RhoGAP DLC-1 tumor suppression and aberrant Rho GTPase activation in lung cancer

4125 The deleted in liver cancer-1 (DLC-1) gene was originally identified as a tumor suppressor gene whose expression was lost in hepatocellular carcinomas. Subsequently, reduced expression of DLC-1 mRNA was reported in breast, colon, prostate, gastric, and lung cancers, with reduced transcript expression of DLC-1 in 95% of primary non-small cell lung carcinomas (NSCLC) and nearly 60% of NSCLC cell lines. DLC-1 encodes a RhoGAP domain (GTPase activating protein for Rho family small GTPases) that is hypothesized to be responsible for the tumor-suppressor function of DLC-1. In vitro studies have demonstrated that DLC-1 inactivates the small GTPase RhoA by catalyzing GTP hydrolysis activity. However, whether the tumor suppressor function of DLC-1 is a consequence of the inhibition of RhoA, and whether RhoA activation is important for the aberrant growth of NSCLCs have not been determined. We have performed western blot analysis on a panel of six NSCLC cell lines, and have demonstrated that DLC-1 expression is lost in A549, H23, H358, and H2228 cells. Ectopic restoration of DLC-1 expression in DLC-1 deficient cell lines resulted in reduced proliferation on plastic as well as reduced anchorage-independent growth. Pull down analyses with the GTP-binding fragment of the RhoA effector Rhotekin has shown that DLC-1 expression results in reduced RhoA-GTP levels in vivo . We have also utilized short interfering RNA (siRNA) to knockdown endogenous DLC-1 in the DLC-1 positive NSCLC lines, and are currently assessing the impact of reduced DLC-1 expression on the transforming properties and Rho signaling pathways of these cells.