ABSTRACT
We examined the methylation status of the Ep-CAM promoter region of human breast cancer cell lines and breast cancer tissue using MethyLight technology and bisulfite sequencing. We found the promoter of Ep-CAM-negative breast cancer cell lines Hs 578T to be methylated to a higher degree as compared to positive cell lines MCF-7. Demethylation of cell lines was performed using 5-aza-2'-deoxycytidine. Ep-CAM RNA and protein expression could be partially restored by treating cells with 5-Aza-2'-deoxycytidine. In most primary breast cancer tissue, methylation of the Ep-CAM gene could be detected at a low level and no correlation was found with Ep-CAM protein expression in tumour tissue. Taken together, these data suggest that methylation of the Ep-CAM promoter is not a crucial mechanism for regulation of Ep-CAM expression in breast cancer. Thus, most important regulatory mechanisms have to be supposed in vivo.
Subject(s)
Antigens, Neoplasm/genetics , Breast Neoplasms/genetics , Cell Adhesion Molecules/genetics , DNA Methylation , Promoter Regions, Genetic/genetics , Antigens, Neoplasm/metabolism , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Breast/metabolism , Breast Neoplasms/pathology , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Colon/metabolism , CpG Islands , Decitabine , Epithelial Cell Adhesion Molecule , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intestinal Mucosa/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The HER-2/neu gene is amplified and overexpressed in 20% to 30% of invasive breast carcinomas and is associated with increased metastatic potential and less tamoxifen sensitivity. We generated the DNA methylation profiles of 143 human breast tumors and found significant differences in HER-2/neu expression and DNA methylation of five genes. For three of these five genes [PGR (coding for the progesterone receptor), HSD17B4 (coding for type 4 17-beta-hydroxysteroid dehydrogenase, an enzyme that mainly degrades active 17-beta-estradiol into inactive metabolites), and CDH13 (coding for H-cadherin)] a higher prevalence of DNA methylation in HER-2/neu-positive cancers was confirmed in an independent set of microdissected primary breast cancers. DNA methylation was not only present in cancer cells but also in the tumor stroma fraction. Of the isolated fractions in HER-2/neu-positive versus -negative cancers, 27.1% versus 10.5%, respectively, showed DNA methylation of the five genes (P = 0.011, Fisher's exact test). In Her-2++/+++ breast cancers, HSD17B4 mRNA expression was inversely associated with HSD17B4 methylation (P = 0.04). These data support the view that in addition to HER-2/neu-associated signaling, epigenetic changes in cancer as well as in tumor stroma cells might attribute to the specific biological features of HER-2/neu-positive cancers.
Subject(s)
Breast Neoplasms/genetics , Receptor, ErbB-2/genetics , 17-Hydroxysteroid Dehydrogenases/genetics , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cadherins/genetics , DNA Methylation , Drug Resistance, Neoplasm , Female , Humans , Immunohistochemistry , Receptor, ErbB-2/biosynthesis , Receptors, Progesterone/genetics , Tamoxifen/pharmacologyABSTRACT
Adjuvant systemic therapy (a strategy that targets potential disseminated tumor cells after complete removal of the tumor) has clearly improved survival of patients with cancer. To date, no tool is available to monitor efficacy of these therapies, unless distant metastases arise, a situation that unavoidably leads to death. We analyzed RASSF1A DNA methylation in pretherapeutic sera and serum samples collected 1 year after surgery from 148 patients with breast cancer who were receiving adjuvant tamoxifen; 19.6% and 22.3% of patients with breast cancer showed RASSF1A DNA methylation in their pretherapeutic and 1-year-after serum samples, respectively. RASSF1A methylation 1 year after primary surgery (and during adjuvant tamoxifen therapy) was an independent predictor of poor outcome, with a relative risk (95% confidence interval) for relapse of 5.1 (1.3-19.8) and for death of 6.9 (1.9-25.9). Measurement of serum DNA methylation allows adjuvant systemic treatment to be monitored for efficacy: disappearance of RASSF1A DNA methylation in serum throughout treatment with tamoxifen indicates a response, whereas persistence or new appearance means resistance to adjuvant tamoxifen treatment. It remains to be seen whether modifications made in adjuvant therapeutic strategies based on detection of circulating nucleic acids will improve survival as well as quality of life.
