Objective 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.
Design 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).
Results 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.
Conclusions 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.
- GENETIC INSTABILITY
- HEPATOBILIARY CANCER
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KR and AG contributed equally.
Contributors VC: study concept and experimental design, cloning experiments, analysis and interpretation of data, drafting of the manuscript and study supervision. ML : immunoblotting and karyotype experiments. SM: immunohistochemistry and animal experiments. TM: immunofluorescence, live cell and bioinformatics analysis. AH: immunohistochemistry analysis. TVL: human tissue evaluation. MP: critical review of the manuscript. KR: experimental design, data analysis and critical review of the manuscript. AG: conceptual and intellectual input, and critical review of the manuscript. All authors read and approved the final manuscript.
Funding This study was in part supported by the University of Florence and Regione Toscana ‘Ricerca regionale in materia di salute D.D. n. 3242’, and by Ministero dell'Istruzione, dell'Università FIRB RBAP10MY35_002 and FiorGen Foundation.
Competing interests None declared.
Patient consent Obtained.
Ethics approval University Hospital of Florence and Royal Free Hospital, London.
Provenance and peer review Not commissioned; externally peer reviewed.
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