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DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

ABSTRACT: Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.

SUBMITTER: Li HK

PROVIDER: S-EPMC4921922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundChronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression.MethodsR-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC.ResultsWe identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo.ConclusionsPSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.

Project description:The aberrant up-regulation of the oncogenic transcription factor Forkhead box M1 (FoxM1) is associated with tumor development, progression and metastasis in a myriad of carcinomas, thus establishing it as an attractive target for anticancer drug development. FoxM1 overexpression in hepatocellular carcinoma is reflective of tumor aggressiveness and recurrence, poor prognosis and low survival in patients. In our study, we have identified the antimalarial natural product, Artemisinin, to efficiently curb FoxM1 expression and activity in hepatic cancer cells, thereby exhibiting potential anticancer efficacy. Here, we demonstrated that Artemisinin considerably mitigates FoxM1 transcriptional activity by disrupting its interaction with the promoter region of its downstream targets, thereby suppressing the expression of numerous oncogenic drivers. Augmented level of FoxM1 is implicated in drug resistance of cancer cells, including hepatic tumor cells. Notably, FoxM1 overexpression rendered HCC cells poorly responsive to Artemisinin-mediated cytotoxicity while FoxM1 depletion in resistant liver cancer cells sensitized them to Artemisinin treatment, manifested in lower proliferative and growth index, drop in invasive potential and repressed expression of EMT markers with a concomitantly increased apoptosis. Moreover, Artemisinin, when used in combination with Thiostrepton, an established FoxM1 inhibitor, markedly reduced anchorage-independent growth and displayed more pronounced death in liver cancer cells. We found this effect to be evident even in the resistant HCC cells, thereby putting forth a novel combination therapy for resistant cancer patients. Altogether, our findings provide insight into the pivotal involvement of FoxM1 in the tumor suppressive activities of Artemisinin and shed light on the potential application of Artemisinin for improved therapeutic response, especially in resistant hepatic malignancies. Considering that Artemisinin compounds are in current clinical use with favorable safety profiles, the results from our study will potentiate its utility in juxtaposition with established FoxM1 inhibitors, promoting maximal therapeutic efficacy with minimal adverse effects in liver cancer patients.

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DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

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