Investigation of DNAmethylation of TWIST Gene in Breast Cancer and Its Relationship to Histopathological Features (original) (raw)
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Cancer Epidemiology, Biomarkers & Prevention, 2008
TWIST1, an antiapoptotic and prometastatic transcription factor, is overexpressed in many epithelial cancers including breast. Only little is known regarding the regulation of TWIST1 in these cancers. Recently, an increase in the TWIST1 promoter methylation has been shown in breast cancers. To correlate the percentage of TWIST1 promoter methylation to the protein levels, we analyzed simultaneously the methylation status as well as the mRNA and the percentage of cells expressing TWIST1 in normal breast tissue and 76 invasive breast cancers. We found that TWIST1 promoter methylation is significantly more prevalent in malignant compared with healthy breast tissue. Furthermore, the percentage of cells expressing TWIST1 was greater in breast malignancy compared with matched healthy tissue from the same patients. There was no correlation, however, between TWIST1 promoter methylation and TWIST1 protein or RNA expression. This indicates that although TWIST1 CpG methylation is useful as a bi...
Anticancer Research, 2010
Background: Promoter hypermethylation and global hypomethylation in the human genome are hallmarks of most cancers. Detection of aberrant methylation in white blood cells (WBC) has been suggested as a marker for cancer development, but has not been extensively investigated. This study was carried out to determine whether aberrant methylation in WBC DNA can be used as a surrogate biomarker for breast cancer risk. Patients and Methods: Promoter hypermethylation of 8 tumor suppressor genes (RASSF1A, APC, HIN1, BRCA1, CYCLIND2, RARβ, CDH1 and TWIST1) and DNA methylation for three repetitive elements (LINE1, Sat2 and Alu) were analyzed in invasive ductal carcinoma of the breast, paired adjacent normal tissue and WBC from 40 breast cancer patients by the MethyLight assay. Methylation in WBC from 40 controls was also analyzed. Results: Tumor and adjacent tissues showed frequent hypermethylation for all genes tested, while WBC DNA was rarely hypermethylated. For HIN1, RASSF1A, APC and TWIST1, there was agreement between hypermethylation in tumor and adjacent tissues (p=0.04, p=0.02, p=0.005 and p<0.0001, respectively). DNA methylation for the three repetitive elements was lower in tumor compared to adjacent tissue and WBC DNA. Significant correlations in the methylation of Sat2M1 between tumor and adjacent tissues and WBC DNA were found (p<0.0001 and p=0.046, respectively). There was also a significant difference in methylation of Sat2M1 between cases and controls (p=0.01). Conclusion: These results suggest that further studies of WBC methylation, including prospective studies, may provide biomarkers of breast cancer risk. Breast cancer is the most common cancer and second cause of cancer-death among females in most Western countries where there is an overall lifetime risk of >10% of developing breast cancer (1). Although diagnosis by screening mammography is believed to be responsible for the significant decline in breast cancer mortality, the limitations of mammography are well recognized, especially for women with premenopausal breast cancer (2). Thus, alternative approaches to breast cancer detection are clearly needed for improving diagnosis/prognosis. Epigenetic alterations, such as DNA methylation are widely accepted as a potential source of early biomarkers for better diagnosis/prognosis (3, 4). Many tumors exhibit excessive methylation of CpG islands that are found near the transcriptional promoters of mammalian genes (5). Indeed, hypermethylation of CpG islands in specific gene promoters is thought to contribute to carcinogenesis through transcriptional silencing of tumor suppressor gene expression, leading to the initiation and progression of cancer (6, 7). Aberrant methylation is frequently found in breast tumors with more than 40 genes shown to be inactivated by promoter CpG island hypermethylation (8). Among these genes, RASSF1A (9, 10), CDH1 (11), RARβ (12, 13), CYCLIND2 (14, 15), TWIST1 (16), BRCA1 (17, 18), HIN1 (19) and APC (20, 21) are frequently methylated. Different studies have confirmed the hypothesis that aberrant methylation at specific genes contributes to the malignant phenotype of breast cancer.
