Study On The Role Of WNT/?-catenin Signaling Pathway In The Development Of Different Cancers

BackgroundWNT signaling pathway plays a crucial role in human organogenesis and tumorigenesis.Wnts are a group of encode secreted cysteine-rich glycoproteins that function as signaling molecules to regulate embryonic development at different stages and participate in adult tissue homeostasis.Disregulation or aberration in Wnt signaling causes a wide variety of human diseases such as tetra-amelia,pulmonary fibrosis,schizophrenia,leukemia,,bone morbidity,kidney damage and different kinds of cancers.The WNT signaling pathway is classified into the canonical(WNT-?-catenin pathway)and the non-canonical pathways(WNT/planar cell polarity(PCP)or WNT/Ca2+ pathway),based on whether ?-catenin is involved.In the canonical pathway,WNT ligands bind to Frizzled receptors induce Dishevelled activation.P-catenin phosphorylation is inhibited by activated Dishevelled via glycogensynthase kinase-3b adenomatous polyposis coli-axin complex that leads to intracellular ?-catenin accumulation and nuclear translocation,subsequently?-catenin operates as a transcriptional coactivator that complexes with TCF/LEF transcription factors and activates the expression of downstream genes such as cyclin Dl,c-myc.The TCF/LEF family is a group of transcription factors that bind DNA by high mobility groups.Upon activation of the VNT signalling pathway its key player P-catenin translocates from the cytoplasm to the nucleus and binds to members of the T-cell factor(TCF)/lymphoid enhancer factor(LEF-1)family namely LEF-1 and TCF4 which are central mediators of transcription.Up to now,nearly 50%of human known tumors have been associated with abnormal regulation of WNT/?-catenin signaling pathway.Thyroid cancer is the most common endocrine cancer with most countries showing mortality rates of 0.2?0.4 per 100,000 men and 0.2?0.6 per 100,000 women.Most countries have displayed a rising incidence of thyroid cancer(mainly papillary carcinomas)over the past several decades,which has been attributed to improved diagnostic techniques over this time period.Papillary thyroid carcinoma(PTC)is the most common histologic type of differentiated thyroid cancer,accounting for about 70%-80%of thyroid cancer.In the year of 2014,approximately 63,000 new cases of thyroid cancer were diagnosed in the United States.From 1975 to 2009,the yearly incidence of thyroid cancer increased from 4.9 per 100,000 to 14.3 per 100,000.Therefore the incidence of papillary thyroid cancer increased from 3.4 to 12.5 per 100,000 in those years,made a big contribution to the growth in thyroid cancer rates.By 2019,one research predicts that papillary thyroid cancer will double in incidence in the United States.It will not only bring serious health effects,but also to the community and the country has brought a huge financial burden.Gastric cancer is a common malignancy,and accounts for about 10%of all invasive cancers worldwide.It may be the second leading cause of cancer death.Although the reported 5-year overall survival rates in advanced GC range from 15%to 52%,those in early gastric cancer(EGC)range from 88%to 97%.Early detection and treatment contribute to decreased mortality rates.In China,the total number of cases and deaths from gastric cancer have increased concomitant with extensive demographic changes and ongoing increase of environmental pollution.A positive correlation of gastric cancer with environmental pollution has been confirmed.Methods and ResultsThis paper aims to investigate the mechanism of WNT signaling pathway in tumors,including the following contents:1 Role of WNT10A/?-catenin Pathway in tumorigenesis of Papillary Thyroid Carcinoma.1.1 To investigate the molecular mechanisms involved in tumorigenesis of papillary thyroid cancer,microarray analysis was used to compare gene expressions in the papillary thyroid cancer and adjacent normal tissues.Methods:To investigate the molecular mechanisms involved in tumorigenesis of papillary thyroid cancer,microarray analysis was used to compare gene expressions in the papillary thyroid cancer and adjacent normal tissues.1.5 fold up-or down regulation were set as cutoff values,and changes in gene expression were analyzed using the Database for Annotation,Visualization and Integrated Discovery(DAVID).In the DAVID analysis,Thyroid cancer,six genes(CCND1,RXRG,LEF1,MYC,CTNNB1,TPM3)were up-regulated more than 1.5 fold in this pathway.As to signaling proteins,WNT10A was the only WNT ligands that were up-regulated more than 1.5 fold and its expression upregulation was found to be>4-fold in the papillary thyroid cancer comparing to adjacent normal tissues.The expression of WNT10A,LEFT,MYC,?-catenin and cycling D1 were verified by QRT-PCR and western blot in papillary thyroid cancer.Results:The results of QRT-PCR and western blot demonstrated that WNT10A/?