DCLK1 Expression Is Correlated With MET And ERK5 Expression,and Associated With Prognosis In Malignant Pleural Mesothelioma

BackgroundMalignant pleural mesothelioma(mesothelioma)is an aggressive form of cancer that originates in the mesothelial cells of the pleura of the lung and is closely associated with asbestos exposure.MPM has been considered very rare,but in the past few decades,the incidence of MPM is increasing year by year.Epidemiological projections indicate that,Mesothelioma incidence will continue to increase and reach its peak in the next 10 years with deaths around 43,000 worldwide annually;furthermore,decreases are not expected by 2020.From asbestos exposure to onset,there is an incubation period of about 30-40 years,at the same time,MPM lacks specific clinical and imaging findings.Although the diagnosis and pathology of thoracoscopy are convenient for definite diagnosis,simple diagnosis is still difficult due to the lack of typical symptoms,signs and imaging findings of MPM.Combined treatments with surgery and chemotherapy has improved survival and quality of life for mesothelioma patients,however,the overall treatment was ineffective.Because of the high malignancy,aggressive and poor prognosis of MPM,most patients survive less than 12 to 18 months and long-term survival is dismal.Therefore,MPM will remain a major public health problem for a long time,and finding new and effective drugs remains the key to the prevention and treatment of MPM.The pathogenesis of MPM is complex,involving a variety of signaling pathways and aberrant regulation of molecules.Mesenchymal-epithelial transition factor,MET is a proto-oncogene MET encoding protein products,is a kind of transmembrane receptors with independent phosphorylation activity,belonging to the Receptor tyrosine kinases,members of the family.Tyrosine kinase receptors play an important role in tumor growth and metastasis.HGF is a polypeptide growth factor,which induces the migration and invasion of epithelial cells,and participates in the regulation of immune activity,tissue regeneration and angiogenesis.The binding of HGF to the MET receptor makes MET phosphorylation of tyrosine residues,and activates protein tyrosine protein kinase in the domain of the receptor's intracellular protein kinase.The downstream signaling pathway is further activated,thereby promoting tumorigenesis,invasion and metastasis,angiogenesis and epithelial-interstitial transformation process.Previous studies have demonstrated that HGF induced mesothelioma cell proliferation through activation of PI3K/ERK5/Fra-1 pathway and HGF has also been shown to phosphorylate ERK5 in human mesothelioma cells.Blocking HGF/MET signaling pathway can effectively inhibit the occurrence,development and metastasis of tumor.Extracellular regulated kinase 5(ERK5)is an im portant primary kinase in the MAPK system,and the ERK5 signaling pathway is also an important component of the MAPK signaling pathway.Through the phosphorylation cascade of ERK5 signaling pathway,a variety of stimuli activate MEKK3 or MEKK2,activate MEK5,and finally activate ERK5,thereby regulating the expression of specific genes.MEK5 is the only specific upstream kinase of ERK5,which is highly specific to the ERK5 kinase and does not activate other members of the MAPK family even in cases where MEK5 overexpression occurs.ERK5 not only promote cell survival and inhibit apoptosis,but also is closely related to the function of blood vessel development and proliferation.Ramos and other studies have found that malignant mesothelioma is associated with the ERK5 signaling pathway.Doublecortin-like kinase 1(DCLK1)is a transmembrane microtubule-associated protein kinase,and the encoded protein has a DCX domain and a serine/threonine kinase domain in the c-terminal.DCLK1 is considered to be a marker of a new normal stem cell in the gastrointestinal tract.Inhibition of expression of DCLK1 inhibits tumor growth.In tumor tissues,a variety of transcription factors,kinases and miRNAs are involved in the process of epithelial mesenchymal transformation.DCLK1 positive cells have stronger EMT,so that tumor cells have stronger invasion and metastasis ability.Although DCLK1 has been linked to various cancers,little is known about its role in the growth of human mesothelioma.Furthermore,the relationship between MET,ERK5,and DCLK1 in human mesothelioma is unknown.In this study,we sought to evaluate the efficacy of MET,ERK5,and DCLK1 as possible biomarkers and potential targets for therapeutic