Loss of NKX6.3 During Gastric Carcinogenesis Enhances Genomic Instability and Promote Tumor Development

Abstract

Background: The maintenance of genomic stability requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Here, we investigated whether NKX6.3 plays a role in maintaining the genomic stability of gastric epithelial cells. Methods: qRT-PCR, immunofluorescent, fluorescence in situ hybridization (FISH) and immunoblot were utilized to analyze the copy number alterations (CNAs) and expression levels of CDKN3, DLGAP5, AurkA, TPX2, SPARCL1, and SORBS2 in NKX6.3 depleted cells and gastric cancer tissues. Fluorescence-activated cell sorting was used for cell cycle and DNA repair analysis. DNA fiber assay, BrdU, and EdU assays were performed to analyze DNA replication stress. DNA fragmentation and Comet assays were used for DNA damage analysis. Chromosomal aberrations were analyzed by metaphase spread and mitotic index assays. Xenograft experiments were performed to determine CNAs and expression levels of genes caused by NKX6.3 depletion in vivo. Whole-genome sequencing was performed to analyze CNAs caused by NKX6.3 depletion. Findings: NKX6.3 depleted HFE-145 immortalized gastric epithelial cells induced a rapidly cell cycle with increased S and G2/M phase cell populations, by altering the expression of cell cycle related proteins. During cell cycle, NKX6.3 depletion induced faster fork progression, fork symmetry, longer gap length, mitotic spindle defects, and chromosome mis-segregation. In DNA repair, depletion of NKX6.3 led to an induction of homologous recombination repression. When we investigated whether NKX6.3 was involved in CNAs of gastric epithelial cells, frequently gained or amplified, and lost or deleted broad chromosomal regions were detected in NKX6.3-depleted cells. Hallmark enrichment analyses of NKX6.3-depleted cells revealed gain of CDKN3, DLGAP5, AurkA, and TPX2, and deletion of SORBS2 and SPARCL1. Moreover, NKX6.3-depletion caused mitotic spindle defects and chromosome mis-segregation in vitro and in vivo through the CNA of genes involved in mitotic spindle formation. In 65 human gastric cancers and xenograft mice models for NKX6.3-depleted HFE-145 cells, expression levels of CDKN3, DLGAP5, AurkA, and TPX2 were markedly increased while expression levels of SPARCL1 and SORBS2 were decreased. In NKX6.3-depleted cells, silencing of CDKN3 and DLGAP5 as well as ectopic expression of SPARCL1 and SORBS2 inhibited cell proliferation and colony formation. Interpretation: These results demonstrate that a novel molecular mechanism for gastric carcinogenesis through the induction of genomic instability by abnormal regulation of DNA replication and repair due to NKX6.3-depletion. Thus, we conclude that NKX6.3 may DNA replication and repair regulation for protecting gastric mucosal epithelial cells from genomic instability such as CNAs. Funding Statement: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017R1D1A1A09000781 to J.H.Y.; 2018R1A2A2A14019713 to W.S.P.). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Written informed consent was obtained from all subjects. The study was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine (MC15SISI0015). Animal experiments were performed in mice maintained under pathogen-free conditions based on approval by the Animal Experiment Ethics Committee of the Catholic University of Korea College of Medicine (CUMC-2017-0018-01).