Functional characterization of FANCD2 and a deleterious variant of BRCA2 in esophageal squamous cell carcinoma (ESCC)

Abstract

Cancer frequently arises following stress from DNA damage. FANCD2 and BRCA2 are two widely known DNA damage repair genes playing roles in the Fanconi Anemia (FA) pathway and homologous recombination pathway, respectively. FA patients with FANCD2 mutations are prone to develop cancers. Mutation-induced silencing of BRCA2 has been reported to promote tumorigenesis. Our next-generation sequencing study concluded that only BRCA2 variants are significantly associated with inherited esophageal squamous cell carcinoma (ESCC). FANCD2 variants show comparable incidences between ESCC patients and healthy individuals. In this current study, I investigated the functional roles of FANCD2 in ESCC progression and validated the pathogenic BRCA2 mutations. FANCD2 expression was up-regulated in clinical samples and ESCC cell lines. To characterize the roles of FANCD2 in ESCC, lentiviral-mediated clustered regularly interspaced short palindromic repeats technology was used to knock out FANCD2 protein expression in ESCC cells for subsequent in vitro and in vivo functional analyses. The FANCD2 knockout suppresses cell proliferation, clonogenic survival and repair of gamma irradiation-induced double-strand breaks (DSBs) in ESCC cells. The mono-ubiquitinated FANCD2 is only observed in the nucleus during S phase, indicative of a role in the regulation of cell cycle. FANCD2 knockout had no effect on cell migration in our assays. In summary, these results suggest that FANCD2 overexpression promotes cell proliferation, clonogenicity, DSB repair, and is crucial for ESCC progression. The study, thus, enhances our understanding of the functions of FANCD2 and offers novel insights into its maintenance roles in ESCC development. Further study of its mechanisms may provide insight into the possibility of FANCD2 targeted therapy. In the study of BRCA2, three mutations, G2508S, C2689fs and D2723H, were constructed in vectors expressing the BRCA2 DSS1-bind-domain. The BRCA2-DSS1 binding controls the essential nuclear localization of BRCA2 for DNA repair. Further experiments are needed to validate the BRCA2 mutations and DSS1 protein-protein interaction.