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PDF file - 856K, Selection of highly invasive oral cancer cells (S1); Overexpressing miR-491-5p inhibited lung metastasis in SCID mice (S2); The GIT1 mRNA level is upregulated in highly invasive OSCC lines (S3); GIT1 was overexpressed in the highly invasive oral cancer lines (S4); Knockdown of GIT1 by siRNA-3 had no affect paxillin mRNA expression (S5); DN-FAK suppresses GIT1-induced cell migration and invasion in OSCC cells (S6); Effect of ERK1/2 on MMP2/9 mRNA expression in OSCC cells (S7); Overexpression of miR-491-5p did not decrease proliferation in OSCC cells before 72h time point (S8); Effect of the DN-MEK or PD98059 on GIT1 induced MMP2/9 mRNA expression in OSCC cells (S9); Depletion of FAK and GIT1 decreased the paxillin and FAK phosphorylation (S10); CGHNC9 cells were transfected with FAM-labeled miR-491-5p (S11).

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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 from poorly 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. miR-491-5p 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 for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-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, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings 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.

PDF file - 38K, Clinicopathologic features of patients (S1); Primers used in this study (S2); Venn diagram showing miR-491-5p targets through the TargetScan and DIANA-microT (S3).