level
Study and compare the NCT01589367105
efficacy of neoadjuvant
therapy of combination vs
Placebo and mono-therapy
along with toxicity profile.
The trail the also investigates
response rate of
breast-conserving surgery
and mammographic density
alter in Ki67 %
Study focus to proof NCT03238495
HER + breast cancer as
2

420mg/m2 with 850 mg/m2 adjuvant therapy after

Metformin for 3 weeks 6 cycles Pathologic complete response
100 patient with cT1c-cT4a-d surgery
node without metastases
women above age of 18
Phase II Combination of 50 mg/m2 Trail focus to study the NCT02488564
randomized trail intravenous infusion efficacy for operable and
st
doxorubicin for 1 day, 30 locally advanced HER2+
mg/m2 Docetaxel on day 2 and breast cancer to evaluate pCR
9 and 4 mg/kg Trastuzumab on and systemic tolerance, with
day 2, 9, 16 along with particular attention to cardiac
Metformin 10 0 0 mg on day 13 toxicity of combination
to 11 for both male and female therapy
HER2 over-expressing 46 cancer
patient age group of 18-75
(continued on next page)

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Table 2 (continued)

Phase Study Design Primary Objective Registration no on

ClinicalTrials.gov
(https://clinicaltrials.gov/,
accessed December 6, 2018

Phase II Metformin 850mg/m2 BID and
Simvastatin 20mg/m2 QHS 28
women 18 years or older with
metastatic or locally advanced
breast cancer
Phase III Monotherapy of metformin for
24 months 400 women had
prior biopsy demonstrating
atypical hyperplasia, lobular in
the age group of 18-55
Phase II Co-delivery of 500 mg/m2
metformin TID along with 75
mg/m2 Docetaxel and
Epirubicin, 500 mg/m2
cyclophosphomide for 21 days
200 women patient with life
span expectancy > 12 months
aged between18- 70.
Phase II Metformin 500 mg/m2 along
with Doxycycline 74 women
localized breast or uterine
cancer above the age of 18
Study the Clinical benefit rate NCT03192293106
(CBR) and toxicity profile of
combination therapy for ER+
breast cancer
Study the efficacy of NCT01905046
monotherapy for prevention
and alteration of RPFNA or
blood biomarkers of Atypical
Hyperplasia in unilateral or
bilateral RPFNA aspirates
breast cancer.
Study pCR after combination NCT01929811
therapy
Study the Change in stromal NCT02874430
cells expressing Caveolin-1 at
an intensity of 1+ by
immuno-histochemistry after
combination therapy

Sites
The oral hypoglycemic drug specifically accumulates in the small intestine and secondly in
stomach, colon, kidney, and liver.73 For breast cancer response of which comparatively deep and
distant sites, metformin is a challenge via oral delivery.

Targeting EMT/CSCs
The study proved that the CSCs are particularly sensitive to metformin. The CSCs are the hall-
mark for chemotherapeutic resistance and relapse of the disease.14 Current research had shown
that metformin inhibits EMT via multiple mechanisms including acting on Wnt/transforming
growth factor β /hedgehog/IL-674 signaling as well as miRNAs. Research reported that the com-
bination therapy was very much effective for prevent relapse and progression of cancer. The local
lymph nodes are more prone to metastatic condition, as a tiny amount of circulating tumor cells
with self-renewal properties may cause lethal metastatic later. To understand the need of pre-
ventive chemotherapy is necessary or not, very much difficult in this situation. Hence, it would
be wise to use no over-toxic metformin dose for preventive treatment after surgical resection
even in the absence of detectable metastasis.

Resistance
Drug resistance for cancer treatment is still a hinder. Metformin is no exception for the drug
resistance. Scherbakov et al75 presented one of the first evidence of the progression of cross-
resistance to metformin and tamoxifen in breast cancer cells due to constitutive activation of
Akt/Snail1/E-cadherin signaling. Alteration in the pathway of Akt/Snail1/E-cadherin may be able
to be the solution for the overcoming of Metformin resistance. Importantly, the finding opens
new perspectives to overcome the metformin resistance of breast cancer.

