Explore the role of tumour supressor genes KDM6A and STK11 in pancreatic cancer phenotypes and chemotherapeutic drug sensitivity with CRISPR/Cas9 system

Background and objectives: Pancreatic cancer is one of the top 5 most deadly cancers in the world. With an extremely low survival rate even after surgery or chemotherapy. While among them, only few patients could have the opportunity to apply surgery, which drive a serious situation of finding an effective method for early screening and prolonging the life and quality of life of patients diagnosed with pancreatic cancer rather than curative treatment. With the development of CRISPR-Cas9 gene editing technology, it is now widely used to explore specific and sensitive genomic targets for diseases including cancer, which brings a new view and hope for the specificities and individualised cancer treatment. This study utilized CRISPR-Cas9 system to dual knock out STK11 and KDM6A in the mouse pancreatic cancer cell model aiming to investigate the early detection, therapeutic, and prognosis potential of this approach. Hypothesis and methods: Given the tumour suppressive roles of STK11 and KDM6A, we hypothesised that their simultaneous deletion would enhance cell proliferation, migration, and invasion, potentially leading to epithelial-mesenchymal transition and a more aggressive pancreatic ductal adenocarcinoma phenotype with enhanced chemoresistance. We used CRISPR-Cas9 to generate STK11-KDM6A double knockout cell lines from TB32047 wt and TB32047-STK11 ko cells. Knockout efficiency was confirmed by Western blot and RT-qPCR. Cellular dynamics were then assessed by wound healing, colony formation and proliferation assays, together with E-cadherin detection by immunofluorescence to evaluate EMT. Chemoresistance was assessed using gemcitabine and oxaliplatin treatments. Results: The findings show that cells deficient in STK11 and KDM6A exhibited significantly reduced proliferation, migration, and colony formation ability, coupled with a downregulation of ZEB1, indicating that the cells did not undergo EMT. IF showed a reduced cell-cell adhesion ability and a lower expression of E-cadherin. Moving to drug resistance, dual ko cells were less sensitive to gemcitabine (GEM), but with less changes in sensitivity to oxaliplatin (OXA). Conclusion: The results contradict the initial hypothesis and show that dual knockout of STK11 and KDM6A in pancreatic cancer cells leads to reduced dynamics and cell adhesion, without EMT but with reduced ZEB1 expression. Interestingly, these cells also showed reduced GEM sensitivity, suggesting drug resistance mechanisms separate from EMT in these cells. This highlights the complexity of drug resistance and the value of CRISPR-Cas9 in exploring the signalling crosstalk in pancreatic cancer.