Integrative Cancer Science and Therapeutics
90% of total breast cancer cases [3]. The majority of breast cancers are sporadic caused by more complicated reasons such as the activation of mitogenic signaling pathways and loss of tumor suppressor expression epigenetically etc., [4]. DNA methylation which is one of the epigenetic mechanisms, which involves the addition of a methyl group to unmodified DNA, is described as an epigenetic change resulting from environmental influence, because it is a chemical modification to DNA not a change brought about by a DNA mutation [5]. Normal methylation of DNA would mark the DNA and enable such actions as turning genes on or off. Thus if epigenomic alteration persists in a tumour suppressor
Advanced biomedical research, 2012
In recent years, DNA methylation as a main epigenetic modification in human cancer is found as a promising biomarker in early detection of breast cancer. Possible applications of numerous hypermethylated genes have been reported in diagnosis of breast cancer but there has been a little comprehensive study on the clinical usefulness of these genes in breast cancer. The aim of the present study was to investigate the promoter methylation status of 14-3-3 sigma gene with the goal of developing a diagnostic application in breast cancer. Totally 40 cases of cancerous and noncancerous tissues were studied. DNA was extracted from tissue samples, and promoter methylation pattern was determined by using methylation-specific polymerase chain reaction. Methylation pattern of 14-3-3 sigma promoter significantly differed between control and malignant breast tissues (P = 0.001), and there was no remarkable correlation between methylation and age (P > 0.05). The relationship of promoter methyla...
Methylated genes in breast cancer
Cancer Biology & Therapy, 2011
Background: hundreds of hypermethylated genes have been described in breast cancer, yet the nature and contribution of these genes in their methylated state to overall risk and prognosis is under-characterized in non-sporadic breast cancers. We therefore compared associations of DNa methylation with tumor stage, hormone/growth receptor status and clinical outcomes in a familial breast cancer cohort. Because few previous methylation studies have considered the oncogenic or tumor suppressor properties of their gene sets, this functional status was included as part of our correlative analysis. Results: We found methylation of oncogenes was associated with better prognostic indicators, whereas tumor suppressor gene methylation was associated with a more severe phenotype in women that were either heR2 + or lymph node positive at diagnosis, and/or tended to recur or develop distant metastases. For example, the methylation of the tumor suppressor gene apC was strongly associated with a specific subset of tumors that were both eR + and heR2 + , while methylation of the TWIsT oncogene was associated with breast cancers that did not metastasize. Methods: This was a retrospective, hospital-based study of n = 99 archival breast tumors derived from women with a germline genetic BRCa1 or BRCa2 mutation and/or familial breast cancer history. DNa methylation was quantified from formalin fixed, paraffin embedded tumors using the established protocol of quantitative multiplex-methylation specific pCR (QM-Msp). Non-parametric statistics were used to analyze candidate gene methylation in association with clinical outcomes. Conclusion: We report several novel, positive associations between percent methylation of the apC, RassF1a, TWIsT, eRα, CDh1 and Cyclin D2 genes and key variables such as tumor stage, hormone and growth receptor status, and a history of recurrent or metastatic disease. Our data suggest the potential utility of parsing gene methylation by functional status and breast tumor subtype.
Intratumor heterogeneity index of breast carcinomas based on DNA methylation profiles
BMC Cancer, 2019
Background: Cancer cells evolve and constitute heterogeneous populations that fluctuate in space and time and are subjected to selection generating intratumor heterogeneity. This phenomenon is determined by the acquisition of genetic/epigenetic alterations and their selection over time which has clinical implications on drug resistance. Methods: DNA extracted from different tumor cell populations (breast carcinomas, cancer cell lines and cellular clones) were analyzed by MS-MLPA. Methylation profiles were used to generate a heterogeneity index to quantify the magnitude of epigenetic heterogeneity in these populations. Cellular clones were obtained from single cells derived of MDA-MB 231 cancer cell lines applying serial limiting dilution method and morphology was analyzed by optical microscopy and flow cytometry. Clones characteristics were examined through cellular proliferation, migration capacity and apoptosis. Heterogeneity index was also calculated from beta values derived from methylation profiles of TCGA tumors. Results: The study of methylation profiles of 23 fresh breast carcinomas revealed heterogeneous allele populations in these tumor pieces. With the purpose to measure the magnitude of epigenetic heterogeneity, we developed an heterogeneity index based on methylation information and observed that all tumors present their own heterogeneity level. Applying the index calculation in pure cancer cell populations such as cancer cell lines (MDA-MB 231, MCF-7, T47D, HeLa and K-562), we also observed epigenetic heterogeneity. In addition, we detected that clones obtained from the MDA-MB 231 cancer cell line generated their own new heterogeneity over time. Using TCGA tumors, we determined that the heterogeneity index correlated with prognostic and predictive factors like tumor size (p = 0.0088), number of affected axillary nodes (p = 0.007), estrogen receptor expression (p < 0.0001) and HER2 positivity (p = 0.0007). When we analyzed molecular subtypes we found that they presented different heterogeneity levels. Interestingly, we also observed that all mentioned tumor cell populations shared a similar Heterogeneity index (HI) mean. Conclusions: Our results show that each tumor presents a unique epigenetic heterogeneity level, which is associated with prognostic and predictive factors. We also observe that breast tumor subtypes differ in terms of epigenetic heterogeneity, which could serve as a new contribution to understand the different prognosis of these groups.
