Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signalling pathway in human pancreatic cancer cells

Over recent decades the incidence of metabolic disorders, such as obesity and type 2 diabetes mellitus, has increased as a consequence of westernized lifestyle and changes in diet. These conditions are in turn associated with an increased risk of developing cancer [1‐3]. Epidemiological studies have demonstrated that obesity and type 2 diabetes are among the top three modifiable risk factors for pancreatic cancer [2, 4‐8]. Almost 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis [9, 10]. The relationship between type 2 diabetes and pancreatic cancer is complex and it remains unclear whether type 2 diabetes contributes to the development of pancreatic cancer or if precancerous cells cause the diabetes. Individuals with elevated fasting glucose and glycated haemoglobin (H_bA_1c) levels [11, 12], or with higher c-peptide or insulin levels have a two to four-fold increase in the risk of pancreatic cancer [1, 7]. Type 2 diabetes patients also demonstrate an increased risk of pancreatic cancer-related death as compared with those without diabetes [13]. Type 2 diabetes is characterized by hyperglycaemia and peripheral insulin resistance with compensatory hyperinsulinemia. Aside from its metabolic actions, insulin can mediate direct mitogenic effects through the insulin receptor (IR) and insulin-like growth factor I (IGF-I) receptor (IGF-IR). Insulin may also affect the cancer risk indirectly via increased production and bioavailability of IGF-I [6, 14]. Additionally, hyperglycaemia can increase the sensitivity to IGF-I [4], thereby enhancing its mitogenic potential and providing an additional link between type 2 diabetes and cancer.

Insulin-sensitizing and glucose lowering drugs, such as metformin, are used as first-line treatment in the management of type 2 diabetes to improve glycaemic control in patients with insulin resistance. The key metabolic action of metformin involves the inhibition of hepatic glucose secretion, which consequently decreases the hyperinsulinemia. This mechanism is mediated via activation of the energy-sensing AMP-activated protein kinase (AMPK) in hepatocytes, through the liver kinase B1 (LKB1) signalling pathway [15]. Although metformin can lower blood glucose, the levels rarely remain within the normal range and as the type 2 diabetes progresses, additional medication such as exogenous insulin is often required to control patients’ hyperglycaemia [16, 17]. In addition to its anti-diabetic effects, metformin has recently been postulated to have a protective role against cancer. Epidemiological and retrospective studies have demonstrated that diabetic patients taking metformin not only have a lower incidence of pancreatic cancer, but also an improved cancer outcome [18‐21]. The indicated anti-neoplastic activity of metformin may relate to reduced plasma insulin concentrations or by direct effects on the tumour cells. Recent studies suggest that metformin-induced AMPK activation at Thr¹⁷² inhibits the central growth control node mammalian target of rapamycin mTOR, thus preventing protein synthesis and cell proliferation [22]. Metformin has recently been shown to possess anti-tumour effects, both in AMPK-dependent and independent manners [23‐25].

Although an increasing number of studies demonstrate the anti-tumour effects of metformin, relatively little is known about the effects and underlying mechanisms of metformin on pancreatic cancer cells. The goal of this study was to examine the direct effects of metformin on human pancreatic cancer cells in the context of normal or elevated glucose levels. Effects on proliferation, apoptosis, AMPK activation and influence on and by the IGF-I pathway were analysed.

Type 2 diabetes or impaired glucose tolerance frequently occurs in pancreatic cancer patients. Compared to other treatments, diabetic patients on metformin have a reduced risk of approximately 40% of developing several types of cancer, including pancreatic cancer [19‐21]. However, the molecular relationships underlying the metabolic and suggested anti-cancer actions of metformin remain poorly understood. Additionally, the importance of optimal glucose control for the anti-tumour effects of metformin has not been fully established. In this study, we describe direct anti-proliferative actions by metformin using in vitro models of pancreatic cancer. In addition, we demonstrate that elevated glucose levels impair AMPK activation and reduce the efficacy of metformin. Importantly, we show a novel role for metformin on human pancreatic cancer cells that may contribute to its indicated anti-cancer actions among type 2 diabetic patients.

Metformin is believed to act mainly through activation of the energy-conserving LKB1-AMPK pathway. Physiological activation of the AMPK metabolic checkpoint in response to nutrient depletion and energy stress suppresses energy-consuming cellular processes such as protein synthesis and cell division. We found that metformin during normal glucose conditions significantly reduced proliferation and promoted apoptosis through PARP cleavage in pancreatic cancer cells with functional LKB1 (MIAPaCa-2, BxPC-3 and PANC-1), while being incapable of suppressing growth under the same conditions in AsPC-1 pancreatic cancer cells. AsPC-1 cells have previously been reported to carry an epigenetic inactivation of LKB1 [27]. Our findings are consistent with prior observations, showing pro-apoptotic actions on breast cancer cells [28, 29] and that a functional LKB1 was required for the in vitro anti-proliferative effect of metformin [25, 30].

