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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 gene promoter is hypermethylated in BRCA1 mutation tumours and its methylation is negatively correlated with its expression. Frequency of FOXA1 methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analyzed using the kConFab database. (a) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or BRCAx mutated tumour. (b and c) FOXA1 expression level inversely correlates with FOXA1 methylation. Box plots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points. GEM, gene expression marker. (d and e) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour. (f and g) Significant lower percentage of FOXA1 expression in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour.
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fig11: FOXA1 gene promoter is hypermethylated in BRCA1 mutation tumours and its methylation is negatively correlated with its expression. Frequency of FOXA1 methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analyzed using the kConFab database. (a) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or BRCAx mutated tumour. (b and c) FOXA1 expression level inversely correlates with FOXA1 methylation. Box plots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points. GEM, gene expression marker. (d and e) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour. (f and g) Significant lower percentage of FOXA1 expression in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour.

Mentions: Having established that BRCA1 regulates FOXA1 methylation and silencing as well as the mechanism involved in human and MMECs, we next examined the association between FOXA1 methylation and expression in vivo in a published methylated DNA immunoprecipitation and gene expression microarray data set derived from familial breast tumour samples collected by kConFab (The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, Melbourne, Australia).23 There are 33 patient samples in the cohort, 11 of which are with BRCA1 mutation. Statistical analysis of the expression and methylation profiles of FOXA1 in these familial breast cancers revealed that there was a strong and significant inverse correlation between FOXA1 methylation and expression using both linear regression model (R2=0.127, P=0.042; Figures 11a and b; Supplementary Figures S7 and S8) and Pearson's correlation model (cutoff is the mean, Pearson correlation coefficient=−0.492, P=0.004; Figure 11c), providing further physiological evidence that FOXA1 methylation negatively regulates its expression in breast cancer patient samples. We also compared the levels of FOXA1 methylation in different familial breast tumours and found that FOXA1 methylation levels were significantly higher in BRCA1-mutated tumours compared with BRCA2, BRCAx (non-BRCA1/2) and BRCA2/x tumours (P=0.006, P=0.046 and P=0.006, respectively; Student's t-tests; Figures 10d and e, Supplementary Figures S9 and S10). Moreover, we also observed that FOXA1 expression was significantly lower in BRCA1-mutated tumours compared with BRCA2, BRCAx and BRCA2/x tumours (P<0.001, P=0.001 and P<0.001, respectively; Student's t-test; Figures 11f and g; Supplementary Figures S11 and S12). These in vivo data provide further proof that DNA methylation is a major mechanism for repressing FOXA1 expression in familial breast cancers and confirm a role for BRCA1 in regulating FOXA1 methylation and 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 gene promoter is hypermethylated in BRCA1 mutation tumours and its methylation is negatively correlated with its expression. Frequency of FOXA1 methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analyzed using the kConFab database. (a) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or BRCAx mutated tumour. (b and c) FOXA1 expression level inversely correlates with FOXA1 methylation. Box plots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points. GEM, gene expression marker. (d and e) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour. (f and g) Significant lower percentage of FOXA1 expression in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour.
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fig11: FOXA1 gene promoter is hypermethylated in BRCA1 mutation tumours and its methylation is negatively correlated with its expression. Frequency of FOXA1 methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analyzed using the kConFab database. (a) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or BRCAx mutated tumour. (b and c) FOXA1 expression level inversely correlates with FOXA1 methylation. Box plots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points. GEM, gene expression marker. (d and e) Significant higher percentage of FOXA1 promoter methylation in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour. (f and g) Significant lower percentage of FOXA1 expression in BRCA1-mutated tumours compared with BRCA2 or/and BRCAx mutated tumour.
Mentions: Having established that BRCA1 regulates FOXA1 methylation and silencing as well as the mechanism involved in human and MMECs, we next examined the association between FOXA1 methylation and expression in vivo in a published methylated DNA immunoprecipitation and gene expression microarray data set derived from familial breast tumour samples collected by kConFab (The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, Melbourne, Australia).23 There are 33 patient samples in the cohort, 11 of which are with BRCA1 mutation. Statistical analysis of the expression and methylation profiles of FOXA1 in these familial breast cancers revealed that there was a strong and significant inverse correlation between FOXA1 methylation and expression using both linear regression model (R2=0.127, P=0.042; Figures 11a and b; Supplementary Figures S7 and S8) and Pearson's correlation model (cutoff is the mean, Pearson correlation coefficient=−0.492, P=0.004; Figure 11c), providing further physiological evidence that FOXA1 methylation negatively regulates its expression in breast cancer patient samples. We also compared the levels of FOXA1 methylation in different familial breast tumours and found that FOXA1 methylation levels were significantly higher in BRCA1-mutated tumours compared with BRCA2, BRCAx (non-BRCA1/2) and BRCA2/x tumours (P=0.006, P=0.046 and P=0.006, respectively; Student's t-tests; Figures 10d and e, Supplementary Figures S9 and S10). Moreover, we also observed that FOXA1 expression was significantly lower in BRCA1-mutated tumours compared with BRCA2, BRCAx and BRCA2/x tumours (P<0.001, P=0.001 and P<0.001, respectively; Student's t-test; Figures 11f and g; Supplementary Figures S11 and S12). These in vivo data provide further proof that DNA methylation is a major mechanism for repressing FOXA1 expression in familial breast cancers and confirm a role for BRCA1 in regulating FOXA1 methylation and 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