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Table 3
Therapy of metformin for management of breast cancer: completed clinical trial evidence
Phase Study Design
Phase II 850 mg/m2 metformin
combined with anthracyclines,
platinum, taxanes or
capecitabine BID for 9 weeks
for 78 women with metastatic
breast cancer
Phase II 500 mg/m2 Metformin for TID
2-3 weeks 40 women who
were about to undergo surgery
in the age of 18-54.
Phase -I Radioactive 400MBq
11C-metformin injected in
cubital vein for 10 male and
female above the age of 50 and
PET image took after 120 min
Phase -II Prior to Paclitaxel therapy 42
both sex patient aged between
18-75 with invasive breast
cancer, stage I – IV treated
with metformin of 500 mg QD
for 5 days and 500 mg BID for
5 days and 1,0 0 0 mg BID for 2
days till 12 weeks
A phase 2 1500mg extended release
single arm Metformin is given to 40
study women above age 18 with
locally advanced breast cancer
and tumor size more than 3
cm for 14-21 days
Phase II Prior administration
mono-therapy of metformin
and then combination of
Doxorubicin and metformin for
30 both sex patients age
greater than or equal to 21
years
Phase I Combination therapy of 850
mg BID metformin and 150 mg
of Erlotinib for 20 female age
between 18-79 years with
TNBC for 5 weeks.
Phase II Metformin QD for 2 weeks,
then BID for remain period of
time for 200 both sex patient
with stage I-III colorectal or
breast cancer above the age of
18
Please cite this article as: A. De and G. Kuppusamy, Metformin in breast cancer: preclinical and clinical evidence,
Current Problems in Cancer, https://doi.org/10.1016/j.currproblcancer.2019.06.003
[mUS1Ga;June 22, 2019;3:8]
Primary Outcome ClinicalTrials.gov Identifier
ClinicalTrials.gov
(https://clinicaltrials.gov/,
accessed December 6,
2018)
Result indicate that the NCT01310231107
combination therapy improves
survival and tumor response by
changing physiological
parameters like insulin, glucose
and Homeostatic model
assessment (HOMA)
The study concluded that NCT0089788412
taking metformin prior to
early-stage breast cancer
surgery reduces cell
proliferation and inhibit tumor
growth
Study confirmed the drug NCT02882581
transportation pathway
through OCT specifically
OCT1-3, MATE 1 &2 and PMAT
via quantitative polymerase
chain reaction (qPCR) as well
as PET imaging confirmed the
drug uptake by breast tissue
Study found a significant NCT02360059
changes specifically for
peripheral neuropathy
(CIPN)during the treatment
with Paclitaxel along with
metformin
Study indicated alteration of NCT01266486
S6K, 4E-BP-1 and AMPK via
immuno-histochemical analysis
and found to be effective to
alter breast cancer metabolism.
Combination therapy reduced NCT02472353
cardiac toxicity in breast cancer
patients by reducing tendency
of significant change in left
ventricle ejection fraction
Combination dose was well NCT01650506
tolerated in TNBC patients and
act as biomarkers to
discovering mechanism of
cancer cells for resistant
Trail concluded that NCT01340300
mono-therapy able to decrease
fasting insulin level. Even
changes in other insulin-related
biomarkers like C peptide,
IGFR, IGF binding protein-3
adiponectin, lectin also found
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Table 3 (continued)

Phase Study Design Primary Outcome ClinicalTrials.gov Identifier

ClinicalTrials.gov
(https://clinicaltrials.gov/,
accessed December 6,
2018)

Early phase 1 Metformin for 24 Monotherapy changed NCT01793948

postmenopausal women 18-75
years
Phase 1 500 mg metformin along with
25 mg Exemestane and 2 mg
Rosiglitazone once a day for 24
obese postmenopausal ER+
and PR+ women breast cancer
with metastatic condition
Phase II 500 mg Metformin QD for 1∼2
weeks followed by placebo in
morning and metformin in the
evening for 3∼24 weeks for
105 operable breast cancer
female patients above the age
of 20
Phase II 10 0 0 mg metformin along with
60 mg/m2 Myocet, and 600
mg/m2 Cyclophosphamide for
112 women with HER2- breast
cancer in age of 18-75
Phase I Combination therapy of
metformin and Temsirolimus
for 28 both sex patients with
histologically confirmed cancer
phosphorylation of proteins
and in ordinal level of breast
density decreases risk for
breast cancer in obese patients
The trial concluded that NCT00933309
combination dose was well
tolerated
Trail indicated that metformin NCT00909506
improve body condition
including weight loss for
overweight or pre-diabetes
mellitus breast cancer patient
The trial concluded that NCT01885013
combination therapy increases
progression-free survival as
well as characterization of
sensitivity in insulin levels
The maximum tolerated dose NCT00659568
for combination therapy
calculated based on antitumor
activity, tumor response rate
and time to progression

OCT1 heterogeneity
Metformin is a hydrophilic drug and the cellular uptake subject on OCT specifically OCT1.76
The heterogeneity of OCT1 expression in the cancer subtype significantly affects metformin sen-
sitivity. In this regard, surface or chemical modification of the anti-diabetic drug itself or a novel
carrier approaches is beneficial to improve the therapeutic efficiency of the drug.

