The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor corepressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized a combined approach of chromatin immunoprecipitation (ChIP)-on-chip and gene expression array studies. By performing ChIP-on-chip on microarrays containing 24,000 promoters, we identified 541 SAFB1/SAFB2-binding sites in promoters of known genes, with significant enrichment on chromosomes 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2 and less were repressed. Interestingly, there was no significant overlap between the genes identified by ChIP-on-chip and gene expression array analysis, suggesting regulation through regions outside the proximal promoters. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in the regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that 12% of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms the primary role of SAFB1/SAFB2 as corepressors and also uncovers a previously unknown role for SAFB1 in the regulation of immune genes and in estrogen-mediated repression of genes. The estrogen receptor ␣ (ER␣)3 plays a pivotal role in both normal and pathophysiological processes, such as osteoporosis and breast cancer (1). The transcriptional activity of ER␣ is not only regulated by its ligand estrogen but also through its interaction with cofactors, which can either enhance (coactivators) or repress (corepressors) its activity (2). Briefly, upon binding of its ligand, ER␣ undergoes major structural rearrangements leading to exposure of binding surfaces that subsequently results in facilitated recruitment of different coactivators, including histone acetyltransferases or histone methyltransferases, and proteins from the SWI/SNF chromatin remodeling complex (3). In the absence of ligand, recruitment of corepressors results in the creation of locally repressed chromatin, subsequently resulting in inhibition of transcription (4).Our group has identified the scaffold attachment factors SAFB1 and SAFB2 as ER␣ corepressors (5, 6). The two proteins are paralogs that share 74% similarity at the amino acid levels, with up to 98% in some functional domains (7). The genes reside in close proximity on chromosome 19p13.3, oriented in a head-to-head orientation, separated by an ϳ500-bp GC-rich promoter (6). The C termini of the proteins harbor a repression domain that can refer repression when transferred to a heterologous DNA-binding protein. This repression domain can be further defined into two ...

We have previously reported on the relevance of the prevalence of CD44 1 /CD24 2/low cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative realtime PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ERa negative MDA-MB-231 cells suggesting that ERa was necessary. This was further confirmed by ERa silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ERa into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ERa corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ERa as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ERa and histone deacetylases. ' 2008 Wiley-Liss, Inc.Key words: CD24; ERa; breast cancer; estrogen-mediated repression CD24 is a small, heavily glycosylated, mucin-like glycosylphophatidyl-inositol-linked cell surface protein that localizes in lipid rafts.1 CD24 has recently generated considerable attention in tumor biology due to its role as a potential breast cancer stem cell marker 2 and its function in cell adhesion and metastatic tumor spread. Particularly, CD24 is expressed in a wide variety of human malignancies and has been associated with an unfavorable prognosis in breast cancer. 1,3 In experimental animals, CD24 has been suggested to regulate numerous processes associated with tumor growth and metastasis. 4 In humans, CD24 has been identified as a molecular marker that allows distinction between luminal epithelial, nonepithelial and myoepithelial cells.5 Moreover, the marker combination CD44 1 /CD24 2/low has been reported to characterize putative breast cancer stem cells.2 Our own studies demonstrated that the high prevalence of CD44 1 /CD24 2/low cells in primary breast cancers favored distant metastasis, especially to the bone. 6 Although the lack of CD24 has been highlighted within the context of putative breast cancer stem cells recent gene expression signatures designed to predict distant metastasis, prognosis and therapeutic planning pointed to a relationship between the breast c...

The scaffold attachment factors SAFB1 and SAFB2 have been shown to function as estrogen receptor (ERalpha) co-repressors in breast cancer cells, and to affect many cellular processes such as stress response, RNA processing, and apoptosis. SAFB1 and SAFB2 have also been implicated in breast tumorigenesis: Their shared chromosomal locus at 19p13 is frequently lost in breast cancer, mutations have been identified, and overexpression results in growth inhibition. The purpose of this study was to determine SAFB1/SAFB2 protein expression in human breast tumors, to correlate their expression with either natural progression ("prognostic factor") or with response to Tamoxifen ("predictive factor"), and to analyze potential correlations with tumor characteristics. SAFB1/SAFB2 protein were measured by immunoblotting using a pan-SAFB antibody in tumor extracts from patients with long-term clinical follow-up (n = 289), a subset of whom had received no adjuvant systemic therapy after breast cancer surgery (n = 117) and another subset of whom were treated with adjuvant Tamoxifen (n = 172). SAFB levels were correlated with clinico-pathological variables and patient outcome. SAFB levels varied widely, with 25 tumors not expressing detectable levels of SAFB. SAFB expression was significantly correlated with ERalpha, HER-2, bcl-2 and with expression of other ERalpha coregulators such as SRC-3. There was no association between SAFB expression and disease free survival, however, low SAFB expression was significantly associated with worse overall survival in patients who did not receive adjuvant therapy. This study shows that low SAFB protein levels predict poor prognosis of breast cancer patients, suggesting critical functions of SAFB1 and SAFB2 in breast cancer cells.

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