RAD51 Is Implicated in DNA Damage, Chemoresistance and Immune Dysregulation in Solid Tumors

SIMPLE SUMMARY: Aims and objectives: Destabilization of genetic material can contribute to development and progression of cancer including development of drug resistance and treatment failure. We have previously shown that a protein RAD51 and its corresponding activity, which maintain stability of genetic material (DNA) in normal cells, are dysregulated in cancer. Purpose of this study was to investigate role of RAD51 and its potential as therapeutic target in cancer. Results: We demonstrate that increased level of RAD51 is associated with poor survival of esophageal, breast and colon cancer patients. Our data indicated that increased RAD51 contributes to growth as well as spontaneous and chemotherapy-induced damage and destabilization of genetic material in cancer cells. Moreover, the treatment of cancer cells with chemotherapeutic drug (cisplatin) led to activation of mechanisms which disrupt immune surveillance and maintenance of genetic material, whereas addition of RAD51 inhibitor prevented activation of such mechanisms. Conclusions: Drugs targeting RAD51 have potential to inhibit growth of cancer cells while preserving integrity of genetic material and immune system. When used in combination, these treatments can increase ability of chemotherapeutic drugs to kill cancer cells while minimizing their harmful impact on genetic material and immune system. ABSTRACT: Background: In normal cells, homologous recombination (HR) is tightly regulated and plays an important role in the maintenance of genomic integrity and stability through precise repair of DNA damage. RAD51 is a recombinase that mediates homologous base pairing and strand exchange during DNA repair by HR. Our previous data in multiple myeloma and esophageal adenocarcinoma (EAC) show that dysregulated HR mediates genomic instability. Purpose of this study was to investigate role of HR in genomic instability, chemoresistance and immune dysregulation in solid tumors including colon and breast cancers. Methods: The GEO dataset were used to investigate correlation of RAD51 expression with patient survival and expression of various immune markers in EAC, breast and colorectal cancers. RAD51 was inhibited in cancer cell lines using shRNAs and a small molecule inhibitor. HR activity was evaluated using a plasmid-based assay, DNA breaks assessed by evaluating expression of γ-H2AX (a marker of DNA breaks) and p-RPA32 (a marker of DNA end resection) using Western blotting. Genomic instability was monitored by investigating micronuclei (a marker of genomic instability). Impact of RAD51 inhibitor and/or a DNA-damaging agent was assessed on viability and apoptosis in EAC, breast and colon cancer cell lines in vitro and in a subcutaneous tumor model of EAC. Impact of RAD51 inhibitor on expression profile was monitored by RNA sequencing. Results: Elevated RAD51 expression correlated with poor survival of EAC, breast and colon cancer patients. RAD51 knockdown in cancer cell lines inhibited DNA end resection and strand exchange activity (key steps in the initiation of HR) as well as spontaneous DNA breaks, whereas its overexpression increased DNA breaks and genomic instability. Treatment of EAC, colon and breast cancer cell lines with a small molecule inhibitor of RAD51 inhibited DNA breaking agent-induced DNA breaks and genomic instability. RAD51 inhibitor potentiated cytotoxicity of DNA breaking agent in all cancer cell types tested in vitro as well as in a subcutaneous model of EAC. Evaluation by RNA sequencing demonstrated that DNA repair and cell cycle related pathways were induced by DNA breaking agent whereas their induction either prevented or reversed by RAD51 inhibitor. In addition, immune-related pathways such as PD-1 and Interferon Signaling were also induced by DNA breaking agent whereas their induction prevented by RAD51 inhibitor. Consistent with these observations, elevated RAD51 expression also correlated with that of genes involved in inflammation and other immune surveillance. Conclusions: Elevated expression of RAD51 and associated HR activity is involved in spontaneous and DNA damaging agent-induced DNA breaks and genomic instability thus contributing to chemoresistance, immune dysregulation and poor prognosis in cancer. Therefore, inhibitors of RAD51 have great potential as therapeutic agents for EAC, colon, breast and probably other solid tumors.