Conclusion

There is a great hope surrounded the very common antidiabetic drug as a potential
anticancer candidate. The epidemiological evidence has linked metformin to decrease the risk of
cancer and cancer-related mortality. The preclinical date provides the encouragement for anti-
cancer mechanisms of metformin. The mTOR inhibition is the basis for the anticancer activity of
breast cancer. The preclinical study also reveals the use of metformin as an immunomodulatory
agent for cancer cells by increases effect for memory T cell population through phenotype
switching of CD8+ TILs. The adjuvant therapy of metformin with cancer vaccine (LmOVA) is
used to increase the number of CD8+ memory T cells that bestowed immunity to carcinoma
cells. The breakthrough research of the immunotherapy for TNBC with checkpoint blockade
monoclonal antibodies (anti-PD-1/L1) has brought forth excitation in the cancer world. The
immunomodulatory attributes of metformin still in the research stage and one of the most in-
teresting field for the oncology scientist. The clinical trial data not only suggested metformin as
therapeutics for breast cancer also has shown that metformin can significantly impact markers

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of tumor proliferation. A total no of 11 ongoing and 13 completed clinical trials has using various
surrogate markers to assess proapoptotic effects of metformin specifically for breast cancer. For
the future clinical trials, the scientist should focus on the different adjuvant agents with met-
formin to have better outcomes in advanced metastatic breast cancer as well as to overcome the
limitation of metformin as a mono-therapeutic. Additional data on survival indices from multiple
ongoing trials will be pivotal to draw a better conclusion. The side effect and the drug resistance
still concern the research scientist. With the evidence of the clinical data, clinicians can easily
assure metformin as a tolerable and safe add-on to a chemotherapy regimen, and should not
limit its practical usefulness. Preclinical and clinical studies make it a very attractive candidate
for drug repurposing for cancer prevention. The advance pathway for cancer management
makes metformin a new approach for promoting signaling pathways. The effect of the glucose is
the key for breast cancer management with the metformin as well as dose and stem cells play
a significant role in the therapeutic efficacy. The heterogeneous nature of the disease makes the
cancer treatment difficult but preclinical and the clinical study strongly supports the use of met-
formin as a new hope for the treatment of breast cancer. Breast cancer is inherent and acquired
resistance to metformin is still to be explored. Much more additional research needed to confirm
the accurate efficacy of the metformin treatment for prevention of cancer and its recurrence
mono or in the adjuvant therapy. If large-scale clinical trials are able to testify to antitumor
results of metformin, this drug may become a valuable repositioned option for cancer therapy.

Acknowledgment

The authors would like to thank Department of Science and Technology – Fund for Improve-
ment of Science and Technology Infrastructure in Universities and Higher Educational Institu-
tions (DST-FIST), New Delhi for their infrastructure support to our department.

