BRCA1-mutation or depletion correlates with basal-like phenotype and poor prognosis in
breast cancer, but the underlying reason remains elusive. RNA and protein analysis of a
panel of breast cancer cell lines revealed that BRCA1-deficiency is associated with
downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown
of BRCA1 by siRNA resulted in downregulation of FOXO3 expression in the BRCA1-
competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-
defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of
FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNAmethylation
inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase EZH2-inhibitor
GSK126, or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect
on the BRCA1-competent MCF-7 cells. Chromatin-immunoprecipitation (ChIP) analysis
demonstrated that BRCA1, EZH2, DNMT1/3a/b, and H3K27me3 are recruited to the
endogenous FOXO3 promoter, further advocating that these proteins interact to modulate
FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1-
depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the
enrichment of the EZH2-mediated transcriptional repressive epigenetic marks, H3K27me3
on the FOXO3 promoter. Methylated DNA immunoprecipitation (MeDIP) assays also
confirmed increased CpG methylation of FOXO3 gene upon BRCA1 depletion. Analysis of
the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that
FOXO3 promoter methylation is significantly associated with BRCA1-mutation. Furthermore,
immunohistochemistry further suggested that FOXO3 expression was significantly
associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3
and EZH2 mRNA levels were significantly associated with good and poor prognosis in breast
cancer, respectively. Together these data suggest that BRCA1 can prevent and reverse
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FOXO3 suppression via inhibiting EZH2 and consequently, its ability to recruit the
transcriptional repressive H3K27me3 histone marks and the DNA methylases DNMT1/3a/3b
to induce DNA methylation and gene silencing on the FOXO3 promoter.