Active DNA end processing in micronuclei of ovarian cancer cells

Abstract

BackgroundOvarian cancer is one of the most deadly gynecological malignancies and inclined to recurrence and drug resistance. Previous studies showed that the tumorigenesis of ovarian cancers and their major histotypes are associated with genomic instability caused by defined sets of pathogenic mutations. In contrast, the mechanism that influences the development of drug resistance and disease recurrence is not well elucidated. Solid tumors are prone to chromosomal instability (CIN) and micronuclei formation (MN). Although MN is traditionally regarded as the outcome of genomic instability, recent investigation on its origin and final consequences reveal that the abnormal DNA metabolism in MN is a driver force for some types of catastrophic genomic rearrangements, accelerating dramatic genetic variation of cancer cells.MethodsWe used Indirect Immunofluorescent staining to visualize micronuclei and activation of DNA repair factors in ovarian cancer cell lines and biopsies.ResultsWe show that ovarian cancer cells are disposed to form micronuclei upon genotoxic insults. Double strand DNA breaks (DSBs)-triggered insurgence of micronuclei is associated with unrepaired chromosomes passing through mitosis. According to their morphology and DNA staining, micronuclei compartments are divided into early and late stages that can be further characterized by differential staining of γH2AX and 53BP1. We also show that MN compartments do not halt controlled DNA metabolism as sequestered nuclear repair factors are enriched at DNA breaks in MN compartments and efficiently process DNA ends to generate single-stranded DNA (ssDNA) structures. Interestingly, unknown factors are required for DNA end processing in MN in addition to the nuclear resection machinery. Finally, these hallmarks of micronuclei evolution depicted in cell culture were recapitulated in different stages of ovarian cancer biopsies.ConclusionsIn aggregate, our findings demonstrate that ovarian cancer cells are inclined to form micronuclei that undergo robust DNA metabolism and generate ssDNA structures, potentially destabilizing genomic structures and triggering genetic variation.

NBS1-CtIP–Mediated DNA End Resection Regulates cGAS Binding to Micronuclei

“…1F) were also positive for γH2AX foci. Since several DNA damage sensing, repair and signaling factors, including ATM, MDC1, NBS1 (component of MRN complex), BRCA1, P53 and RPA32, are shown to localized in the micronuclei, 35,36 it is reasonable to speculate that one of these factors may modulate the recruitment of cGAS to micronuclei in response to DNA damage. Initially, we examined the localization of MDC1, ATM, MRE11, and NBS1 to the micronuclei and found that ~18-40% of micronuclei were positive for MDC1, phosphorylated ATM (S1981), MRE11 and NBS1 in response to 6-thio-dG ( Figs.…”

Small but strong: Mutational and functional landscapes of micronuclei in cancer genomes

“…Micronuclei generated from laggards are less condensed than those generated from chromatin bridges 37 . Some micronuclei undergo active DNA end processing to generate single‐strand DNA (ssDNA) 44 …”

“…Similarly, lamin‐B positive DMs‐derived micronuclei are as actively transcribed as main nuclei 46 . Spontaneous‐occurring and ionizing radiation‐induced micronuclei exhibit opposite capacity to recruit DNA damage binding proteins γH2AX and 53BP1, 44 indicating that micronuclei of different origins display heterogeneity in DDR signaling. Similarly, the γH2AX and 53BP1 binding capacity is also different between ssDNA‐enriched and ssDNA‐absent micronuclei, 44 suggesting chromatin structure also impact DDR signaling in micronuclei.…”

“…37 Some micronuclei undergo active DNA end processing to generate single-strand DNA (ssDNA). 44 Different types of mNE defects cause heterogeneity in genetic activities among micronuclei. DNA replication occurs predominately in lamin-B positive micronuclei rather than in lamin-B negative ones.…”

An Automated, Single Cell Quantitative Imaging Microscopy Approach to Assess Micronucleus Formation, Genotoxicity and Chromosome Instability

“…In the past, many genotoxic studies have sought to determine the impact compounds have on genome stability by manually assessing the presence of micronuclei following a treatment [4,5], while more recent studies have employed micronucleus formation assays to uncover genes with pathogenic implications in cancer [6][7][8]. In this regard, numerous cytological studies have identified micronuclei within various cancer contexts including head and neck, ovarian, and breast cancers [9][10][11]. Interestingly, the presence of micronuclei in precursor lesions is associated with an increased risk of developing certain cancers [12][13][14][15], suggesting that the presence and frequency of micronuclei may hold clinical value as diagnostic and prognostic biomarkers.…”