Breast cancer is the second most common cancer and cause of cancer death among women in the United States. The American Cancer Society estimates approximately 231,840 new cases of invasive breast cancer and 40,730 breast cancer deaths in
2016 (1). Epidemiological studies suggest that women with type 2 diabetes are at a greater risk of developing breast cancer. A meta-analysis study showed that type 2 diabetes is associated with 23% increased risk of breast cancer, especially in post-menopausal women (2-5).
Evidence indicates that the frontline antidiabetic drug for type 2 diabetes, namely metformin, acts as an anticancer agent in several cancers, including breast cancer (6-11). Studies have also shown that diabetic women on long-term metformin treatment have a lower risk of breast cancer compared to those not on metformin therapy, and that diabetic breast cancer patients on metformin have a lower risk of distant metastases compared to those not receiving
metformin (12, 13). Pre-operative metformin treatment of non-diabetic women with operable invasive breast cancer results in down-regulation of Ki-67, a biomarker of cell proliferation and a predictive marker for clinical or pathological response to neoadjuvant
therapy (11). Retrospective analyses showed higher pathological complete response rates (24%) in diabetic patients on metformin undergoing neoadjuvant chemotherapy for breast cancer versus diabetic patients not on metformin (8%) or non-diabetic patients (16%) (14). An association was observed between survival in diabetic cancer patients and metformin therapy, but not between survival and sulfonylurea or insulin therapy (15). Preclinical studies in xenograft mouse models of breast, prostate and lung cancer showed that metformin and the
chemotherapeutic agent doxorubicin (DOX) were more effective in blocking tumor growth and preventing relapse than DOX alone (6). In vitro studies also showed that metformin inhibited
growth and colony formation of breast cancer cells, and induced cell cycle arrest and apoptosis (10).
As an anticancer agent, metformin is thought to exert its antiproliferative effects via an extracellular indirect pathway (insulin-dependent) and an intracellular direct pathway (insulin- independent). Insulin can bind to the insulin receptor that is highly expressed in cancer cells, and induce cell proliferation. It is suggested that metformin, by lowering circulating insulin levels, can induce anticancer effects by intercepting insulin-dependent tumor growth (16, 17). The direct antiproliferative effects of metformin in cancer cells are thought to be mediated via activation of intracellular adenosine monophosphate-activated protein kinase (AMPK), which leads to down regulation of the mammalian target of rapamycin (mTOR) and its downstream target, p70S6K (18, 19). In hepatocytes, AMPK and its upstream regulator liver kinase B1 (LKB1) (20) are key mediators in the glucose-lowering effect of metformin. Metformin activates AMPK via LKB1, leading to inhibition of liver gluconeogenesis (21) and lowering of circulating glucose and insulin levels (22). Hence, AMPK appears to be a common intracellular target both for the antidiabetic and anticancer effects of metformin.
Metformin is hydrophilic (logD of -6.13 at pH 6.0) and charged at all physiological pH values (pKa 12.4) (23). Therefore, it cannot enter cells via passive diffusion across the cell membrane (24), and relies on cation-selective transporters to enter the cell where it can activate its intracellular target, AMPK. Transport proteins such as organic cation transporters (OCT 1-3)
(SLC22A1-3), plasma monoamine transporter (PMAT) (SLC29A4), and multidrug and toxin
extrusion proteins (MATE1 and 2) (SLC47A1 and SLC47A2) facilitate metformin trafficking in different organs and tissues (25, 26), such as the intestine, liver, and kidney, and thus drive the disposition of metformin. In the liver, metformin is taken up into hepatocytes predominantly via OCT1, and thus this transporter plays a critical role in the antidiabetic effect of metformin. We have recently reported that as many as four cation-selective transporters, namely OCT1, PMAT, serotonin reuptake transporter (SERT), and a high affinity choline transporter (CHT) contribute
to the intestinal uptake and absorption of metformin (27). These four transporters define the systemic exposure to orally administered metformin, and consequently, its pharmacologic behavior.
Emerging literature on the antiproliferative and anticancer efficacy of metformin in cancer cell lines and preclinical models of the disease either ignores the role of transporters or often suggests that a single transporter is responsible for metformin trafficking through tumor cells/tissues (28, 29, 30). Based on our studies on metformin transport in the intestinal tissue, we anticipate that multiple transporters, and interplay among them, may affect the uptake of metformin into tumor cells and tissues, and therefore influence its antiproliferative and antitumor efficacy. Hence, in any preclinical or clinical study in which the anticancer efficacy of metformin is evaluated, one must consider the expression of one or more metformin transporters in tumor cells for appropriate interpretation of the mechanisms underlying the antitumor effect of
metformin.
In the present study, we have characterized the expression of cation-selective transporters in human breast cancer tissues and in nine commonly studied human breast cancer cell lines. The nine human breast cancer cell lines analyzed in this study were selected based on the two main subtypes of breast cancer, namely luminal and basal. Luminal breast cancer accounts for >70% of tumors that express estrogen receptors (ER) and/or progesterone receptors (PR), and low or no human epidermal growth factor receptors (EGFR) 1 and 2, the latter also known as HER2 (31). Basal-like breast cancers are generally triple negative as they lack ER, PR, and HER2, but express EGFR1 (32) and exhibit enhanced hypoxia and high tumor grade (33, 34). Further, we have compared transporter expression profiles between breast tissues and breast cancer cell lines, and evaluated an association between transporter expression levels and the antiproliferative efficacy of metformin in human breast cancer cells. The most definitive role of cation-selective transporters in the uptake and antiproliferative efficacy of metformin was obtained in this study by engineering an OCT3-overexpressing cell
line (i.e., OCT3-BT20) from the BT-20 cell line that does not express detectable levels of cation- selective transporter genes. Gene and protein expression of OCT3 in BT-20 and OCT3-BT20 cells are related to metformin uptake, its antiproliferative efficacy, and its modulation of the AMPK-mTOR signaling pathway. It is important to emphasize that OCT3 was chosen for overexpression simply as a representative of cation-selective transporters. Thus, this is the first comprehensive study in which the expression of cation-selective transporter genes and proteins has been characterized in several commonly used human breast cancer cell lines, and an unequivocal relationship has been established between cation transporter expression in human breast cancer cells and the antiproliferative efficacy of metformin.