Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
Department of Experimental and Clinical Biomedical Sciences, General Pathology Unit, University of Florence, Florence, Italy.
Gut. 2018 Feb;67(2):348-361. doi: 10.1136/gutjnl-2016-313114. Epub 2017 Mar 30.
Chromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is an important factor of DNA damage, sustaining structural chromosome abnormalities but the underlying mechanisms are unknown.
DNA damage response protein checkpoint kinase 2 (Chk2) expression was evaluated in an animal model of diethylnitrosamine-induced HCC characterised by DNA damage and elevated mitotic errors. Chk2 was also determined in two discrete cohorts of human HCC specimens. To assess the functional role of Chk2, gain on and loss-of-function, mutagenesis, karyotyping and immunofluorescence/live imaging were performed by using HCT116, Huh7 and human hepatocytes immortalised with hTERT gene (HuS).
We demonstrate that mitotic errors during HCC tumorigenesis cause lagging chromosomes/DNA damage and activation of Chk2. Overexpression/phosphorylation and mislocalisation within the mitotic spindle of Chk2 contributes to induce lagging chromosomes. Lagging chromosomes and mitotic activity are reversed by knockdown of Chk2. Furthermore, upregulated Chk2 maintains mitotic activity interacting with Aurora B kinase for chromosome condensation and cytokinesis. The forkhead-associated domain of Chk2 is required for Chk2 mislocalisation to mitotic structures. In addition, retinoblastoma protein phosphorylation contributes to defective mitoses. A cohort and independent validation cohort show a strong cytoplasm to nuclear Chk2 translocation in a subset of patients with HCC.
The study reveals a new mechanistic insight in the coinvolvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients.
染色体不稳定性（CIN）是最常见的基因组不稳定性形式，通过增强肿瘤异质性、药物耐药性和免疫逃逸，促进肝细胞癌（HCC）的进展。CIN 本身是 DNA 损伤的一个重要因素，维持结构染色体异常，但潜在机制尚不清楚。
在二乙基亚硝胺诱导的 HCC 动物模型中评估 DNA 损伤反应蛋白检查点激酶 2（Chk2）的表达，该模型以 DNA 损伤和有丝分裂错误增加为特征。还在两个不同的 HCC 标本队列中确定了 Chk2。为了评估 Chk2 的功能作用，使用 HCT116、Huh7 和用 hTERT 基因（HuS）永生化的人肝细胞进行了增益和失活、诱变、核型分析和免疫荧光/活细胞成像。
我们证明 HCC 肿瘤发生过程中的有丝分裂错误导致滞后染色体/DNA 损伤和 Chk2 的激活。Chk2 的过度表达/磷酸化和有丝分裂纺锤体中的错误定位有助于诱导滞后染色体。Chk2 的敲低可逆转滞后染色体和有丝分裂活性。此外，上调的 Chk2 通过与 Aurora B 激酶相互作用维持有丝分裂活性，用于染色体浓缩和胞质分裂。Chk2 的叉头相关结构域是 Chk2 错误定位到有丝分裂结构所必需的。此外，视网膜母细胞瘤蛋白磷酸化有助于有丝分裂缺陷。一个队列和独立的验证队列显示，在 HCC 患者的亚组中，Chk2 从细胞质到细胞核的易位强烈。
该研究揭示了 Chk2 在 HCC 进展中涉及的新机制见解。这些发现提出 Chk2 作为一种潜在的生物标志物来检测 HCC 中的 CIN，为患者的临床/治疗管理提供了有价值的支持。
