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Identification of the ?-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

ABSTRACT: Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). ?-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both ?-catenin and PTMA in a dose-dependent manner. Silencing ?-catenin reduces PTMA level and sensitizes HCC cells to sorafenib. In contrast, ectopic expression of ?-catenin induces PTMA expression and cell resistance to the drug. Sorafenib inhibits PTMA expression at the transcriptional level by inhibiting the ?-catenin pathway. Nucleotide deletion analysis of the PTMA gene promoter reveals that a DNA segment lying 1,500-1,600 bp upstream of the PTMA transcription start site represents an AP-1-binding site that is critical for ?-catenin modulation of gene transcription in response to sorafenib. In addition, chemical inhibitors that target JNK abrogate ?-catenin/AP-1 binding to the endogenous PTMA gene and reduces PTMA transcription and protein expression. Silencing of ?-catenin or c-Fos induces similar effects on gene regulation and these are reversed by ectopic expression of ?-catenin. Mutations in the PTMA promoter at the predicted ?-catenin/AP-1 binding site partly abrogate sorafenib's effects on PTMA transcription. These results indicate that PTMA is induced by the oncoprotein ?-catenin and protects HCC cells against sorafenib-induced cell death. The ?-catenin/JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

SUBMITTER: Lin YT

PROVIDER: S-EPMC4770752 | biostudies-other | 2015 Nov

REPOSITORIES: biostudies-other

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Identification of the β-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

Lin Yi-Te YT Chao Chuck C-K CC

Oncotarget 20151101 36

Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). β-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both β-catenin and PTMA in a dose-dependent manner. Silencing ...[more]

PMID: 26517516

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Project description:Hepatocellular carcinoma (HCC) is a global health problem. Its incidence and mortality are increasing. Exploring novel therapeutic targets against HCC is important and urgent. We here explored the expression and potential function of Gαi2 (G protein subunit alpha i2) in HCC. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database shows that the number of Gαi2 transcripts in HCC tissues is significantly higher than that in the normal liver tissues. Moreover, Gαi2 overexpression in HCC correlates with poor prognosis of the patients. Gαi2 mRNA and protein expression are also elevated in local HCC tissues and different human HCC cells. In patient-derived primary HCC cells and immortalized HepG2 cells, Gαi2 silencing (by targeted shRNA) or knockout (KO, by the dCas9-sgRNA method) largely suppressed cell proliferation and motility, while inducing cell cycle arrest and caspase-apoptosis activation. Moreover, Gαi2 silencing or KO-induced reactive oxygen species (ROS) production and oxidative injury in primary and HepG2 HCC cells. Whereas different antioxidants ameliorated Gαi2-shRNA-induced anti-HCC cell activity. Using a lentiviral construct, Gαi2 overexpression further augmented proliferation and motility of primary and immortalized HCC cells. Further studies revealed that the binding between the transcription factor early growth response zinc finger transcription factor 1 (EGR1) and Gαi2 DNA promoter was significantly increased in HCC tissues and cells. In vivo, intratumoral injection of Gαi2 shRNA adeno-associated virus significantly hindered HCC xenograft growth in nude mice. Moreover, the growth of Gαi2-KO HCC xenografts in the nude mice was remarkably slow. Gαi2 depletion, oxidative injury, and apoptosis induction were detected in Gαi2-silenced or Gαi2-KO HCC xenografts. Together, overexpressed Gαi2 is required for HCC cell growth in vitro and in vivo, representing as a novel and promising diagnosis marker and therapeutic target of HCC.

Project description:BackgroundSorafenib is the first-line treatment for advanced-stage hepatocellular carcinoma (HCC). Several studies have shown that the up-regulation of β-catenin plays a role in sorafenib resistance in HCC; however, the mechanism associated with this phenomenon remains elusive.MethodsWestern blotting, flow cytometry, and an evaluation of IC50 values were used to confirm the role of β-catenin in HCC sorafenib resistance. Immunoprecipitation and western blotting were then performed to identify regulatory interactions between β-catenin and Nek2. Further, western blotting, flow cytometry, and an in vivo xenograft model were used to evaluate the function of Nek2 in HCC sorafenib resistance, whereas rescue experiments were performed to confirm that Nek2 induces sorafenib resistance via β-catenin. Finally, western blotting and immunohistochemistry were used to evaluate the expression level of Nek2 in paired HCC and non-tumor tissues.ResultsWe showed that β-catenin could suppress sorafenib-induced apoptosis and cell growth inhibition in HCC cell lines. By screening β-catenin-interacting proteins, we found that Nek2 could bind β-catenin in sorafenib-treated HCC cell lines. Our results also showed that Nek2 stabilizes β-catenin and promotes its translocation to the nucleus, consequently activating the transcription of downstream target genes. We further confirmed that Nek2 could induce sorafenib resistance in HCC cell lines, and that β-catenin was the key element involved in this process. Further, a xenograft tumor model showed that Nek2 knockdown could improve the anti-tumor effect of sorafenib, whereas an analysis of tumor proteins showed that Nek2 regulates β-catenin protein levels and its nuclear translocation in vivo. In addition, Nek2 was found to be up-regulated in HCC tissue, and especially in advanced-stage disease.ConclusionsOur study proves that Nek2 induces HCC sorafenib resistance via β-catenin and suggests a novel therapeutic strategy to improve the anti-tumor effects of sorafenib in HCC.

Dataset Information

Identification of the ?-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

Publications

Identification of the β-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

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