Dishevelled-1 And -3 Affect Lung Cancer Cell Invasion Through β-catenin And P120ctn, Which Is Associated With Poor Prognosis

IntroductionCarcinogenesis is a mutistep pathological process involving multiple gene abnormalities, in which many signaling transduction pathways participate. Increasing evidence has linked the aberrant activation of Wnt signaling pathway to variety of human cancers. Dishevelled (Dvl) is family of positive regulatory factors located upstream of the Wnt pathway, which mediate three intracellular Wnt signal pathways, including the Wnt/β-catenin pathway, non-canonical Wnt pathway and the Wnt/Ca2+ pathway. The human Dishevelled family proteins consists of three members, Dvl-1, Dvl-2 and Dvl-3, which are all over-expressed in non-small cell lung cancer (NSCLC), but the correlations between Dvls and P-catenin, p120ctn as well as prognosis are still not clear. The underlying mechanisms by which Dvl-1 and Dvl-3 affect invasiveness and metastasis in lung cancer require our further research.Materials and Methods1. Patients and specimensTumor specimens from 80 patients with NSCLC who underwent surgical resection in the First Affiliated Hospital of China Medical University between 1998 and 2005 were randomly selected from the archival files of the Department of Pathology. All specimens are from patients with complete follow-up information.2. Immunohistochemical study and result assessmentAll resected specimens were fixed with 10% neutral-buffered formalin and embedded in paraffin blocks. Tissue blocks were cut into 4μm slides, deparaffinized, rehydrated, and incubated with mouse anti-Dvl-1 and Dvl-3 monoclonal antibodies mouse anti-β-catenin monoclonal antibody or p120ctn mouse monoclonal antibody. The sections were evaluated at low magnification (×100) to identify areas where Dvls, P-catenin or pl20ctn was evenly stained. We counted 400 tumor cells perspecimen and calculated the percentage of positively stained cells. All specimens were evaluated by two investigators who were unaware of the clinical data. For the Dvls, we only considered samples with more than 10% positive cells as expressing the proteins.Forβ-catenin, specimens in which 90% or more of the tumor cells showed membranous staining were defined as having normal P-catenin expression, whereas those with 90% or fewer of the tumor cells have membranous expression, with or without cytoplasmic staining were defined as having reduced P-catenin expression. Reduced or aberrant nuclear expression also was considered abnormalβ-catenin expression. For p120ctn, which is localized to the membrane in normal bronchial epithelium, samples in which fewer than 90% of the cells had membranous expression were identified as having reduced membranous expression. Either reduced membranous expression or cytoplasmic expression was defined as abnormal expression of p120ctn.3. RT-PCRRT-PCR was performed with the RNA PCR Kit (AMV) Version 3.0, according to the manufacturer's instructions.4. Western Blot50μg proteins were separated by SDS-PAGE. After transferring to polyvinylidene fluoride membrane, the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody. After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours, the proteins were visualized using DAB or ECL5. Cell culture and transfectionThe human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection. The human lung adenocarcinoma cell line LTEP-a-2 was obtained from the Cell Bank of Chinese Academy of Science. The cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum,2 g/L NaHCO3,100 U/ml penicillin G and 100 mg/ml streptomycin at 37℃in a humidified atmosphere (5% CO2 and 95% air).6. Plasmid construction and transfectionThe cells were stably transfected with the Dvl-1 plasmids or Dvl-3 plasmids using Lipofectamine 2000, following the manufacturer's instructions. The empty plasmid was used as a negative control. Transfected cells were cultured in RPMI 1640 medium containing 10% fetal calf serum.7. Dual-luciferase AssayLipofectamine2000 was used for transfections according to the manufacturer's instructions. Cells were plated in 24-well plates 24h prior to transient transfection with pGL3-OT or pGL3-OF (0.5μg) reporter gene plasmids, together with the control plasmid pRL-TK (50 ng). After incubation for 30 h at 37℃, reporter gene expression was detected by the Dual-Luciferase Assay System. Tcfmediated gene transcription activity was determined by the ratio of pGL3-OT to pGL3-OF luciferase activity, which was normalized to Renilla luciferase activity from the control plasmid pRL-TK. To analyze the effect of Dvls onβ-catenin signalling, pCS2-Myc-Dvll or pMyc-cyto-Dvl3 (0.5μg) were cotransfected