Interaction of deleted in liver cancer 1 (DLC1) with tensin2 and its implications in tumor suppression

Abstract

Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide and the second commonest fatal cancer in Asia and Hong Kong. Deleted in liver cancer 1 (DLC1), encoding a Rho GTPase activating protein (RhoGAP), is a recently identified tumor suppressor gene. DLC1 is widely expressed in normal human tissues, but we have shown it is frequently underexpressed in HCC. Rho small GTPases are important regulators in actin cytoskeleton reorganization, cell proliferation, metastasis and tumorigenesis. RhoGAP proteins serve their tumor suppressor function by down-regulating Rho GTPase activity. We have previously characterized DLC1 to be a growth suppressor in HCC cell lines and documented its in vitro RhoGAP activity. Our recent findings have revealed that inhibition of Rho-mediated actin stress fiber formation by DLC1 is associated with its growth suppressive effect. In the present study we have identified tensin2 as the novel binding partner of DLC1 in a yeast two-hybrid library screening. Tensin2 belongs to a new family of focal adhesion proteins that play key roles in cytoskeleton organization and signal transduction. Specific in vitro and in vivo interaction between DLC1 and tensin2 was confirmed by GST pull-down assay and co-immunoprecipitation, respectively. We mapped the minimal interacting domains between two proteins to the phosphotyrosine binding domain of tensin2 and residues 375-509 of DLC1. Dysregulation of tensin proteins has previously been implicated in human cancers. Tensin2 is highly expressed in human liver. Introduction of tensin2 into HCC cell lines with low expression of tensin2 caused significant growth inhibition in colony formation assay and induction of apoptosis. Interaction between DLC1 and tensin2 is of particular interest because both interacting partners exert growth suppressive effect on HCC cell lines. Using reporter assay to investigate the effect of DLC1 and tensin2 on the serum response element (SRE), which is one of the Ras effectors, we found that expression of either DLC1 or tensin2 alone could not inhibit or only slightly inhibited the activation of SRE. The effect of inhibition was enhanced when DLC1 and tensin2 were co-expressed. Our results showed that DLC1 and tensin2 cooperatively inhibited Ras signaling based on their specific interaction. Our findings presented here will derive new insights into the biological functions of tensin2. More importantly, our work suggests a new model for the action of DLC1 in hepatocytes, whereby DLC1-tensin2 complex interacts to effect cytoskeletal reorganization and signaling pathway. We believe that a better understanding of the functional roles of DLC1 and tensin2 will have profound implications for diagnosis and therapeutic intervention of HCC. (This study was funded in part by Association for International Cancer Research and Kadoorie Charitable Foundation - Michael Kadoorie Cancer Genetic Research Program)