Metformin promotes progesterone receptor expression via inhibition of mammalian target of rapamycin (mTOR) in endometrial cancer cells

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Abstract

Progesterone has been used in the hormonal treatment of endometrial cancer (EC) for many years, but the response rates are unsatisfying. The down-regulated progesterone receptor (PR) is the main reason for treatment failure. The insulin-like growth factor (IGF) system is related to EC risk, and IGF-I can inhibit PR transcription in breast cancer. Recent evidence suggests that metformin-combined oral contraceptives may reverse progesterone-resistant atypical endometrial hyperplasia, but the mechanism is unclear. We attempt to investigate the interaction of metformin, PR and IGF-II expression, and identify whether metformin can enhance the antitumor effect of medroxyprogesterone acetate (MPA) using Ishikawa and HEC-1B EC cell lines. We found that both IGF-I and IGF-II inhibit PR A/B mRNA and protein expression, whereas metformin markedly promotes PR expression. In parallel, IGF-II increases phosphorylation of AKT and p70S6K, while metformin increases AMPK phosphorylation and decreases p70S6K phosphorylation. The effects of metformin on PR A/B and p70S6K are partially reversed by an AMPK inhibitor. Furthermore, metformin synergistically antiproliferates MPA in two cell lines, with the peak synergy occurring with 10 μM metformin combined with 1 μM MPA (CI = 0.20448 for Ishikawa, CI = 0.12801 for HEC-1B). Our results demonstrate that metformin promotes PR expression, which can be inhibited by overexpressed IGF-II in EC. This effect is partially mediated through activating AMPK followed by inhibiting the overactivated mTOR pathway.

Highlights

► Metformin promotes PR expression via AMPK activation, which is followed by mTOR pathway inhibition in endometrial cancer. ► IGF-I and IGF-II inhibit PR expression via mTOR pathway activation. ► Metformin has a synergistic antitumor effect with MPA in endometrial cancer cells. ► MPA combined with metformin may be a novel treatment strategy in endometrial cancer.

Introduction

In Western countries, endometrial cancer (EC) is the most common gynecological malignancy, accounting for 6% of all cancers in women [1], [2]. Approximately 80% of EC patients are diagnosed in Stage I and are usually cured with hysterectomy [3]. However, a subset of young women present with EC in a setting of obesity, irregular menses, chronic anovulation and polycystic ovarian syndrome (PCOS). This group of women poses a therapeutic dilemma, since preservation of fertility is a major concern for these individuals. Thus, reproductive-sparing treatment is crucial in this scenario.

Endometrial carcinogenesis is related to estrogen overexposure without progesterone modulation. The role of progesterone in the endometrium is primarily to induce cellular differentiation and to antagonize estrogen-mediated cell proliferation [4]. Progesterone and its synthetic form (medroxyprogesterone acetate, or MPA) have been used for the treatment of EC in advanced or recurrent cases, and in those who wish to preserve their fertility [5], [6]. Progesterone binds to its receptor and activates the transcription of several genes which are involved in cross-talk with other signaling pathways, such as growth factors and cytokines [7]. The antitumor effect of progesterone is in its binding to the human progesterone receptors (hPR-A, hPR-B), belonging to the steroid hormone superfamily of nuclear receptors [8]. Unfortunately, PR expression decreases during EC progression, resulting in the loss of progesterone-regulated growth inhibition [9]. Down-regulated progesterone receptors frequently lead to carcinogenesis and treatment failure, as evidenced by the overall response rate of PR-rich or PR-poor tumors (72% vs 12%, respectively) [5]. Unfortunately, progesterone treatment also leads to depletion of PRs within the target tissue.

