Rev1-Rev7 Complex Inhibitor JH-RE-06 Enhances Mitomycin C Chemosensitivity in HCT116 Colorectal Cancer Cells

Abstract Background: Cancer cells can develop resistance to DNA interstrand crosslinker agents through a DNA repair bypass pathway called TLS. JH-RE-06, a TLS-targeting inhibitor, has been shown to increase melanoma cell susceptibility to cisplatin. Nevertheless, whether JH-RE-06 can be used in combination with Mitomycin C (MMC) to benefit Colorectal Cancer (CRC) patients receiving hyperthermic intraperitoneal chemotherapy (HIPEC) treatment remains unknown. Methods: Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) data were obtained from The Cancer Genome Atlas (TCGA) database, and the expression of Rev1-associated proteins in normal and malignant tissues were compared to generate receiver operating characteristic curves (ROC) . The association between Rev1 and Rev7 expression and the prognosis of CRC patients was derived from the PrognoScan database. Expression at the protein level was verified with a tissue microarray. Western blot was performed to identify alterations in the protein levels of Rev1 and Rev7 following MMC treatment of HCT116 cells, whereas CCK8 revealed alterations in the IC50 value of MMC following the knockdown of Rev7 and Rev1. Co-Immunoprecipitation for the targeting of JH-RE-06. EdU demonstrated the inhibitory effect of JH-RE-06 and MMC on cancer cell growth; Wound healing, and clone formation assays were carried out to evaluate the cell migration and clone formation abilities, respectively. Flow cytometry analysis was performed to detect cell apoptosis, and a commercial reagent kit was used to detect ROS and NAD + /NADH changes. Immunofluorescence was used to analyze cellular DNA damage. Finally, the potential mechanism of action and targets of JH-RE-06 in the treatment of CRC were investigated by network pharmacology. Results: Analysis of bioinformatics data revealed high expression of Rev1 and Rev1-associated proteins Rad18, Rev3, and Rev7 in CRC tumor tissues compared to normal tissues, with Rad18 and Rev7 showing high diagnostic values for CRC. High Rev1 expression was associated with a poor prognosis, whereas high Rev7 expression was associated with a favorable prognosis. The protein-level expression of Rev1 and Rev7 was verified by immunohistochemistry, indicating that the downregulation of Rev1 and Rev7 may increase HCT116 susceptibility to MMC treatment. Co-treatment with JH-RE-06 may augment the therapeutic efficacy of MMC in CRC cells, increase cell apoptosis, mitochondrial and DNA damage, and limit cancer cell migration and clone formation. Results from network pharmacology revealed that JH-RE-06 treatment may also involve the MAPK, PI3K, and Akt signaling pathways. Conclusions: Rad18 and Rev7 can be employed as predictive biomarkers for CRC. Targeting TLS renders HCT116 sensitive to MMC treatment, and JH-RE-06 has the potential to serve as a combination therapy medication for the MMC treatment of peritoneal metastatic CRC in HIPEC.