Hepatocellular carcinoma (HCC) is one of the major malignancies in the world. The prognosis of HCC is poor, due to frequent intrahepatic metastasis and tumor recurrence. P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Here, we show that Pak1 gene was overexpressed in human HCCs. Overexpression of Pak1 in human HCCs was associated with more aggressive tumor behavior in terms of more metastatic phenotype and more advanced tumor stages. In addition, HCC cell line stably expressing Pak1 displayed increased cell motility rates and, conversely, knockdown of endogenous Pak1 expression by small interfering RNA reduced the migration rates of HCC cells. In an established metastatic HCC cell line, we found that Pak1 was overexpressed compared with its primary HCC cell line and this overexpression was associated with higher cell motility. Importantly, we found that c-Jun NH 2 -terminal kinase (JNK) was activated in HCC cell lines overexpressing Pak1. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of HCC cells via attenuation of paxillin phosphorylation at Ser 178 . In conclusion, our results document that Pak1 is overexpressed in HCCs and plays an important role in the metastasis of HCC. The mechanism by which Pak1 induces cancer metastasis may involve activation of JNK and phosphorylation of paxillin. [Cancer Res 2007;67(8):3601-8]

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.

Supplementary Figure 2 from P21-Activated Protein Kinase Is Overexpressed in Hepatocellular Carcinoma and Enhances Cancer Metastasis Involving c-Jun NH₂-Terminal Kinase Activation and Paxillin Phosphorylation

Supplementary Figure 1 from P21-Activated Protein Kinase Is Overexpressed in Hepatocellular Carcinoma and Enhances Cancer Metastasis Involving c-Jun NH₂-Terminal Kinase Activation and Paxillin Phosphorylation