References
1. Zhou M, Xia L, Wang J. Metformin transport by a newly cloned proton-stimulated organic cation transporter (PMAT)
expressed in human intestine. Drug Metab Dispos. 2007;35:1956–1962.
2. Bailey CJ. Metformin: historical overview. Diabetologia. 2017;60:1566–1576.
3. Viollet B, Guigas B, Garcia NS, Leclerc J, Foretz M, Andreelli F. Cellular and molecular mechanisms of metformin: an
overview. Clin Sci. 2012;122:253–270.
4. Scheen A, Esser N, Paquot N. Antidiabetic agents: potential anti-inflammatory activity beyond glucose control. Dia-
betes Metab. 2015;41:183–194.
5. Calvert JW, Gundewar S, Jha S, et al. Acute metformin therapy confers cardioprotection against myocardial infarction
via AMPK-eNOS–mediated signaling. Diabetes. 2008;57:696–705.
6. Mujica-Mota M. Otoprotection of Metformin in Radiation-Induced Sensorineural Hearing Loss. McGill University; 2012.
7. Nasri H, Baradaran A, Ardalan M, Mardani S, Momeni A, Rafieian-Kopaei M. Bright renoprotective properties of
metformin beyond blood glucose regulatory effects. Iran J Kidney Dis. 2013;7:423–428.
8. Chukwunonso Obi B, Chinwuba Okoye T, Okpashi VE, Nonye Igwe C, Olisah Alumanah E. Comparative study of the
antioxidant effects of metformin, glibenclamide, and repaglinide in alloxan-induced diabetic rats. J Diabetes Res.
2016;2016:1–5.
9. Brem RF. Radiofrequency Ablation of Breast Cancer: A Step Forward. Radiological Society of North America; 2018.
10. Warburg O. On the origin of cancer cells. Science. 1956;123:309–314.
11. Phan LM, Yeung S-CJ, Lee M-H. Cancer metabolic reprogramming: importance, main features, and potentials for
precise targeted anti-cancer therapies. Cancer Biol Med. 2014;11:1.
12. Dowling RJ, Niraula S, Chang MC, et al. Changes in insulin receptor signaling underlie neoadjuvant metformin
administration in breast cancer: a prospective window of opportunity neoadjuvant study. Breast Cancer Res.
2015;17:32.
13. Jiralerspong S, Palla SL, Giordano SH, et al. Metformin and pathologic complete responses to neoadjuvant
chemotherapy in diabetic patients with breast cancer. J Clin Oncol. 2009;27:3297.
14. Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together
with chemotherapy to block tumor growth and prolong remission. Cancer Res. 2009;69:7507–7511.
15. Giovannucci E, Harlan DM, Archer MC, et al. Diabetes and cancer: a consensus report. CA Cancer J Clin.
2010;60:207–221.
16. Camacho L, Dasgupta A, Jiralerspong S. Metformin in breast cancer-an evolving mystery. BioMed Central; 2015.
17. Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin is an AMP kinase–dependent growth in-
hibitor for breast cancer cells. Cancer Res. 2006;66:10269–10273.

Please cite this article as: A. De and G. Kuppusamy, Metformin in breast cancer: preclinical and clinical evidence,
Current Problems in Cancer, https://doi.org/10.1016/j.currproblcancer.2019.06.003
 ARTICLE IN PRESS
JID: YMCN [mUS1Ga;June 22, 2019;3:8]
A. De and G. Kuppusamy / Current Problems in Cancer xxx (xxxx) xxx 15

