Wnt/β-catenin Signal Pathway Regulates Mesothelin In Epithelial Ovarian Cancer

Epithelial ovarian cancer（EOC） arises from the ovarian surface epithelium（OSE） and account for greater than 90% of all ovarian cancers. Among the gynecological cancers, ovarian cancer is less common than cancer of the cervix and the uterus, but it has the highest fatality-to-case ratio, accounting for almost half of the deaths from cancers of the female genital tract. Despite advances in screening and treatment, the 5-year survival rate is less than 30%. Compared with the progress in surgery and chemotherapy, the progress in understanding the pathogenesis of EOC grows very slowly. We have demonstrated that mesothelin（MSLN） gene is one of significantly up-regulated genes in EOC.MSLN gene encodes a precursor protein that is cleaved into two products, megakaryocyte potentiating factor（33Kda） and mesothelin（40Kda）. Megakaryocyte potentiation factor functions as a cytokine that can stimulate colony formation in bone marrow megakaryocytes. Mesothelian is a glycosylphosphatidylinositol-anchored cell-surface protein that may function as a cell adhesion protein. Mesothelin is a newly identified tumor-cell surface antigen, it is present on normal mesothelin cells lining the body cavities and is highly expressed in several cancers, including mesotheliomas, ovarian cancer and pancreatic cancers. Approximately 70% of ovarian cancers, including up to 100% of serous papillary ovarian cancers, express mesothelin.The regulatory mechanism of mesothelin expression in EOCs is not known. Using the mouse mammary epithelial cell line C57 mg, Prieve and Moon showed that mesothelin was up-regulated by canonical Wnt /β-catenin pathway. Wnt signaling regulates a variety of developmental processes including cell growth, cell differentiation, cell polarity, and apoptosis. Mis-regulation of Wnt signaling leads to the development of several human cancers. Canonical Wnt signaling initiates in the binding of a Wnt ligand（Wnt-1,-3A, and-8） to its cell surface receptor, members of the frizzled gene family, along with the co-receptor LRP5/6, leading to changes in the activities of cytoplasmic effectors resulting in the stabilization of β-catenin protein. Î’-catenin then accumulates in the nucleus where it interacts with members of the Lymphoid Enhancer Factor/T Cell Factor（LEF/TCF） family to regulate downstream gene expression, e.g. c-myc、cyclin D1、MMPs and VEGF. The Wnt pathway controls biological processes via the regulation of these direc target genes expression. Indirect target genes are regulated via transcription regulators, which are targets of the Wnt pathway. Evidence in support of activated Wnt signalling in ovarian tumours has been reported, but whether Wnt signalling regulates MSLN gene in EOC is not clear.In this study, The expression of Wnt-1, β-catenin and mesothelin m RNA in EOC and in normal ovarian tissues were evaluated by real time PCR, protein expression were evaluated by immunohistochemical stainingand the Correlation analysis between the expression of Wnt signalling and mesothelin was done to explore the association of canonical Wnt signaling and regulation of mesothelin.Partâ…  The expression and correlation of Wnt signalling and mesothelin in epithelial ovarian cancersObjective: To investigate the expression and correlation of Wnt signalling and mesothelin in epithelial ovarian cancer.Methods: Real-time PCR was conducted to identify Wnt-1, β-catenin and mesothelin in 31 pithelial ovarian cancers（31samples）, benign ovarian epithelial tumour（20 samples） and normal ovarian epithelium tissue（10 samples）. Immunohistochemical staining with Eli Vison method was conducted to identify the expression rates of mesothelin, Wnt-1 and β-catenin in epithelial ovarian cancer（57 samples）, benign ovarian epithelial tumour（20 samples） and normal ovarian epithelium tissue（16 samples）. The different expression of Wnt-1, β-catenin and mesothelin was contrasted within different histologic types, surgery- pathologic stages. And correlation between mesothelin and aberrant Wnt signalling was analyzed.Results: 1 The expression of Wnt-1 m RNA in EOCs. Wnt-1 m RNA expressed in 22.6%（7/31） of EOCs, the ralative expression evel was 0.64±0.06. 