with pGL3-OT or pGL3-OF for luciferase asssays. Empty pCS2+or pMyc-cyto (0.5μg) vector was added to control for the amount of plasmid DNA.8. Matrigel invasion assayFollowing the manufacturer's instructions, in the upper chambers,5×106 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix. The lower chambers were filled with RPMI 1640 medium containing 10% fetal calf serum. After incubation at 37℃in a humid atmosphere of 5% CO2 and 95% air, the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin. Then the number of cells counted visually using microscope in five different fields under 200×magnifications per filter.9. Statistical analysisAll statistical calculations were performed by SPSS 13.0 for Windows software. p values less than 0.05 were considered statistically significant.Results1,Normal bronchial epithelial cells had no Dvl-1 or-3 expression. In contrast, in the 80 lung cancer tissue specimens, Dvl-1 and-3 showed positive staining with diffuse distribution in the cytoplasm. Normal bronchial epithelial cells showed strong p120ctn and P-catenin membranous expression, while lung cancer tissues showed reduced membranous expression or abnormal expression. Dvl-1 and-3 were expressed more frequently in adenocarcinoma than in squamous cell carcinoma. Positive expression of Dvl-1 and-3, and the abnormal expression ofβ-catenin and p120ctn, correlated with poor tumor differention. Abnormal expression was significantly more frequent in samples from patients with lymph node metastasis than those without metastasis. The positive expressions of Dvl-1, and abnormal expression ofβ-catenin and p120ctn, were more likely in stage III and IV tumors than in stage I and II tumors; however, the expression of Dvl-3 in stage III and IV tumors was not statistically different compared to stage I and II tumors.2,Dvl-1 expression correlated with abnormalβ-catenin expression, while Dvl-3 expression correlated with p120ctn expression in NSCLCs, but not with the expression ofβ-catenin expression.3,The expression of Dvl-3 and its relation to survival time were analyzed further among 80 NSCLC patients. For patients with normal p120ctn expression, the mean survival time was significantly different from patients with abnormal p120ctn expression. The mean survival time for patients lacking Dvl-3 expression was significantly different from patients with abnormal Dvl-3 expression. Dvl-1 over-expression and abnormalβ-catenin expression had no correlation to survival. In contrast, abnormal p120ctn expression, Dvl-3 expression, TNM stage and lymph node metastasis were associated with poor survival and were independent risk factors for NSCLC prognosis.4,we chose the lung adenocarcinoma cell lines of A549 and LTEP-a-2, which have stable Dvl-2 expression but low Dvl-1 and Dvl-3 expression.β-catenin mRNA levels did not change significantly before and after Dvl-1 or Dvl-3 transfection. Western blotting revealed that Dvl-1-transfected lung cancer cells had increased P-catenin expression, and Dvl-3-transfected lung cancer cells had reducedβ-catenin protein expression. Over-expression of Dvl-1 enhancedβ-catenin-dependent Tcf transcription activity, but over-expression of Dvl-3 reducedβ-catenin-dependent Tcf transcription activity when compared with the control group.5,Over-expression of Dvl-1 and Dvl-3 in A549 and LTEP-a-2 cells both increased p120ctn protein expression. RT-PCR revealed that p120ctn mRNA did not change significantly after transfection of Dvl-1, while p120ctn mRNA level increased significantly after Dvl-3 transfection.6,A549 cells and LTEP-a-2 cells transfected with Dvl-1 or Dvl-3 were far more invasive than control cells. Whenβ-catenin antibody (100 ng/ml) was added to Dvl-1 transfected cells, and p120ctn antibody (100ng/ml) to Dvl-3 transfected cells, the invasiveness of both was significantly inhibited after 18 h. However, addition of theβ-catenin antibody to Dvl-3 transfected cells, or the p120ctn antibody to Dvl-1 transfected cells, did not affect the invasiveness of the cells. Thus, Dvl-1 and Dvl-3 enhanced the invasiveness of lung adenocarcinoma cells throughβ-catenin and via p120ctn, respectivelyConclusion1,Dvl-1 and Dvl-3 was expressed in primary tumors. Their expressions were associated with poor tumor differentiation, high TNM stage and lymph node metastasis.2,Dvl-1 expression is related to abnormal P-catenin expression in NSCLC, and Dvl-3 to abnormal expression of p120ctn and poor prognosis.3,Dvl-1 may affect the biological behavior of lung cancer cells throughβ-catenin (canonical Wnt pathway), while Dvl-3 may act through p120ctn (non-canonical Wnt pathway). Dvl-3 expression could be a useful indicator of NSCLC prognosis.