Characterization of novel putative tumor suppressor gene, DLC2 (deleted in liver cancer 2), in hepatocellular carcinoma

Abstract

Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene located at chromosome 13q12.3, was first identified by our group (Ching et al., J Biol Chem 2003;278:10824-10830). The DLC2 gene has striking homology to another tumor suppressor gene, DLC1, located at chromosome 8p22-8p21.3. DLC2 encodes a Rho GTPase activating protein (RhoGAP) with GAP activity specific for RhoA and Cdc42. The chromosomal region of the DLC2 gene shows frequent deletion with loss of heterozygosity in human hepatocellular carcinoma (HCC). By RT-PCR analysis, DLC2 is also frequently underexpressed in HCCs compared with the corresponding nontumorous livers. These suggest that the DLC2 gene may play a role in liver carcinogenesis. In this study, we aimed to further characterize DLC2 biochemically and functionally. From the bioinformatics search of DLC2 gene in humans, we found that there were four different isoforms of DLC2 gene, which we designate as DLC2α, β, γ, and δ. The four isoforms of DLC2 gene were identified in silico. With RT-PCR using four pairs of isoform specific primers performed on 10 HCC cell lines, DLC2α, γ, and δ were found to be expressed in all hepatoma cell lines tested, with different levels of expression. In contrast, the mRNA expression of the DLC2β isoform could be detected in only 4 of these 10 HCC cell lines. Direct sequencing of the PCR products of these isoforms confirmed the presence of these isoforms. In addition, we analyzed the localization and the in vivo function of DLC2 gene. Transfection of DLC2α and DLC2γ into Swiss3T3 cells showed their predominant cytoplasmic localization. Interestingly, over-expression of DLC2α and DLC2γ induced morphological alterations in the transfected Swiss3T3 cells. The shape of transfected Swiss3T3 cells was changed from angular and spindle to round when compared with the non-transfected parent cells. Confocal microscopy revealed that the morphological changes were associated with remarkable inhibition of actin stress fiber formation. In conclusion, these results were in keeping with the functions of DLC2 as a RhoGAP and that it induced morphological alterations through regulation of cytoskeletal reorganization. DLC2 likely exerts its tumor suppressor function partly via its RhoGAP function to inhibit RhoA activity, resulting in suppression of stress fiber formation. Further studies on the functional characterization of the different isoforms of DLC2 may give insight into its tumor suppressor function.