18. Goodwin PJ, Pritchard KI, Ennis M, Clemons M, Graham M, Fantus IG. Insulin-lowering effects of metformin in
women with early breast cancer. Clin Breast Cancer. 2008;8:501–505.
19. Zhuang Y, Chan DK, Haugrud AB, Miskimins WK. Mechanisms by which low glucose enhances the cytotoxicity of
metformin to cancer cells both in vitro and in vivo. PLoS One. 2014;9.
20. De A, Kuppusamy G, Karri VVSR. Affibody molecules for the molecular imaging and targeted drug delivery in the
management of breast cancer. Int J Biol Macromol. 2017;107:906–919.
21. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin and reduced risk of cancer in diabetic
patients. BMJ. 2005;330:1304–1305.
22. Gershell L. Type 2 Diabetes Market. Nature Publishing Group; 2005.
23. Werner EA, Bell J. CCXIV.—The preparation of methylguanidine, and of ββ -dimethylguanidine by the interac-
tion of dicyanodiamide, and methylammonium and dimethylammonium chlorides respectively. J Chem Soc Trans.
1922;121:1790–1794.
24. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nat Rev Drug Discovery.
2013;12:829.
25. Martin-Castillo B, Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA. Metformin and cancer: doses, mechanisms
and the dandelion and hormetic phenomena. Cell Cycle. 2010;9:1057–1064.
26. Currie CJ, Poole CD, Jenkins-Jones S, Gale EA, Johnson JA, Morgan CL. (2012) Mortality after incident cancer in
people with and without type 2 diabetes: impact of metformin on survival. Diabetes care. DC_111313.
27. Iliopoulos D, Hirsch HA, Struhl K. (2011) Metformin decreases the dose of chemotherapy for prolonging tumor
remission in mouse xenografts involving multiple cancer cell types. Cancer research. Canres. 3471.2010.
28. Yang Y. (2015) Targeting downstream effectors of IGF/insulin signaling system in human breast cancer.
29. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer.
Oncogene. 2010;29:4741.
30. Xue F, Michels KB. Diabetes, metabolic syndrome, and breast cancer: a review of the current evidence–. Am J Clin
Nutr. 2007;86:823S–835S.
31. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of
cell proliferation. Science.. 2009;324:1029–1033.
32. Ryu TY, Park J, Scherer PE. Hyperglycemia as a risk factor for cancer progression. Diabetes Metab J. 2014;38:330–336.
33. Ge J, Chen Z, Wu S, et al. Expression levels of insulin-like growth factor-1 and multidrug resistance-associated
protein-1 indicate poor prognosis in patients with gastric cancer. Digestion. 2009;80:148–158.
34. Sun HZ, Wu SF, Tu ZH. Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated
cytotoxicity. Cell Res. 2001;11:107.
35. Thomas F, Holly JM, Persad R, Bahl A, Perks CM. Fibronectin confers survival against chemotherapeutic agents but
not against radiotherapy in DU145 prostate cancer cells: Involvement of the insulin like growth factor-1 receptor.
Prostate. 2010;70:856–865.
36. Eckstein N, Servan K, Hildebrandt B, et al. Hyperactivation of the insulin-like growth factor receptor I signaling
pathway is an essential event for cisplatin resistance of ovarian cancer cells. Cancer Res. 20 09;69:2996–30 03.
37. Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer. 2008;8:915.
38. Karnevi E, Said K, Andersson R, Rosendahl AH. Metformin-mediated growth inhibition involves suppression of the
IGF-I receptor signalling pathway in human pancreatic cancer cells. BMC Cancer. 2013;13:235.
39. Gonzalez-Angulo AM, Meric-Bernstam F. Metformin: a therapeutic opportunity in breast cancer. Clin Cancer Res.
2010;16:1695–1700.
40. Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA. (2010) Metformin prevents tobacco
carcinogen–induced lung tumorigenesis. Cancer prevention research. 1940-6207. CAPR-10-0055.
41. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: an evolving paradigm. Nat Rev
Cancer. 2013;13:714.
42. Longley D, Johnston P. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–292.
43. Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview.
Adv Drug Deliv Rev. 2012;64:138–153.
44. Pernicova I, Korbonits M. Metformin—mode of action and clinical implications for diabetes and cancer. Nat Rev
Endocrinol. 2014;10:143.
45. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: from mechanisms of action to therapies. Cell Metab.
2014;20:953–966.
46. Fletcher JI, Haber M, Henderson MJ, Norris MD. ABC transporters in cancer: more than just drug efflux pumps. Nat
Rev Cancer. 2010;10:147.
47. Peng M, Darko KO, Tao T, et al. Combination of metformin with chemotherapeutic drugs via different molecular
mechanisms. Cancer Treat Rev. 2017;54:24–33.
48. Kim HG, Hien TT, Han EH, et al. Metformin inhibits P-glycoprotein expression via the NF-κ B pathway and CRE
transcriptional activity through AMPK activation. Br J Pharmacol. 2011;162:1096–1108.
49. Berstein LM, Yue W, Wang J-P, Santen RJ. Isolated and combined action of tamoxifen and metformin in wild-type,
tamoxifen-resistant, and estrogen-deprived MCF-7 cells. Breast Cancer Res Treat. 2011;128:109–117.
50. Ma J, Guo Y, Chen S, et al. Metformin enhances tamoxifen-mediated tumor growth inhibition in ER-positive breast
carcinoma. BMC Cancer. 2014;14:172.
51. Qu C, Zhang W, Zheng G, Zhang Z, Yin J, He Z. Metformin reverses multidrug resistance and epithelial–mesenchymal
transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells. Mol Cell Biochem.
2014;386:63–71.
52. Cioce M, Valerio M, Casadei L, et al. Metformin-induced metabolic reprogramming of chemoresistant ALDHbright
breast cancer cells. Oncotarget. 2014;5:4129.

