The major vault protein mediates resistance to epidermal growth factor receptor inhibition in human hepatoma cells

Abstract

To better understand the response of HCC to EGFR inhibition, we analyzed factors connected to the resistance of HCC cells against gefitinib. Sensitive HCC3 cells co-expressed EGFR and ErbB3 but lacked kinase-domain mutations in EGFR. Interestingly, expression of MVP was restricted to resistant cell lines, whereas ABCB1 and ABCC1 showed no association with gefitinib resistance. Moreover, ectopic MVP expression in HCC3 cells decreased gefitinib sensitivity, increased AKT phosphorylation and reduced the expression of inflammatory pathway-associated genes, whereas silencing of MVP in Hep3B and HepG2 cells increased sensitivity. These findings suggest MVP as a novel player in resistance against EGFR inhibition.

Introduction

Liver cancer is the fifth most common cancer worldwide, with more than 500,000 new cases per year [1]. Complete surgical resection of tumors or liver transplantation is only possible in a minority of patients; for patients with advanced disease, the prognosis is extremely poor, with an overall median survival of only a few months. Response rates to classical chemotherapy are low, and even with combination regimens, durable remission has remained elusive [2]. Thus, there is a strong need for additional therapeutic options.

In recent years, rationally designed, molecularly targeted drugs have become available. These agents are designed to target growth or survival pathways hyper-activated in cancer cells. Tyrosine kinases are thought to be excellent molecular oncology targets because they transduce growth and survival signals and are hyper-activated in most, if not all, human malignancies [3]. The ErbB receptor tyrosine kinase family, comprising EGFR (=ErbB1) and ErbB2, -3, and -4, has been of central interest in the development of targeted anticancer strategies. Trastuzumab (Herceptin), a monoclonal antibody against ErbB2, is successfully being used in patients with ErbB2-overexpressing breast cancer, and overexpression of ErbB2 through gene amplification is a good predictor of favorable response [4]. Several preclinical and clinical studies have addressed the efficacy of EGFR-targeting agents, including tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa, ZD1839) and erlotinib (Tarceva, OSI-774), as well as monoclonal anti-EGFR antibodies, such as cetuximab, for the treatment of non-small cell lung cancer (NSCLC), head and neck cancer, colon carcinoma, glioblastoma, and other tumors [5], [6]. Although NSCLC patients with activating mutations in the kinase domain of EGFR respond favorably to EGFR TKIs, leading to their approval for this subset of malignancies, the molecular basis determining the response of tumor cells to EGFR-targeting drugs in other settings is only partially understood and is discussed controversially.

In HCC, the small molecule inhibitor sorafenib (Nexavar, BAY 43-9006), which targets multiple kinases, including Raf, c-kit, vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR), is the first agent that has been found to improve survival of patients with advanced disease [7], which demonstrates that targeted agents may improve the treatment opportunities for this devastating disease [8]. Several lines of evidence also argue for an important role of deregulated EGFR signaling in HCC development. EGFR and its ligands TGFα, HB-EGF, amphiregulin and betacellulin are elevated in HCC [9], [10], [11], and have been demonstrated to drive liver tumorigenesis in cell culture and animal models [12], [13], [14]. Hepatoma cells have been shown to respond with growth inhibition and cell death induction to inhibition of EGFR with gefitinib [15], [16], erlotinib [17] or cetuximab [18], and gefitinib prevented HCC development in cirrhotic rat livers [19]. Clinical phase II trials with EGFR inhibitors, however, have shown that cetuximab has no antitumor activity and that erlotinib improved disease control in only a subset of patients [20], [21], [22], raising the question as to which mechanisms determine the response of HCC to EGFR inhibition.

In the present study, we have used a panel of extensively characterized HCC-derived cell lines [23], [24] to identify molecular factors contributing to resistance against the EGFR inhibitor gefitinib. Our data demonstrate a previously unrecognized role of the major vault protein (MVP) for EGFR-inhibitor resistance in hepatoma cells.