Metformin inhibits epithelial–mesenchymal transition in prostate cancer cells: Involvement of the tumor suppressor miR30a and its target gene SOX4

https://doi.org/10.1016/j.bbrc.2014.08.154Get rights and content

Highlights

  • Metformin inhibits TGF-β-induced EMT in prostate cancer (PCa) cells.

  • Metformin upregulates tumor suppressor miR30a and downregulates SOX4 in PCa cells.

  • SOX4 is a target gene of miR30a.

Abstract

Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have been shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-β-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p = 0.013), Vimentin (p = 0.002) and the decrease of E-cadherin (p = 0.0023) and β-catenin (p = 0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P < 0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4.

Introduction

Metformin (1,1-Dimethylbiguanide) is one of the most commonly used drugs for type II diabetes. It reduces glucose levels through activation of the AMP-activated protein kinase (AMPK) pathway and inhibition of hepatic gluconeogenesis [1]. Recently, multiple epidemiological studies have shown that metformin may reduce cancer risk and/or improve cancer prognosis [2]. In addition, metformin affects the progression and relapse of breast, prostate, and lung cancer mouse xenografts, when combined with suboptimal doses or standard chemotherapeutic agents [3]. So far, possible mechanisms by which metformin exerts its potential antineoplastic properties include activation of AMPK, and thereby inhibition of m-TOR signaling pathway; selective impairment of P53-deficient cells; decrease of Cyclin D1 level and downregulation of C-myc [4], [5], [6].

Prostate cancer (PCa) is a common heterogeneous disease with marked variability in progression [7]. Tumor metastasis is the leading cause of mortality in PCa patients. Epithelial–mesenchymal transition (EMT), characterized by the loss of epithelial characteristics and acquisition of a mesenchymal phenotype, plays a critical role in cancer progression and metastasis [8]. Multiple transcription factors such as Snail, Slug and ZEB, act as molecular switches, respond to the known signaling pathways and regulate the EMT program [8]. Previously, we have reported that SOX4 gene, an oncogenic transcription factor, may contribute to PCa metastasis by initiating a transcriptional program that enables EMT phenotype [9].

Although preliminary, several studies suggested metformin may suppress EMT in malignancy [10], [11], [12]. Vazquez-Martin et al. demonstrated metformin can prevent generation of breast cancer stem cell phenotype by downregulating some EMT regulators (“EMT status”) independently of changes in EMT functioning (“EMT phenotype”) [13]. Additionally, Qu et al. observed that metformin reversed EMT phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells [12]. However, the link between metformin and the EMT process in PCa progression remains unclear.

MicroRNAs (miRNAs) are short, stable, noncoding RNAs that target the mRNA of coding genes for degradation, thus effectively reducing the levels of the gene products [14]. Of note, a growing number of miRNAs were reported to regulate tumor invasion/metastasis through EMT-related and/or non-EMT-related mechanisms [15]. Interestingly, metformin has been shown to upregulate tumor suppressive miRNA let-7a and miRNA96 and downregulate oncogenic miRNA181a [16]. This can be the key preventing self-renewal of cancer-initiating cells arising from EMT. Given the potential ability of metformin to broadly affect multiple pathways, we hypothesized that miRNA modulation could be part of its mechanism of reversing EMT in cancer. In the current study, we investigated the effects of metformin on EMT in PCa cells and analyzed the possible microRNA (miRNA)-based mechanisms.

Section snippets

Reagents

Metformin was obtained from Sigma–Aldrich (St. Louis, MO, USA). TGF-β was purchased from RD Biosciences (San Diego, CA, USA).

Cell culture and treatment

Human PCa cell lines Vcap, DU-145, Lncap, PC-3, normal prostate epithelial cell line RWPE and Kidney HEK293T cells were from American Type Culture Collection (Rockville, MD, USA) and cultured following the manufacturer’s recommendations. For experiments, PCa cells were treated with metformin or vehicle at indicated concentration for up to 24, 48 and 72 h. TGF-β (5 ng/ml for

Metformin inhibits cellular proliferation, migration and invasion of PCa cell lines

Human PCa cell lines Vcap and PC-3 as well as normal prostate epithelial cell line RWPE were utilized to determine metformin regulation of cell proliferation. Consistent with previous findings [4], metformin was growth inhibitory for both Vcap and PC-3 cells through time- and dose-dependent manner. Metformin at 5 mM significantly inhibited the proliferation of Vcap and PC-3 cells at 48 and 72 h, whereas there was no effect on RWPE cells, accordingly (Fig. 1A).

Wound healing assay indicated that

Discussion

Although earlier epidemiological studies strongly suggest a role for metformin in reducing PCa risk, the mechanism by which metformin inhibits PCa carcinogenesis and progression remains unclear. Previous studies suggested two possible mechanisms underlying the anti-proliferative of metformin on neoplastic cells in vitro and in vivo: reduction of systemic insulin levels and a direct action involving AMPK activation within neoplastic cells [19]. Other possible mechanisms involving p53 and cyclin

Conflict of interest

None.

Acknowledgment

Supported by the National Natural Science Foundation of China (Grant Nos. 81072110 and 81171951); Foundation of heath care development of Shandong Province.

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