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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.

SUBMITTER: Gong C

PROVIDER: S-EPMC4430311 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The heterogeneous breast cancers can be classified into different subtypes according to their histopathological characteristics and molecular signatures. Foxa1 expression is linked with luminal breast cancer (LBC) with good prognosis, whereas Twist1 expression is associated with basal-like breast cancer (BLBC) with poor prognosis owing to its role in promoting epithelial-to-mesenchymal transition (EMT), invasiveness and metastasis. However, the regulatory and functional relationships between Twist1 and Foxa1 in breast cancer progression are unknown. In this study, we demonstrate that in the estrogen receptor (ER?)-positive LBC cells Twist1 silences Foxa1 expression, which has an essential role in relieving Foxa1-arrested migration, invasion and metastasis of breast cancer cells. Mechanistically, Twist1 binds to Foxa1 proximal promoter and recruits the NuRD transcriptional repressor complex to de-acetylate H3K9 and repress RNA polymerase II recruitment. Twist1 also silences Foxa1 promoter by inhibiting AP-1 recruitment. Twist1 expression in MCF7 cells silenced Foxa1 expression, which was concurrent with the induction of EMT, migration, invasion and metastasis of these cells. Importantly, restored Foxa1 expression in these cells largely inhibited Twist1-promoted migration, invasion and metastasis. Restored Foxa1 expression did not change the Twist1-induced mesenchymal cellular morphology and the expression of Twist1-regulated E-cadherin, ?-catenin, vimentin and Slug, but it partially rescued Twist1-silenced ER? and cytokeratin 8 expression and reduced Twist1-induced integrin ?5, integrin ?1 and MMP9 expression. In a xenografted mouse model, restored Foxa1 also increased Twist1-repressed LBC markers and decreased Twist1-induced BLBC markers. Furthermore, Twist1 expression is negatively correlated with Foxa1 in the human breast tumors. The tumors with high Twist1 and low Foxa1 expressions are associated with poor distant metastasis-free survival. These results demonstrate that Twist1's silencing effect on Foxa1 expression is largely responsible for Twist1-induced migration, invasion and metastasis, but less responsible for Twist1-induced mesenchymal morphogenesis and expression of certain EMT markers.

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

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