Abstract P5-10-04: Metformin mediated upregulation of microRNA-193 triggers apoptosis by decreasing fatty acid synthase

Abstract

Abstract Background: We have previously shown that the anti-diabetic drug metformin kills triple negative (TN) breast cancer cells lines via apoptosis, at lower IC50 than luminal and HER2 subtypes (presented at ENDO, manuscript in preparation). Metformin reportedly shows greater efficacy against stem cells than differentiated cells, reducing mammosphere formation in in non-adherent culture. Fatty acid synthase (FASN) is an enzyme critical for de novo fatty acid synthesis that is overexpressed in breast cancer. Hypothesis: We hypothesized that miRNAs might be involved in the ability of metformin to preferentially kill TN cell lines at a lower IC50 than luminal A lines. Methods: TN and luminal A (LA) breast cancer cell lines (MDA-468, HCC70 and MCF7, T47D, respectively) were used to study the effects of 10mM metformin action in vitro, at physiological and hyperglycemic culture conditions. Affymetrix chips Human Gene 1.1 and the miRNA 2.0 were utilized to profile gene and miRNA expression at 6 and 24 hrs. Mimics and lentiviral expression vectors were utilized to manipulate miRNA expression and a luciferase reporter was used to confirm miR-193 direct targeting of the FASN 3′ UTR. Results: miR-193 is significantly higher in untreated LA as compared to TN cells. MiR-193 increases 2–4 fold within 6 hrs of metformin treatment in both TN, but not LA cell lines. A predicted target of miR-193, fatty acid synthase (FASN), is decreased by 8 fold following 24 hrs 10mM metformin treatment of the TN breast cancer cells. miR-193 directly targets FASN via a binding site in the 3′ UTR, downregulating gene expression. Restoration of miR-193 to TN lines, causes a dramatic decrease in FASN protein in a dose dependent fashion. Conclusions and future directions: Metformin stimulates an increase in mature miR-193, which mediates the dramatic downregulation of FASN. This occurs coincident with apoptotic cell death. It remains to be determined if FASN is the only relevant direct target of miR-193 in TN cell lines. The addition of exogenous non-targetable FASN, lacking its 3′ UTR, will demonstrate whether this pathway is a primary mechanism of metformin action in TN cells. Future studies will investigate the ability of metformin to inhibit FASN in non-adherent mammosphere (stem cell-like) subpopulations, the role of fatty acid metabolism in mammosphere maintenance and anoikis resistance. Supported by Komen Breast Cancer Foundation Grants KG100575 (ADT, JKR, SME, NSS) and KG090415 (JKR, DRC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-04.