Molecular and Cellular Pathobiology Lysophosphatidic Acid Receptor LPAR6 Supports the Tumorigenicity of Hepatocellular Carcinoma

The aberrant processes driving hepatocellular carcinoma (HCC) are not fully understood. Lysophosphatidic acid receptors (LPAR) are commonly overexpressed in HCC, but their contributions to malignant development are not well established. In this report, we show that aberrant expression of LPAR6 sustains tumorigenesis and growth of HCC. Overexpression of LPAR6 in HCC specimens associated with poor survival in a cohort of 128 patients with HCC. We took a genetic approach to elucidate how LPAR6 sustains the HCC tumorigenic process, including through an expression profiling analysis to identify genes under the control of LPAR6. RNAi-mediated attenuation of LPAR6 impaired HCC tumorigenicity in tumor xenograft assays. Expression profiling and mechanistic analyses identified Pim-3 as a pathophysiologically relevant LPAR6 target gene. In nonmalignant cells where LPAR6 overexpression was sufficient to drive malignant character, Pim-3 was upregulated at the level of transcription initiation through a STAT3-dependent mechanism. A further analysis of HCC clinical specimens validated the connection between overexpression of LPAR6 and Pim-3, high proliferation rates, and poorer survival outcomes. Together, our findings establish LPAR6 as an important theranostic target in HCC tumorigenesis. Cancer Res; 75(3); 1–12. 2014 AACR. Introduction Hepatocellular carcinoma (HCC) is a very frequent malignancy, being the sixth most common cancer worldwide (1). In addition, the incidence of HCC has been increasing over the last years, especially in western countries (2). The etiology of HCC is multifactorial, and the disease is very often preceded by other conditions, including chronic inflammation, liver fibrosis, and cirrhosis, that are frequently associated with hepatitis B virus or hepatitis C virus infection, carcinogen/toxin exposure including alcohol and aflatoxin; gender and metabolic conditions are other risk factors (3, 4). Given the multifactorial etiology of HCC, a better understanding of the common molecular pathogenic mechanisms underlying the tumorigenic process of this disease is essential to help identify more effective therapeutic targets. In this context, different molecular mechanisms driving the process evolving from chronic inflammation and cirrhosis to the formation of dysplastic nodules have been identified (5). These mechanisms may not only involve hepatocytes but also liver microenvironment elements, which contribute to HCC growth and progression (6–8). The HCC microenvironment is considered as a metastasis-promoting element in itself, and although the presence of distant metastases in HCC is rarely observed, intrahepatic metastases are very frequent, being a clear sign of tumor progression and an adverse prognostic factor. Although different mechanisms and predisposing factors for the development of intrahepatic metastases have been investigated (9, 10), intrahepatic metastasis remains a serious clinical problem and the main cause of HCC recurrence. Lysophosphatidic acid (LPA) receptors are G protein–coupled receptors (GPCR) that bind the bioactive phospholipid LPA and activate multiple cellular responses, including cell proliferation, cytoskeletal rearrangements, motility, and apoptosis (11–13). At least five LPAR (LPAR1-5) are relatively well characterized and currently under investigation (14). LPAR6 is a newly identified receptor, originally referred to as purinergic receptor P2Y5 that has been involved in inherited forms of hair loss, specifically hypotrichosis simplex (15) and woolly hair (16). However, other than these reports, the role of LPAR6 in human disease including cancer has not been investigated. Here, we show that LPAR6 is essential for maintaining the proliferation capacity and the tumorigenic phenotype of HCC, and that the tumorigenicity control exerted by LPAR6 occurs through the transcriptional activation of proto-oncogene Pim-3. This mechanism sustains HCC growth and progression. Interdisciplinary Department ofMedicine, University of Bari School of Medicine, Bari, Italy. IRCCS "S. de Bellis", National Institute for Digestive Diseases, Bari, Italy. Department of Emergency andOrgan Transplantation, Section of Internal Medicine, Allergology and Clinical Immunology, University of Bari School of Medicine, Bari, Italy. Institut Curie, Centre de Recherche, Pole de Biologie du D eveloppement et Cancer, Paris, France. InstituteofHumanGenetics,UniversityofBonn, Bonn, Germany. Cancer Research Institute, Fudan University Shanghai Cancer Center, Shanghai, China. Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. GenXPro GmbH, Altenh€ oferallee 3, Frankfurt Main, Germany. Department of Biomedical Sciences and Human Oncology, University of Bari School of Medicine, Bari, Italy. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). G. Giannelli and C. Sabb a contributed equally to this article. Corresponding Author: Antonio Mazzocca, University of Bari School of Medicine, Piazza G. Cesare 11, 70124 Bari, Italy. Phone: 39-080-5593-593; Fax: 39080-5478-126; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-1607 2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 28, 2017. © 2015 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 14, 2015; DOI: 10.1158/0008-5472.CAN-14-1607