title: FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.
abstract: FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: C
authors.initials: C
authors.lastname: Gong
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.|Department of Obstetrics & Gynecology, Faculty of Medicine, Juntendo University, Bunkyoku, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fujino
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: L J
authors.initials: LJ
authors.lastname: Monteiro
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: A R
authors.initials: AR
authors.lastname: Gomes
authors.affiliation: Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Drost
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
authors.forename: H
authors.initials: H
authors.lastname: Davidson-Smith
authors.affiliation: Department of Obstetrics & Gynecology, Faculty of Medicine, Juntendo University, Bunkyoku, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: U S
authors.initials: US
authors.lastname: Khoo
authors.affiliation: Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Jonkers
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
authors.forename: D
authors.initials: D
authors.lastname: Sproul
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: E W-F
authors.initials: EW
authors.lastname: Lam
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D019398:Genes, BRCA1; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000235:genetics*; D006801:Humans; D051379:Mice; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D011401:Promoter Regions, Genetic; D000097584:DNA Methyltransferase 3B
chemical_list: D002843:Chromatin; D004248:DNA (Cytosine-5-)-Methyltransferases; D000071221:Enhancer of Zeste Homolog 2 Protein; D051541:Hepatocyte Nuclear Factor 3-alpha; D063151:Polycomb Repressive Complex 2; D000097584:DNA Methyltransferase 3B; C496499:FOXA1 protein, human; C498409:EZH2 protein, human