Construction And Identification Of KRAS-mutant Cell Lines

Cancer is a major disease that threatens human health.RAS has a high mutation rate in all human cancers,especially in highly lethal tumors,such as pancreatic cancer,lung cancer and colon cancer.Therefore,drug discovery by targeting RAS protein has become an important area of cancer therapy.Isogenic cell lines and animal disease models provide effective tools for targeted drug research.The construction of cell and animal models are based on gene editing technology.Currently,the gene editing technologies which have been widely used are the TALENs technology and the CRISPR/Cas9 technology.The theory of gene targeting is based on homologous recombination and non-homologous end joining which can make gene knock-out,knock-in and over-expression.In this paper,we used adeno-associated virus-mediated gene editing to construct four different types of KRAS-mutant cell lines(KRASG12D,KRASG12C,KRASG12S,KRASG12V).The KRAS-mutant cell lines were identified at the protein level,and we found that there was a significant increase of KRAS both in protein level and protein activation level(KRAS-GTP).KRAS mutation could activate downstream signaling pathways.Our results showed that compared with wild-type cell line,the levels of phosphorylated ERK1/2(p-ERK1/2-Thr202/Tyr204),phosphorylated cRaf(p-cRaf-ser338),phosphorylated AKT(p-AKT-ser473),and phosphorylated MEK(p-MEK-ser217/221)were all significantly increased in KRAS-mutant cell lines.These results indicate that mutant KRAS protein play a role in the activation of signal transduction.In view of the activation of KRAS signaling pathway in mutant cell lines,we further studied the difference of cell growth in KRAS-mutant and wild-type cells.The results showed that the growth rate of mutant cells were significantly increased by KRAS point-mutations,and the cell number in S phase of cell cycle in KRAS-mutant cells was two times higher than that in wild-type cell.The protein expression levels ofcell cycle related protein and kinase in S phase(CyclinE,CyclinA and phosphorylated CDK2)were significantly increased in mutant cell lines.These results suggest that KRAS mutation can promote the over-activation of downstream signaling pathways,leading to fast cell proliferation.We further investigated the drug sensitivity of KRAS-mutant cell lines.In this paper,about 20 kinds of clinical small-molecule inhibitors targeting RAS signal transduction pathway were selected,and drug sensitivity was detected by analyzing the half maximal inhibitory concentration(IC50).The result showed that KRAS-mutant cell lines had significantly tolerance to MEK inhibitors(AZD6244,Trametinib,RO5126766).KRAS-mutant cell lines have sensitivity to Weel inhibitor(MK1775)and AKT inhibitor(Uprosertib)to some extent.In summary,we successfully constructed four different types of KRAS-mutant cell lines,which provide good cell models for drug discovery and drug target identification.