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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 Jun 26;176(15):2724–2735. doi: 10.1111/bph.14694

Efficacious dose of metformin for breast cancer therapy is determined by cation transporter expression in tumours

Hao Cai 1,[Link], Ruth S Everett 1, Dhiren R Thakker 1,✉
PMCID: PMC6609538  PMID: 31032880

Abstract

Background and Purpose

It has been extensively reported that the leading anti‐diabetic drug, metformin, exerts significant anticancer effects. This hydrophilic, cationic drug requires cation transporters for cellular entry where it activates its intracellular target, the AMPK signalling pathway. However, clinical results on metformin therapy (used at antidiabetic doses) for breast cancer are ambiguous. It is likely that the antidiabetic dose is inadequate in patients that have breast tumours with low cation transporter expression, resulting in non‐responsiveness to the drug. We postulate that cation transporter expression and metformin dose are key determinants in its antitumour efficacy in breast cancer.

Experimental Approach

Antitumour efficacy of metformin was compared between low cation transporter‐expressing MCF‐7 breast tumours and MCF‐7 tumours overexpressing organic cation transporter 3 (OCT3‐MCF7). A dose–response relationship of metformin in combination with standard‐of‐care paclitaxel (for oestrogen receptor‐positive MCF‐7 breast tumours) or carboplatin (for triple‐negative MDA‐MB‐468 breast tumours) was investigated in xenograft mice.

Key Results

Metformin had greater efficacy against tumours with higher cation transporter expression, as observed in OCT3‐MCF7 versus MCF‐7 tumours and MDA‐MB‐468 versus MCF‐7 tumours. In MCF‐7 tumours, a threefold higher metformin dose was required to achieve intratumoural exposure that was comparable to exposure in MDA‐MB‐468 tumours and enhance antitumour efficacy of standard‐of‐care in MCF‐7 tumours versus MDA‐MB‐468 tumours. Antitumour efficacy correlated with intratumoural AMPK activation and metformin concentration.

Conclusions and Implications

An efficacious metformin dose for breast cancer varies among tumour subtypes based on cation transporter expression, which provides a useful guide for dose selection.

Abbreviations

ER

oestrogen receptor

mTOR

mammalian target of rapamycin

OCT

organic cation transporter

OCT3‐MCF7

OCT3‐overexpressing MCF7 cells

P70S6K

ribosomal protein S6 kinase β‐1

PMAT

plasma monoamine transporter

T2D

type II diabetes

TN

triple‐negative

What is already known

  • Metformin (antidiabetic drug) has shown beneficial effects against breast cancer in some clinical studies.

  • Cation‐selective transporters are required for metformin uptake into breast cancer cell lines.

What this study adds

  • Breast tumours with higher expression of metformin transporters respond better to the anticancer effect of metformin.

  • Breast tumours expressing higher metformin transporters achieve greater intratumoural metformin concentrations and greater AMPK activation.

What is the clinical significance

  • Our findings suggest that a higher metformin dose may be needed for anticancer versus antidiabetic efficacy.

  • Cation transporter expression in tumours may serve as a biomarker for anticancer efficacy of metformin.

1. INTRODUCTION

Breast cancer is the second most frequently diagnosed cancer among women in the United States (American Cancer Society, 2017). Based on patterns of cell surface receptor expression, breast cancer can be categorized into different subtypes, including oestrogen receptor‐positive (ER+) and triple‐negative (TN; Carey et al., 2007; Marcotte et al., 2016). This wide variability in the expression of, and mutations in, oncogenes and tumour suppressor genes among different subtypes of breast cancer poses a significant challenge in developing an efficacious therapeutic agent for breast cancer (Carey et al., 2007; Howlader et al., 2014; Marcotte et al., 2016; Oh et al., 2006).

Epidemiological studies have shown that patients with type II diabetes (T2D) have an increased risk of developing breast cancer (Alokail, Al‐Daghri, Al‐Attas, & Hussain, 2009). Metformin, a first‐line therapeutic agent for T2D, has beneficial effects against breast cancer and other types of cancer. Retrospective studies revealed that diabetic patients on long‐term metformin treatment had a significantly lower risk of developing breast cancer compared to those on insulin and sulfonylurea (Bosco, Antonsen, Sorensen, Pedersen, & Lash, 2011; Chae et al., 2016; Chlebowski et al., 2012; Evans, Donnelly, Emslie‐Smith, Alessi, & Morris, 2005; Ruiter et al., 2012). Hence, this safe, widely administered, and affordable antidiabetic drug is being investigated as an agent for cancer therapy. Although studies have shown that pre‐operative metformin treatment inhibits tumour cell proliferation (Hadad et al., 2011; Schuler et al., 2015) and improves pathological complete response in cancer patients (Jiralerspong et al., 2009), a consensus on the beneficial effects of metformin against breast cancer is lacking, as several clinical trials failed to demonstrate its anticancer effects (Cazzaniga et al., 2013; Kalinsky et al., 2014). One likely explanation for these mixed results is that, in most studies, metformin dose for breast cancer therapy was not optimized as the dose selection was based on its antidiabetic use (e.g., a daily dose of 850 mg). Therefore, to improve the efficacy of metformin as a therapeutic agent for breast cancer, the key question that needs to be answered is: what drives the differences in response to metformin among patient populations?

