Mechanism Of Kras Mutation Up-regulating Pank2 And Pank3 Mediated Pancreatic Cancer Cells Resistance To Ferroptosis

Objective:Ferroptosis is a novel regulatory cell death modality and a potential therapeutic strategy for pancreatic cancer,the mechanisms of which are still being further explored.In this study,we investigated the mechanism by which KRAS mutations upregulate PANK2 and PANK3 to mediate ferroptosis resistance in pancreatic cancer cells,which is expected to provide a potential therapeutic target for pancreatic cancer with high KRAS mutation rate.Methods:(1)Analysis of the sensitivity of KRAS mutant pancreatic cancer cells to BSO-induced ferroptosis using bioinformatics methods,CCK8 cell proliferation assay and flow cytometry;(2)Analysis of changes in the sensitivity of pancreatic cancer cells to BSO after inhibition of KRAS mutation using Western blot,PCR quantitative analysis,divalent iron ion assay,malondialdehyde assay,GSH content assay and clonogenic assay;(3)Analysis of changes in m RNA and protein expression of PANK2 and PANK3 after BSO treatment of pancreatic cancer cells using bioinformatics methods,quantitative PCR analysis and Western blot;(4)Changes in PANK2 and PANK3 expression in pancreatic cancer cells after overexpression of KRAS mutations using bioinformatics methods,quantitative PCR analysis and Western blot;(5)Validation of PANK2 and PANK3 catalytic source of CoA promoting pancreatic cancer cells against BSO-induced ferroptosis using Western blot and lipid reactive oxygen species assay.Results:(1)High KRAS mutation rate is an important characteristic of pancreatic cancer cells,and KRAS expression is significantly upregulated after pancreatic tissue carcinogenesis,resulting in significantly lower survival rates in pancreatic cancer patients.At the same time,there was a high correlation between KRAS and ferroptosis-related molecules.When KRAS mutant and wild-type pancreatic cancer cells were treated with the drug BSO separately,KRAS mutant pancreatic cancer cells were significantly resistant to BSO-induced ferroptosis.(2)In KRAS-mutant pancreatic cancer cells,knockdown of KRAS did sensitize BSOinduced ferroptosis and there was a decrease in the reducing substance GSH and an increase in the levels of lipid peroxides and lipid reactive oxygen species.However,the levels of divalent ferric ions were reduced,suggesting that knockdown of KRAS does not create a complete system for ferroptosis to occur.(3)KRAS mutations cause pancreatic cancer cells to be resistant to BSO in addition to BSO treatment,which also promotes PANK2 and PANK3 expression.(4)Overexpression of KRAS alone does not increase the expression of PANK2 and PANK3,but rather overexpression of KRAS with mutant sites promotes the expression of these two pantothenate kinases.(5)CoA is a direct metabolite of resistance to BSO-induced ferroptosis,and KRAS mutations promote the expression of pantothenic acid kinases also to increase the abundance of CoA in pancreatic cancer cells,which is the main reason why KRAS mutated pancreatic cancer cells are more resistant to BSO-induced ferroptosis.Conclusions:In pancreatic cancer cells,KRAS mutations upregulate PANK2 and PANK3 to mediate BSO-induced ferroptosis resistance.KRAS mutations increase the abundance of CoA in pancreatic cancer cells by promoting the expression of PANK2 and PANK3,and CoA decreases the intracellular iron ion concentration by upregulating the expression of FTH1 and decreasing the expression of TFRC,ultimately resisting BSOinduced of ferroptosis.