Hedgehog Signaling Antagonist Promotes Regression of Both Liver Fibrosis and Hepatocellular Carcinoma in a Murine Model of Primary Liver Cancer George M. Philips1., Isaac S. Chan1,2., Marzena Swiderska1, Vanessa T. Schroder3, Cynthia Guy4, Gamze F. Karaca1, Cynthia Moylan 1 , Talaignair Venkatraman 5 , Sebastian Feuerlein5, Wing-Kin Syn 1 , Youngmi Jung1,6, Rafal P. Witek1, Steve Choi1, Gregory A. Michelotti1, Fatima Rangwala7, Elmar Merkle5, Christopher Lascola5, Anna Mae Diehl1* 1 Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America, 2 Department of Genetics, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America, 3 Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America, 4 Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States of America, 5 Department of Radiology, Duke University, Durham, North Carolina, United States of America, 6 Department of Biological Science, Pusan National University, Pusan, Korea, 7 Divisions of Cell Therapy, Hematology and Medical Oncology, Duke University Medical Center, Durham, North Carolina, United States of America Abstract Objective: Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogen- esis. We used Mdr22/2 mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC. Methods: Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr22/2 mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging. Results: Unlike controls, Mdr22/2 mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra- hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality. Conclusions: Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced. Citation: Philips GM, Chan IS, Swiderska M, Schroder VT, Guy C, et al. (2011) Hedgehog Signaling Antagonist Promotes Regression of Both Liver Fibrosis and Hepatocellular Carcinoma in a Murine Model of Primary Liver Cancer. PLoS ONE 6(9): e23943. doi:10.1371/journal.pone.0023943 Editor: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America Received June 13, 2011; Accepted July 27, 2011; Published September 2, 2011 Copyright: 2011 Philips et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported, in part, by National Institutes of Health (NIH) RO1 DK077794, NIH RO1 AA010154, NIH T32 DK007568, and NIH T32 CA071341. A portion of the imaging was performed at the Duke Center for In Vivo Microscopy, supported, in part, by National Center for Research Resources National Biomedical Technology Research Center (P41 RR005959) and National Cancer Institute Small Animal Imaging Resource Program (U24 CA092656). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: annamae.diehl@duke.edu . These two authors contributed equally to the work. Introduction Hepatocellular carcinoma (HCC) is an insidious cancer that accounts for up to 1 million deaths a year and is the third leading cause of cancer deaths worldwide [1]. Cirrhosis, a consequence of progressive liver injury and fibrosis, is the single largest risk factor for HCC [2]. An altered wound healing response to chronic liver injury, with resultant dysregulated activation of myofibroblasts and progenitor cell populations, has been implicated in cirrhosis pathogenesis and eventual carcinogenesis [3]. Loss-of-function mutations in the hepatocyte canalicular phos- pholipid flippase MDR3 (ABCB4) have been associated with a wide range of human biliary diseases including progressive familial intrahepatic cholestasis type 3 (PFIC3), cholestasis of pregnancy, drug induced cholestasis and an adult biliary cirrhosis with fea- tures similar to primary sclerosing cholangitis (PSC) [4,5,6,7]. Mice with a targeted deletion of Mdr2 (the murine ortholog of MDR3) lack the liver-specific P-glycoprotein that transports phosphatidylcholine (PC) across the canalicular membrane. The absence of phospholipids in bile results in progressive sclerosing PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e23943