Please cite this article as: A. De and G. Kuppusamy, Metformin in breast cancer: preclinical and clinical evidence,
Current Problems in Cancer, https://doi.org/10.1016/j.currproblcancer.2019.06.003
 ARTICLE IN PRESS
JID: YMCN [mUS1Ga;June 22, 2019;3:8]
16 A. De and G. Kuppusamy / Current Problems in Cancer xxx (xxxx) xxx

53. Li Y, Wang M, Zhi P, You J, Gao J-Q. Metformin synergistically suppress tumor growth with doxorubicin and re-
verse drug resistance by inhibiting the expression and function of P-glycoprotein in MCF7/ADR cells and xenograft
models. Oncotarget. 2018;9:2158.
54. Menendez JA, Oliveras-Ferraros C, Cufí S, et al. Metformin is synthetically lethal with glucose withdrawal in cancer
cells. Cell Cycle. 2012;11:2782–2792.
55. Wahdan-Alaswad R, Fan Z, Edgerton SM, et al. Glucose promotes breast cancer aggression and reduces metformin
efficacy. Cell Cycle. 2013;12:3759–3769.
56. Zordoky BN, Bark D, Soltys CL, Sung MM, Dyck JR. The anti-proliferative effect of metformin in triple-negative
MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: implications for cancer therapy
and prevention. Biochimica et Biophysica Acta (BBA)-General Subjects. 2014;1840:1943–1957.
57. Wahdan-Alaswad R, Edgerton S, Salem H, Thor A. Metformin targets glucose metabolism in triple negative breast
cancer. J Oncol Transl Res. 2018;4(1):129.
58. Liu H, Scholz C, Zang C, et al. Metformin and the mTOR inhibitor everolimus (RAD001) sensitize breast cancer cells
to the cytotoxic effect of chemotherapeutic drugs in vitro. Anticancer Res. 2012;32:1627–1637.
59. Rocha GZ, Dias MM, Ropelle ER, et al. Metformin amplifies chemotherapy-induced AMPK activation and antitumoral
growth. Clin Cancer Res. 2011;2243:010.
60. Sahra IB, Laurent K, Giuliano S, et al. Targeting cancer cell metabolism: the combination of metformin
and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells. Cancer Res. 2010:0 0 08–5472
CAN-09-2782.
61. Rico M, Baglioni M, Bondarenko M, et al. Metformin and propranolol combination prevents cancer progression and
metastasis in different breast cancer models. Oncotarget. 2017;8:2874.
62. Falah RR, Talib WH, Shbailat SJ. Combination of metformin and curcumin targets breast cancer in mice by angio-
genesis inhibition, immune system modulation and induction of p53 independent apoptosis. Therap Adv Med Oncol.
2017;9:235–252.
63. Xintaropoulou C, Ward C, Wise A, Marston H, Turnbull A, Langdon SP. A comparative analysis of inhibitors of the
glycolysis pathway in breast and ovarian cancer cell line models. Oncotarget. 2015;6:25677.
64. Lau Y-KI, Du X, Reyannavar V, et al. Metformin and erlotinib synergize to inhibit basal breast cancer. Oncotarget.
2014;5:10503.
65. Chatterjee S, Thaker N, De A. Combined 2-deoxy glucose and metformin improves therapeutic efficacy of sodi-
um-iodide symporter-mediated targeted radioiodine therapy in breast cancer cells. Breast Cancer. 2015;7:251.
66. Cuyas E, Martin-Castillo B, Bosch-Barrera J, Menendez JA. Metformin inhibits RANKL and sensitizes cancer stem
cells to denosumab. Cell Cycle. 2017;16:1022–1028.