-catenin signaling pathway(WNT10A,CCND1,RXRG,LEF1,MYC,CTNNB1,TPM3)was activated in papillary thyroid cancer tissues.1.2 Activation of WNT10A/?-catenin signaling pathway affects papillary thyroid cancer cell proliferation.Methods:We first examined the dose-dependent effects of WNT10A overexpression plasmid on papillary thyroid carcinoma by CCK8 assay.Next,cultured K1 cells were transfected with pEnter-WNT10A,pEnter-LEF1 and control plasmid pEnter-mock.siRNA-WNT10A and siRNA-LEF1 were employed to knockdown the expression of this two genes,and the proliferations of the treated cells were quantified with the Cell Counting Kit-8 assay.The expression of WNT10A,?-catenin,LEF1,MYC and cyclin D1 in the cell lines was confirmed by RT-PCR and western blot.Results:CCK8 assay showed the value of WNT10A/p-catenin signaling pathway on papillary thyroid carcinoma cells(K1 cells)increased with the increase of WNT10A plasmid concentration.CCK8 results confirmed that the WNT10A and LEF1 overexpressing cells exhibited significant higher level of proliferation as compared with the empty vector controls and siRNA-WNT10A.RT-PCR and western blot showed that overexpression of WNT10A leaded to an increase in?-catenin,up-regulated cyclin D1 and c-myc expression and it was the same with overexpression of LEF1.The opposite was expression of WNT10A,CCND1,LEF1,MYC,CTNNB1 protein was down-regulated in K1 cells.In contrast,siRNA-LEF1 and siRNA-WNT10A in K1 cells,the expression of WNT10A,CCND1,LEF1,MYC and CTNNB1 protein was significantly reduced.1.3 Activation of WNT10A/?-catenin signaling pathway has an effect on late apoptosis of papillary thyroid carcinoma cells.Methods:K1 cells were examined by flow cytometry to test whether cell cycle and cell apoptosis were modulated concomitant with WNT10A and LEF1 administration.Following transfection with the pEnter-WNT10A,pEnter-LEF1 plasmid,siRNA-WNT10A and siRNA-LEF1 respectively.Results:flow cytometry results indicated a lower percentage of late apoptotic cells after WNT10A and LEF1 over-expression.As expected,higher percentage of late apoptotic cells were detected after reduction of WNT10A and LEF1 with siRNA.Cell cycle results showed that the siRNA-WNT10A induced thyroid cancer cell lines K1 arrest in the S phase.1.4 Activation of WNT10A/?-catenin signaling pathway has an effect on migration of papillary thyroid carcinoma cells.Methods:the directional migration of K1 cells was examined with the wound-healing assay under the circumstance of K1 cells were transfected with pEnter-WNT10A,pEnter-LEF1 and control plasmid pEnter-mock,siRNA-WNT10A and siRNA-LEF1.Results:K1 cells grew into the wound as time passed,and the speeds of the WNT10A and LEF1 over-expressing cells migrating into the wound were significantly increased compared to the control cells In line with this result,knockdown of WNT10A and LEF1 expression with their siRNA reduced the migration ability of K1 cells respectively.2 PFDA plays a role in gastric cancer by acting the WNT/?-catenin signaling pathway.2.1 cDNA microarray analysis was employed in compared PFDA treated AGS cells with DMSO treated AGS cells.Methods:To investigated the molecular mechanism of PFDA-induced cell proliferation,cDNA microarrays analysis was was employed.In the DAVID analysis,the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway with the lowest p-value(p=0.011)was the vascular endothelial growth factor(VEGF)signaling pathway.In that pathway,the expression of sPLA2-?A(PLA2G2A)was 21.4%of that in controls,and it was the third most changed among all the genes analyzed.The upstream transcription factor of sPLA2-?A,TCF4,was the second most changed gene,with a decreased in expression to 19.9%of controls after PFDA treatment.Furthermore,the differences of TCF4 and sPLA2-?A between the experimental group and the control group were verified by qRT-PCR and western blot.The above indicates that the WNT/?-catenin signaling pathway plays an important r ole in the procession.Result:The decreased expression of TCF4 and sPLA2-IIA was verified by RT-qPCR and in western blots.The down-regulation of sPLA2-?A and TCF4 expression was also observed in Bel-7402 cells,sPLA2-?A and TCF4 mRNAs decayed to undetected levels after PFDA treatments.2.2 Effects of PFDA on gastric cell proliferation.Methods:To assess effects on in vitro cell proliferation,we treated AGS gastric epithelial cells with PFDA and monitored growth with a Cell Counting Kit-8(CCK8)and a colony forming assay.Result:CCK8 assays found that cells incubated with certain concentration of PFDA had significantly increased cell amount compared with DMSO-treated control cells.Moreover,the growth response of AGS cells varied in response to stimulation by different PFDA concentrations,this dose-dependent effect was verified by hepatic cell line Bel-7402.More evidence was obtained from colony forming assay,PFDA enhanced colony forming ability by more than 70%compared with control cells,which was significantly higher than other perfluorinated compound.2.3 PFDA enhanced gastric epithelial cells via suppressing senescence?Methods:To identify the cellular processes involved in PFDA-induced cell proliferation,we used flow cytometry,western blots,and SA-?