The estrogen receptor alpha (ER), and its natural ligand, 17beta-estradiol (E2) have long been implicated in the etiology of breast cancer. The aims of this research were to investigate the role of ER and its cooperating factors in gene regulation in human breast cancer cells.
Here we report on the genome-wide identification of genes regulated by ER, when occupied by agonists and selective estrogen receptor modulators (SERMs). We also describe an E2 regulated transcriptional network critical for estradiols control of breast cancer cell proliferation.
In addition, we report on the regulation of the transcription factor PITX1 by ER, and a new role for PITX1 in coordinating gene-specific control in response to the E2-ER complex.-To improve our understanding of ER gene regulation in breast cancer cells, ERalpha was stably integrated into the ER negative human breast cancer cell line MDA-MB-231 creating 231ER+ cells.
Transcription profiling using DNA microarrays revealed that presence of the ER significantly altered the expression of several hundred genes. Transcriptional profiling of the 231ER+ cells treated with E2 in the presence or absence of SERMs defined hormone-dependent gene networks and pathways. In addition, the antagonist and agonist character of the SERMs were determined for E2 regulated genes.-Estrogens generally stimulate the proliferation of ER-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors.
Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to E2, we sought to identify genes involved in E2-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite E2-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by E2. We identified a set of 70 genes regulated by E2 in both cells, with most being oppositely regulated by hormone. Using a variety of bioinformatic approaches, we found the E2F binding site to be over-represented in the potential regulatory regions of many cell-cycle related genes stimulated by E2 in MCF-7 but inhibited by estrogen in 231ER+ cells.
Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon E2 treatment. Furthermore, RNAi-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of E2 regulation of proliferation. These results demonstrate that estrogen regulation of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells.-We also report that paired-like homeodomain transcription factor (PITX1), a tumor suppressor and member of the homeobox transcription factor family, is robustly up-regulated by ERalpha.
PITX1 also interacts with ERalpha to suppress ER transcriptional activity and the association of this nuclear hormone receptor with genomic binding sites of ER target genes. PITX1 mRNA is under the primary transcriptional control of ERalpha in several ER-positive breast cancer cell lines, and E2 induces ER-dependent communication between the proximal promoter and a 5 upstream enhancer ER binding site of the PITX1 gene.
The consensus PITX1 binding motif is present in 23% of ER binding sites and is enriched in a subset of ER genomic binding sites, and E2 treatment enhances PITX1 recruitment only to these binding sites. Overexpression of PITX1 selectively inhibited the transcriptional activity of ERalpha and ERbeta while PITX1 enhanced the activities of other nuclear hormone receptors, such as the glucocorticoid receptor and progesterone receptor.
Reduction of PITX1 expression by siRNA enhances ERalpha recruitment only to ER binding sites that contain the PITX1 motif, which results in increased basal mRNA expression in this subset of ER target genes. These studies identify the tumor suppressor PITX as a new transcriptional target of ERalpha and define a novel function of PITX1 to selectively repress the transcriptional activity of the ER.-These studies provide new insights into gene regulation by the estrogen receptor and cooperating factors such as PITX1.
They also highlight the importance of this nuclear hormone receptor in controlling cellular functions including proliferation of breast cancer cells. These studies should assist in the development of novel targeted therapies for breast cancer prevention and treatment.