In addition to clinical trials, studies have been conducted to understand the molecular mechanism(s) of the anticancer effects of metformin in breast cancer. In diabetic cancer patients, metformin appears to exert its anticancer effect indirectly by decreasing circulating glucose and insulin levels (Ikeda, Iwata, & Murakami, 2000; Musi et al., 2002; Shaw et al., 2005) and reducing insulin‐mediated tumour growth stimulus (Dalmizrak et al., 2007; Quinn et al., 2013). However, in non‐diabetic cancer patients, the insulin‐lowering effects of metformin were contradictory (DeCensi et al., 2014; Goodwin et al., 2008; Hadad et al., 2015), suggesting that mechanisms other than lower insulin levels may also contribute to the anticancer effect of metformin. It has been reported that the anticancer efficacy of metformin in non‐diabetic cancer patients may also be mediated via direct inhibition of the respiratory‐chain complex 1 in tumour cells (Batandier et al., 2006) leading to an elevated AMP/ATP ratio and increased phosphorylation and activation of its intracellular target, AMPK. It is thought that subsequent down‐regulation of the mammalian target of rapamycin (mTOR) and the downstream ribosomal protein S6 kinase β‐1 (P70S6K; Dowling, Zakikhani, Fantus, Pollak, & Sonenberg, 2007; Rocha et al., 2011) results in the suppression of cell proliferation.

It is well established that metformin is unable to cross the cell membrane via passive diffusion due to its hydrophilicity (logD −6.13 at pH 6.0) and positively charged state at physiological conditions (pKa 12.4) and that it requires cation transporters to traverse cell membranes and enter the cells (Han et al., 2013; Han et al., 2015; Kimura, Okuda, & Inui, 2005; Nies et al., 2009). Cation transporters are expressed in multiple organs and play a critical role in the absorption, distribution, and elimination of metformin. Our group had previously demonstrated that organic cation transporter (OCT)1, plasma membrane monoamine transporter (PMAT), the 5‐HT transporter, and choline transporter that are expressed in human enterocytes are responsible for metformin intestinal absorption (Han et al., 2013). The uptake of metformin into its primary target organ, the liver, is mediated by OCT1. In the liver, metformin (<20%) is secreted into the bile by the multidrug and toxin extrusion protein (MATE)1 transporter. Metformin is taken up into the kidney proximal tubules via OCT2 and secreted into the urine via MATE1 and MATE2 (Chen et al., 2009). Hence, the knockout of both Oct1 and Oct2 in mice altered the pharmacokinetics of metformin (Higgins, Bedwell, & Zamek‐Gliszczynski, 2012). Published reports on the effect of genetic polymorphisms of cation transporters (e.g., OCT1 and OCT2) on metformin efficacy in T2D are controversial, with some results suggesting that these polymorphisms have little impact on the clinical efficacy of metformin (Shikata et al., 2007) and other studies demonstrating their contribution to variability in response to metformin (Mato, Guewo‐Fokeng, Essop, & Owira, 2018). Cation transporter polymorphisms were also shown to impact the glucose‐lowering effects of metformin (Becker et al., 2009).

Our previous study showed that, while multiple cation transporters are expressed with a high degree of variability in breast tumours and several human breast cancer cell lines (Cai, Zhang, Han, Everett, & Thakker, 2016), OCT3 is the predominant cation transporter in several breast tumour tissues, with the PMAT also showing significant expression in breast tumours. We also observed that metformin treatment induced greater activation of the intracellular AMPK pathway in cation transporter‐expressing breast cancer cells compared to transporter‐deficient breast cancer cells. In addition, cation transporter‐expressing breast cancer cells are more responsive to the antiproliferative effects of metformin compared to transporter‐deficient breast cancer cells (Cai et al., 2016).

Based on these findings, we hypothesize that (a) cation transporter expression and transporter‐mediated accumulation of metformin in breast tumour tissues are an important determinant of their responsiveness to metformin and as a corollary (b) metformin is not efficacious in some breast cancer patients because low cation transporter expression in their breast tumours results in insufficient metformin uptake leading to insufficient intracellular exposure and activation of the AMPK pathway, thus and making the antidiabetic metformin dose inadequate in these patients. Our first hypothesis was tested in mice bearing human MCF‐7 tumours that overexpress a single cation transporter (i.e., OCT3) that is highly expressed in human breast cancer tissue (Cai et al., 2016). This strategy, which uses two tumour types that are derived from genetically identical cells and differ only in cation transporter expression without any alterations to their physiology, enabled us to investigate the critical role of transporters in the sensitivity of tumours to the antitumour effects of metformin. The second hypothesis is tested with the experiments described in this study, in which we evaluated a relationship between dose and the anticancer efficacy of metformin in combination with standard‐of‐care chemotherapy (i.e., paclitaxel for ER+ tumours and carboplatin for TN tumours) in ER+ MCF‐7 tumours with low transporter expression and TN MDA‐MB‐468 tumours with high transporter expression. To evaluate if an antidiabetic dose of metformin is efficacious against breast tumours of different subtypes and transporter expression profiles, the range of metformin dose that was used in this study was such that the lowest dose for breast cancer therapy would be equivalent in mouse to a dose that is lower than the starting dose used in humans for T2D and the highest dose for breast cancer treatment would be equivalent in mouse to the maximum daily dose administered to T2D patients.