development for human mesothelioma.Objects:1.To investigate the expression levels of MET,ERK5 and DCLK1 in MPM cells,and to elucidate the possibility of MET,ERK5 and DCLK1 as diagnostic biomarkers for MPM.2.To investigate the relationship between the expression of DCLK1 and the survival of MPM patients,and to elucidate the possibility that DCLK1 may be a prognostic factor in MPM patients.3.To observe the effect of MET and ERK5 inhibitors on the proliferation of MPM cells by cell viability assay.4.To observe the effect of MET inhibitor or ERK5 inhibitor of MET/ERK5/DCLK1 pathway at the level of transcription and translation level by Western blot technology and RT-PCR technology.5.To study the relationship between inhibitors of MET or ERK5 and invasion of MPM,and to provide a new target for the effective prevention and treatment of MPM.Methods:1.Fresh mesothelioma and adjacent normal pleural tissues were obtained from patients with malignant plural mesothelioma who were undergoing surgical resection of the primary tumor at University of California,San Francisco Medical Center from July 1997 to December 2008.All human tissue samples were obtained and analyzed in accordance with procedures approved by the institutional review board of the University of California,San Francisco(IRB H8714-22 942-01),and written informed consent was obtained from all subjects.2.Immunohistochemical technique.73 cases of specimens from tissue microarray sections and mesothelioma cell lines H290,H513,H28,211H,MS-1,H2052 and normal mesothelial cell line LP9 were stained by immunohistochemical,to clear MPM tissues and cells in MET,ERK5 and DCLK1 expression.3.Cell proliferation activity was detected by Celltiter-GloTM assay.Cells were cultured in a 96-well plate and treated with different doses of XL184 and XMD8-92.After 48 hours of incubation,cells were lysed and luminescent signaling was generated by a CellTiter-Glo Luminescent Cell Viability Assay reagent.Luminescent signaling was measured on the GloMax-96 Microplate Luminometer.Proportional cell viability was analyzed with GraphPad Prism6 software,which was used to calculate dose-response curves and IC50.4.Transwell invasion assayThe transwell invasion assay was performed in a 6-well plate transwell system.The transwell inserts were coated with 300?l matrigel.Cells were harvested and resuspended in serum-free medium supplemented with XL184,XMD8-92 or DMSO to the upper chamber of the transwell.The lower chamber was infused with 2.6ml complete growth medium(10%FBS).The transwell was incubated at 37? for 20 hours.After formalin fixation,the membrane was stained by Crystal Violet for 20 minutes.Phase contrast images were taken and the cells on the lower side of the membrane were counted under a 20 x objective lens.5.Tumorsphere Assays1 ×103 number single-cell suspensions after treated with XL 184,XMD8-92,or DMSO were plated in 24-well ultra-low attachment plates in Serum free medium.Tumorspheres were cultured for 7 days.Tumorspheres formed in non-adherent cultures were counted under a 10 x objective lens.The cut-off size for the spheres counted is 60?m.6.Quantitative real-time PCRAfter treatment,the cells were extracted with Rneasy Mini kit kit,and the total RNA was detected by reverse transcription.Quantitative real-time PCR was used to detect the change of the expression level of target gene mRNA.7.Western blot.After treatment,the cells were lysed using cell lysate,and the protein concentration was determined after high temperature denaturation.Gel electrophoresis was performed with SDS-PAGE gel,and the expression level of the target protein was detected by Western blot.8.Statistical analysisData are expressed as mean ± standard deviation(SD)from three independent experiments.The chi-square independence test was used to compare IHC results between the staining intensity of MET,ERK5,and DCLK1 in the same mesothelioma tumors.Student's t-test was used for comparison between two groups.One-way ANOVA followed by Scheffe multiple comparisons were used to compare the differences among multiple groups.The other statistical analyses were performed using the GraphPad Prism.Survival analysis in patients was performed by Kaplan-Meier analysis with GraphPad Prism.A significant difference was considered when the P value from a two-tailed test was P<0.05.Results:1.MET,ERK5 and DCLK1 were overexpressed in MPM tissue simples and cells.