WWC3 Regulates The Wnt And Hippo Pathways Via Dishevelled Proteins And Large Tumour Suppressor 1,to Suppress Lung Cancer Invasion And Metastasis

Objective: Lung cancer is a common cancer and the incidence and mortality are the highest in the world.Although there has been great progress in the treatment of lung cancer in the past decades,the long-term survival rate of the patients is still not satisfactory due to the complexity of various genetic backgrounds.Therefore,it is important to find out the new oncogene / tumor suppressor gene and explain its molecular mechanism to prevent and treat malignant tumors.In recent years,Hippo pathway was found to be a signaling pathway which regulates cell proliferation and organ volume control,and plays an important role in embryonic development and tumor formation.It is reported that in Drosophila,WW and C2 domain containing protein family(WWCs family)could affect cell proliferation and organ development by activating Hippo pathway,however,the expression level of WWC3 in human tumors and the mechanisms underlying its role in cellular signal transduction have not yet been reported.Therefore,the purpose of this study is to explore the mechanisms underlying the effects of WWC3 on the proliferation and invasion of lung cancer cells,and to reveal the important role of WWC3 in the crosstalk between Hippo pathway and Wnt pathway.Methods: We collected 127 non-small cell lung cancer and 20 adjacent normal lung tissues specimens(excluding 5cm and paraffin embedded specimens),which were treated surgically and included complete follow-up data in the First Affiliated Hospital of China Medical University.In addition,20 cases of fresh lung cancer tissues were obtained from the First Affiliated Hospital of China Medical University.We applied immunohistochemical staining method to detect the location and expression of WWC3 in non-small cell lung cancer,and performed statistical analysis(including: Chi-square test,Kaplan-Meier method and Cox regression)for the correlation between WWC3 and clinical pathological factors,prognosis and risk factors of the patients with non-small cell lung cancer.Western blotting was used to detect the expression of WWC3 in six lung cancer cell lines and an immortalized normal human bronchial epithelial cell line HBE;Western blotting and real-time PCR(RT-q PCR)were used to detect the protein and of m RNA level of WWC3 in 20 lung cancer tissues and adjacent normal lung tissue;Colony formation assay and Transwell assay,MTT assay and subcutaneous xenografts in nude mice and tumor metastasis assay via tail vein were used to performed to explore the impact of WWC3 on lung cancer cell proliferation and invasion and metastasis in vitro and in vivo.Dual-luciferase assay,Western blotting,RT-q PCR and immunofluorescence was applied to detect the Wnt/Hippo pathway activity,main molecules phosphorylation levels including Dishevelled(DVLs),Ste-20 family of protein kinase(MST1),Large tumor supressor(LATS1)and Yes-associated protein(YAP),the m RNA and protein expression level of target genes and the level of beta-catenin/YAP nuclear entry.Immunoprecipitation,immunofluorescence and GST-pulldown were used to detect the binding of key molecules DVL2 and WWC3.Co-immunoprecipitation and Western blotting were used to detect the competitive binding of WWC3,DVL2 and LATS1,and the changes of Hippo pathway and Wnt pathway activity induced by the crosstalk of them.The SPSS22.0 statistical analysis software was used to analyze the experimental data by t-test,and P<0.05 showed that the results were statistically significant.Results: Our results indicate that low WWC3 expression is detected in both lung cancer cell lines and lung cancer specimens and is associated with low differentiation,advanced p-TNM stage,positive lymph node metastasis,as well as poor prognosis in patients with lung cancer.Meanwhile,overexpression of WWC3 has an inhibitory role in proliferation and invasiveness of lung cancer cells.We disclose that WWC3 perform these functions by inhibiting and stimulating Wnt and Hippo pathway,respectively,in vitro and in vivo.WWC3 interacts with Dvls,prevents CK1? kinase from phosphorylating Dvls,inhibits b-catenin nuclear translocation so as to diminish Wnt pathway.Transfection of WWC3-?WW&ADDV plasmid abrogated these effects.Moreover,the interaction of WWC3 and Dvls causes reduction of WWC3 and LATS1 interaction and decreases phosphorylated LATS1,thereby increases YAP nuclear importation and attenuates Hippo pathway.WWC3-?WW plasmid transfection abrogates this effect.Conclusion: These findings elucidate the molecular interplay between WWC3,Dvls and LATS1,meanwhile reveal a yet unknown link between Wnt and Hippo pathways,providing us a potential target of clinical intervention for patients suffering lung cancer.