2. METHODS

2.1. Cell culture

MCF‐7 and MDA‐MB‐468 cells were obtained from the Tissue Culture Facility at the University of North Carolina at Chapel Hill (UNC‐Chapel Hill) and authenticated in 2015 by the Tissue Culture Facility through forensic Short Tandem Repeat Analysis techniques. MCF‐7, OCT3‐overexpressing MCF7 cells (OCT3‐MCF7), and MDA‐MB‐468 cells were cultured using previously published methods (Cai et al., 2016).

2.2. Generation and characterization of OCT3‐overexpressing MCF7

MCF‐7 cells were transfected with a vector containing the OCT3 gene, and a single clone with high levels of OCT3 gene expression (OCT3‐MCF7) was isolated (Figure 1). High OCT3 gene expression in the isolated clone was accompanied by high OCT3 protein expression as determined by Western blot analysis (Figure 1a, inset). OCT3 protein expression was compared in MCF‐7 cells and OCT3‐MCF7 cells to confirm significantly higher OCT3 protein expression in the OCT3‐MCF7 clone compared to wild‐type MCF‐7 cells, in which OCT3 protein expression was barely detectable (Figure 1a, inset). Metformin uptake in the absence and presence of the pan transporter inhibitor, quinidine, was evaluated to confirm the functional activity of OCT3 in OCT3‐MCF7 cells, and the data were compared to uptake in MCF‐7 cells (Figure 1b).

Figure 1.

Figure 1

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Characterization of OCT3‐MCF7 cells. (a) Relative expression of three predominant cation transporter genes (OCT3, PMAT, and MATE1) in OCT3‐MCF7 and MCF‐7 cells was determined by RT‐PCR and normalized to 18s rRNA. OCT3 protein expression in OCT3‐MCF7 and MCF‐7 cells was assessed by Western blot analysis (shown in inset). (b) [14C]Metformin uptake in OCT3‐MCF7 and MCF‐7 cells was evaluated in the presence or absence of the pan transporter inhibitor, quinidine, and normalized to protein content. Data represent mean ± SD; n = 3. NS, not significant. *P < .05

2.3. Generation of xenograft mice

Female 6‐ to 8‐week‐old athymic nude mice (RRID:RGD_5508395) with body weight from 20 to 25 g were purchased from the Animal Studies Core at UNC‐Chapel Hill. Two million MCF‐7 cells, OCT3‐MCF7 cells, or MDA‐MB‐468 cells were resuspended in 50% Matrigel™ (BD Biosciences, San Jose, USA) and injected s.c. or orthotopically into 8‐week‐old female nude mice. To stimulate growth of MCF‐7 and OCT3‐MCF7 tumours, oestrogen pellets (Innovative Research of America, Sarasota, USA) were s.c. implanted into mice. Mice were assigned to treatment groups after the tumour size was >100 mm3. The animals were housed, and animal studies were conducted in the animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology.

2.4. Treatment of xenograft mice with metformin and determination of metformin effect on tumour size and volume

Mice bearing MCF‐7 tumours or OCT3‐MCF7 tumours were randomized and injected i.p. with saline or 50 mg·kg−1·day−1 metformin (Sigma Aldrich, St. Louis, USA) daily for 20 days and killed on Day 20. Tumour size was measured externally with a calliper, and tumour volume was calculated using the equation: tumour volume = 0.5 × (length) × (width)2.

2.5. Metformin dose–response relationship in MCF‐7 tumours and MDA‐MB‐468 tumours

Mice bearing tumours were randomized and treated with (a) saline, (b) 30 mg·kg−1·week−1 paclitaxel (MCF‐7 tumours), or 50 mg·kg−1·week−1 carboplatin (MDA‐MB‐468 tumours) alone, (c) 360 mg·kg−1·day−1 metformin alone, (d) 30 mg·kg−1·week−1 paclitaxel or 50 mg·kg−1·week−1 carboplatin plus metformin at 12, 36, 120, or 360 mg·kg−1·day−1. Saline (Hospira, Lake Forest, USA), paclitaxel (APP Pharmaceuticals, Los Angeles, USA), carboplatin (Toronto Research Chemicals, Toronto, CA), and metformin were obtained from the indicated sources. Paclitaxel and carboplatin were administered via i.v. injections every week, and metformin was administered daily by oral gavage.