-gal staining to determine whether cell apoptosis,autophagy,and senescence were modulated by PFDA treatment.Result:Flow cytometry showed no significant difference between in the percentages of apoptotic cells with DMSO-treated control cells and PFDA-treated cells.Western blot data showed no differences in degradation of autophagy substrates(p62)or lipidation of LC3(LC3-II)in response to PFDA treatment compared with controls cell.These results suggested that neither apoptosis nor autophagy were key factors in the PFDA-induced cell growth promotion.However,cell senescence-associated P-galactosidase(SA-?-gal)activity decreased following PFDA treatment,which was confirmed by a reduction in both the number of SA-?-gal-stained cells and in the staining intensity.Overall,these results implied that cell senescence played an important role in PFDA-induced promotion of AGS cell growth.2.4 Effects of TCF4 and sPLA2-?A on proliferation of gastric cancer cells in PFDA treated.Methods:To investigate the effect of TCF4 and sPLA2-?A gene expression on cell proliferation,CCK8,western blot and qQT-PCR were employed.Results:To investigate the effects of TCF4 and sPLA2-?A gene expression on cell proliferation,we transfected AGS gastric epithelial cells with TCF4 and sPLA2-?A expression plasmids(pENTER-TCF4 and pENTER-PLA2G2A)to stimulate TCF4 and sPLA2-?A expression concomitant with PFDA treatment.CCK8 assays found that AGS cells were evaluated for changes in cell growth following transfection with pENTER-TCF4 and pENTER-PLA2G2A,the growth rate of the transfected cells was reduced by 60%by pENTER-TCF4 and 30%.However,proliferation rates of cells transfected with pENTER-TCF4 and siRNA-sPLA2-?A were higher than in cells with only pENTER-TCF4 transfection.Western blot and qQT-PCR data showed that TCF4 and sPLA2-?A expression was restored and expression levels of each gene were increased in dose-dependent manner after transfection.However,transfection of pENTER-PLA2G2A did not affect TCF4 expression.Overall,these results indicated that TCF4 regulation of gastric cell growth by sPLA2-IIA is influenced by PFDA treatment.2.5 sPLA2-IIA expression restored cell senescence and inhibited cell proliferation.Methods:The effect of sPLA2-IIA expression on cell senescence of pENTER-TCF4 and pENTER-PLA2G2A transfected AGS cells was observed byResults:pENTER TCA4 and pENTER-PLA2G2A were transfected into AGS cells,the expression of PLA2G2A was restored,the number of senescent cells increased significantly and the intensity of SA-?-gal staining was increased.After adding siRNA-PLA2G2A,the number of senescent cells decreased and the intensity of SA-?-gal staining was decreased.PFDA induced suppression of cell senescence and then stimulated cell proliferation through regulation of sPLA2-IIA protein expression.Interestingly,PFDA significantly stimulated IL-1? and IL18 secretion and their mRNA levels compared with control cells.By RT-PCR and western-blot we found that up-regulation of NLRP3 were associated with promotion of IL-1? and IL-18 production.PFDA can not only promote AGS cell proliferation by regulation of sPLA2-?A,but also stimulate the aggregation of NLRP3 inflammatory cells in AGS cells.Conclusion:Part 1:Role of WNT/?-catenin Pathway in tumorigenesis of Papillary Thyroid Carcinoma.? WNT10A/?-catenin signaling pathway was activated in papillary thyroid cancer.? Molecules involved in WNT10A/?-catenin signaling pathway were up-regulated in papillary thyroid cancer tissues.? WNT10A/?-catenin signaling pathway activation promoted proliferation of thyroid cells.? WNT10A/?-catenin signaling pathway activation suppressed late apoptosis of thyroid cancer cells.? WNT10A/p-catenin signaling pathway activation enhanced migration of the thyroid cells.Part 2:PFDA plays a role in gastric cancer by acting the WNT/?-catenin signaling pathway.? The cDNA chip analysis showed that PLA2-?A and its transcription factor TCF4,are down-regulated in PFDA-treated AGS.? PFDA had an effect on the growth of AGS.?PFDA enhanced the proliferation of AGS via suppressing senescence.? TCF4 regulation of gastric cell growth by sPLA2-?A is influenced by PFDA treatment.? sPLA2-IIA expression restored cell senescence and inhibited cell proliferation.Significance1.WNT signaling pathway plays an important role in papillary thyroid carcinoma,WNT10A providing new targets for cancer clinical treatment.2.PFDA promotes the proliferation of AGS cells through sPLA2-IIA and TCF4 in the WNT/?-catenin signaling pathway,and stimulates the accumulation of NLRP3 inflammatory cells in AGS cells,suggesting that environmental pollution is associated with cancer and inflammation.