CpG Island-Containing BRCA1 Distal Promoter Is Associated with Breast Stem Cells and Resistance to Doxorubicin
Rulong Shen, Wenchao Zhou, Zhengang Peng, Mingxiang Teng, Yunlong Liu, Amanda Toland, Kun-Yu Teng, Jennifer R Chao, Suling Liu, Max Wicha, Huey-Jen L Lin. The Ohio State University, Columbus, OH; Indiana University, Indianapolis, IN; University of Arizona, Phoenix, AZ
Background: Multiple lines of evidence have implicated the existence of somatic normal and cancer stem cells but little is known about the underlying mechanisms.
Design: Cancer stem cells were obtained from SUM159 cells by capturing subpopulation displaying elevated ALDH1 using ALDEFLUOR assay. Afterwards, MBD-seq (Methyl-binding domain sequencing) method was implemented to seek stem cell-associated key regulators controlled by DNA methylation in a genome-wide scale, among which BRCA1 ranked of the greatest interest. Thus combined bisulfite restriction analysis (COBRA) and quantitative DNA methylation MassARRAY were applied to confirm methylation levels of BRCA1 in various cell contexts. Finally, in vitro DNA methylation followed by luciferase assay was used to assess promoter activity of regions flanking BRCA1 promoter and its response to DNA methylation.
Results: We demonstrated that an unreported distal promoter region of BRCA1 was hypermethylated in ALDH1+ SUM159 cells, which was confirmed in other breast cancer cell lines, nonmalignant breast tissues and a xenografted breast tumor. Moreover, this region was validated to bear promoter activity comparable to the previously reported BRCA1 proximal promoter, which was sensitive to DNA methylation. While the latter was reported to be infrequently methylated in breast tumors, its methylation was undetectable in this study. Conversely, methylation at this novel distal promoter occurred in all 16 primary tumors we tested with 6 of which displayed a positive relationship between levels of ALDH1 and methylation of BRCA1. It was further proven by inverse staining pattern of BRCA1 and ALDH1 in immunohistochemistry. Surprisingly, methylation at this promoter was also elevated in survivor cells after a treatment with chemotherapeutic agent Doxorubicin, but not with Placitaxel, causing gene suppression.
Conclusions: Our findings unveiled that hypermethylation at a novel BRCA1 promoter might epigenetically regulate cancer stem-like cell population and cell resistance to Doxorubicin.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 278, Tuesday Morning