2.6. Effect of metformin treatment on the activation of AMPK pathway in xenograft tumours

At the end of the treatment period, mice were killed, and ~10 mg of tumour tissues was isolated, washed, and lysed in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, USA). Protein in tissue lysates was measured by the bicinchoninic acid assay (Santa Cruz Biotechnology) and subjected to Western blot analyses (40 μg) as previously described (Cai et al., 2016; Han et al., 2015) using a rabbit monoclonal IgG primary antibody against phospho‐AMPK (Cell Signaling Technology, Danvers, USA, RRID:AB_2799368). GAPDH rabbit monoclonal antibody (Santa Cruz Biotechnology, RRID:AB_629536) was used as a loading control. An anti‐rabbit IgG, HRP‐linked antibody was used as secondary antibody (Cell Signaling Technology, RRID:AB_2099233). The intensity of the phospho‐AMPK band was normalized to the GAPDH band to reduce variation in loading. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

2.7. Plasma and intratumoural concentrations of metformin

Mice bearing OCT3‐MCF7, MCF‐7, or MDA‐MB‐468 tumours were generated using the method described above. When tumours were >100 mm3, mice received a single dose of [14C]metformin 12, 36, 120, and 360 mg·kg−1, 50 μCi) i.p. or p.o. Blood was collected from the tail vein at 5 min, 15 min, 50 min, 2 hr, 8 hr, and 24 hr post‐metformin administration, and plasma was isolated. Hepatic, renal, and tumour tissues were harvested and lysed in PBS. [14C]Metformin concentration in plasma and tissue lysates was measured by liquid scintillation spectrometry.

2.8. Data and statistical analyses

All data are expressed as mean ± SD. The number of animals for each experimental group is reported in the figure legends. ANOVA followed by Tukey's test was performed to determine the statistical significance for the differences in transporter gene expression and tumour weight among different treatment groups. Statistical significance for the differences in survival rates was assessed by log–rank (Mantel–Cox) test. Kruskal–Wallis test was performed to determine the significant differences in AMPK phosphorylation by different treatments. P values <.05 were used to indicate statistical significance. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. All statistical analyses were performed with GraphPad Prism (GraphPad Software Inc., La Jolla, USA, RRID:SCR_002798).

2.9. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Cidlowski et al., 2017; Alexander, Fabbro et al., 2017; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. Antitumour efficacy of metformin correlates with cation transporter expression in tumour cells

The results described below show that, in two very different experimental models, (a) mice bearing OCT3‐MCF7 versus MCF‐7 tumours and (b) mice bearing MDA‐MB‐468 versus MCF‐7 tumours, metformin exhibits greater antitumour potency against tumours with higher cation transporter expression compared to tumours that express lower levels of these transporters.

Xenograft mice bearing MCF‐7 and OCT3‐MCF7 tumours were generated by injecting the same number of MCF‐7 and OCT3‐MCF7 cells into athymic nude mice. The two types of tumours are expected to be identical in all aspects except in OCT3 expression, with OCT3‐MCF7 tumours expressing higher levels of OCT3 proteins compared to MCF‐7 tumours (Figure S1). The growth rate of both tumour types was similar (Figure 2), suggesting that the transfection of OCT3 into MCF‐7 cells did not introduce any growth‐related alterations in the OCT3‐overexpressing tumours. These data support our rationale that any differences observed in the antitumour efficacy of metformin between MCF‐7 and OCT3‐MCF7 tumours will be due to the differences in OCT3 expression. Although metformin, at a dose of 50 mg·kg−1·day−1, attenuated the growth of both MCF‐7 and OCT3‐MCF7 tumours, it was more potent against OCT3‐MCF7 tumours (70.9% reduction of tumour volume) versus wild‐type MCF‐7 tumours (34.3% reduction of tumour volume) and completely arrested OCT3‐MCF7 tumour growth (Figure 2). Metformin treatment did not significantly change the body weight of mice bearing OCT3‐MCF7 tumours or MCF‐7 tumours (Figure S2).

Figure 2.

Figure 2

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Effect of saline (n = 6) and 50 mg·kg−1·day−1 metformin (n = 8) on OCT3‐MCF7 tumours and MCF‐7 tumours over a 20‐day treatment period. Both saline and metformin were administered via i.p. injection. Results are shown as the mean ± SD

Recognizing that metformin will likely be used as an adjuvant therapy in the clinic, we designed studies to evaluate if metformin, when used in combination with the standard‐of‐care chemotherapeutic agents, such as paclitaxel for ER+ breast cancer and carboplatin for TN breast cancer, improves the efficacy of these agents. Mice bearing MCF‐7 tumours or MDA‐MB‐468 tumours were chosen as models for ER+ and TN breast cancer, respectively. These two tumour types also differ in the level of cation transporter expression (Figure S3), with MCF‐7 tumours expressing lower levels of cation transporters compared to MDA‐MB‐468 tumours (Cai et al., 2016).

In this combination therapy study, mice were treated with a range of metformin doses (mouse‐equivalent of sub‐therapeutic to supra‐therapeutic doses relative to doses used for T2D) in combination with a fixed dose of paclitaxel (30 mg·kg−1·week−1) or carboplatin (50 mg·kg−1·week−1). Initially, the therapeutic efficacies of paclitaxel, carboplatin, and metformin as monotherapies were assessed. Our data showed that all three chemotherapeutic agents, when used alone, were efficacious in suppressing tumour growth (Figure 3a–d). The combination therapy study showed that the antitumour efficacy of carboplatin against MDA‐MB‐468 tumours was significantly enhanced by metformin at 120 and 360 mg·kg−1·day−1 doses, whereas the two lower doses of metformin (i.e., 12 and 36 mg·kg−1·day−1) produced no improvement (Figure 3a,c). In contrast, in MCF‐7 tumours, only the highest dose of metformin (i.e., 360 mg·kg−1·day−1) significantly improved the antitumour efficacy of paclitaxel (Figure 3b,d). Thus, lower minimum doses of metformin are required in TN MDA‐MB‐468 tumours to enhance the antitumour efficacy of carboplatin compared to the metformin doses required to boost the effect of paclitaxel in ER+ MCF‐7 tumours.

