title: GSTP1 promoter haplotypes affect DNA methylation levels and promoter activity in breast carcinomas.
abstract: 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.
authors.affiliation: Department of Genetics, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, Oslo, Norway.
authors.forename: Jo Anders
authors.initials: JA
authors.lastname: Rønneberg
authors.forename: Jörg
authors.initials: J
authors.lastname: Tost
authors.forename: Hiroko K
authors.initials: HK
authors.lastname: Solvang
authors.forename: Grethe I G
authors.initials: GI
authors.lastname: Alnaes
authors.forename: Fredrik E
authors.initials: FE
authors.lastname: Johansen
authors.forename: Elen M
authors.initials: EM
authors.lastname: Brendeford
authors.forename: Zohar
authors.initials: Z
authors.lastname: Yakhini
authors.forename: Ivo G
authors.initials: IG
authors.lastname: Gut
authors.forename: Per Eystein
authors.initials: PE
authors.lastname: Lønning
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Børresen-Dale
authors.forename: Odd S
authors.initials: OS
authors.lastname: Gabrielsen
authors.forename: Vessela N
authors.initials: VN
authors.lastname: Kristensen
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D051549:Glutathione S-Transferase pi / Q000235:genetics* / Q000502:physiology; D006239:Haplotypes; D006801:Humans; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D012044:Regression Analysis; D020218:Response Elements
chemical_list: D020598:Proto-Oncogene Proteins c-myb; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi