Metformin inhibits β-catenin phosphorylation on Ser-552 through an AMPK/PI3K/Akt pathway in colorectal cancer cells
Introduction
Despite advances in early detection, surgery and chemotherapy, colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the U.S. and other developed countries. Novel targets and agents for therapy and chemoprevention are urgently needed. They most likely will arise from a clear understanding of the signaling pathways involved and the consequent repurposing of currently used drugs.
Considerable evidence has linked metabolic syndrome, obesity and type 2 diabetes mellitus (T2DM), conditions characterized by peripheral insulin resistance and hyperinsulinemia, with an increase in the incidence of multiple malignancies, including CRC (Gonzalez et al., 2017). Consequently, antidiabetic therapies have emerged as possible new strategies in the prevention and treatment of different types of cancer such as CRC (Chang et al., 2018; Gonzalez et al., 2017; Ikhlas and Ahmad, 2017; Jackson and Garcia-Albeniz, 2018; Klil-Drori et al., 2017; Kobiela et al., 2018). In epidemiological studies, the administration of the biguanide metformin, the most commonly prescribed anti-diabetic agent (He and Wondisford, 2015; Rena et al., 2013), has been associated with lower CRC incidence and mortality in T2DM patients (Zhang et al., 2011). Furthermore, a recent double-blind placebo-controlled, randomized trial that showed that low-dose metformin significantly decreased colorectal adenoma recurrence (Higurashi et al., 2016). Despite its potential clinical importance, the cellular and molecular mechanism(s) by which metformin acts as a preventive agent in CRC and other malignancies remains poorly understood (Li et al., 2018).
It is recognized that aberrant activation of β-catenin signaling plays a central role in the majority of sporadic colorectal tumors (Clevers, 2006; Clevers and Nusse, 2012; McDonald et al., 2006). In most cases, the Wnt/β-catenin pathway undergoes an early deregulation that leads to the nuclear accumulation of β-catenin and the constitutive activation of its target genes (Cheah, 2009; Huels et al., 2015; Kinzler and Vogelstein, 1996; Krausova and Korinek, 2014; Polakis, 2012; Sansom et al., 2004; Walther et al., 2009). Phosphorylation cascades that are dependent and independent of Wnt signaling play a critical role in the control of β-catenin stability, intracellular distribution and transcriptional activity (Clevers, 2006; Fang et al., 2007; He et al., 2007; Taurin et al., 2006, 2008; Vermeulen et al., 2010). For example, the canonical sequential phosphorylation of β-catenin in its N-terminal domain targets β-catenin for proteosomal degradation (Clevers and Nusse, 2012) whereas its non-canonical phosphorylation at Ser552 and Ser675 promotes its nuclear translocation and transcriptional activity (Fang et al., 2007; Rey et al., 2012; Taurin et al., 2006). We hypothesized that phosphorylation of β-catenin at Ser552 in response to insulin and IGF-1 provides a plausible mechanism by which T2DM and metabolic syndrome potentiates the promotion of CRC. As a corollary of this hypothesis, we considered that metformin targets this pathway, thus inhibiting β-catenin signaling and progression of CRC. In an effort to test these hypotheses, we examined whether metformin affects β-catenin phosphorylation in human colorectal adenocarcinoma-derived cells stimulated with insulin or IGF-1. Our results show that metformin inhibits β-catenin Ser552 phosphorylation by a mechanism that involves 5′ AMP-activated protein kinase (AMPK)-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling inhibition.
Section snippets
Cell culture and transfections
The human colorectal adenocarcinoma-derived cell lines SW-480 and HT-29 were obtained from the American Type Culture Collection (Manassas, Virginia, USA) and maintained as recommended. Transfections were performed with Lipofectamine Plus (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as previously described (Rey et al., 2001). Dulbecco’s Modified Eagle’s medium (DMEM) containing physiological (5.0 mM) concentration of glucose was employed for all the experimental procedures.
Western blot analysis
The
Metformin inhibits β-catenin Ser552 phosphorylation induced by insulin and IGF-1 in human colorectal cell lines
The phosphorylation of β-catenin on Ser552 has been linked to non-canonical positive regulation of β-catenin intracellular distribution and transcriptional activity (Fang et al., 2007; Rey et al., 2012; Taurin et al., 2006). Consequently, we initially determined whether insulin and IGF-1, which promote CRC development (Aleman et al., 2014; Cohen and LeRoith, 2012; Kant and Hull, 2011), have any impact on phosphorylation of β-catenin on Ser552 in CRC-derived cells. Stimulation of human SW-480
Concluding remarks
It is well established that over 90% of CRCs are promoted by activation of the Wnt/β-catenin pathway leading to robust β-catenin nuclear accumulation (Walther et al., 2009). Consequently, we hypothesized that the chemopreventive properties of the antidiabetic agent metformin are mediated, at least in part, by direct interference with β-catenin signaling. In support of this hypothesis, our results indicate that metformin inhibited β-catenin Ser552 phosphorylation and promoted its plasma membrane
Acknowledgements
This work was supported by FONCYT, Secretaría de Ciencia Tecnología e Innovación Productiva, Argentina, PICT 2012-0875 and PICT 2013-0891 to O. R. and PICT 2015-2996 to C. D. E. R. is supported by NIH P30DK041301 and VA I01BX003801. G. A. and E. M. L. are recipients of fellowship awards from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
References (55)
- et al.
Mechanisms of obesity-induced gastrointestinal neoplasia
Gastroenterology
(2014) - et al.
Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases
Pharmacol. Ther.
(2015) - et al.
Dose-dependent relationship between metformin and colorectal Cancer occurrence among patients with type 2 Diabetes-A nationwide cohort study
Trans. Oncol.
(2018) Recent advances in colorectal cancer genetics and diagnostics
Crit. Rev. Oncol. Hematol.
(2009)Wnt/beta-catenin signaling in development and disease
Cell
(2006)- et al.
Wnt/beta-catenin signaling and disease
Cell
(2012) - et al.
Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I
J. Biol. Chem.
(2000) - et al.
Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity
J. Biol. Chem.
(2007) - et al.
Metformin action: concentrations matter
Cell Metab.
(2015) - et al.
Metformin for chemoprevention of metachronous colorectal adenoma or polyps in post-polypectomy patients without diabetes: a multicentre double-blind, placebo-controlled, randomised phase 3 trial
Lancet Oncol.
(2016)