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EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

ABSTRACT: Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.

SUBMITTER: Kusakabe Y

PROVIDER: S-EPMC8560765 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:Sorafenib, a broad tyrosine kinase inhibitor, is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC) but provides limited survival benefits. Recently, immunotherapy has emerged as a promising treatment strategy, but its role remains unclear in HCCs, which are associated with decreased cytotoxic CD8(+) T-lymphocyte infiltration in both murine and human tumors. Moreover, in mouse models after sorafenib treatment intratumoral hypoxia is increased and may fuel evasive resistance. Using orthotopic HCC models, we now show that increased hypoxia after sorafenib treatment promotes immunosuppression, characterized by increased intratumoral expression of the immune checkpoint inhibitor programmed death ligand-1 and accumulation of T-regulatory cells and M2-type macrophages. We also show that the recruitment of immunosuppressive cells is mediated in part by hypoxia-induced up-regulation of stromal cell-derived 1 alpha. Inhibition of the stromal cell-derived 1 alpha receptor (C-X-C receptor type 4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment, inhibited tumor growth, reduced lung metastasis, and improved survival. However, the combination of AMD3100 and sorafenib did not significantly change cytotoxic CD8(+) T-lymphocyte infiltration into HCC tumors and did not modify their activation status. In separate experiments, antibody blockade of the programmed death ligand-1 receptor programmed death receptor-1 (PD-1) showed antitumor effects in treatment-naive tumors in orthotopic (grafted and genetically engineered) models of HCC. However, anti-PD-1 antibody treatment had additional antitumor activity only when combined with sorafenib and AMD3100 and not when combined with sorafenib alone. CONCLUSION:Anti-PD-1 treatment can boost antitumor immune responses in HCC models; when used in combination with sorafenib, anti-PD-1 immunotherapy shows efficacy only with concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors.

Project description:PurposeHepatocellular carcinoma (HCC), a worldwide leading cause of morbidity and mortality, is the most frequent primary liver tumor. Most HCC patients are diagnosed with advanced liver cancer, resulting in a very low 5-year survival rate. Thus, there is an urgent need for the development of targeted therapies. In this study, we aimed to investigate the effect and mechanism of the miR-20a/EZH1 axis on the proliferation and metastasis of HCC and the inhibitory effect of the EZH1/EZH2 inhibitor UNC1999 on HCC.Materials and methodsThe expression of miR-20a in human HCC tissues and cell lines was detected using quantitative real-time PCR (qRT-PCR). The expressions of proteins were analyzed with immunohistochemistry and Western blotting. Luciferase assay was used to verify whether miR-20a targets EZH1 or EZH2. The effect of miR-20a on HCC progression was studied in vivo and in vitro. The tumor inhibitory effect of UNC1999 was confirmed in vivo. CCK8 assay, wound healing assay, cell migration and invasion assay were used to evaluate the synergistic effect of UNC1999 with sorafenib. RNA sequencing (RNA-seq) was performed to screen the differentially expressed genes in the Huh7 and SMMC7721 cell lines after UNC1999, sorafenib, and combination treatments.ResultsIn this study, miR-20a showed a lower expression in both HCC tissues and cell lines. MiR-20a inhibited the proliferation and migration of SMMC7721 and Huh7 cells. The results of the luciferase assay and Western blot analysis revealed that miR-20a directly targeted EZH1, a histone methyltransferase. We demonstrated that miR-20a negatively regulated the expression of EZH1 and inhibited the proliferation and metastasis of HCC by reducing H3K27 methylation. We found UNC1999 inhibited tumor cells proliferation and enhanced the inhibitory effect of sorafenib.ConclusionWe demonstrated that miR-20a suppresses the tumor proliferation and metastasis in HCC by directly targeting EZH1. UNC1999 can inhibit tumor proliferation in vivo and increase the sensitivity of hepatoma cell lines to sorafenib.

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EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

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