Molecular and Cellular Pathobiology miRNA-491-5p and GIT1 Serve as Modulators and Biomarkers for Oral Squamous Cell Carcinoma Invasion and Metastasis

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols frompoorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein—coupled receptor kinase-interacting protein 1 (GIT1)—as a direct target gene formiR-491-5p control. GIT1 overexpressionwas sufficient to rescuemiR-491-5p–mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal–regulated kinase (ERK1/2) activation, andMMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph nodemetastasis, with expression levels of miR-491-5p and GIT1 correlated inversely inOSCCs, where they informed tumor grade. Together, ourfindings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer. Cancer Res; 74(3); 751–64. 2013 AACR. Introduction Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide and accounts for more than 95% of all head and neck cancer (1). The incidence of oral cancer in Taiwan increased by 30% in the last 5 years, mortality rate grew by 25% with 30 to 49 years old men having the highest rate. More than 44,000 patients annually require sustained medical treatment (2, 3). Unlike other types of solid tumors, the metastasis of OSCCs mostly involves local invasion and often is restricted to the head and neck area. Cervical lymph node metastasis is a critical prognostic factor for OSCCs, the patients without such metastasis usually have higher survival rates (4–6). Like other cancers, oral cancer metastasis requires an extensive remodeling and degradation of extracellular matrix (ECM), in part, via increased expression of matrix metalloproteinases (MMP; ref. 7). However, the molecular mechanism regulating the invasion and metastasis of OSCCs is still largely unclear. MicroRNAs (miRNA) are an evolutionarily conserved group of small RNAs of 18 to 24 nucleotides that inhibit or stimulate gene expression. microRNAs have been known to play important roles in various cancer progression and metastasis (8, 9); however, study of their roles in OSCC metastasis is relatively scarce (10). GIT1 is a multifunctional scaffold protein found to be associated with paxillin and capable of stimulating lamellipodia formation and spreading of cells (11–13). Although the GIT1/paxillin complex is known to play a role in regulating focal adhesion formation and cell migration (14), the precise role of GIT1 in metastasis of cancer especially in OSCCs is Authors' Affiliations: Institute of Molecular and Genomic Medicine, Division of Environmental Health and Occupational Medicine, National Health Research Institute, Miaoli; Department of Life Sciences, National Central University, Jhongli; Environment-Omics-Disease Research Center, China Medical University Hospital, Taichung; Departments of Medical Biotechnology, Chang Gung University; Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital; Head and Neck Oncology Group, Chang GungMemorial Hospital, Taoyuan; Department of Oral and Maxillofacial Section, Chi-Mei Medical Center, Liouying; and Department of Oral Hygiene, Kaohsiung Medical University, Kaohsiung, Taiwan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). W.-C. Huang and S.-H. Chan contributed equally to this work and are cofirst authors. Corresponding Author: Lu-Hai Wang, National Health Research Institute and National Central University, No. 35, Keyan Road, Zhunan, Miaoli County 35053, Taiwan. Phone: 886-37-246166, ext. 31010; Fax: 88637-585242; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-1297 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org 751 on June 6, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1297