15%（3/20） of benign ovarian epithelial tumours expressed Wnt-1 m RNA and the ralative expression level was 0.47±0.04. There was no m RNA expression in normal ovarian epithelium tissues. There was no difference of Wnt-1 m RNA expression between EOCs and benign ovarian epithelial tumours. Because of few positive expression of Wnt-1 in EOCs, we did not analyse the difference of Wnt-1 m RNA expression within different histologic types and pathologic stages.2 The expression of β-catenin m RNA in EOCs. Î’-catenin mRNA expressed in all samples. The ralative expression evel in EOCs was 0.77±0.46, 0.70±0.46 in enign ovarian epithelial tumours and0.71±0.45 in normal ovarian epithelium tissues. There was no difference of β-catenin expression within different histologic types. The expression of β-catenin m RNA in III^IV stage EOCs was higher than that in I^IIstage（t=-0.189, P<0.05）.3 The expression of mesothelin m RNA in EOCs. Mesothelin m RNA expressed in 93.5%（29/31） of EOCs and 0% in benign ovarian epithelial tumours and normal ovarian epithelium tissues. There was no difference of mesothelin expression within different histologic types（F=2.942, P=>0.05） and pathologic stages（t=-0.788, P>0.05）.There was no correlation between Wnt signalling and mesothelin expression in m RNA level.4 The expression of Wnt-1 protein in EOCs. Wnt-1 was expressed in the cellular membranes and was found in 43.9%（25/57） EOCs and 20%（4/20） in benign ovarian epithelial tumours investigated（x2=3.590, P>0.05）. Whreas, only 6.2%（1/16） normal ovarian tissues showed Wnt-1 staining（x2=10.637, P<0.05）. There was no difference of Wnt-1 protein expression within different histologic types and pathologic stages.5 Î’-catenin staining in EOCs. Cytoplasmic and/or nuclear β-catenin immunoreactivity to was seen in 75.4%（43/57） EOCs and 5%（1/20） in benign ovarian epithelial tumours investigated（x2=29.995, P<0.01）and most of them showed a cytoplasmic localization. All normal ovarian tissues showed cellular membranes β-catenin staining.Cytoplasmic and/or nuclearβ-catenin staining was seen in 88.9% of endometrioid adenocarcinomas and almost higher than that of 50% of mucinous adenocarcinomas（P=0.051）. Cytoplasmic and/or nuclearβ-catenin immunoreactivity in in III^IV stage EOCs was 88.2% and higher than 56.5% in I^IIstage.6 The mesothelin staining in EOCs. Mesothelin was expressed in the cellular membranes and cytoplasm. Positive mesothelin expression was found in 80.7%（46/57） EOCs, and 100% negative in benign ovarian epithelial tumours and normal ovarian tissues. There was no difference of mesothelin protein expression within different histologic types. The mesothelin staining was seen in 91.2% of III^IV stage EOCs and was higher than that in 65.2% of I^IIstage.7 The correlation between Wnt signalling and mesothelin expression ing protein level. Correlation analysis showed a significant, positive association between cytoplasmic and/or nuclear β-catenin and mesothelin expression（r=0.405, P<0.05）. No association was found between wnt-1 and mesothelin expression（P>0.05）.Conclusions: There are aberrant expressions of Wnt signalling and mesothelin in EOCs. The expression of mesothelin and aberrant canonical wnt signal pathway may be responsible for higher surgery- pathologic stage. The expression of mesothelin may be caused by aberrant canonical Wnt signal pathway in EOCs.Part II Wnt/β-Catenin Signal Pathway Regulates Mesothelin In SKOV-3 cellsObjective: By respectively activation and inhibition Wnt signalling in SKOV-3 cell in vitro, the expression of mesothelin was evaluated to examine if the Wnt cascade is involved in mesothelin expressionMethods: 1 The basic expression of mesothelin in SKOV-3 cells was evaluated. 