PAK4 Phosphorylates p53 at Serine 215 to Promote Liver Cancer Metastasis.

PAK4 kinase contributes to signaling pathways controlling cancer cell transformation, invasion, and survival, but its clinicopathological impact has begun to emerge only recently. Here we report that PAK4 overexpression in hepatocellular carcinoma (HCC) conveys aggressive metastatic properties. A novel nuclear splice isoform of PAK4 lacking exon 2 sequences was isolated as part of our studies. By stably overexpressing or silencing PAK4 in HCC cells, we showed that it was critical for their migration. Mechanistic investigations in this setting revealed that PAK4 directly phosphorylated p53 at S215, which not only attenuated transcriptional transactivation activity but also inhibited p53-mediated suppression of HCC cell invasion. Taken together, our results showed how PAK4 overexpression in HCC promotes metastatic invasion by regulating p53 phosphorylation. Cancer Res; 76(19); 5732-42. ©2016 AACR.

IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-κB activation.

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.

CDK5RAP3 is a novel repressor of p14ARF in hepatocellular carcinoma cells.

CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14(ARF) tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14(ARF). Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14(ARF) promoter in vivo. Furthermore, knockdown of p14(ARF) in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14(ARF).

The centrosomal protein Tax1 binding protein 2 is a novel tumor suppressor in hepatocellular carcinoma regulated by cyclin-dependent kinase 2.

UNLABELLED: Deregulation of cellular-signaling pathways by the inactivation of tumor-suppressor genes is one of the major causes of hepatocellular carcinoma (HCC). In this study, we identified Tax1 binding protein 2 (TAX1BP2) as a novel tumor-suppressor gene in HCC. TAX1BP2 transcript was frequently underexpressed (42.2% with T/NT <0.5; P < 0.03) in HCCs, and underexpression of TAX1BP2 was associated with poorer overall survival rates in patients after surgical resection. An effector domain (ED) for TAX1BP2 tumor-suppressor activity was mapped to the amino-acid residues 267-756. Transient or stable expression of either full-length or ED of TAX1BP2 significantly suppressed HCC cell tumorigenicity through the activation of the p38/p53/p21 pathway. In contrast, silencing of TAX1BP2 by short interfering RNA remarkably suppressed the activation of the p38/p53/p21 pathway. Finally, phosphorylation of TAX1BP2 at serine-763 by cyclin-dependent kinase (CDK)2 abolished the TAX1BP2-mediated p38 activation and tumor-suppressive activity, indicating that TAX1BP2 can adapt CDK2 signaling to the p38/p53/p21 pathway. CONCLUSION: Taken together, our data provide the first evidence that TAX1BP2 is a CDK2-regulated tumor-suppressor gene in HCC and is a novel activator of the p38/p53/p21 pathway.

The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells.

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has been tested in clinical trials as a cancer chemopreventive drug. 4HPR is cytotoxic to cancer cells but the underlying molecular mechanisms are at present only partially understood. Here we demonstrate that in the human cervical cancer cell line HeLa and the human leukemia cell line HL-60, 4HPR caused rapid, Reactive Oxygen Species (ROS)-dependent activation of the Unfolded Protein Response (UPR). In HeLa cells, 4HPR was shown to induce cell death and activation of procaspases. These effects of 4HPR could be abolished by the over-expression of dominant negative mutants of PERK or eIF2 alpha. HeLa cells incubated with 4HPR were found to form autophagosomes that were also mediated by the PERK/eIF2 alpha pathway. While 4HPR-induced cell death could be significantly prevented by the presence of specific caspase inhibitors, 3-methyladenine (3-MA) that inhibits autophagosome formation enhanced 4HPR-induced cell death. Examination of individual 4HPR-treated HeLa cells revealed that those without the development of autophagosomes hence exhibiting an incomplete UPR were caspase-active and were not viable, while those with autophagosomes were caspase-inactive and retained cell viability. Our data suggest that the PERK/eIF2 alpha pathway is essential for the cytotoxicity of 4HPR that targets on cancer cells with malfunctional UPR.

ROS mediates 4HPR-induced posttranscriptional expression of the Gadd153 gene.

All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that can induce apoptosis in many cancer cell lines. The cytotoxicity of 4HPR is dependent on the production of ROS but the underlying reasons are not entirely certain. We have investigated the role of 4HPR-induced production of ROS in mediating the expression of the recently identified 4HPR-responsive gene Gadd153. In 4HPR-treated cells, the elevation of Gadd153 protein level was prevented by vitamin C, which had no effect on the activation of the Gadd153 gene promoter. The 4HPR-induced elevation of Gadd153 mRNA level persisted even after transcription was blocked with actinomycin D, but declined rapidly upon the addition of antioxidants to the transcription-arrested cells. The mRNA expressed from the full-length Gadd153 cDNA was degraded constitutively in cells in the absence but not in the presence of 4HPR. Such an inhibitory effect of 4HPR was abolished by antioxidants and by inhibitors of 12-lipoxygenase, baicalein (specific) and esculetin (panspecific). The inhibition of 4HPR-induced expression of Gadd153 protein by vitamin C was independent of intracellular proteasome activity and vitamin C had no effect on the intracellular decay of Gadd153 protein. Our data provide the first evidence that the posttranscriptional expression of the Gadd153 gene can be regulated by ROS produced by 4HPR.