CHK2 overexpression and mislocalisation within mitotic structures enhances chromosomal instability and hepatocellular carcinoma progression

Abstract

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.

SIRT1 modulates cell cycle progression by regulating CHK2 acetylation−phosphorylation

“…The expression of p-CHK2 revealed an association with lagging chromosome in SIRT1-deficient cells, consistent with a previous study (Fig. S5A) [34]. In addition, cell cycle profile in HCT116 cells under EX527 treatment conditions with or without additional selective CHK2 inhibitor (C3742) were examined.…”

“…Previous studies have demonstrated that CHK2 hyperactivation (phosphorylation), during mitosis, is responsible for increasing the frequency of lagging chromosomes [33,34]. We then sought to address the functional significance of dysregulated activation of CHK2 in cell response to SIRT1 depletion.…”

Cell-targeted c(AmpRGD)-sunitinib molecular conjugates impair tumor growth of melanoma

“…Cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies) (1 μg/mL for 10 min). Following two washes in PBS, coverslips were mounted with 1:1 PBS:Glycerol on glass slides and the cells were observed with an inverted confocal Nikon Eclipse TE2000 microscope equipped with a 960S-Fluor oil immersion lens (FITC filter for sunitinib, Cy3 filter for Rab-7, DAPI filter for nuclear stain) [40]. A single composite image was obtained by superimposition of 6 optical sections for each sample observed.…”

Immunohistochemistry analysis of checkpoint kinase 2 in oral squamous cell carcinoma

“…Chk2 is considered a promising target for therapeutic and prognostic purposes. Many studies have associated different types of cancers with Chk2, such as breast carcinoma [15], colorectal cancer [16], hepatocellular carcinoma [17], ovarian carcinoma [18] and bladder cancer [19]. However, the relationship between Chk2 and OSCC is still poorly understood.…”