RASSF10 Prohibits Proliferation And Migration Of NSCLC By Inhibiting Wnt Signaling Pathway

Lung cancer is the leading cause of cancer-related death worldwide,accounting for 25.3% of cancer deaths in 2018(according to the SEER program of NIH).Owing to its intricate nature,recent advancements in lung cancer therapies have helped little in survival improvement,and the 5-year survival of lung cancer remains low(18.6%,2008-2014).Wnt signaling is crucial during embryo development,and Wnt overactivation is one of the most common causes of malignancy.Canonical Wnt signaling acts through modulating β-catenin translocation,eventually influence transcription factor TCF4.Ras-association domain family proteins(RASSFs)are known to regulate proliferation and apoptosis through diverse pathways.Classical RASSFs,with Cterminal Ras-association domains(RA domains),function as tumor suppressors.By interacting with MST1/2 via their centrally located SARAH domains,classical RASSFs can activate Hippo signaling,eventually induce apoptosis.Unlike classical RASSFs,RASSF7~10 have RA domains at the N-termini and no SARAH domain.RASSF7,8 and 10 are predicted to share similar regions called coiled-coils,emerging evidence had proved that coiled-coil containing proteins have an impact on the Wnt pathway.However,the significance of RASSF10 in non-small cell lung cancer(NSCLC)remained elusive.In this study,we explored the role of RASSF10 among NSCLC,in the hope of uncovering RASSF10’s role and its corresponding mechanisms in NSCLC.Objective: In this study,we determined the low expression of RASSF10 in nonsmall cell lung cancer by detecting the expression of RASSF10 in pathological specimens of non-small cell lung cancer and the relationship between clinicopathological factors and prognosis;The cell line was transfected with RASSF10 and a variety of splices,and the changes in cell biological behavior and changes in Wnt pathway were observed to elucidate the possible mechanism of RASSF10 functioning in non-small cell lung cancer.Methods: 136 cases of non-small cell lung cancer specimens were collected from the First Affiliated Hospital of China Medical University(patients receive no chemoradiotherapy before surgery,with complete follow-up data)and 43 cases of adjacent normal tissues(>3cm from cancer).The expression of RASSF10 in pathological specimens was detected by immunohistochemistry,and the relationship between its expression and clinicopathological factors and prognosis was analyzed.Twenty-eight fresh lung cancer tissue specimens were obtained after surgical resection in 2016.The expression of RASSF10 in cancer tissues and corresponding adjacent tissues was detected by Western blot.Clinical information mining was performed on the TCGA lung cancer database,and the mRNA level of RASSF10 and the survival time of patients were statistically analyzed.The expression of RASSF10 in lung cancer cell lines was detected by Western blot,and RASSF10 was interfered or overexpressed in cell lines with high or low expression,respectively.MTT,clone formation assay and invasion assay were used to investigate the influencet of RASSF10 on lung cancer cells’ biological behavior.Western blot,luciferase report and qPCR were used to detect the transcriptional and translational levels of Wnt signaling pathway and downstream target genes c-Myc,cyclinD1 and MMP7 in lung cancer cells after interference or overexpression of RASSF10.By constructing and transfecting splicing mutants such as RASSF10,RASSF10-△RA and RASSF10-△CC,the effects of RASSF10 splicing mutants on lung cancer cells were observed,and the possible mechanism of RASSF10 function was explored.Results: 1.Compared with the low expression of RASSF10 in cancer tissues,RASSF10 was highly expressed in normal lung tissues,and RASSF10 was expressed in both nucleus and cytoplasm.Low expression of RASSF10 was positively correlated with poor differentiation,high TNM stage,and positive lymph node metastasis in lung cancer.Kaplan-Meier survival analysis showed that the survival time of lung cancer patients with low expression of RASSF10 was significantly shorter than that of patients with high expression of RASSF10.COX regression analysis showed that low expression of RASSF10 is an independent risk factor for the prognosis of patients with lung cancer.In the TCGA lung cancer database,the prognosis of the RASSF10 high transcription was significantly better than that of the low transcription(P=0.003),and the proportion of RASSF10 high transcription in the tumor-free patients was significantly higher than that in the with-tumor patients(P = 0.01).2.A549 cell with relatively high expression of RASSF10 were selected for transfection and interference.H460 and H292 cell with low expression of RASSF10 were selected for transfection,and HBE and H1299 cell lines with relatively high expression of RASSF10 were selected for interference.Cell function experiments showed that transfection of RASSF10 inhibited the clonality and migration ability of A549,H460 and H292 cells,while interference with RASSF10 promoted the clonality of A549 and HBE,and also promoted the migration of A549 and H1299.3.The activity of TOPFlash of Wnt pathway was significantly decreased after transfection of RASSF10.The mRNA and protein levels of Wnt target genes c-Myc,cyclinD1,cJun,MMP7 and Axin2 downstream of Wnt pathway were also decreased after transfection of RASSF10.The total protein levels of the main members of the classical Wnt pathway,LRP6,Axin1,GSK3β,and Dvl2 did not change,while the level of 1490 phosphorylated LRP6 was significantly decreased.After transfection of RASSF10,although the total amount of β-catenin did not change significantly,the levels of phosphorylation at 33,37,and 45 increased.The results of nucleoplasm separation experiments showed that the level of β-catenin in the nucleus was significantly reduced after transfection of RASSF10,and the above results were also confirmed by immunofluorescence experiments.However,the phenomenon of decreased transcriptional activity of TOPFlash disappeared after transfection of RASSF10 in cells interfering with LRP6 expression,indicating that the inhibition of Wnt pathway by RASSF10 was achieved by LRP6.4.Immunoprecipitation experiments confirmed that both endogenous and exogenous RASSF10 bind to LRP6.Pull-down experiments confirmed the structural basis for the binding of RASSF10 to LRP6.Immunofluorescence results confirmed the colocalization of RASSF10 with LRP6.The LRP6-M5 mutant is LRP6 which cannot be phosphorylated.Under Wnt3 A stimulation,LRP6-M5 cannot bind to RASSF10,while wild-type LRP6 binds to RASSF10,and the binding increases under Wnt3 A stimulation.5.Based on the above experimental results,we analyzed the domains of RASSF10 and then constructed four RASSF10 splicing mutants.After transfection of RASSF10 mutants into A549 cells,both cell function assays and luciferase reporter assays demonstrated the greatest difference in the function of ΔCC mutant and wild-type RASSF10.The ΔCC mutant was unable to bind to p-LRP6 and the most significant increase in p-LRP6 levels was observed when transfected with the ΔCC mutant.The above results indicate that the coiled-coil structure of RASSF10 is the key to its function.Conclusion: 1.Compared with normal lung tissue,RASSF10 is poorly expressed in non-small cell lung cancer tissues,and its low expression is positively correlated with poor prognosis of patients.2.RASSF10 can inhibit the activity of Wnt pathway and the malignant biological behavior in lung cancer cells;3.RASSF10 binds to LRP6 to play a role in inhibiting Wnt pathway;4.The biological function of RASSF10 depends on the coiled-coil structure.