(1)Seventy-three tissue microarray sections and mesothelioma cell lines H290,H513,H28,211H,MS-1,and H2052 and mesothelial cell line LP9 were immunostained stained with the properly diluted antibodies:anti-MET;anti-ERK5 and anti-DCLK1.Our analysis also showed that,in 38.4%of the mesothelioma tumor samples,moderate to strong expression of all three proteins was detected(n=28).Statistical analysis revealed a significant association between MET,ERK5,and DCLK1 expression in these mesothelioma samples(P<0.05).Moreover,Statistical analysis indicated a significant higher expression of MET,ERK5 and DCLK1 in mesothelioma samples compared to their expression in normal pelural tissues obtained from the sample 8 patients(P<0.05).(2)We analyzed the IHC staining score of MET,ERK5,and DCLK1 of mesothelioma samples and normal pleural tissues obtained from 8 patients.Statistical analysis indicated a significant higher expression of MET,ERK5 and DCLK1 in mesothelioma samples compared to their expression in normal pelural tissues obtained from the sample 8 patients2.DCLK1 up-regulation correlates with poor prognosis in mesothelioma(1)DCLK1 expression was significantly higher in MPM patients' tissues than normal pleural tissues(Table ?)(P=0.000409).suggesting a possible oncogenic role of DCLK1 in MPM.(2)Analyzing the associations between DCLK1 expression and clinicopathologic characteristics,we did not find significantly correlations between DCLK1 and age,gender,smoke status or TNM stage(P>0.05)(3)The correlation between DCLK1 expression and overall survival(OS)in MPM patients.The OS times with high DCLK1 staining is significantly shorter than those with low DCLK1 staining(P=0.0235),which indicated DCLK1 level is a potential indicator of OS survival of MPM patients.3.MET and ERK5 inhibitors suppresses cell viability of human mesothelioma cells.Cell viability was assayed and IC50 of each cell line was calculated based on the dose-response curves.The IC50 values indicate different levels of inhibitory effects of XL184 and XMD8-92 among the cell lines.A higher dose of XL184 and XMD8-92 resulted in lower viability of the mesothelioma cell lines.4.MET or ERK5 inhibition causes down-regulation of MET/ERK5/DCLK1 signaling(1)We analyzed phospho-MET,MET,phospho-ERK5,ERK5 and DCLK1 expression in H290 and H513 cell lines treated with the MET inhibitor XL184 or the ERK5 inhibitor XMD8-92 by Western blot assay.(2)Significant decrease in mRNA levels of DCLK1 was detected in MPM cells after XL184 or XMD8-92 treatments analyzed by quantitative real-time PCR.These results suggest that MET or ERK5 inhibition decreased the expression of phospho-ERK5 and DCLK1 in MPM cells.It is further explained that DCLK1 lies downstream of the MET/ERK5 signaling pathway.5.MET or ERK5 inhibition impaired invasion and tumor sphere formation ability of mesothelioma cells H290.(1)To test the effect of MET or ERK5 inhibition on the invasion ability of mesothelioma cells,a transwell assay was performed using H290 cells.The number of the cells that invaded to the lower side of the membrane decreased significantly compared to that in the control group treated with DMSO.Research showed that inhibitors of MET or ERK5 could inhibit the migration and invasion of H290 cells.(2)To measure the effect of EMT or ERK5 inhibition on the self-renewal of cancer stem cells in mesothelioma,we used a tumorsphere assay.Tumorsphere formation efficiency decreased significantly in a dose-dependent manner after XL 184 or XMD8-92 treatment.Conclusion:(1)In this study,we found that MET,ERK5 and DCLK1 were highly expressed in MPM tumor tissues and cells,and the expression levels of MET,ERK5 and DCLK1 in mesothelial tissue samples were significantly higher than those in normal pleural mesothelial tissues.(2)In patients with MPM,the survival of patients with high DCLK1 expression was significantly shorter than those with low expression,and DCLK1 could be a prognostic factor for malignant pleural mesothelioma.(3)Our results suggest that DCLK1 is regulated by MET/ERK5 signaling in human mesothelioma,and demonstrate that DCLK1 is located downstream of the MET/ERK5 signaling pathway.MET and ERK5 could be further developed into a promising therapeutic target against mesothelioma.(4)Our results show that this MET/ERK5/DCLK1 signaling activity could be important for cell growth in mesotheliomas.MET,ERK5 and DCLK1 may serve as diagnostic markers for malignant pleural mesothelioma,providing new target sites for MPM immunotherapy,and elucidating that small molecule inhibitors may provide new therapeutic tools for malignant pleural mesothelioma.