Acid-adapted Fusobacterium nucleatum inhibits necroptosis and promotes oxaliplatin chemotherapy resistance in colorectal cancer

Abstract:

[Background] Drug resistance is a major clinical challenge impacting the prognosis of colorectal cancer. The resistance to oxaliplatin, a first-line chemotherapeutic agent for colorectal cancer, is a pressing issue in clinical settings. Fusobacterium nucleatum has been found to be closely related to chemotherapy resistance in colorectal cancer. Acidification of the tumor microenvironment is one of the key features of tumor metabolism. However, whether the acidic microenvironment regulates the adaptive evolution of microorganisms to affect the drug resistance of tumors remains to be studied. [Objective] To investigate the role of F. nucleatum evolved in the acidic tumor microenvironment in promoting oxaliplatin resistance of colorectal cancer cells and decipher the potential mechanism. [Methods] We cultured the original F. nucleatum strain ORI in an acidic environment for adaptive evolution and obtained an acid-adapted F. nucleatum strain LA57 at pH 5.7. The relative survival rates of colorectal cancer cells HCT-116 and HT-29 co-cultured with strain ORI or strain LA57 were examined. The original HCT-116 cells as well as the cells co-cultured with strain ORI or strain LA57 were subjected to transcriptome sequencing for identifying the key functions of relevant genes. An in vitro cell chemotherapy model was constructed by oxaliplatin treatment, and the cell proliferation and the mRNA and protein levels of relevant genes were determined after co-culture with strain ORI or strain LA57. Furthermore, a subcutaneous tumor model in nude mice was constructed with HCT-116 cells, with oxaliplatin being injected intraperitoneally and strain ORI or strain LA57 intratumorally injected at multipoints for interventions. The immunohistochemical assay was employed to examine the expression of tumor tissue-associated proteins, and quantitative immunohistochemical scoring was performed. [Results] strain LA57 could grow stably at pH 5.7, while strain ORI could not survive under this circumstance. The differentially expressed genes between the strain LA57 group and the strain ORI group were mainly enriched in necroptosis, among which RIPK1/RIPK3/MLKL demonstrated a significant difference in expression. The cell experiment results showed that the cell proliferation ability of the oxaliplatin and strain LA57 co-treatment group was significantly higher than that of the oxaliplatin and strain ORI co-treatment group. The RT-qPCR and Western blotting results showed that compared with strain ORI, strain LA57 demonstrated enhanced inhibitory effects on the oxaliplatin-induced up-regulation of the necroptotic molecules RIPK1/RIPK3/MLKL at both mRNA and protein levels. The results of in vivo experiments in mice showed the same trend. [Conclusion] Acid-adapted F. nucleatum promotes the resistance of colorectal cancer cells to oxaliplatin chemotherapy by inhibiting necroptosis.