Figure 3.

Figure 3

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Antitumour efficacy of p.o. administered metformin at different doses (12, 36, 120, and 360 mg·kg−1·day−1) as a monotherapy and in combination with i.v. injected 50 mg·kg−1·week−1 carboplatin (for MDA‐MB‐468 tumours) and 30 mg·kg−1·week−1 paclitaxel (for MCF‐7 tumours). The progression of (a) MDA‐MB‐468 tumours and (b) MCF‐7 tumours under different treatments was assessed by measuring tumour volume. Monotherapy is represented as solid lines and combination therapy as dashed lines. Weights of (c) MDA‐MB‐468 tumours and (d) MCF‐7 tumours isolated from mice at the end of the treatment period were compared among different treatment groups. Data represent mean ± SD; n = 6. *P < .05

3.2. Metformin is more potent in activating the AMPK pathway in transporter‐expressing tumours

Since AMPK is regarded as the primary intracellular target of metformin for its antitumour effect, AMPK activation (reflected by its phosphorylation) in tumour tissues may prove to be a useful biomarker to assess the responsiveness of tumours to metformin treatment. This is supported by our data which showed greater activation of the AMPK pathway by metformin in the high cation transporter‐expressing OCT3‐MCF7 tumours compared to the low cation transporter‐expressing MCF‐7 tumours, as evidenced by greater AMPK phosphorylation and suppression of P70S6K phosphorylation in OCT3‐MCF7 tumours compared to MCF7 tumours (Figure S4). The extent of AMPK activation in both MDA‐MB‐468 and MCF‐7 tumours increased with an increase in metformin dose (Figure 4). With the same metformin dose, greater AMPK phosphorylation was observed in MDA‐MB‐468 tumours which express higher levels of cation transporters, compared to MCF‐7 tumours that have low levels of cation transporters. In MDA‐MB‐468 tumours, metformin (120 and 360 mg·kg−1·day−1) in combination with carboplatin caused significantly greater AMPK activation compared to saline plus carboplatin treatment, whereas in MCF‐7 tumours, only the highest dose of metformin (360 mg·kg−1·day−1) in combination with paclitaxel caused significantly greater AMPK phosphorylation compared to saline plus pactitaxel treatment. Carboplatin and paclitaxel, as monotherapies, did not activate AMPK. These data establish a clear association between metformin dose/cation transporter expression and intratumoural AMPK activation/antitumour efficacy, either as a monotherapy or in combination with other chemotherapeutic agents.

Figure 4.

Figure 4

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Activation of AMPK by p.o. administered metformin at different doses (12, 36, 120, and 360 mg·kg−1·day−1) and/or i.v. administered 50 mg·kg−1·week−1 carboplatin and 30 mg·kg−1·week−1 paclitaxel in (a, b) MDA‐MB‐468 tumours and (c, d) MCF‐7 tumours. Tumour tissues from mice killed at the end of treatment were lysed and analysed by Western blot analyses to assess AMPK phosphorylation, using GAPDH as a loading control. Western blot image (i.e., a and c) and the quantifications of Western blot band intensities (i.e., b and d) are provided. Data are presented as scatter points, and the mean value of each group is presented as a dashed line. n = 3. *P < .05

3.3. Cation transporters enhance the anticancer efficacy of metformin by enabling metformin cellular uptake and achieving sufficient intratumoural concentrations

We evaluated the impact of OCT3 overexpression on metformin uptake in tumour tissues (Figure 5), and the results showed that intratumoural metformin concentration in OCT3‐MCF7 tumours, following i.p. administration, was five‐fold higher than that observed in MCF‐7 tumours at 15 min (T max) post‐administration (403 vs. 77.4 μM). These results suggest that in tumours with high OCT3 expression, there is rapid exposure of tumour tissue to metformin and more efficient metformin uptake. Even at 24 hr post‐metformin administration, when the drug is virtually eliminated from the body, its intratumoural concentration in OCT3‐MCF7 tumours was approximately eight‐fold higher than that in MCF‐7 tumours (5.61 vs. 0.71 μM; Figure 5). Thus, the initial higher exposure of OCT3‐MCF7 tumours to metformin persists through the 24‐hr period. In contrast, metformin concentrations in the liver (the pharmacological target organ for the antidiabetic effects of metformin) and kidney (the organ of metformin elimination) in mice bearing OCT3‐MCF7 and MCF‐7 tumours were comparable (Figure 5). Thus, higher levels of OCT3 expression in OCT3‐MCF7 tumours compared to MCF‐7 tumours appeared to have contributed to greater sensitivity of OCT3‐MCF7 tumours to metformin treatment.

