Metformin requires 4E-BPs to induce apoptosis and repress translation of Mcl-1 in hepatocellular carcinoma cells

// Mamatha Bhat 1, 2, 3 , Akiko Yanagiya 1 , Tyson Graber 4 , Nataliya Razumilava 3, 5 , Steve Bronk 3 , Domenick Zammit 1 , Yunhao Zhao 6 , Chadi Zakaria 1 , Peter Metrakos 7 , Michael Pollak 6 , Nahum Sonenberg 1 , Gregory Gores 3 , Maritza Jaramillo 8 , Masahiro Morita 1, * and Tommy Alain 4, * 1 Goodman Cancer Centre, Department of Biochemistry, McGill University, Montreal, Canada 2 Division of Gastroenterology, University Health Network and University of Toronto, Toronto, Canada, USA 3 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 4 Children’s Hospital of Eastern Ontario Research Institute, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada 5 Division of Gastroenterology, University of Michigan, Ann Arbor, Michigan, USA 6 Departments of Medicine and Oncology, Lady Davis Institute for Medical Research and Segal Cancer Center, Montreal, Canada 7 Department of Surgery, McGill University Health Centre, Montreal, Canada 8 INRS Institut Armand-Frappier Research Centre, Laval, Quebec, Canada * These authors have contributed equally to this work Correspondence to: Mamatha Bhat, email: mamatha.bhat@uhn.ca Masahiro Morita, email: masahiro.morita@mail.mcgill.ca Tommy Alain, email: tommy@arc.cheo.ca Keywords: metformin, hepatocellular carcinoma, mRNA translation, mTORC1, 4E-BPs Received: October 04, 2015     Accepted: July 06, 2016     Published: July 18, 2016 ABSTRACT Metformin inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which is frequently upregulated in hepatocellular carcinoma (HCC). Metformin has also been shown to induce apoptosis in this cancer. Here, we investigate whether metformin-induced apoptosis in HCC is mediated by the downstream mTORC1 effectors eukaryotic initiation factor 4E and (eIF4E)-binding proteins (4E-BPs). Further, we ask whether changes in 4E-BPs activity during metformin treatment negatively regulate translation of the anti-apoptotic myeloid cell leukemia 1 ( Mcl-1 ) mRNA. A genetic HCC mouse model was employed to assess the ability of metformin to reduce tumor formation, induce apoptosis, and control 4E-BP1 activation and Mcl-1 protein expression. In parallel, the HCC cell line Huh7 was transduced with scrambled shRNA (control) or shRNAs targeting 4E-BP1 and 4E-BP2 (4E-BP knock-down (KD)) to measure differences in mRNA translation, apoptosis, and Mcl-1 protein expression after metformin treatment. In addition, immunohistochemical staining of eIF4E and 4E-BP1 protein levels was addressed in a HCC patient tissue microarray. We found that metformin decreased HCC tumor burden, and tumor tissues showed elevated apoptosis with reduced Mcl-1 and phosphorylated 4E-BP1 protein levels. In control but not 4E-BP KD Huh7 cells, metformin induced apoptosis and repressed Mcl-1 mRNA translation and protein levels. Immunostaining of HCC patient tumor tissues revealed a varying ratio of eIF4E/4E-BP1 expression. Our results propose that metformin induces apoptosis in mouse and cellular models of HCC through activation of 4E-BPs, thus tumors with elevated expression of 4E-BPs may display improved clinical chemopreventive benefit of metformin.