Tamoxifen, a selective oestrogen receptor modulator, has been used in the treatment of all stages of hormone-responsive breast cancer. However, tamoxifen shows partial oestrogenic activity in the uterus and its use has been associated with an increased incidence of endometrial cancer. The molecular explanation for these observations is not known. Here we show that tamoxifen and oestrogen have distinct but overlapping target gene profiles. Among the overlapping target genes, we identify a paired-box gene, PAX2, that is crucially involved in cell proliferation and carcinogenesis in the endometrium. Our experiments show that PAX2 is activated by oestrogen and tamoxifen in endometrial carcinomas but not in normal endometrium, and that this activation is associated with cancer-linked hypomethylation of the PAX2 promoter.
Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, an adipocyte-derived cytokine, elicits proliferative effects in some cell types and potentially stimulates the growth of mammary epithelium. Here we show that leptin induced time-and dose-dependent signal transducer and activator of transcription 3 (STAT3) phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 kinase activation in breast carcinoma cells. Blocking STAT3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation of MCF-7 cells, whereas blocking ERK1/2 activation by a specific ERK1/2 kinase inhibitor, U0126, did not result in any significant changes in leptin-induced cell proliferation. Our experiments also showed that one member of the p160 family of steroid receptor coactivators, steroid receptor coactivator (SRC)-1, but not glucocorticoid receptor interacting protein 1 (GRIP1) or amplified in breast cancer 1 (AIB1), also functioned in gene transactivation in response to leptin treatment. Glutathione S-transferase pull-down experiments showed that SRC-1 physically interacted with the activation domain of STAT3 and that chromatin immunoprecipitation experiments detected the occupancy of SRC-1, but not GRIP1 or AIB1, on the promoter of STAT3 target genes. Our experiments collectively showed that SRC-1 is involved in STAT3 signaling pathway that is implicated in leptin-stimulated cell growth.
SRCs (steroid receptor coactivators) are required for nuclear receptor-mediated transcription and are also implicated in the transcription initiation by other transcription factors, such as STATs and NFB. Despite phenotypic manifestations in gene knockout mice for SRC-1, GRIP1, and AIB1 of the SRC (Steroid Receptor Coactivator) family indicating their differential roles in animal physiology, there is no clear evidence, at the molecular level, to support a functional specificity for these proteins. We demonstrated in this report that two species of SRC coactivators, either as AIB1:GRIP1 or as AIB1:SRC-1 are recruited, possibly through heterodimerization, on the promoter of genes that contain a classical hormone responsive element (HRE). In contrast, on non-HRE-containing gene promoters, on which steroid receptors bind indirectly, either GRIP1 or SRC-1 is recruited as a monomer, depending on the cellular abundance of the protein. Typically, non-HRE-containing genes are early genes activated by steroid receptors, whereas HRE-containing genes are activated later. Our results also showed that SRC proteins contribute to the temporal regulation of gene transcription. In addition, our experiments revealed a positive correlation between AIB1/c-myc overexpression in ER + breast carcinoma samples, suggesting a possible mechanism for AIB1 in breast cancer carcinogenesis.
AIB1, a member of the steroid receptor coactivator (SRC) family that participates in gene transcriptional activation by nuclear receptors and other transcription factors, is required for animal growth and reproductive development and implicated in breast carcinogenesis. The mechanisms underlying the AIB1 pleiotropic functions are not fully understood and neither is the regulation of its activity. Here, we showed that AIB1 was a sumoylated protein and the sumoylation attenuated the transactivation activity of AIB1, which is in contrast to the sumoylation of its paralogs, GRIP1 and SRC-1. The transactivation activity of AIB1 is enhanced by its phosphorylation by several kinases, including mitogen-activated protein kinase. We demonstrated in this report that estrogen treatment led to an increased phosphorylation and decreased sumoylation of AIB1 and that the sumoylation coordinated with phosphorylation in regulating the transcriptional activity of AIB1, providing a mechanism for post-translational modifications in regulating the transcriptional output of AIB1.Nuclear receptors play critical roles in animal development and homeostasis through their bimodal function as repressors or activators of gene transcription (1). The repression and activation activity of nuclear receptors are determined by their association with corepressors and coactivators, respectively. Among the coactivators, the steroid receptor coactivator (SRC) 2 family is both necessary and sufficient for nuclear receptor-mediated gene activation as we previously demonstrated (2). The SRC family of coactivators includes three distinct but related p160 members: SRC-1, GRIP1 (or SRC-2), and AIB1 (also named RAC3, ACTR, SRC-3, or p/CIP in mice) (3-6). They share 40% overall sequence similarity, and they all contain, in the N terminus, a basic helix-loop-helix domain that is known for protein dimerization-DNA interaction as well as a Per-Arnt-Sim domain, which is known to be involved in protein-protein interaction. Three LXXLL motifs, also known as nuclear receptor boxes (for nuclear receptor binding), are centrally located in all three proteins (7-9). In the C terminus, the SRC proteins contain two activation domains, AD1 and AD2, which are associated with histone acetyltransferases (CREB-binding protein/p300 and pCAF) and coactivator-associated arginine methyltransferase 1, respectively, and function to modify the configuration of the chromatin structure (10, 11).Despite the structural similarities among the members of the SRC family of coactivators, evidence has been accumulated to suggest different physiological functions for SRC-1, GRIP1, and AIB1. Genetic studies with gene ablation showed that, although both male and female SRC1-null mice are viable and fertile, they exhibit partial resistance to several hormones, including estrogen, progestin, androgen, and thyroid hormones (12, 13). Elimination of GRIP1 impairs fertility in both male and female mice (14), and GRIP-null mice are protected against obesity and display enhanced adaptive thermogenesis, ...
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