The Potential Molecular Mechanism Of TOPK Activation On Epithelial-mesenchymal Transition In RAS-mutated Lung Cancer

Objectives:Epithelial-Mesenchymal Transition plays a key role in tumor invasion and metastasis-a complex biological process closely related to anomalous activation of oncogene.RAS is one of the most frequently mutated oncogenes in human cancer,including lung cancer.According to relevant reports and our previous research,activation of RAS is of special importance on EMT in RAS mutated lung cancer cells.On account of the unrevealed molecular mechanism,further research was employed to uncover the role of RAS on modulation of tumor metastasis in order to provide new target for RAS mutated lung cancer.Methods:(1)Cell line MCF10A ER:HRAS V12 and HKe3ER:HRAS V12 was used to verify the influence of RAS mutation on EMT,which can conditionally express mutant RAS when 4-hydroxytamoxifen(OHT)was used.(2)K-RAS was knocked down in K-RAS mutant NSCLC cell line A549 and H157,and the changes in EMT were detected by WB,Trans-Well,and scratch tests.(3)Through Protein Kinase Array analysis,we can screen which kinases’function has changed after RAS activated and find possible target molecules.(4)Through drug experiments,it was found that T-lymphocyte-derived protein kinase(TOPK/PBK)played a key role in the process of EMT.(5)Data download from TCGA database was used to analysis the function of TOPK on lung cancer patients and its relationship with P62.(6)Pull-down and COIP was used to confirm p62 can be combined by TOPK and vice versa.(7)P62 protein was synthesized and purified for further use.Kinase experiment confirmed that P62 can be phosphorylated by TOPK.(8)Netphos2.0 was applied to predict the possible phosphorylation site of P62,and the corresponding peptides were synthesized in vitro to identify the specific phosphorylation sites.Results:(1)Activation of RAS in tumor cells can promote EMT,and knock down KRAS in KRAS mutant non-small cell lung cancer cells can reverse EMT.(2)The activity of ERK,MEK,AKT and TOPK were significantly up-regulated after mutant RAS expressed.(3)TOPK has an effect on the tumor grade,metastasis,and prognosis of lung cancer patients.(4)The expression of TOPK was positive correlated with P62.(4)TOPK can interact with P62 and phosphorylate P62 at S24.Conclusion:The activity of TOPK is increased in NSCLC with mutant RAS,and inhibition of TOPK can reverse the EMT of tumor cells,and TOPK can phosphorylate the autophagy-related molecule P62 at S24.