2 SKOV-3 cells were stimulated with Li Cl or with recombinant Wnt3 a protein. The expression of β-catenin was detected by Real-time PCR and Western blot analysis to evaluate our cell models with activated Wnt pathway.3 Lentiviral vectors were constructed to allow an efficient expression of anti-β-catenin si RNA. The cell line SKOV-3 was transfected by lentivirus, and β-catenin gene silencing effect was identified using q RT-PCR and western blot method, lentiviral vector containing invalid RNAi sequence As control.4 Mesothelin mRNA and protein expression was was evaluated by real-time PCR and western blot after respectively activation and inhibition Wnt signalling in SKOV-3 cell.Results: 1 β-catenin m RNA relative expressio was detected by real-time PCR in SKOV-3 cell model with activated Wnt pathway. The expression level of β-catenin was 2.10±0.23 in cells treated with Li Cl, and 1.75±0.19 in cell treated with Wnt-3a, which were higher than that of 0.97±0.33 in cells treated with KCl and 0.97±0.33 in cell treated with DMEM（F=22.635,P<0.01）. It confirmed that Li Cl and Wnt3 a could activate Wnt/β-catenin signal pathway.2 RNAi lentiviral vectors againstβ-catenin were succesfully constructed. After SKOV-3 cells were transfected with RNAi lentiviral vectors, the relative β-catenin m RNA expression level decreased by 77% and protein expression decreased by 75%.3 In the cell models with activated Wnt pathway, the relative expression of β-catenin m RNA was 0.57±0.07 in cells treatedwith Li Cl and 0.50±0.02 in cells treatedwith Wnt-3a, which was higher than that of 0.25±0.04 in cells treated with KCl and 0.24 ±0.07 in cells treated with DMEM（F=27.576,P<0.01）. Thenβ-catenin expression was detected, the OD value of β-catenin was 1.279 in cells treatedwith Li Cl and 1.146 in cells treated with Wnt-3a, which were approximately 2 fold higher than that of 0.513 in cells treated with KCl and 0.550 in cells treated with DMEM.4. By inhibiting Wnt pathway in SKOV-3 cells via β-catenin RNAi, the relative expression of β-catenin m RNA was 0.15±0.03 in cells transfected withβ-catenin-RNAi-LV, which was not lower than that of 0.25±0.07 in cells transfected with NC-RNAi-LV. Then β-catenin expression was detected, the OD value of β-catenin was 0.415 transfected withβ-catenin-RNAi-LV and 0.550 n cells transfected with NC-RNAi-LV. There was no differece of mesothelin expression between two groups.Conclusion: Wnt/β-catenin signal pathway can regulate the expressionof mesothelin gene in SKOV-3 cell. The mesothelin expression was up regulated by actevating Wnt signalling, but by inhibiting Wnt signalling the expressionof mesothelin was not decreased.Part â…¢ The cilico analysis of MSLN gene 5’ untranslated region and identification of related regulatory elementsObjective: to analyze MSLN gene 5’ UTR in cilico by using bioinformatics databases, predict and identificate MSLN gene promoter region and Wnt signal pathway related transcription factor binding sites and demonstrate the way of Wnt singalling regulate mesothelin expression.Methods: 1 Updated MSLN gene information was obtained from Gene Bank and UCSC Genome Bioinformatics. 4Kb region upstream translation start site was obtained, MSLN gene promoter region and Wnt signal pathway related transcription factor binding sites were predicted by using bioinformatics databases.2 Based on the locations of MSLN gene promoter and TCF/LEF transcription factor binding sites, various promoter constructs were constructed and were cloned upstream of the luciferase gene, then were transfected into ovarian cancer cell lines, SKOV-3 and 3Ao. Luciferase activity was determined 24 hour later using. As controls, transfections were performed using the SV40 promoter（p GL3 promoter） or a promoterless construct（p GL3 Basic）. Luciferase activity was determined using reporter assays.3 Electrophoretic mobility shift assay（EMSA） was done to identificate Wnt signal pathway related transcription factor binding sitesResults: 1 At present, the knowledge abou MSLN gene demonstrate that it have three variants: variant 1（Gen Bank accession NM₀05823） encoding MPF; variant 2（NM₀13404） encoding mesothelin; and variant 3（AF180951）, was a partial alternatively spliced c DNA with a disrupted GPI-anchor motif. The updated informations about MSLN gene believe that variant 1 represents the predominant transcript and encodes isoform 1. Variant 3 differs in the 5’ UTR, compared to variant 1. Both variants 1 and 3 encode the same isoform isoform 1. Variant 2 uses an alternate splice site in the coding region, compared to variant 1. Variant 2 encodes isoform 2 which is longer than isoform 1.2 The translation start site of MSLN gene was marked as +1. 