Head and neck cancer is the sixth most common cancer world wide (1
). As per the statistical report of the American Cancer Society, about 70,000 new head and neck squamous cell carcinoma will be diagnosed this year in the United States (2
). In contrast to other epithelial cancers for which effective screening exists, most of the patients with head and neck cancer are diagnosed at a very late stage (stage III and IV). Despite the advancement in surgical procedures, chemotherapy, and radiation therapy, survival rates have not improved in the last several decades (3
). Furthermore, it has been shown that the high rate of morbidity is due to both locoregional recurrence and distant metastases.
In the past decade, numerous studies have shown that the Rho family of GTPases, (RhoA, RhoB, RhoC, Rac1, Rac2, Rac3 and CDC42) is involved in instilling a metastatic phenotype into localized cancerous cells that are localized to the organ of origin. RhoA and RhoC are over expressed in a number of tumor types (4
) suggesting an oncogenic role. Among the Ras homology protein family, RhoC (molecular mass of 21 kDa) has been implicated in a wide range of cellular activities, including downstream expression of inflammatory genes and chemokines, cell proliferation, intracellular signaling, and cytoskeletal organization (6
). More significantly, RhoC plays a central role in the assembling focal adhesion, by modulating the orientation of cytoskeletal fibers, resulting in cell polarity, increased cell motility and, consequently, increased invasiveness (7
). In addition, signaling mediated by Rho proteins through Rho activating kinase (ROCK) regulate proteins that in turn regulate actin polymerization such as cofilin, profilin and formin homology (FH) proteins (10
). Interestingly, high levels of RhoC and ROCK are also associated with membrane blebbing, a phenomenon that is observed in motile or invasive cells (10
RhoC over expression is now well documented in a wide range of malignant cancers suggesting an important role in changing non-invasive carcinomas into invasive forms. Interestingly, over expression of RhoC has been reported in inflammatory breast cancer and exclusively in invasive breast carcinoma (12
). Other tumor types where over expression of RhoC has been reported are ovarian carcinoma (16
), esophageal squamous cell carcinoma (17
), pancreatic cancer (18
), gastric cancer (17
) and human melanoma (11
). In addition, functional studies have shown that RhoC can act as a transforming oncogene when it is over expressed in human mammary epithelia converting these normally immobile cells into highly motile and invasive malignant cells (12
). Thus, a wide range of current studies reveal an important role of RhoC in cancer metastasis.
However, very few studies to date have investigated the role of RhoC in head and neck cancer. Studies on gene expression profiling of stage III and IV regionally metastatic head and neck squamous cell carcinoma (HNSCC) showed that there is elevated levels of RhoC when compared to stage I and II localized malignancy (22
). Furthermore, in our laboratory, we have shown that there is elevated RhoC expression in tumors of patients with HNSCC when compared to normal squamous cell epithelium (21
). More importantly, our study showed that increased RhoC expression is strongly associated with lymph node metastasis and could also be used to predict metastasis even in small (T1, T2) primary tumors (23
). In the present study, we investigated the role of RhoC in head and neck metastasis by inhibiting its function using RNA interference (RNAi). Our in vitro
findings determined that inhibiting RhoC function strongly reduced cell motility and invasion. Furthermore, we observed a remarkable reduction in tumor metastasis and microvessel density in SCID mice injected with RhoC knockdown cell lines. These findings suggest that inhibition of RhoC function in head and neck squamous cell carcinoma can diminish a tumor's aggressive behavior, thus opening new possibilities for future drug therapies targeting this pathway.