Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

https://doi.org/10.1016/j.bbrc.2012.11.010 Get rights and content

Abstract

Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser79 and Raptor at Ser792, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05–0.1 mM) that were 10–100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α1 and α2 catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

Highlights

► Metformin inhibits cancer cell growth but the mechanism(s) are not understood. ► We show that the potency of metformin is sharply dependent on glucose in the medium. ► AMPK activation was enhanced in cancer cells incubated in physiological glucose. ► Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. ► Metformin, at low concentrations, inhibited DNA synthesis through AMPK.

Introduction

Pancreatic ductal adenocarcinoma (PDAC), which comprises 90% of all human pancreatic cancers, is one of the most lethal human diseases, with overall 5-year survival rate of only 3–5% and a median survival period of 4–6 months. The incidence of this disease in the US has increased to more than 44,000 new cases in 2011 and is now the fourth leading cause of cancer mortality in both men and women [1]. As the current therapies offer very limited survival benefits, novel molecular therapeutic targets and strategies are urgently needed to treat this aggressive disease.

G protein-coupled receptors (GPCRs) and their cognate agonists are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors, including small cell lung cancer, colon, prostate, breast and pancreas [2], [3], [4]. We showed that PDAC cell lines express multiple functional GPCRs [5] and that a variety of GPCR agonists, including neurotensin, stimulated DNA synthesis and proliferation in PDAC cells, such as PANC-1 cells [5], [6], [7]. Furthermore, a broad-spectrum GPCR antagonist inhibited the growth of PDAC cells either in vitro or xenografted into nu/nu mice [8]. More recently, we identified crosstalk between insulin/IGFI receptors and GPCR signaling systems in PDAC cells, leading to enhancement of GPCR-induced mitogenic signaling [7], [9], [10]. Insulin-induced potentiation of GPCR signaling was mediated through the phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1 signaling module [7], [9], a key pathway in insulin/IGF action [11]. These findings assume an added importance in view of the large number of epidemiological studies linking obesity and long standing type-2 diabetes mellitus (T2DM), characterized by peripheral insulin resistance and compensatory overproduction of insulin, with increased risk for developing PDAC [see [12] for review].

Metformin (1,1-dimethylbiguanide hydrochloride), the most widely prescribed drug for treatment of T2DM worldwide, is emerging as a potential anticancer agent. At the cellular level, metformin indirectly stimulates AMP-activated protein kinase (AMPK) activation [13], [14], though other mechanisms of action have been proposed, especially using high concentrations of this biguanide [15]. Major downstream targets of AMPK include the tumor suppressor TSC2 (tuberin) and the substrate binding subunit Raptor of mTORC1 [16], [17], the phosphorylation of which inhibits mTORC1 activity [18]. Converging epidemiological, clinical and preclinical studies support the use of metformin in cancer prevention and therapeutics [10], [19]. Strikingly, T2DM patients who had taken metformin had a 62% lower adjusted incidence and better survival of PDAC compared with those who had not taken this drug [20], [21]. Conversely, T2DM patients who received insulin or insulin secretagogues had a significantly higher risk of PDAC compared with diabetic patients who had not taken these drugs [20], [22]. In preclinical studies, metformin disrupted crosstalk between insulin/IGF receptor and GPCR signaling systems in PDAC cells and inhibited the growth of PDAC xenografts [7]. Other studies showed that metformin inhibited the proliferation of breast, colon, lung and prostate cancer cells [10], [19]. All these reports suggest that the antidiabetic drug metformin may provide a novel approach for the prevention and treatment of PDAC and other malignancies. Although the effects of metformin on cancer cells in vitro suggest a direct mechanism, most previous studies used very high concentrations of this agent to demonstrate inhibitory outcomes (e.g. 5–30 mM [15], [23], [24], [25], [26], [27]) and consequently, their significance has been questioned.

We noticed that many studies examining effects of metformin in vitro were carried out with cancer cells cultured in media supplemented with supra-physiological concentrations of glucose. Here, we demonstrate that PDAC cells cultured in medium containing physiological concentrations of glucose (5 mM) display a marked increase in their sensitivity to metformin. The biguanide inhibited stimulation of mTORC1, DNA synthesis and cell proliferation in PDAC cells, at concentrations 10–100-fold lower than those used in most previous reports. At low concentrations, metformin prevented stimulation of DNA synthesis, at least in part, through an AMPK-dependent pathway.

Section snippets

Cell culture

The human pancreatic cancer cell lines PANC-1, MiaPaCa-2 and the immortalized ductal cell line hTERT-HPNE were obtained from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 and MiaPaCa-2 cells were grown in Dulbecco’s modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS). hTERT-HPNE cells were grown in the medium containing 75% DMEM without glucose, 25% Medium M3 Base (Incell Corp. Cat# M300F-500), 5% FBS, 10 ng/ml human recombinant EGF, 5.5 mM d-glucose (1 g/L) and 750 

Stimulation of AMPK by metformin in PDAC cells: dependence on glucose concentration in the culture medium

In order to determine whether AMPK activation in response to metformin in PDAC cells depends on the ambient glucose concentration, cultures of MiaPaCa-2 and PANC-1 cells were incubated in medium containing glucose at 5 mM (a physiological concentration) or 25 mM (as in regular DMEM) and then treated with increasing concentrations of metformin. Cell lysates were analyzed by immunoblotting using antibodies that detect the phosphorylated state of acetyl-CoA carboxylase (ACC) at Ser79 or Raptor at Ser

Discussion

Many studies have demonstrated that metformin inhibits proliferation of cells in culture, suggesting that the anticancer action of this drug could be mediated, at least in part, through a direct effect on cancer cells. However, an important shortcoming of most previous studies in vitro is that metformin was used at very high concentrations. Consequently, the clinical significance of inhibitory effects obtained using metformin at ∼10 mM has been questioned. In order to understand the anticancer

Acknowledgments

This work was supported by NIH Grants R21CA137292, RO1DK55003, P30DK41301 and P01CA163200.

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