4Kb region upstream mesothelin translation start site was obtained to predict and analyze related bioinformations. Blast N demonstrate that available EST sequences to 4Kb region are located in-1900^-1850 and-1500^-1430. Blast X showed that corresponding sequences encoding protein are located in-2700^-4000.Cp G island searcher predicted two postulated Cp G islands which are locate in-2830^-2590 and-1706^-1497 region. Promoter searching software predicted a postulated promoter located in-2168^-1599 region,-1668 as transcription start site, and a postulated core promoter located in-2875-2825 region,-2835 as transcription start site. Compared with mouse MSLN gene, the evolutionary conserved regions of human MSLN gene are located in-2360^-2060,-1910^-1760 and-1150^-1. Within the 4Kb region, the evolutionary conserved transcription factor binding sites are lcated in-2005^-1994.Comprehensive analysis of the predictive bioinformation, postulated transcription start site-1668 is located near the-1706^-1497 Cp G island,-1910^-1760 evolutionary conserved region and the first exon of MSLN Ref Seq NM₀13404.4. it may be the transcription start site of mesothelin transcript variant 2. postulated transcription start site-2835 is located near the-2830^-2590 Cp G island, but not located near the evolutionary conserved region and there is no no EST sequence matches for it. So there is unlikely promoter sequence within upstream region of-2835. evolutionary conserved transcription factor binding sites are lcated in-2005^-1994, which is located in the promoter-2168^-1599 region and near by the first exons of MSLN Ref Seqs NM₀05823.5 and NM₀01177355.1. It is likely that three mesothelin transcript variants are regulated by the same promoter region.3 Transcription factor binding site searching software predicted two postulated TCF/LEF binding sites within the 2Kb（-1608^-3608） region upstream MSLN transcript variant 2, a LEF-1 binding site is located in-3036^-3029 and a TCF-4 binding site is located in-1935^-1926.4 Based on the locations of MSLN gene promoter and TCF/LEF transcription factor binding sites, three sequences, 1764bp（-3371 ^-1607）,472bp（-2079^-1607） and 261bp（-1868^-1607） were amplified from the 5’ end of the MSLN gene and were was subcloned into the p GL3-luciferase vector. Each construct was transfected into SKOV-3 and 3AO cell. Reporter activity of p GL3-1764 in SKOV-3 cells was 76.3967±6.9207 and that of p GL3-472 was 62.1667±5.003, reporter activityof p GL3-261 was 7.900±0.9440. In 3AO cells, reporter activities of sequences were 64.4833±4.2174, 48.3800±7.7385,and 7.5433±1.4883. Both in SKOV-3 and 3AO cells, Reporter activity of 472 bp constructs did not differ from those of 1764 bp, but was significantly higher than that of 261 bp. A 211bp（-2079^-1868） region contains the core promoter of MSLN gene.5 EMSA showed that labeled TCF-4 probe bound to nuclear extracts of SKOV-3 cell and exhibit a retarded band, while mutated probe could not bind to nuclear extracts. Unlabeled competitor probe inhibited the binding of labeled probe to nuclear extracts, but not unlabeled mutated probe. Labeled probe. Aretarded band was also present when labeled probe was incubated with recombinant TCF-4 protein.Conclusion:1 Updated MSLN Ref Segs have corrected the sequences of transcript variant 2 and 3. Both variants 1 and 3 encode the same isoform isoform 1. Variant 2 uses an alternate splice site in the coding region, compared to variant 1. Variant 2 encodes isoform 2 2 in cilico analysis of 5’ end of MSLN gene shows that the promoter of MSLN may located in 2835^-1668 region（translation start site was marked as +1）. There are two TCF/LEF binding site within 2Kb region of 5’ end of MSLN gene: a LEF binding site located in-3036^-3029 and a TCF-4 binding site located in-1935^-1926.3 Based on the locations of promoter and TCF/LEF transcription factor binding sites, there fragments were constructed and transfected into ovarian cancer cells. In transient transfection assays, we identified a 211 bp region that activated transcription from a minimal promoter, which cotain a TCF-4 binding site.4 EMSA showed that the TCF-4 in the nuclear extracts of SKOV-3 cell binds to the TCF-4 consensus Sequence of the MSLN promoter.5 MSLN gene is a direct target of Wnt/β-catenin signalpathway.