Functional role of estradiol in breast cancer through stimulation of the expression of the prolactin receptor via transcriptional activation of its generic promoter.
Endocrinology and Reproduction Research Branch, NICHD, MD, Bethesda
AIM: Prolactin (PRL) exerts diverse functions in several target tissues through specific receptors and is a potent mitogen in normal and neoplastic breast tissues and cells. PRL and estradiol (E2) exhibit synergistic effects on proliferation of breast cancer cells. Prolactin receptors (PRLR) are expressed in the human breast and in most breast cancer cells. Expression of the human PRLR (hPRLR) is controlled by a complex regulatory system at the transcriptional level. (one generic promoter hPIII, and five human specific which direct transcription of alternative non-coding exons 1). Prolactin gene expression of the alternative exon-1 driven by the hPRLR hPIII promoter (the most commonly used in all tissues and cells examined) is induced by E2. This study identified the elements and transfactor complexes in PRLR hPIII associated with E2-induced expression of the hPRLR. METHODS: PRLR hPIII was cloned into an expression vector with a luciferase reporter gene, wild type or with deletions or mutations transfected in MCF-7 cells. Analysis of activities and EMSA, Western Blots, DNA affinity precipitation assay (DAPA), Coimmunopreciptation (CoIP) studies. RESULTS: E2 increased luciferase activity 6-8 fold in MCF7 cells transfected with the 5’ flanking region (-931 /-112 bp) of hPIII construct (hPIII, lacks ERE). Deletion/mutation analysis identified CEBP and Sp1 sequences within the promoter domain (-480/-112 bp). Mutation of either element abolished the E2 effect, indicating that cooperation of these factors bound to DNA is required for estrogen action. EMSA showed that Sp1/Sp3 and C/EBP beta bound to their cognate elements within the hPIII promoter. DAPA revealed that E2/ER alpha was bound to the Sp1/Sp3 and C/EBP beta DNA-protein complex. Similar results were observed by CoIP. All E2/ER alpha interactions were inhibited by the E2 antagonist ICI 182,780. CONCLUSIONS: These findings demonstrate that E2/ER alpha, Sp1 and C/EBP beta complexes are required for transcriptional expression of the hPLR through the generic promoter III in MCF-7 cells. Estrogen from breast stromal and adipose tissue cells, major sources of estradiol in post-menopausal women, in addition to circulating E2, could up-regulate hPRLR gene expression and stimulate breast tumor growth.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 994 (Signaling pathways - Part II).