Figure 5.

Figure 5

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Plasma and tissue concentrations of metformin in xenograft mice bearing OCT3‐MCF7 tumours and MCF‐7 tumours post‐i.p. injection of 50 mg·kg−1 of [14C]metformin. (a) Metformin plasma concentration‐time profiles. Metformin concentrations in the liver, kidney, and tumours at (b) 15 min and (c) 24 hr post‐administration. Data represent mean ± SD. n = 3. NS, not significant. *P < .05

The intratumoural concentrations of metformin in mice bearing MCF‐7 and MDA‐MB‐468 tumours were also compared to the corresponding systemic levels. Plasma concentrations of metformin administered at the same dose were similar in mice bearing MDA‐MB‐468 and MCF‐7 tumours and increased linearly with an increase in metformin dose. In contrast, a two‐fold to four‐fold higher intratumoural concentration of metformin was observed at 1 and 24 hr post‐p.o. administration in MDA‐MB‐468 tumours compared to MCF‐7 tumours (Figure 6a–d), which correlates with higher cation transporter expression in MDA‐MB‐468 tumour cells compared to that in MCF‐7 tumour cells (Cai et al., 2016). These results reveal that the differences in the sensitivities of MDA‐MB‐468 and MCF‐7 tumours to metformin treatment are likely due to the differences in intratumoural exposure to drug and that systemic exposure does not correspond to the responsiveness of tumours to metformin.

Figure 6.

Figure 6

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Assessment of metformin pharmacokinetics and animal survival rates in mice administered metformin p.o. at different doses (12, 36, 120, and 360 mg·kg−1·day−1). Metformin plasma concentrations‐time profiles post‐p.o. administration in mice bearing (a) MDA‐MB‐468 tumours and (b) MCF‐7 tumours. Plasma concentration data points are represented as open circles and time profiles of the mean plasma concentration of metformin are represented as dashed lines. Intratumoural concentrations of metformin in (c) MDA‐MB‐468 tumours and (d) MCF‐7 tumours are represented as mean ± SD; n= 3. Kaplan–Meier survival curves showing the effect of p.o. administered metformin as a monotherapy and in combination with i.v. administered (e) 50 mg·kg−1·week−1 carboplatin or (f) 30 mg·kg−1·week−1 paclitaxel on the survival of mice bearing MDA‐MB‐468 or MCF‐7 tumours. Monotherapy is represented as solid lines and combination therapy as dashed lines. n = 8 mice per treatment group

3.4. Low doses of metformin in combination with standard‐of‐care chemotherapy improves survival of xenograft mice

Animal survival rate during chemotherapy, generally regarded as a critical indicator of treatment outcomes, was evaluated in this study. Overall, xenograft mice on combination therapy (metformin plus paclitaxel/carboplatin) had higher survival rates compared to those on paclitaxel/carboplatin monotherapy (Figure 6e,f). A metformin dose of 120 mg·kg−1·day−1 as a combination therapy was most effective in enhancing survival rates. In contrast, the highest metformin dose of 360 mg·kg−1·day−1 when used in combination with paclitaxel/carboplatin resulted in lower survival rates despite inducing highest antitumour efficacy among all treatment groups (Figure 6e,f). Additionally, metformin doses that did not significantly enhance the antitumour efficacy of paclitaxel and carboplatin were also effective in improving the survival rates of mice.

4. DISCUSSION

While there is mounting clinical evidence on the anticancer effects of metformin, a consensus on its beneficial effects against breast cancer is lacking. As with most chemotherapeutic agents for breast cancer, metformin doses are not optimized based on breast tumour subtypes; hence, it is possible that the commonly administered metformin dose, which is based on its antidiabetic dose, is not optimal for certain breast cancer subtypes. Previously, we demonstrated that both a functional intracellular AMPK pathway and a cation transporter expression are required for the antiproliferative effects of metformin against breast cancer cell lines (Cai et al., 2016). We also showed a wide heterogeneity in cation transporter expression among various breast cancer tissues and cell lines, with OCT3 and PMAT being the two predominant cation transporters in breast tumour cells (Cai et al., 2016). The present study takes the next step towards understanding the relationship between transporter expression and antitumour efficacy of metformin; and examines if transporter expression levels in breast tumour tissues can be used to (a) select the most suitable breast cancer patients for metformin therapy and (b) determine an efficacious metformin dose for breast cancer treatment.

Our initial proof‐of‐concept study was to evaluate the impact of cation transporter expression on the antitumour efficacy of metformin. This was accomplished by overexpressing one of the predominant metformin transporters (i.e., OCT3) in MCF‐7 breast cancer cells (Cai et al., 2016), and comparing antitumour efficacy of metformin against tumours derived from MCF‐7 cells and OCT3‐MCF7. MCF‐7 cells were selected for this study because they (a) are widely used to develop xenograft mouse models of breast cancer, (b) have relatively low overall metformin transporter expression (Cai et al., 2016), (c) are highly tumorigenic, and (d) have a functional AMPK‐mTOR‐P70S6K pathway. This is the most straightforward approach for elucidating the role of cation transporters in the antitumour efficacy of metformin, because the same genetic origin of OCT3‐MCF7 tumours and MCF‐7 tumours, which differ only in OCT3 expression levels, enabled direct comparison of the impact of OCT3 expression on tumour response to metformin. Our approach is in contrast to that used by others, where the antiproliferative efficacy of metformin was compared between genetically different cell lines (Patel et al., 2013).