Assessment of DNA methylation status in early stages of breast cancer development
Breast Diseases: A Year Book Quarterly, 2013
Background: Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development. Methods: To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARb2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n ¼ 52), ductal hyperplasia (n ¼ 23), atypical ductal hyperplasia (n ¼ 31), ductal carcinoma in situ (DCIS, n ¼ 95) and AJCC stage I invasive ductal carcinoma (IDC, n ¼ 34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed. Results: Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, o0.0001 and o0.0001 for MINT17, MINT31, RARb2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics. Conclusion: DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.
Association of aberrant DNA methylation with clinicopathological features in breast cancer
Oncology Reports, 2011
Aberrant DNA methylation is responsible for the epigenetic silencing of genes associated with tumourigenesis and progression of cancer. In this study, we assessed the methylation status of eight genes in 49 snap-frozen primary breast tumours. Epigenetic alterations of 8 genes were analysed with methylation-specific polymerase chain reaction (MS-PCR) (DCR1, DAPK1, RASSF1A and DCR2) or methylationsensitive high-resolution melting analysis (MS-HRM) (APC, MGMT, GSTP1 and PTEN). MS-HRM performance was validated by bisulfite pyrosequencing regarding the methylation levels of MGMT. Promoter methylation was observed in APC 54.34%, 40.4% DCR1, 37.5% DAPK1, 33.3% RASSF1A, 22.44% MGMT, 16.6% GSTP1, 6% PTEN and 0% DCR2 promoters, respectively. Interestingly, 37 out of 49 cases (75.5%) displayed aberrant promoter methylation in at least one gene. An association of MGMT promoter methylation with age and tumour grade was recorded. Moreover, a correlation with advanced T-category was elicited for GSTP1, RASSF1 and DAPK1 promoter methylation. Finally, concurrent methylation of several genes showed a marginal statistical relationship with N-category. We conclude that APC, DCR1, DAPK1 and RASSF1A promoter methylation represents a common event in breast cancer tumourigenesis. Our results suggest that GSTP1, RASSF1, DAPK1 and MGMT may be implicated in the acquisition of a more aggressive phenotype in breast cancer.
Asian Pacific Journal of Cancer Prevention, 2015
Background: Fragile histidine triad (FHIT) gene is a tumor suppressor gene which involved in breast cancer pathogenesis. Epigenetics alterations in FHIT contributes to tumorigenesis of breast cancer. Objective: Our objective was to study FHIT promoter region hypermethylation in Egyptian breast cancer patients and its association with clinicopathological features. Materials and Methods: Methylation-specific polymerase chain reaction was performed to study the hypermethylation of FHIT promoter region in 20 benign breast tissues and 30 breast cancer tissues. Results: The frequency of hypermethylation of FHIT promoter region was significantly increased in breast cancer patients compared to bengin breast disease patients. The Odd´s ratio (95%CI) of development of breast cancer in individuals with FHIT promoter hypermethylation (MM) was 11.0 (1.22-250.8). There were also significant associations between FHIT promoter hypermethylation and estrogen, progesterone receptors negativity, tumor stage and nodal involvment in breast cancer pateints. Conclusions: Our results support an association between FHIT promotor hypermethylation and development of breast cancer in Egyptian breast cancer patients. FHIT promoter hypermethylation is associated with some poor prognostic features of breast cancer.