Accumulating evidence indicates that obesity, diabetes and insulin resistance are strong risk factors for EC, and the insulin-like growth factor (IGF) system plays a vital role in carcinogenesis and disease progression [10]. IGF-II and IGF-IR (IGF-I receptor) were found to be much higher in EC than in normal endometrium [11]. Both IGF-I and IGF-II are mitogenic and antiapoptotic. IGF-IR binds to the ligands IGF-II, IGF-I, or insulin, triggering autophosphorylation. This in turn leads to activation of distinct signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)-AKT/mammalian target of rapamycin (mTOR) pathway [12]. On the contrary, phosphatase and tensin homolog deleted on chromosome ten (PTEN) exerts its tumor-suppressive function through its activity as a phospholipid phosphatase, leading to inhibition of PI3K signaling and inactivation of downstream kinases such as AKT and mTOR. Unfortunately, loss of PTEN is found in 30–83% of EC, which leads to overactivation of the mTOR pathway, ultimately contributing to dysregulation of cell proliferation, growth, differentiation, and survival [13]. A recent study suggests that IGF-I inhibits PR gene transcription via the PI3K/AKT/mTOR pathway in breast cancer [14]. Thus, IGF-I may weaken the antitumor effect of progesterone through reduction of PR levels in breast cancer, although this has not been identified in EC.

A recent case report has shown that combination therapy with metformin and oral contraceptives may reverse progesterone-resistant atypical endometrial hyperplasia [15]. Therefore, metformin may enhance the effect of progesterone on atypical endometrial hyperplasia, but this mechanism is unclear. The mechanism of action of metformin is by activating AMP-activated protein kinase (AMPK) via Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1), the kinase responsible for phosphorylating and activating AMPK [13]. This process leads to the regulation of multiple signaling pathways involved in cellular proliferation, including the mTOR pathway. Loss of LKB1 expression has been documented in up to 65% of ECs, which stimulates mTOR pathway overactivation in ECs [16], [17].

Based upon the preceding evidence, we investigated the interaction of metformin, IGF-II and PR expression, explored the cell signaling pathway targets, and identified whether metformin can enhance the antitumor effect of MPA using Ishikawa and HEC-1B EC cell lines.

Section snippets

Cell lines and reagents

The human EC cell lines Ishikawa (IK, well differentiated) and HEC-1B (moderately differentiated), generously provided by Prof. Wei LH (Perking University People's Hospital, China), were maintained in phenol red-free DMEM/F12 with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. The cell cultures were routinely passaged every 3–5 days. Metformin, MPA (medroxyprogesterone17-acetate), dextran-coated charcoal were purchased from Sigma. Insulin-like growth factor-I (IGF-I) and IGF-II were purchased

IGF-I and IGF-II down-regulate PR mRNA and protein levels while metformin up-regulates PR levels

At 48 h treatment, IGF-I and IGF-II significantly decreased PRA/B mRNA and protein levels in IK and HEC-1B cell lines (p < 0.05, Fig. 1). The maximal reduction of PR protein levels occurred at 10 ng/ml of IGF-II (p < 0.01), the physiologic concentration in females. This finding suggests that IGF-II has a down-regulatory effect on PR levels in EC cells.

At 72 h, metformin significantly increased PRA/B mRNA and protein levels in two cell lines (p < 0.05 for 10 μM, p < 0.01 for 100 μM, Fig. 2). Additionally,

Discussion

This study is the first to identify the function of metformin in promoting PR expression in EC cells. Conversely, IGF-I and IGF-II inhibit PR expression. We demonstrate that metformin promotes PR expression via by AMPK activation, which is followed by mTOR pathway inhibition in EC cells. We also show that metformin synergistically enhances the anti-proliferative effect of MPA on EC cells.

Conclusion

Our results demonstrate that metformin promotes PR expression via inhibition of the mammalian target of rapamycin (mTOR) pathway in EC cells and identified that metformin had a synergistic antitumor effect with MPA in EC. Because PR expression is important in EC prevention and in successful progesterone therapy, the combination of metformin and MPA may be a potentially effective way to control EC, especially in PR poor cancers. We hope that our data will provide a scientific foundation for

Acknowledgements

This study was supported by a Grant from National Natural Science Foundation of China (30801223). We appreciate Dr. Ting-Chao Chou (Memorial Sloan-Ketter Cancer Center, USA) for the CalcuSyn software.

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