Our study shows that the antitumour efficacy of metformin, as a monotherapy, was significantly greater in OCT3‐MCF7 tumours versus MCF‐7 tumours (Figure 2), reflected by greater activation of the AMPK pathway in OCT3‐MCF7 tumours (Figure S4). This difference in efficacy can be directly linked to OCT3 overexpression in OCT3‐MCF7 tumours and consequent five to eight‐fold higher intratumoural metformin concentrations in OCT3‐MCF7 tumours (Figure 5). This is a remarkable finding, as the selective modulation of cation transporter expression, without alterations in tumour physiology, can significantly impact tumour response to metformin.

Since metformin will most likely be used in combination with standard chemotherapy in clinic, we evaluated the dose–response relationship of metformin, as a combination therapy. To simulate the clinical setting, we evaluated metformin antitumour efficacy in two wild‐type breast tumours, namely, ER+ and TN, rather than using genetically modified breast tumours. The extent of improvement in the anti‐cancer efficacy of standard chemotherapy, in combination with different metformin doses, was compared between ER+ and TN tumours. Orthotopic xenograft mice bearing ER+ tumours were generated using low transporter‐expressing MCF‐7 cells, and TN tumours were generated with MDA‐MB‐468 cells that express high levels of multiple cation transporters. Mice with ER+ tumours received metformin with paclitaxel, and mice with TN tumours received metformin with carboplatin. Four different metformin doses (360, 120, 36, and 12 mg·kg−1·day−1) were administered to evaluate its antitumour efficacy. The doses used were equivalent to human doses used for T2D. Conversion from human doses to mouse doses was based on body surface area, as described in the FDA guidance (U.S. Food and Drug Administration Center for Drug Evaluation and Research, 2005). The 360 mg·kg−1·day−1 is comparable to the maximum recommended human daily dose of 2,550 mg·day−1, and the 120 and 36 mg·kg−1·day−1 doses are comparable to two common human doses, 850 and 250 mg·day−1, respectively. A metformin dose close to the sub‐therapeutic dose of 85 mg·day−1 in humans was also included in this study. A metformin monotherapy group (360 mg·kg−1·day−1) was included to enable interpretation of results.

Our data showed that the minimum metformin dose (in the combination therapy) required to observe significant antitumour efficacy varied depending on tumour type and cation transporter expression levels. For high transporter‐expressing TN tumours, the minimum required metformin dose was 120 mg·kg−1·day−1 (equivalent to the human dose of 850 mg·day−1) in combination with carboplatin (50 mg·kg−1·day−1), whereas for low cation transporter‐expressing ER+ tumours, a metformin dose of 360 mg·kg−1·day−1 (equivalent to the 2,550‐mg antidiabetic dose) in combination with paclitaxel (30 mg·kg−1·day−1) was needed (Figure 3). However, the most frequently used daily metformin dose in cancer clinical trials is 850 mg, which, according to our results, is sub‐therapeutic for low transporter‐expressing tumours. This could explain the lack of antitumour efficacy in some clinical studies which used 850 mg·day−1 of metformin in combination with chemotherapy.

Although standard‐of‐care chemotherapy in combination with 360 mg·kg−1·day−1 metformin resulted in greatest antitumour efficacy in both tumour types, only limited improvement in survival rates was observed at this metformin dose compared to chemotherapy alone (Figure 6e,f). This may be due to the increased toxicity at such a high dose. Interestingly, metformin as a combination therapy at lower doses (e.g., 36 mg·kg−1·day−1) improved survival rates compared to chemotherapy alone. Since metformin is less toxic than paclitaxel and carboplatin, our study suggests that one approach to optimizing breast cancer therapy is to test whether combining a chemotherapeutic agent at lower doses than the current standard‐of‐care dose with 360 mg·kg−1·day−1 metformin can reduce toxicity and improve survival rates.

To investigate the molecular mechanism underlying the differences in metformin dose–response relationship between MDA‐MB‐468 tumours and MCF‐7 tumours, metformin‐induced activation of the AMPK pathway in tumour tissues was evaluated. AMPK phosphorylation increased with higher metformin doses. The extent of AMPK phosphorylation was greater in MDA‐MB‐468 tumours compared to MCF‐7 tumours, resulting in greater antitumour efficacy in MDA‐MB‐468 tumours (Figure 4). Interestingly, a combination of paclitaxel and metformin induced higher AMPK phosphorylation in tumours compared to metformin or paclitaxel monotherapy, which was corroborated by other groups (Rocha et al., 2011). This could be due to a synergistic effect of the two drugs on the activation of the AMPK pathway, as studies suggest that paclitaxel induces p53 activation in cancer cells, subsequently leading to activation of the AMPK pathway (Rocha et al., 2011). Since biomarkers have been widely used to guide and monitor tumour response to chemotherapy, our study suggests that AMPK activation in breast tumour cells can be a reliable biomarker for assessing the efficacy of metformin against breast cancer.

