The Signaling Regulation Role Of Tnk1 In Prostate Cancer Cells

Prostate cancer is the fourth most common cancer in the world and the second most common cancer among men,and the most common malignant tumor among men.In 2018,the number of prostate cancer deaths was 35.9 million and 23,000 in the world and in China respectively,with metastasis the leading cause of cancer death.Currently,the clinical treatment of prostate cancer is limited and has serious side effects;moreover,some patients acquire resistant to the treatment.Therefore,in-depth study of the molecular mechanism of the development,progress and metastasis of prostate cancer to provide new drug targets for its clinical treatment is important in both theory and clinic.Tyrosine kinases are divided into receptor tyrosine kinases and non-receptor tyrosine kinases;they play important roles in regulating the growth,proliferation,differentiation and survival of normal cells.Abnormal activation of tyrosine kinase and its downstream signaling pathways,such as PI3K/Akt,MAPK and JAK/STAT,is closely related to the development,progress and metastasis of tumors,which is a hot field in the research of tumor-targeted therapy.TNK1 is one of the non-receptor tyrosine kinases that have been found to have oncogenic activity in recent years.Studies have shown that TNK1 is related to tumor development.Increased expression of TNK1 in human tumor samples can promote the growth and survival and inhibit apoptosis of tumor cells via PI3K/Akt and JAK/STAT pathways.However,these studie did not elucidate the activation mechanism TNK1 and its downstream target molecules,which will be addressed in this thesis.Western blot experiments showed that TNK1 was expressed in human prostate PC-3,DU145 and LNCaP cell lines,and the expression of TNK1 in human prostate cancer tissues was significantly higher than that in adjacent tissues,suggesting that TNK1 may play an important role in the development and progress of prostate cancer.In order to investigate the role of TNK1 in prostate cancer cells,CRISPR/Cas technology was used to establish TNK1 deficient cell line.3 sgRNAs were designed,with sgRNA1 targeting the N-terminal exon and sgRNA2 and sgRNA3 targeting the kinase coding region.Preliminary study showed that the gene editing efficiency of sgRNA2 and sgRNA3 was superior to that of sgRNA1 based on the expression level of TNK1 in G418 resistance cell pools.Correspondingly,plasmids containing sgRNA2 or sgRNA3 were transfected into PC-3 cells,and G418 resistant cell clonses were selected with 48 clones for each sgRNA.The clones were numbered by 2CX and 3CX(X=1-48),respectively.Western blot results showed that the gene editing efficiency of sgRNA2 or sgRNA3 was similar,and 25% of the cell clones did not express TNK1.Genomic DNA extracted from some TNK1 deficient clones was amplificated by PCR,and the products sequencing results indicated that TNK1 deficiency was caused by frame-shift mutation.Clones 2C29 and 2C40 with different frame-shift mutations were selected for subsequent experiments.Clonogenicity experiments showed that TNK1 deficiency had no significant effect on the number of PC-3 cell colonies,but the average area of the colonies was significantly reduced,suggesting that TNK1 deficiency inhibited cell proliferation but did not affect the density-dependent growth of cells.Transwell migration experiment showed that TNK1 deficiency significantly inhibited cell migration,changed the morphology of the migrated cells,and inhibited the spreading of the migrated cells on the membrane.In addition,TNK1 deficiency down-regulated the expression of mesenchymal markers,i.e.vimentin and N-cadherin,indicating that TNK1 deficiency inhibited the EMT process of tumor cells.Analysis of the signaling pathway in PC-3 cells showed that TNK1 deficiency significantly inhibited the phosphorylation of STAT3 active site Y705,but had no significant effect on the phosphorylation of Akt and ERK1/2,suggesting that TNK1 regulated PC-3 cells through JAK/STAT signaling pathway.Taken together,this study identified for the first time that TNK1,a non-receptor tyrosine kinase,can promote the proliferation,migration and EMT of PC-3 cells by inuding the phosphorylation of STAT3 Y705 site,which reveales a new mechanism of TNK1 regulating tumor cell response and provides a new drug target and new strategies for the clinical treatment of prostate cancer.