Previous work indicates that metformin functions by activating AMPK at Thr¹⁷² with subsequent downstream inhibition of the growth promoting PI3K/Akt/mTOR pathway [29, 31, 32]. Similarly, we also found the growth inhibitory properties of metformin to be associated with the activation of AMPK^Thr172 in pancreatic cancer cells. Under hyperglycaemic conditions, the efficacy of metformin was reduced with less anti-proliferative and pro-apoptotic activity observed. Other investigators have reported that lung and colon carcinoma cells were more sensitive to metformin-induced growth inhibition at low glucose concentrations, while no significant effect of metformin on cell death was observed in high glucose conditions [30]. Similarly, a recent study demonstrated anti-proliferative effects on pancreatic cancer cells by metformin at the low 0.05-1 mM range at normal (5 mM) glucose conditions [33]. This study is in concordance with our data demonstrating direct anti-tumour effects of metformin and supports our findings of enhanced sensitivity at physiological normal glucose levels. We have now shown that the lower anti-proliferative effect of metformin on pancreatic cancer cells at higher glucose levels correlates to an impaired AMPK^Thr172 activation and a shifted balance from AMPK^Thr172 towards AMPK^Ser485 activation. The role of AMPK^Ser485 in the complex AMPK signaling network is at present not completely clear and conflicting reports exist. A recent study indicated that endogenous protein kinase A (PKA)-induced activation of AMPK^Ser485 in pancreatic beta cells did not affect the phosphorylation status of AMPK^Thr172. However, the activation of Thr¹⁷² and Ser⁴⁸⁵ were inversely correlated in response to glucose [34]. Other studies have proposed that PKB/Akt-induced phosphorylation of AMPK^Ser485 can counteract AMPK^Thr172 activation, thereby reducing the effects of metformin [31, 32, 35].

Hyperinsulinemia with resulting increased circulating levels of IGF-I have been suggested to play a role in the connection between type 2 diabetes and cancer [36]. Activation of the IR and IGF-IR result in receptor autophosphorylation and recruitment of insulin receptor substrate (IRS) 1, which in turn activates the PI3K/Akt pathway leading to protein synthesis and cell survival [14]. The responsiveness to IGF-I can be enhanced by exposure to high glucose concentrations [4], which may then further promote cancer progression. In pancreatic cancer cells, IGF-I stimulated a pronounced phosphorylation of Akt and also AMPK^Ser485. However, at physiologically normal glucose levels, IGF-I stimulated AMPK^Ser485 phosphorylation did not appear to antagonize pharmacological activation of AMPK^Thr172 by metformin. Instead, we established that metformin under these conditions suppressed IGF-IR/IR phosphorylation causing a downstream inhibition of both basal and IGF-I stimulated Akt phosphorylation. It is well established that IGF-IR via activation by its ligands transmits mitogenic signals leading to the survival and proliferation of multiple types of cancer. Mechanisms by which metformin inhibits these pathways may thus contribute to the anti-tumour effects previously observed in response to metformin. Studies in other cell types have shown that during normal glucose conditions, AMPK^Thr172 can phosphorylate inhibitory serine residues on IRS-1, which prevents signalling through the PI3K/Akt pathway [29, 37]. However, studies have also shown that Akt at high glucose conditions can inhibit AMPK by phosphorylation of Ser⁴⁸⁵, which prevents activation of Thr¹⁷² and thereby the action of metformin [29, 31, 35, 37, 38]. In keeping with this, we observed a strong activation of Akt and AMPK^Ser485 following IGF-I stimulation at high glucose, which was sustained after exposure to metformin. At high glucose, IGF-I induced Akt and AMPK^Ser485 phosphorylation appeared to correlate with a further reduction of the already-impaired AMPK^Thr172 phosphorylation by metformin.

The findings of the current study using human pancreatic cancer cells add novel information to the indications of direct anti-tumour actions by metformin on transformed epithelial cells. Metformin mediated its effects through activation of AMPK^Thr172 together with inhibition of the IR/IGF-IR signalling pathway (Figure 7). Hyperglycaemia, with and without IGF-I, reduced the sensitivity to metformin and counteracted the growth inhibitory effect otherwise exerted by the drug. Our data suggests that metformin may have beneficial effects on tumour prevention or protection in non-diabetic patients with normal glucose levels. Importantly, these data indicate that optimizing glucose control in type 2 diabetic patients may improve the beneficial anti-tumour actions provided by metformin and should thus be further investigated. Due to the strong associations between type 2 diabetes and pancreatic cancer, evaluating the potential beneficial effects by metformin, as well as the impact by different glucose levels, should be considered of utmost importance. Increased understanding of the relationship between the two conditions may improve both future treatment strategies as well as potentially providing possibilities of novel early diagnostic approaches.