CRISPR/Cas9 screening reveals PFKFB3 gene as a key contributor to 5-Fluorouracil resistance in esophageal cancer

Abstract Background and objectives: Esophageal cancer (EC) is the eighth most common cancer and the sixth most common cause of death worldwide. Esophageal squamous cell carcinoma (ESCC) comprised the majority of esophageal cancers globally. Chemotherapy is an important part of treating esophageal cancer, and 5-Fluorouracil (5-FU) is one of the commonly used chemotherapy drugs. However, chemoresistance to drugs is a main obstacle to the successful treat this malignancy. In this study, we used the CRISPR/Cas9 screening method to find out the target gene which is related to 5-FU drug resistance in esophageal cancer. We also explore the mechanism between the target gene and 5-FU resistance, the result may lead to a breakthrough in the personalized treatment of esophageal cancer. Methods: In this project, human esophageal squamous cell carcinoma cell lines KYSE-70 and KYSE-270 were transfected with human protein kinase library. The IC90 of 5-FU for 3 weeks as a drug resistance screening condition, and collected the vehicle control and treatment groups DNA for next generation sequencing to identify the candidate genes. Target gene was knocked out using CRISPR-Cas9 technology in KYSE-70, KYSE-270 and KYSE-150 cell lines individually. Verify the drug resistance phenotype by compared the IC50 value between before and after knocked out target gene and some related mechanism behind them were observed. Results: Our research findings indicate that the loss of PFKFB3 can increase the resistance of different human esophageal squamous cell carcinoma cell lines to 5-FU through various pathways. Specifically, in KYSE-70 cells, loss of PFKFB3 can induce epithelial-mesenchymal transition (EMT) and prolong the S phase of the cell cycle, allowing cancer cells to evade the effects of 5-FU and develop resistance. In KYSE-270 and KYSE-150 cell lines, loss of PFKFB3 can upregulate the expression of Slug and Mcl-1, indirectly regulate Chk1 and promote its autophosphorylation, which in turn inhibits apoptosis, thus counteracting the effects of 5-FU. Conclusion: Our research not only enriches our understanding of the biological characteristics of different ESCC cell lines but also provides new clinical insights for future personalized treatments. Assessing the status of PFKFB3 can help predict resistance to 5-FU in ESCC patients with different genetic backgrounds, allowing more precise treatment planning. This personalized approach has the potential to improve treatment efficacy, reduce unnecessary drug use and side effects, and ultimately improve patient survival rates and quality of life.