RhoC expression and head and neck cancer metastasis.

RhoC protein, a known marker of metastases in aggressive breast cancers and melanoma, has also been found to be overexpressed in certain head and neck cancers, thus we investigated the correlation between RhoC expression and the metastatic behavior of head and neck squamous cell carcinoma. Selective inhibition of RhoC expression was achieved using lentiviral small hairpin RNA (shRNA) transduced and tracked with green fluorescent protein to achieve 70% to 80% RhoC inhibition. Fluorescence microscopy of the RhoC knockdown stable clones showed strong green fluorescence in the majority of cells, signifying a high efficiency of transduction. Importantly, quantitative real-time PCR showed no significant decrease in the mRNA expression levels of other members of the Ras superfamily. Cell motility and invasion were markedly diminished in RhoC-depleted cell lines as compared with control transduced lines. H&E staining of lung tissue obtained from severe combined immunodeficiency mice, which had been implanted with RhoC knockdown cells, showed a marked decrease in lung metastasis and inflammation of the blood vessels. The cultured lung tissue showed a significant decrease in cell growth in mice implanted with RhoC-depleted cell lines as compared with shRNA-scrambled sequence control lines. Microscopic studies of CD31 expression revealed substantial quantitative and qualitative differences in the primary tumor microvessel density as compared with parental and shRNA-scrambled controls. This study is the first of its kind to establish the involvement of RhoC specifically in head and neck metastasis. These findings suggest that RhoC warrants further investigation to delineate its robustness as a novel potentially therapeutic target.