Functional characterization of FANCD2 in esophageal squamous cell carcinoma

Abstract

Introduction: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value. Methods: We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability. Results: The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome. Conclusion: These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance.