Abstract P5-10-05: Novel Mechanisms of Metformin Action in TN Breast Cancer: Upregulation of miRNA 141 and 192, are Associated with a Decrease in Targets GRB2 and MSN Involved in Signaling and Motility Respectively
Authors: Edgerton SM , Richer JK , Fan Z , Spoelstra NS , Wahdan-Alsawad RS , Arnadottir SS , Thor AD 
Published: August 2014

Abstract
BACKGROUND: We have previously shown that the anti-diabetic agent, metformin, inhibits cellular proliferation, colony formation and cell signaling, with an induction of S-phase arrest and apoptosis in triple negative (TN) breast cancer cell lines in vitro, and cell outgrowth and proliferation in vivo. These changes are associated with profound shifts in phosphorylated cell signaling intermediates, including EGFR, IGF-1R, Akt, MAPK and others (Liu, et al Cell Cycle 2009). We have also shown that Stat3 activity (via phosphorylation at serine and tyrosine sites) is critical to the effects of metformin against TN cells (Deng, et al Cell Cycle 2012). In unpublished studies, we have noted that metformin significantly inhibits cellular motility in vitro. In this study we focus on mechanisms underlying the effects of metformin in TN cancer cell signaling and motility.
METHODS: TN breast cancer cell lines MDA-MB-468 and MDA-MB-231were used to study mechanisms of metformin action in vitro. Invasion chamber and motility assays were used to quantitate the effects of metformin as compared to untreated controls, with and without a variety of inhibitors at normal or supraphysiological glucose concentrations. Affymetrix chips Human Gene 1.1 and miRNA 2.0 were used to identify genes and miRNA up or down regulated by metformin treatment at 6 and 24 hr at glucose concentrations of 5, 10 or 17 mM. Appropriate primers and antibodies were used to validate these data at the transcript and protein levels respectively by qRT-PCR and western blot analyses. Lentiviral expression vectors and mimics were used to manipulate miRNA expression levels. Direct targeting of the GRB2 and MSN 3′ UTR's were performed using luciferase reporters.
RESULTS: TargetScan and Diana miR-Path analysis of miRNA's up-regulated at 6 hr following metformin treatmetns identified genes that were down regulated with metformin treatment such as GRB2 (a predicted target of miR-141) and MSN (a predicted target of miR-192), as possible mechanisms of drug action. GRB2 regulated the phosphorylation of numerous receptor tyrosine kinase (RTK) gene pathway intermediates, including those downstream of EGFR, IGF-1R and MAPK. Knock-down of miR-141 by an antagomir showed that miR-141 regulated GRB2. Invasion chamber and motility assays confirm that the metformin associated reduction in TN cell motility and invasion capacity involved miR-193 and MSN.
CONCLUSIONS: Metformin acts by diverse molecular mechanisms; some of these may be cell type specific. In TN breast cancer cells, metformin causes a rapid increase in miR-141, which targets GRB2, a gene that encodes a critical regulator upstream of many growth factors. Metformin treatment also upregulates miR-192, which represses MSN to reduce motility and invasion.
Supported by Komen Breast Cancer Foundation Grant KG100575 (SME, JKR, ZF, NSS, ADT)
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-05.