Dishevelled Family Proteins Are Expressed In Non-Small Cell Lung Cancer And Function Differentially On Tumor Progression

IntroductionThe Wnt/β-catenin pathway has critical roles in the embryonic development and stem cell maintenance of animals from Hydra to human.The key of this signaling cascade is the regulation of the level of cytoplasmicβ-catenin.In the absence of Wnt signal,β-catenin is assembled to a multiprotein destruction complex typically consisting of axis inhibition protein(Axin),adenomatous polyposis coli(APC), glycogen synthase kinase-3β(GSK3β)and casein kinase Ia(CKIa),and targeted for degradation through the proteasomal machinery,thereby maintaining the cytoplamicβ-catenin at exceedingly low level.Upon Wnt stimulation,the activated cell surface receptor Frizzled(Fz)triggers the aggregation and polymerization of Dishevelled(Dvl) at cytoplasmic membrane by unknown mechanisms.The Dvl polymer in turn synergizes with low-density-lipoprotein receptor-related proteins 5 and 6(LRP5/6)to recruit Axin to the cytoplasmic membrane,leading to the dissociation or reduced formation of the destruction complex and subsequently the breakdown ofβ-catenin degradation.This results in the cytoplasmic accumulation ofβ-catenin,which then translocates into the nucleus,where it complexes with members of the TCF/LEF family of transcription factors and induces the transcription of Wnt-responsive genes. Increasing evidence has linked the aberrant activation of Wnt/β-catenin signaling to a variety of human diseases,including cancers,such as colon cancer and hepatocarcinoma.Althoughβ-catenin has been shown to be abnormally expressed in a variety of tumors,the mutation of it occurs infrequently.Thus,other components of the Wnt/β-catenin signaling pathway upstream ofβ-catenin may contribute to the activation of this signaling cascade.Dishevelled(Dvl)family proteins are cytoplasmic mediators of the Wnt/β-catenin signaling pathway.Human DVL gene family consists of three members,DFL-1,DFL-2 and DVL-3,which locate on chromosomes lp36, 17p13.2 and 3q27,respectively.Their encoded proteins(Dvl-1,Dvl-2 and Dvl-3, accordingly)contain three highly conserved domains(DIX,PDZ and DEP).Dvl family proteins have recently been linked to cancers.However,the involvement of Dvl in tumorigenesis is largely unknown.Several studies regarding the expression of Dvls in cancers have revealed controversial results.Furthermore,the roles of individual Dvls and their expression in human cancers are poorly defined.Our study aimed to characterize the expression of Dvls and their correlation to clinicopathological factors andβ-catenin expression,as well as exploring the roles of Dvl in tumor progression in non-small cell lung cancer(NSCLC).MethodsTumor specimens from 113 patients with NSCLC who underwent surgical resection in the First Affiliated Hospital of China Medical University between 2003 and 2006 were randomly selected from the archival files of the Department of Pathology. Among the 113 cases,the nodal metastases of 39 patients were available.Of the patients,68 are male and 45 are female,with a 1.5:1 ratio of male to female.Patients' ages at the time of surgery ranged from 20 to 81(median age:57 years).According to the TNM staging system of the International Union Against Cancer(UICC),patients were categorized into stagesⅠ(n=27),Ⅱ(n=24),Ⅲ(n=56)andⅣ(n=6).All specimens were reevaluated for diagnosis following the criteria for classification of lung cancer by the World Health Organization(WHO),and 48 squamous cell carcinomas and 65 adenocarcinomas were confirmed.No patient had received radio-or chemotherapy before tumor excision.This study was conducted under the regulations of the Institutional Review Board of China Medical University.Informed consent was obtained prior to surgery