The deleted in liver cancer 2 (DLC2) gene, located at chromosome 13q12.3, is a recently identified tumor suppressor gene. The gene is frequently underexpressed in human hepatocellular carcinoma, and its chromosomal region shows frequent deletion. DLC2 encodes a unique RhoGTPase-activating protein (RhoGAP) specific for small RhoGTPases, RhoA, and Cdc42. With bioinformatic analysis, we have identified four different isoforms of DLC2, which we named DLC2␣, DLC2␤, DLC2␥, and DLC2␦. Three of the isoforms contain the RhoGAP domain, namely, DLC2␣, DLC2␤, and DLC2␥. Ectopic expression of these three isoforms in mouse fibroblasts showed cytoplasmic localization. Of interest, overexpression of these isoforms suppressed the lysophosphatidic acid-induced stress fiber formation in mouse fibroblasts and changed the morphology of the transfected cells from angular and spindle to round. Furthermore, the RhoA pull-down assay demonstrated a remarkable reduction in RhoA activity in the DLC2 transiently transfected cells. In contrast, cells transfected with inactive DLC2 GAP-mutant remained unchanged in cell morphology, actin stress fiber formation, and RhoA activity. HepG2 hepatoma cells stably transfected with the DLC2␥ isoform also changed to a round morphology, as in mouse fibroblasts. Of significance, these DLC2␥ stable transfectants showed marked suppression in cell proliferation, motility, and transformation, and there was a remarkable reduction in in vivo RhoA activity in these cells. These results suggest that DLC2 exhibits its tumor suppressor functions in vivo as a GAP specific for RhoA, exerting its effects in suppression of cytoskeleton reorganization, cell growth, cell migration, and transformation. H epatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in Southeast Asia and Hong Kong. The risk factors contributing to HCC include chronic hepatitis B and C virus infection, cirrhosis, and prolonged exposure to aflatoxin B1. Although the risk factors are well defined, the molecular mechanisms of hepatocarcinogenesis are unclear.The small RhoGTPases are members of the Ras superfamily of small GTP-binding proteins and are key regulators of cytoskeleton dynamics in eukaryotic cells. RhoGTPases act as molecular switches, cycling between the active GTP-bound state and the inactive GDP-bound state, and regulate a number of cellular processes such as cytoskeleton reorganization, gene transcription, and cell cycle progression (1). The best characterized Rho family members in mammalian cells are RhoA, Cdc42, and Rac1. RhoA is involved in the formation of stress fibers and focal adhesions, whereas Cdc42 and Rac1 work together at the leading edges of cells to direct the formation of lamellipodia and filopodia, respectively (2-4).In addition to the regulation of the cytoskeleton reorganization, RhoGTPases are involved in cell proliferation and cell motility (5). Active RhoGTPases promote cells to progress from G 1 to S phase, whereas inhibition of RhoGTPases blocks serum-induced G 1 progression (6).I...

The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA-mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that overexpression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation. Cancer Res; 71(8); 2949-58. Ó2011 AACR.

Oncogenic activation of the WNT/b-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Dishevelled (Dvl), a key activator of the pathway, inhibits the adenomatous polyposis coli complex, and this leads to the accumulation of b-catenin and promotes tumorigenesis. Recently, a novel inhibitor of Dishevelled, namely Dapper (Dpr), was isolated in Xenopus. To explore whether HDPR1, the human homologue of Dpr, has an anti-oncogenic role in hepatocarcinogenesis, we studied the expression of this gene in HCCs. We found that there were two alternatively spliced transcripts of HDPR1, designated as a and b forms, in human liver. Downregulation of the gene expression was observed in 31 (43%) of the 72 human HCC samples using the primer pair that amplified both transcripts. Furthermore, the HDPR1a was downregulated in 42 (58%) of 72 human HCCs and the downregulation significantly correlated with accumulation of b-catenin. Also, downregulation of HDPR1 by RNA interference in HLE cells led to cytoplasmic accumulation of b-catenin. Furthermore, a CpG island located at the promoter region and exon 1 of the HDPR1 gene was methylated in 22 (51%) of human HCCs. We showed that downregulation of HDPR1, in hepatoma cell lines, was associated with methylation of this CpG island using bisulfite sequencing and 5-aza-2 0 -deoxycytidine demethylation experiment. In addition to methylation-mediated downregulation of HDPR1, allelic loss (13-28% of informative cases) was detected using microsatellite markers flanking the HDPR1 locus. To conclude, downregulation of HDPR1 is common in HCCs, frequently involves hypermethylation of the promoter region, and allelic loss of the HDPR1 locus may also play a role.