NRAGE is involved in homologous recombination repair to resist the DNA-damaging chemotherapy and composes a ternary complex with RNF8–BARD1 to promote cell survival in squamous esophageal tumorigenesis

Abstract

NRAGE, a neurotrophin receptor-interacting melanoma antigen-encoding gene homolog, is significantly increased in the nucleus of radioresistant esophageal tumor cell lines and is highly upregulated to promote cell proliferation in esophageal carcinomas (ECs). However, whether the overexpressed NRAGE promotes cell growth by participating in DNA-damage response (DDR) is still unclear. Here we show that NRAGE is required for efficient double-strand breaks (DSBs) repair via homologous recombination repair (HRR) and downregulation of NRAGE greatly sensitizes EC cells to DNA-damaging agents both in vitro and in vivo. Moreover, NRAGE not only regulates the stability of DDR factors, RNF8 and BARD1, in a ubiquitin-proteolytic pathway, but also chaperons the interaction between BARD1 and RNF8 via their RING domains to form a novel ternary complex. Additionally, the expression of NRAGE is closely correlated with RNF8 and BARD1 in esophageal tumor tissues. In summary, our findings reveal a novel function of NRAGE that will help to guide personalized esophageal cancer treatments by targeting NRAGE to increase cell sensitivity to DNA-damaging therapeutics in the long run. In mammalian cells, DNA lesions are always endogenously and exogenously induced, 1 among which double-strand breaks (DSBs) are the most lethal lesions.2 Therefore, efficient DNA-damage response (DDR) is essential to maintain genomic integrity.1,3 However, DDR is a double-edged sword. In normal organisms, DDR protects cells from tumorigenesis, whereas in tumor cells, DDR enables cells to grow and resist DNA-damaging therapeutic agents. 4 Thus, in order to correctly use DDR factors in cancer diagnosis and targeted therapy, it is important to study molecular pathways underlying DDR in cancer cells. 5NRAGE is a member of the melanoma-associated antigen (MAGE) family. 6 Previous studies reveal that it is possibly a DDR factor: (1) upregulation of NRAGE in the nucleus is highly associated with the development of esophageal carcinoma (EC) by interacting with the proliferating cell nuclear antigen (PCNA); 7 (2) the level of NRAGE is significantly increased in the nucleus of radioresistant EC cells; 8 (3) NRAGE knockout (KO) mice exhibit depression-like behavior 9 and disorder in circadian clock, 10 both of which are analogous to DDR defective scenarios.11,12 However, to our knowledge, there has been no direct report concerning the role of NRAGE in DDR.In the present study, we aim to study the role and the underlying mechanisms of NRAGE in DDR. Our data support that NRAGE is a positive regulator in homologous recombination (HR) and regulates the chemoresistance of EC cells both in vivo and in vitro. From mechanism, it regulates the stability of RNF8 and BARD1 via the ubiquitin-proteasome pathway and interacts with the RING domains of the two proteins as a chaperon to form a novel ternary complex to participate in DDR. Furthermore, clinical characterization of NRAGE, RNF8, and BARD1 in squamous EC tissues shows that the expression of NRAGE protein is closely related wi...

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