Therapeutic Discovery Sorafenib-Mediated Targeting of the AAA þ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death Ping Yi 1,2,5 , Arisa Higa 1,2 , Said Taouji 1,2 , Mariana G. Bexiga 1,2,6 , Esther Marza 1,2 , Daniela Arma 1,2 , Claire Castain 3 , Brigitte Le Bail 1,2,3 , Jeremy C. Simpson 6 , Jean Rosenbaum 1,2 , Charles Balabaud 1,2 , Paulette Bioulac-Sage 1,2,3 , Jean-Fr ed eric Blanc 1,2,4 , and Eric Chevet 1,2 Abstract The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer–derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal–regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA þ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 2610–20. Ó2012 AACR. Introduction Hepatocellular carcinoma (HCC) is globally the third cause of cancer-related death, with the majority of patients dying within 1 year of diagnosis (1). Recently sorafenib, a multikinase inhibitor of the Raf/MEK/ERK pathway and of tyrosine kinase receptors, was shown to induce apo- ptosis as well as inhibiting tumor cell proliferation and angiogenesis in a variety of tumors (2). The molecular involvement of the Raf-1 and tyrosine kinase signaling pathways is well established in the pathogenesis of HCC and provided a rationale for the use of sorafenib in HCC treatment. Indeed, sorafenib was the first targeted therapy to improve the outcome of patients with HCC (3) and has become a standard treatment of such disease. In preclin- ical experiments, sorafenib reduced tumor angiogenesis and increased tumor apoptosis in a mouse xenograft model of human HCC (4). However, the precise mechan- isms through which sorafenib induces cell death in HCC remain to be identified. Reports have indicated that sorafenib treatment may induce cell death signaling pathways through the activa- tion of endoplasmic reticulum (ER) stress (5–10). The ER is the first compartment of the secretory pathway and is composed of a system of interconnected membranous tubules and vesicles in which proteins and lipids are synthesized and intracellular calcium levels are regulated. In addition, the ER functions in the maintenance of protein homeostasis (proteostasis) of secreted and transmem- brane proteins. Misfolded or toxic proteins present in the ER are retrotranslocated to the cytosol and are subse- quently degraded by the proteasome, in a process known as ER-associated degradation (ERAD). When the levels of misfolded protein overwhelms the ER folding and degrading capacity, an evolutionarily conserved cellular adaptive response, named the unfolded protein response Authors' Affiliations: 1 Institut National de la Sant e et de la Recherche Medicale (INSERM) U1053; 2 Universit e Bordeaux-Segalen; 3 Service d'Anatomie Pathologique, H^ opital Pellegrin; 4 Service d'H epatologie, H^ opital St Andr e, CHU Bordeaux, Bordeaux, France; 5 Sino-France Labo- ratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China; and 6 School of Biology and Environmental Science and Conway Institute of Biomolec- ular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Eric Chevet, Institut National de la Sant e et de la Recherche Medicale (INSERM) U1053, Universit e Bordeaux-Segalen, 146 rue Leo Saignat 33076 Bordeaux, France. Phone: 33-0-557579253; Fax: 33-0-556514077; E-mail: eric.chevet@inserm.fr doi: 10.1158/1535-7163.MCT-12-0516 Ó2012 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 11(12) December 2012 2610 on June 2, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst October 5, 2012; DOI: 10.1158/1535-7163.MCT-12-0516