67. Pearce EL, Walsh MC, Cejas PJ, et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature.
2009;460:103.
68. Luo Z, Zang M, Guo W. AMPK as a metabolic tumor suppressor: control of metabolism and cell growth. Future
Oncol. 2010;6:457–470.
69. Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of
the mitochondrial respiratory chain. Biochem J. 20 0 0;348:607–614.
70. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest.
2001;108:1167–1174.
71. Chen X-G, Park H-J. Chemical characteristics of O-carboxymethyl chitosans related to the preparation conditions.
Carbohydr Polym. 2003;53:355–359.
72. Chen S, Zhu X, Lai X, Xiao T, Wen A, Zhang J. Combined cancer therapy with non-conventional drugs: all roads lead
to AMPK. Mini Rev Med Chem. 2014;14:642–654.
73. Wilcock C, Bailey C. Accumulation of metformin by tissues of the normal and diabetic mouse. Xenobiotica.
1994;24:49–57.
74. Zhao M, Wang Y, Du C, Liu Y, Zhang N, Luo F. Aspirin and metformin exhibit antitumor activity in murine breast
cancer. Oncol Rep. 2018;39:1414–1422.
75. Scherbakov AM, Sorokin DV, Tatarskiy Jr VV, et al. The phenomenon of acquired resistance to metformin in breast
cancer cells: the interaction of growth pathways and estrogen receptor signaling. IUBMB Life. 2016;68:281–292.
76. Nies AT, Hofmann U, Resch C, Schaeffeler E, Rius M, Schwab M. Proton pump inhibitors inhibit metformin uptake
by organic cation transporters (OCTs). PLoS One. 2011;6:e22163.
77. Lu Z, Long Y, Cun X, et al. A size-shrinkable nanoparticle-based combined anti-tumor and anti-inflammatory strat-
egy for enhanced cancer therapy. Nanoscale. 2018;10(21):9957–9970.
78. Shafiei-Irannejad V, Samadi N, Salehi R, et al. Reversion of multidrug resistance by co-encapsulation of doxoru-
bicin and metformin in poly (lactide-co-glycolide)-d-α -tocopheryl polyethylene glycol 10 0 0 succinate nanoparticles.
Pharm Res. 2018;35:119.
79. Shafiei-Irannejad V, Samadi N, Yousefi B, Salehi R, Velaei K, Zarghami N. Metformin enhances doxorubicin sensitivity
via inhibition of doxorubicin efflux in P-gp-overexpressing MCF-7 cells. Chem Biol Drug Des. 2018;91:269–276.
80. El-Ashmawy NE, Khedr NF, El-Bahrawy HA, Abo Mansour HE. Metformin augments doxorubicin cytotoxicity in
mammary carcinoma through activation of adenosine monophosphate protein kinase pathway. Tumor Biol. 2017;39.
81. Xue C, Wang C, Sun Y, et al. Targeting P-glycoprotein function, p53 and energy metabolism: combination of
metformin and 2-deoxyglucose reverses the multidrug resistance of MCF-7/Dox cells to doxorubicin. Oncotarget.
2017;8:8622.
82. Cooper AC, Fleming IN, Phyu SM, Smith TA. Changes in [18F] Fluoro-2-deoxy-D-glucose incorporation induced by
doxorubicin and anti-HER antibodies by breast cancer cells modulated by co-treatment with metformin and its
effects on intracellular signalling. J Cancer Res Clin Oncol. 2015;141:1523–1532.
83. Xiao Y, Wang S, Zong Q, Yin Z. Co-delivery of metformin and paclitaxel via folate-modified pH-sensitive micelles
for enhanced anti-tumor efficacy. AAPS PharmSciTech. 2018;19(5):2395–2406.

