Clinical Studies Of Abnormal Breast Cancer Gene Methylation

Part 1:The Canonical Wnt Antagonist Sox17 Is Epigenetically Inactivated by Promoter Methylation in Human Breast CancerObjective:Sox17 is a transcription factor involved in many developmental processes and can act as an antagonist of canonical Wnt/β-catenin signaling pathway. This study was aimed to investigate the relationship between Sox17 gene expression and its methylation status,and its potential role in Wnt/β-catenin signaling in breast cancer.Methods:The expression levels of Sox17 andβ-catenin were examined by RT-PCR and real-time PCR in seven breast cancer cell lines treated with or without demethylation agent 5-aza-dC and 31 paired breast tissue samples.Methylation status was detected by bisulfite sequencing and methylation-specific PCR.Correlation of Sox17 methylation status with clinicopathologic characteristics was evaluated in 113 breast cancers.Results:The expression level of Sox17 mRNA was dramatically decreased in five different breast cancer cell lines and 23 of 31 primary breast tumor samples, which significantly correlated with its methylation status.Aider treated with 5-aza-2'-deoxycytidine(5-aza-dC,a demethylation agent),the expression levels of Sox17 mRNA and protein were obviously increased.Restored expression of Sox17 by 5-aza-dC treatment decreased the expression level ofβ-catenin in breast cancer cell lines.Furthermore,small interfering RNA(siRNA)-mediated knockdown of Sox17 in SK-BR-3 and Bacp-37 cells enhancedβ-catenin expression.In 31 paired tissue samples,a significant difference between the expression level of Sox17 andβ-catenin was also observed(P＜0.001).Clinically,Sox17 methylation was detected in 74.3% breast tumors(84/113) and 31.9%(36/113) paired normal tissues respectively (P＜0.0001).Sox17 methylation was also associated with tumor stage(P=0.028) and lymph node metastasis(P=0.013).Conclusion:These findings indicate that silencing of Sox17 due to promoter hypermethylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer.Sox17 may be a valuable biomarker for the study of breast cancer carcinogenesis and progression. Part 2:Frequent Epigenetic Inactivation of Receptor Tyrosine Kinase EphA5 by Promoter Methylation in Human Breast CancerObjective:In an effort to identify new genes that are functionally affected by altered methylation status in breast cancer,a microarray based methylation analysis showed that EphA5 gene is higher hypermethylated in tumor samples.The aim of the present study was to investigate EphA5 gene expression profiles,methylation status, and clinical significances in breast cancer.Methods:Gene expression level of EphA5 was detected by RT-PCR and realtime RT-PCR with or without 5-aza-2'-deoxycytidine treated in breast cancer cell lines. Methylation status was tested by Bisulfite genomic sequencing and MSP.Further, correlation between expression level and methylation status of EphA5 gene were assessed in 31 paired tissue samples.Finally,the methylation status of EphA5 gene was examined in total of 117 paired tissue samples by MSP assay and the correlation with patients' clinicopathologic features were also evaluated.Results:The expression level of EphA5 mRNA was dramatically decreased in five different breast cancer cell lines.Aider treated with 5-aza-2'-deoxycytidine (5-aza-dC,a demethylation agent),the expression levels of EphA5 mRNA and protein were obviously increased.Bisulfate sequencing and methylation-specific PCR detection showed that decreased expression of EphA5 was associated with its methylation status.We also found a significant correlation(P=0.017) between reduction of EphA5 mRNA abundance and aberrant methylation of EphA5 in 31 paired tissue samples.In clinical samples,EphA5 methylation was detected in 64.1% breast tumors(75/117) and 28.2%(33/117) paired normal tissues respectively (P＜0.001),which was associated with higher tumor grade(P=0.024),lymph node metastasis(P=0.004),and PR negative status(P=0.008).Conclusion:Our findings indicate that EphA5 is likely a potential target for epigenetic silencing in primary breast cancer.EphA5 may be a valuable molecular marker of breast cancer carcinogenesis and progression. Part 3:Multiple Gene Methylation in Chinese women with Sporadic Breast CancerObjective:Multiple gene methylation is common in breast cancer and may be a useful biomarker for breast cancer detecting.However,the correlation between multiple genes methylation status and clinicopathological parameters,and diagnostic value in Chinese breast cancer women are unknown.Material and Methods:Using mutiplex nested methylation specific PCR,we analyzed methylation status of eight critical genes:RASFF1A,APC,MGMT,GSTP1, p16,ESR1,RAR-β₂ and BRCA1,in 106 paired breast tumors and corresponding normal tissues,and 96 plasma DNA samples including 38 benign breast diseases and 58 breast cancer patients.Results:The frequency of methylation of these genes varied from 18.9%for MGMT to 77.4%for RASSF1A.Methylation in at least one of the genes was found in 94.3%of the breast cancer.And the mean number of genes hypermethylated in each tumor and paired normal breast tissues was 3.53 and 1.32 respectively.The strongest and most consistent association observed of specific gene methylation was with higher tumor grade,there are four genes APC,MGMT,RAR-β2 and p16 reached statistical significance.High frequency simultaneous methylation of multiple genes was more often in these with higher tumor grade,lymph node metastasis,or ER negative patients.In plasma samples,86.2%(50/58) tumors DNA showed hypermethylation of at least one of the eight genes.Logistic regression models analysis showed that the odds ratio for diagnosis of breast cancer was 4.3(95%CI 1.4～13.4,P=0.01) in patients with 1～3 methylated gene and 20.1 in patients with more than 3 genes methylation(95%CI was 5.9-69.2,P＜0.0001).Conclusions:Hypermethylation of these genes may be linked to various known clinicopathological features of breast cancer in Chinese women,and increasing multiple gene methylation in tumors may indicate a aggressive phenotype for breast cancer.Detection of hypermethylation in plasma DNA using a combination of these epigenetic markers may be useful for identifying patients at high risk for breast cancer.