ERK5-IN-1 Prevents The Progression Of Nonsmall Cell Lung Cancer Via Inhibiting A Novel Target DCLK1 Kinase

Lung cancer is the leading cause of cancer deaths worldwide,and nonsmall cell lung cancer is the main type of lung cancer.The current treatment for early NSCLC is mainly surgical treatment,and advanced NSCLC often chooses combination therapies including targeted therapy and radiotherapy and chemotherapy.Doublecortin-like kinase 1(DCLK1)is a cancer stem cell(CSC)marker highly expressed in various human cancers and an increasing interest protein kinase target for cancer therapy.Although a variety of small molecule kinase inhibitors have been confirmed to have inhibitory effects on DCLK1 kinase.However,to date there are no drug candidates targeting DCLK1 kinase have been developed for neither clinical use nor even in clinical trials yet.Through in vitro kinase experiments,we discovered that ERK5-IN-1can bind to DCLK1 kinase and inhibit its activity.As reported in the literature,DCLK1 is related to lung cancer.Therefore,we took experiments to further evaluate the effect of ERK5-IN-1 on the expression of DCLK1 and cell proliferation activity in non-small cell lung cancer(NSCLC)cells.The experimental results show that ERK5-IN-1 can inhibit the expression of DCLK1 and cell proliferation activity.At the same time,experiments verified its inhibitory effect on the expression and phosphorylation of ERK5,indicating the possibility of ERK5-IN-1 as a dual kinase inhibitor of ERK5 and DCLK1.In addition,in order to explore whether ERK5-IN-1 affects cell proliferation activity through DCLK1.We constructed the Plenti vector inserted into DCLK1 c DNA and transfected it into the genome of lung cancer A549 cells.Through MTT experiment,observe the effect of ERK5-IN-1 on the proliferation activity of transfected A549 cells.The results show that the up-regulation of DCLK1 can resist the inhibitory effect of ERK5-IN-1 on cell proliferation.In addition,because epithelial-mesenchymal transition(EMT)is closely related to cell stemness.We used western blot to observe the changes in the expression of EMT-related markers in lung cancer cells treated with drugs.Experimental data showed that ERK5-IN-1 reduced the expression of mesenchymal-like marker ZEB1 in NSCLC cells,and up-regulated the expression of epithelial-like marker E-cadherin,showing an inhibitory effect on EMT.In addition,we also conducted tumor sphere formation experiments and flow cytometry to study the effect of ERK5-IN-1 on stemness.The results of the sphere formation experiment showed that ERK5-IN-1 reduced the sphere formation efficiency of lung cancer cells.However,the results of flow cytometry found that the percentage of ALDHhigh in NSCLC cells treated with ERK5-IN-1 increased.We speculated that this may be caused by EMT inhibition and changes in the tumor microenvironment.In addition,we found that lung cancer cells treated with ERK5-IN-1 have reduced expression of the stemness marker β-catenin.It is further verified that ERK5-IN-1 can inhibit the stemness of lung cancer cells.In general,ERK5-IN-1 inhibits the activity of DCLK1 kinase,the IC50 in the in vitro kinase experiment is only 8.84 n M;and in lung cancer cells,it inhibits the expression of DCLK1,cell proliferation,EMT,and stemness.In summary,ERK5-IN-1 has tumor treatment potential and the possibility of becoming a lead compound for DCLK1 targeted kinase inhibitors.