Defective Chromosome Segregation Causes DNA Damage and Chromosome Abnormalities In Human Hepatocellular Carcinoma

Background: High levels of genomic instability correlate with progression in hepatocellular carcinoma (HCC) of which the most common form is chromosomal instability (CIN), result- ing in chromosomal aberrations leading to a heterogeneously tumour cell population. This has the ability to undergo selective evolution represented by drug resistance. Chromosomal insta-bility per se is an important factor of DNA damage sustaining numerical and structural chromosome abnormalities but the underlying causes and mechanisms are unknown. Methods: Proteins known to be activated in the presence of DNA dam- age were screened by using an animal model of diethylnitrosa- mine-induced HCC. The DNA damage response protein Chk2 expression and localization was determined in a cohort and an additional independent cohort of HCC specimens in humans. To assess the functional role of Chk2, gain on-and loss-of-func- tion, mutation analysis, karyotyping, immunofluorescence and live imaging were performed in vitro by using HCT116, Huh7 and human hepatocytes immortalized with TERT gene (HuS). Results: DEN-treated animals showed nuclear upregulation of Chk2 and P-Histone H2A.X as DNA damage response pro- teins in tumour tissue and this reflected a bona fide response triggered by missegregation of chromosomes. Overexpression and activation of the DNA damage response protein Chk2 and its mislocalization within the mitotic spindle induces itself a chromosome missegregation-self-perpetuating DNA damage. Moreover, Retinoblastoma protein phosphorylation contributes to CIN in HuS cells and CIN-induced DNA damage promotes Chk2 phosphorylation and localization with mitotic structures. The Chk2 forkhead-associated (FHA) domain mediates Chk2 localization to mitotic structures and is essential for the DNA damage response. Up-regulated Chk2 contributes to sustained mitotic activity exploiting Aurora B kinase for chromosome con- densation and cytokinesis. Two cohorts showed a strong cyto- plasmic staining in Grade I HCC patients, with a perinuclear translocation in Grade II and a strong nuclear translocation in grade III HCC patients. Conclusions: These findings pro- pose Chk2 as a putative biomarker that can be used to detect chromosomal instability and DNA damage in HCC providing a valuable support for clinical management of HCC patients.