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FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

Gong C, Fujino K, Monteiro LJ, Gomes AR, Drost R, Davidson-Smith H, Takeda S, Khoo US, Jonkers J, Sproul D, Lam EW - Oncogene (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.

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.

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FOXA1 expression is lower and its promoter more hypermethylated in the BRCA1-deficient murine mammary epithelial cell line. (a) KB1PR-3.12 E3, KB1PR-3.12 F4 and KB1P-3.12 murine mammary epithelial cell lines were collected and analyzed by western blotting and (b) quantitative reverse transcription-PCR (qRT–PCR) to determine the expression levels of BRCA1 and FOXA1. qRT–PCR results are presented as the mean±s.d. of three independent experiments in triplicates. (c) Schematic representation, as presented on UCSC genome web browser, of the locations of CpG islands in mouse Foxa1 gene and the positions of primers used for pyrosequencing analysis. (d) DNA extracts from KB1PR-3.12 E3 and KB1P-3.12 cell lines were bisulphite converted and methylation status of Foxa1 promoter region were analyzed by pyrosequencing. Average Foxa1 methylation values of the two analyzed regions within the CpG island 86, located in the Foxa1 promoter region, are shown for both cell lines. Results are expressed as the mean±s.d. from two independent experiments in triplicates. *P⩽0.05 **P⩽0.01, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
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fig3: FOXA1 expression is lower and its promoter more hypermethylated in the BRCA1-deficient murine mammary epithelial cell line. (a) KB1PR-3.12 E3, KB1PR-3.12 F4 and KB1P-3.12 murine mammary epithelial cell lines were collected and analyzed by western blotting and (b) quantitative reverse transcription-PCR (qRT–PCR) to determine the expression levels of BRCA1 and FOXA1. qRT–PCR results are presented as the mean±s.d. of three independent experiments in triplicates. (c) Schematic representation, as presented on UCSC genome web browser, of the locations of CpG islands in mouse Foxa1 gene and the positions of primers used for pyrosequencing analysis. (d) DNA extracts from KB1PR-3.12 E3 and KB1P-3.12 cell lines were bisulphite converted and methylation status of Foxa1 promoter region were analyzed by pyrosequencing. Average Foxa1 methylation values of the two analyzed regions within the CpG island 86, located in the Foxa1 promoter region, are shown for both cell lines. Results are expressed as the mean±s.d. from two independent experiments in triplicates. *P⩽0.05 **P⩽0.01, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.

Mentions: To confirm further that the loss of FOXA1 expression is a result of BRCA1 depletion in mammary epithelial cells, we next investigated the effect of re-expressing BRCA1 on FOXA1 expression in Brca1-deficient mouse mammary epithelial cells (MMECs). Analysis of the expression levels of Foxa1 in Brca1-deficient KB1P-3.12 (K14cre;Brca1F/F;p53F/F, clone 12) and BRCA1-reconstituted KB1PR-3.12 E3 and KB1PR-3.12 F4 (K14cre;Brca1F/F;p53F/F BRCA1-reconstituted clones E3 and F4) MMECs,18 revealed that Foxa1 expression was significantly higher at both the mRNA and protein levels in the BRCA1-reconstituted cell lines compared with the parental Brca1-deficient, KB1P-3 12 cells (Figures 3a and b), further confirming that BRCA1 has a role in modulating FOXA1 expression.


FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

Gong C, Fujino K, Monteiro LJ, Gomes AR, Drost R, Davidson-Smith H, Takeda S, Khoo US, Jonkers J, Sproul D, Lam EW - Oncogene (2014)

FOXA1 expression is lower and its promoter more hypermethylated in the BRCA1-deficient murine mammary epithelial cell line. (a) KB1PR-3.12 E3, KB1PR-3.12 F4 and KB1P-3.12 murine mammary epithelial cell lines were collected and analyzed by western blotting and (b) quantitative reverse transcription-PCR (qRT–PCR) to determine the expression levels of BRCA1 and FOXA1. qRT–PCR results are presented as the mean±s.d. of three independent experiments in triplicates. (c) Schematic representation, as presented on UCSC genome web browser, of the locations of CpG islands in mouse Foxa1 gene and the positions of primers used for pyrosequencing analysis. (d) DNA extracts from KB1PR-3.12 E3 and KB1P-3.12 cell lines were bisulphite converted and methylation status of Foxa1 promoter region were analyzed by pyrosequencing. Average Foxa1 methylation values of the two analyzed regions within the CpG island 86, located in the Foxa1 promoter region, are shown for both cell lines. Results are expressed as the mean±s.d. from two independent experiments in triplicates. *P⩽0.05 **P⩽0.01, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
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fig3: FOXA1 expression is lower and its promoter more hypermethylated in the BRCA1-deficient murine mammary epithelial cell line. (a) KB1PR-3.12 E3, KB1PR-3.12 F4 and KB1P-3.12 murine mammary epithelial cell lines were collected and analyzed by western blotting and (b) quantitative reverse transcription-PCR (qRT–PCR) to determine the expression levels of BRCA1 and FOXA1. qRT–PCR results are presented as the mean±s.d. of three independent experiments in triplicates. (c) Schematic representation, as presented on UCSC genome web browser, of the locations of CpG islands in mouse Foxa1 gene and the positions of primers used for pyrosequencing analysis. (d) DNA extracts from KB1PR-3.12 E3 and KB1P-3.12 cell lines were bisulphite converted and methylation status of Foxa1 promoter region were analyzed by pyrosequencing. Average Foxa1 methylation values of the two analyzed regions within the CpG island 86, located in the Foxa1 promoter region, are shown for both cell lines. Results are expressed as the mean±s.d. from two independent experiments in triplicates. *P⩽0.05 **P⩽0.01, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
Mentions: To confirm further that the loss of FOXA1 expression is a result of BRCA1 depletion in mammary epithelial cells, we next investigated the effect of re-expressing BRCA1 on FOXA1 expression in Brca1-deficient mouse mammary epithelial cells (MMECs). Analysis of the expression levels of Foxa1 in Brca1-deficient KB1P-3.12 (K14cre;Brca1F/F;p53F/F, clone 12) and BRCA1-reconstituted KB1PR-3.12 E3 and KB1PR-3.12 F4 (K14cre;Brca1F/F;p53F/F BRCA1-reconstituted clones E3 and F4) MMECs,18 revealed that Foxa1 expression was significantly higher at both the mRNA and protein levels in the BRCA1-reconstituted cell lines compared with the parental Brca1-deficient, KB1P-3 12 cells (Figures 3a and b), further confirming that BRCA1 has a role in modulating FOXA1 expression.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.

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.

Show MeSH
Related in: MedlinePlus