Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

Abstract

BackgroundAberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression.MethodsQuantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV.ResultsSignificant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival.ConclusionsIn the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

RASSF tumor suppressor gene family: Biological functions and regulation

“…RASSF1A promoter methylation correlated inversely with K-Ras and B-Raf mutation status in cervical adenocarcinomas [122] and thyroid cancers [123][124][125]. RASSF1A methylation was also shown recently to inversely correlate with TP53 mutations [120], suggesting that they likely have independent mechanisms in tumourigenesis. A positive correlation between the degree of RASSF1A methylation and higher tumor grade [51,120], the ability of deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes in endometrial cancer was reported [51].…”

“…RASSF1A methylation was also shown recently to inversely correlate with TP53 mutations [120], suggesting that they likely have independent mechanisms in tumourigenesis. A positive correlation between the degree of RASSF1A methylation and higher tumor grade [51,120], the ability of deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes in endometrial cancer was reported [51]. Interestingly RASS-F1A hypermethylation correlated with chromosome instability in Wilm's tumors [126] supporting a role for RASS1A in controlling genomic stability.…”

“…RASSF1A was the first to be identified and is considered to be one of the most methylated genes in human cancer. Not only is it epigenetically silenced in numerous cancers but it is considered as one of the earliest detectable changes in cancer [120]. Reports show that epigenetic silencing of RASSF1A occurs at a prevalence of 10-30% in cervical cancer, 60% in breast cancers to 80% in small cell lung cancer and up to 99% in prostate cancer (see Table 3 for the complete list and references).…”

DNA Methylation Status of Key Cell-Cycle Regulators Such as CDKNA2/p16 and CCNA1 Correlates with Treatment Response to Doxorubicin and 5-Fluorouracil in Locally Advanced Breast Tumors

“…CDKN2A promoter methylation has been detected in breast and many other human cancers (25). We previously showed that hypermethylation of CDKN2A is a late event during breast carcinogenesis, leading to its inactivation in late stage breast cancers (26). Expression of CCND2 is lost in the majority of breast cancers, and a mechanism underlying this loss is the promoter hypermethylation of cyclin D2 (27).…”

Promoter Methylation Status of the Retinoic Acid Receptor-Beta 2 Gene in Breast Cancer Patients: A Case Control Study and Systematic Review

“…Reduced expression of RARB2 by genetic or epigenetic mechanisms is linked to many human cancers such as cervical [9,10], head and neck [11], non-small cell lung [12], prostate [13], bladder, brain, and breast cancers [4,14,15,16]. Knowledge of the DNA methylation status in the promoter regions of some candidate genes such as RARB2 may be useful for early detection, prognosis, and drug response prediction in breast cancer patients [6,17].…”