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GSTP1 Promoter Haplotypes Affect DNA Methylation Levels and Promoter Activity in Breast Carcinomas

¹ Department of Genetics, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, ² Faculty Division The Norwegian Radium Hospital, University of Oslo, and ³ Department of Molecular Biosciences, University of Oslo, Oslo, Norway ⁴ Laboratory for Epigenetics, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France; ⁵ Section of Oncology, Institute of Medicine, University of Bergen and Department of Oncology, Haukeland University Hospital, Bergen, Norway; ⁶ Department of Computer Science, Technion-Israel Institute of Technology, Technion city, Haifa, Israel; and ⁷ Faculty Division Ahus University Hospital, University of Oslo, Oslo, Norway

Requests for reprints: Vessela Nedelcheva Kristensen, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, 0310 Oslo, Norway. Phone: 47-22-93-44-17; Fax: 47-22-93-44-40; E-mail: Vessela.N.Kristensen{at}rr-research.no.

Key Words: GSTP1 • tandem repeat polymorphism • single nucleotide polymorphism • DNA methylation • transcriptional activation

The CpG island spanning the transcription start of the glutathione S-transferase P1 becomes methylated in a variety of human cancers including breast cancer. To study the effect of sequence variation on hypermethylation of the GSTP1 promoter, we analyzed the genetic and epigenetic variability in 90 tumors from patients with locally advanced breast cancer. High-resolution quantitative analysis revealed large variability in the DNA methylation levels. Lack of methylation was more often observed in the basal and normal-like estrogen receptor (ER)-negative tumors, and methylated GSTP1 was associated with better overall survival (P = 0.00063). Studies of the genetic variation identified 14 different haplotypes. The distribution of methylation levels of tumors homozygous for the most frequent haplotype was significantly different from other haplotype combinations (P = 0.011), the difference being more pronounced in ER-positive (P = 0.005) and progesterone receptor–positive (P = 0.008) tumors. Regression modeling identified the ER status and haplotype as the main determinants of DNA methylation variability. We identified a putative c-Myb response element (MRE) that was present in one of two minimal promoter haplotypes. In vitro analysis showed that c-Myb binds to the MRE, but binding was weakened by the two polymorphisms. Transient cotransfections in luminal-type and basal-like breast cancer cell lines confirmed cell-specific differential binding of c-Myb to the polymorphic sites, leading to a change in the expression from the GSTP1 promoter in vivo. GSTP1 expression was moderately but significantly (P = 0.01) reduced after siRNA-mediated knockdown of c-Myb. Our results indicate that haplotype structure of a promoter is important for the extent of DNA methylation. [Cancer Res 2008;68(14):5562–71]

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