UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation.
UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation.
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UBE2T 调节的 H2AX 单泛素化通过促进 CHK1 激活诱导肝细胞癌放射抗性
DOI：
10.1186/s13046-020-01734-4
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发表时间：
2020-10-21
期刊：
影响因子：
--
通讯作者：
Wu D
中科院分区：
文献类型：
--
作者：
Sun J;Zhu Z;Li W;Shen M;Cao C;Sun Q;Guo Z;Liu L;Wu D
背景放射抵抗是肝细胞癌（HCC）放射治疗的主要障碍. DNA损伤反应（DDR）的失调，包括DNA修复和细胞周期检查点激活，导致肝癌患者的放射抵抗和限制放射治疗的疗效。然而，其潜在的机制尚未清楚understood.MethodsWe获得了7对肝癌组织和相应的非肿瘤组织，和UBE 2 T被确定为最上调的基因之一。通过体外集落形成试验和体内异种移植肿瘤模型检查UBE 2 T的放射抗性作用。采用彗星实验、流式细胞术和γ H2 AX焦点测定等方法研究UBE 2 T介导DDR的机制。染色质分级和免疫荧光染色用于评估细胞周期检查点激酶1（CHK 1）的激活。结果UBE 2 T在肝癌组织中表达上调，且UBE 2 T表达水平越高，肝癌患者预后越差。功能研究表明，UBE 2 T在体外和体内增加肝癌的放射抗性。在机制上，UBE 2 T-RNF 8被鉴定为E2-E3对，在辐射暴露后与单泛素化组蛋白变体H2 AX/γ H2 AX物理结合。UBE 2 T调节的H2 AX/γ H2 AX单泛素化促进CHK 1的磷酸化以激活CHK 1，并促进CHK 1从染色质释放到细胞质中以降解。阻断UBE 2 T介导的H2 AX/γ H2 AX上的单泛素化，包括UBE 2 T的E2酶缺陷突变（C86 A）和H2 AX的单泛素化位点缺陷突变（K119/120 R），不能有效激活CHK 1。此外，CHK 1的遗传和药理学抑制损害了UBE 2 T在HCC中的放射抗性作用。结论UBE 2 T介导的H2 AX/γ H2 AX单泛素化在促进细胞周期阻滞激活，为辐射诱导的DNA修复提供足够的时间，从而赋予肝癌放射抵抗性方面具有新的作用。本研究表明，阻断UBE 2 T-H2 AX-CHK 1通路可能是克服肝癌放射抗性的一个有希望的潜在策略。
BackgroundRadioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood.MethodsWe obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T.ResultsHere, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy.ConclusionOur results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.
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