from all enrolled patients.We used immunohistochemistry to assess the presence of the three Dvl family proteins in the 113 individual NSCLC specimens.Thirty-nine of the 113 cases were examined further for Dvl andβ-catenin protein expression in matched primary growths and autologous nodal metastases.We also examined the effect of Dvl-1 and Dvl-3 overexpression onβ-catenin expression and the invasive ability of A549 and QG56 lung cancer cells by using Immunofluorescence,RT-PCR,Western blotting and Matrigel invasion assay.ResultsThe positive expression rate in primary tumors was 53.1%(60/113)for total Dvl, 36.3%(41/113)for Dvl-1,36.3%(41/113)for Dvl-2 and 41.6%(47/113)for Dvl-3, while normal adult bronchial and alveolar epithelia showed negative expression of all these proteins.The expression levels of total Dvl and all three Dvl proteins were significantly higher in adenocarcinomas than in squamous carcinomas(p＜0.01),and were associated with poor tumor differentiation(p＜0.05).The positive expression of total Dvl,Dvl-1 and Dvl-2 proteins was significantly correlated to advanced pTNM stages(Ⅲ-Ⅳvs.Ⅰ-Ⅱ,p＜0.05).Of the 39 pairs of matched data,the positive expression rate was 87.2%(34/39)for total Dvl,69.2%(27/39)for Dvl-1,51.3%(20/39)for Dvl-2, and 69.2%(27/39)for Dvl-3 in nodal metastases,and 64.1%(25/39)for total Dvl, 48.7%(19/39)for Dvl-1,35.9%(14/39)for Dvl-2,and 48.7%(19/39)for Dvl-3 in primary tumors.The expression levels of total Dvl,Dvl-1 and Dvl-3 were significantly higher in nodal metastases than in primary growths(p＜0.05),with total Dvl and Dvl-1 expression correlating toβ-catenin expression in metastases(p＜0.05).There was no difference in the expression of Dvl-2 between paired primary tumors and metastases (p＞0.05).However,Dvl-2 expression correlated significantly withβ-catenin expression in primary growths(p＜0.05).Ectopic expression of Dvl-1 in A549 and QG56 resulted in the nuclear localization of Dvl-1 proteins in both cell lines while there was no obvious nuclear localization of Dvl-3 protein in A549 or QG56 cell transfected with Dvl-3 expression plasmid.Overexpression of Dvl-1 and Dvl-3 did not change the levels ofβ-catenin mRNA in A549 or QG56 cells(p＞0.05).However,overexpression of Dvl-1 significantly increased theβ-catenin protein levels of both A549 and QG56 cells (p＜0.05).Interestingly and supprisingly,overexpression of Dvl-3 did not alter theβ-catenin protein level of QG56 cells(p＞0.05)while did decrease theβ-catenin protein level of A549 cells(p＜0.05).Exogenous expression of Dvl-1 and Dvl-3 both significantly enhanced the invasive ability of A549 and QG56 cells,as compared with controls(p＜0.01).Conclusions1.Expression of Dvl family proteins,Dvl-1,Dvl-2 and Dvl-3,is common in NSCLCs.The adult normal bronchial and alveolar epithelia show negative expression for all Dvl family proteins.The expression of Dvls is higher in adenocarcinomas than in squamous carcinomas and correlated with poor differentiation of NSCLCs.2.The expression of Dvl-1 and Dvl-2 correlates with high pTNM stages of NSCLCs.The expression of Dvl-1 and Dvl-3 is higher in nodal metastases than in primary growths.The Dvl-2 expression correlates withβ-catenin in primary tumors while Dvl-1 expression correlates toβ-catenin in nodal metastases.3.Overexpression of Dvl-1 and Dvl-3 increases the invasive ability of lung cancer cells.Overexpression of Dvl-1 and Dvl-3 has no effect onβ-catenin mRNA level of A549 and QG56 cells.However,exogenous expression of Dvl-1 increases,while exogenous expression of Dvl-3 does not change or even decreases,the protein levels ofβ-catenin of both cell lines.4.Dvl-1 may function through the Wnt/β-catenin signaling pathway,while Dvl-3 may function via non-canonical Wnt or other signaling cascades,on tumor progression in lung cancers.