Our data show that greater metformin‐mediated activation of the AMPK pathway and tumour inhibitory efficacy in MDA‐MB‐468 tumours compared to MCF‐7 tumours is due to higher cation transporter expression, leading to greater transporter‐mediated metformin uptake in MDA‐MB‐468 tumours. Metformin plasma concentration‐time profiles were comparable between mice bearing the two tumour subtypes (Figure 6a,b). However, the peak plasma metformin concentrations in mice at two high metformin doses (i.e., 120 and 360 mg·kg−1·day−1) were much higher than peak plasma concentrations in humans at the commonly used clinical dose (i.e., 850 mg·day−1). The higher peak plasma concentrations achieved with the highest dose used in this study are likely two‐fold to threefold higher than the peak plasma concentrations achieved with the highest clinical dose (2,550 mg·day−1). Metformin at high doses does not cause hypoglycaemia, and the risk of lactic acidosis is small. Further, high metformin doses were used to test the hypothesis that the doses for anticancer efficacy may be different than those used for T2D. This is important because in most cases, the anticancer efficacy of metformin is evaluated at the standard clinical dose for T2D. Although metformin plasma concentrations were comparable between mice bearing MDA‐MB‐468 and MCF‐7 tumours, intratumoural metformin concentrations were ~two‐fold higher in high cation transporter‐expressing MDA‐MB‐468 tumours at 1 hr post‐administration and >four‐fold higher at 24 hr post‐administration, compared to low transporter‐expressing MCF‐7 tumours, at all four metformin doses used (Figure 6c,d). This provides a rationale as to why different minimum metformin doses are required to activate the AMPK pathway and improve chemotherapeutic efficacy in MDA‐MB‐468 tumours and MCF‐7 tumours. MCF‐7 tumours required ~three‐fold higher metformin dose to achieve an intratumoural metformin exposure that was comparable to its exposure in MDA‐MB‐468 tumours (Figure 6c,d).

Our findings suggest that breast cancer patients with high cation transporter‐expressing tumours are more suitable for metformin treatment. This study provides the first evidence that the metformin dose used for T2D may not be sufficient to achieve the intratumoural exposure needed for antitumour efficacy and suggests that increasing the metformin dose may enhance its efficacy against low cation transporter‐expressing breast tumours.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

H.C. designed and performed the experiments and prepared the manuscript. R.S.E. participated in the study design and revised the manuscript. D.R.T. provided guidance and supervision in the design and implementation of the study as well as in the preparation of the manuscript.

Supporting information

Figure S1. Immunohistochemical staining of tissues from OCT3‐MCF7 and MCF‐7 tumors to evaluate OCT3 expression. Images were taken at 40X amplification. OCT3 protein expression in OCT3‐MCF7 and MCF‐7 tumors is shown as brown‐red staining.

Figure S2. Change of body weight of mice bearing MCF‐7 or OCT3‐MCF7 tumors on saline or 50 mg kg‐1 day‐1 metformin treatment. Data represent mean ± SD.

Figure S3. Expression of six cation transporter genes in MCF‐7 cells and MDAMB‐468 cells. Data represent mean ± SD, N=3.

Figure S4. Activation of AMPK pathway in tumor tissues from mice treated with 50 mg kg‐1 day‐1 metformin or saline. Tumor tissues from mice euthanized at the end of treatment were lysed and analyzed by Western blot analyses to assess AMPK and P70S6K phosphorylation, using GAPDH as a loading control. Intensity of Western blot bands were provided.

Click here for additional data file. (2.3MB, pdf)

ACKNOWLEDGEMENTS

The OCT3 plasmid was a generous gift from Dr. Ganapathy at Georgia Regents University. We would like to thank the University of North Carolina Animal Studies Core Facility for performing xenograft mouse studies. This work was partially supported by funding from the North Carolina Translational and Clinical Sciences Institute, University of North Carolina at Chapel Hill.

Cai H, Everett RS, Thakker DR. Efficacious dose of metformin for breast cancer therapy is determined by cation transporter expression in tumours. Br J Pharmacol. 2019;176:2724–2735. 10.1111/bph.14694

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Immunohistochemical staining of tissues from OCT3‐MCF7 and MCF‐7 tumors to evaluate OCT3 expression. Images were taken at 40X amplification. OCT3 protein expression in OCT3‐MCF7 and MCF‐7 tumors is shown as brown‐red staining.

Figure S2. Change of body weight of mice bearing MCF‐7 or OCT3‐MCF7 tumors on saline or 50 mg kg‐1 day‐1 metformin treatment. Data represent mean ± SD.

Figure S3. Expression of six cation transporter genes in MCF‐7 cells and MDAMB‐468 cells. Data represent mean ± SD, N=3.

Figure S4. Activation of AMPK pathway in tumor tissues from mice treated with 50 mg kg‐1 day‐1 metformin or saline. Tumor tissues from mice euthanized at the end of treatment were lysed and analyzed by Western blot analyses to assess AMPK and P70S6K phosphorylation, using GAPDH as a loading control. Intensity of Western blot bands were provided.

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