Please cite this article as: A. De and G. Kuppusamy, Metformin in breast cancer: preclinical and clinical evidence,
Current Problems in Cancer, https://doi.org/10.1016/j.currproblcancer.2019.06.003
 ARTICLE IN PRESS
JID: YMCN [mUS1Ga;June 22, 2019;3:8]
A. De and G. Kuppusamy / Current Problems in Cancer xxx (xxxx) xxx 17

84. Soo JS-S, Ng C-H, Tan SH, et al. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combi-
nation therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells. Apoptosis.
2015;20:1373–1387.
85. Farajzadeh R, Pilehvar-Soltanahmadi Y, Dadashpour M, et al. Nano-encapsulated metformin-curcumin in PLGA/PEG
inhibits synergistically growth and hTERT gene expression in human breast cancer cells. Artif Cells Nanomed Biotech-
nol. 2018;46:917–925.
86. Zheng G, Shen Z, Xu A, et al. Synergistic chemopreventive and therapeutic effects of Co-drug UA-Met: implication
in tumor metastasis. J Agric Food Chem. 2017;65:10973–10983.
87. Arbe MF, Fondello C, Agnetti L, et al. Inhibition of bioenergetic metabolism by the combination of metformin and
2-deoxyglucose highly decreases viability of feline mammary carcinoma cells. Res Vet Sci. 2017;114:461–468.
88. Bizjak M, Malavašič P, Dolinar K, Pohar J, Pirkmajer S, Pavlin M. Combined treatment with Metformin and 2-deoxy
glucose induces detachment of viable MDA-MB-231 breast cancer cells in vitro. Sci Rep. 2017;7:1761.
89. Wokoun U, Hellriegel M, Emons G, Gründker C. Co-treatment of breast cancer cells with pharmacologic doses of
2-deoxy-D-glucose and metformin: starving tumors. Oncol Rep. 2017;37:2418–2424.
90. Ariaans G, Jalving M, De Vries EGE, De Jong S. Anti-tumor effects of everolimus and metformin are complementary
and glucose-dependent in breast cancer cells. BMC Cancer. 2017;17:232.
91. Wang Y, Wei J, Li L, Fan C, Sun Y. Combined use of metformin and everolimus is synergistic in the treatment of
breast cancer cells. Oncol Res. 2015;22:193–201.
92. Amaral MEA, Nery LR, Leite CE, de Azevedo Junior WF, Campos MM. Pre-clinical effects of metformin and aspirin
on the cell lines of different breast cancer subtypes. Invest New Drugs. 2018;36(5):782–796.
93. Talarico G, Orecchioni S, Dallaglio K, et al. Aspirin and atenolol enhance metformin activity against breast cancer
by targeting both neoplastic and microenvironment cells. Sci Rep. 2016;6:18673.
94. Haugrud AB, Zhuang Y, Coppock JD, Miskimins WK. Dichloroacetate enhances apoptotic cell death via oxida-
tive damage and attenuates lactate production in metformin-treated breast cancer cells. Breast Cancer Res Treat.
2014;147:539–550.
95. Hong S-E, Jin H-O, Kim H-A, et al. Targeting HIF-1α is a prerequisite for cell sensitivity to dichloroacetate (DCA)
and metformin. Biochem Biophys Res Commun. 2016;469:164–170.
96. Banala VT, Sharma S, Barnwal P, et al. Synchronized ratiometric codelivery of metformin and topotecan through
engineered nanocarrier facilitates in vivo synergistic precision levels at tumor site. Adv Healthc Mater. 2018.
97. Guo L-S, Li H-X, Li C-Y, et al. Synergistic antitumor activity of vitamin D3 combined with metformin in human
breast carcinoma MDA-MB-231 cells involves m-TOR related signaling pathways. Pharmazie. 2015;70:117–122.
98. Yeo SK, Paul R, Haas M, Wang C, Guan J-L. Improved efficacy of mitochondrial disrupting agents upon inhibition of
autophagy in a mouse model of BRCA1-deficient breast cancer. Autophagy. 2018;14:1214–1225.
99. Chatran M, Pilehvar-Soltanahmadi Y, Dadashpour M, et al. Synergistic anti-proliferative effects of metformin
and silibinin combination on T47D breast cancer cells via hTERT and cyclin D1 inhibition. Drug Res.
2018;68(12):710–716.
100. Rasouli S, Zarghami N. Synergistic growth inhibitory effects of chrysin and metformin combination on breast cancer
cells through hTERT and Cyclin D1 suppression. Asian Pac J Cancer Prev. 2018;19:977–982.
101. Zheng Z, Zhu W, Yang B, et al. The co-treatment of metformin with flavone synergistically induces apoptosis
through inhibition of PI3K/AKT pathway in breast cancer cells. Oncol Lett. 2018;15:5952–5958.
102. Bojková B, Kajo K, Kisková T, et al. Metformin and melatonin inhibit DMBA-induced mammary tumorigenesis in
rats fed a high-fat diet. Anticancer Drugs. 2018;29:128–135.
103. Hou J, Li F, Li X, Mei Q, Mi M. Lily polysaccharide 1 enhances the effect of metformin on proliferation and apoptosis
of human breast carcinoma cells. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi= Chin J Cell Mol Immunol. 2016;32:780–783.
104. Martinez JA, Chalasani P, Thomson CA, et al. Phase II study of metformin for reduction of obesity-associated breast
cancer risk: a randomized controlled trial protocol. BMC Cancer. 2016;16:500.
105. Kim J, Lim W, Kim E-K, et al. Phase II randomized trial of neoadjuvant metformin plus letrozole versus placebo
plus letrozole for estrogen receptor positive postmenopausal breast cancer (METEOR). BMC Cancer. 2014;14:170.
106. Massarweh S, Romond E, Black EP, et al. A phase II study of combined fulvestrant and everolimus in patients with
metastatic estrogen receptor (ER)-positive breast cancer after aromatase inhibitor (AI) failure. Breast Cancer Res
Treat. 2014;143:325–332.
107. Lega IC, Austin PC, Gruneir A, Goodwin PJ, Rochon PA, Lipscombe LL. Association between metformin therapy and
mortality after breast cancer: a population-based study. Diabetes Care. 2013;36:3018–3026.

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Current Problems in Cancer, https://doi.org/10.1016/j.currproblcancer.2019.06.003