Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) a expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERa cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17b-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER a and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

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The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E 2 ) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E 2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based geneselection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E 2 , OH-Tam or both E 2 and OH-Tam. Compiled data revealed a hyper-response to E 2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor a on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E 2 . Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.

Receptor interacting protein 140 (RIP140) is a negative transcriptional regulator of nuclear hormone receptors that is required for the maintenance of energy homeostasis and ovulation. In this study, we investigated the mechanisms by which RIP140 expression is controlled by estrogens in breast cancer cells. We first analyzed by real time reverse transcription-polymerase chain reaction the regulation of RIP140 mRNA accumulation by estrogen receptor (ER) ligands in MCF-7 cells. We showed that the induction by estradiol (E2) was rapid and did not affect the apparent stability of the mRNA, suggesting a direct transcriptional regulation. To further study the underlying regulatory mechanisms, we then characterized the human RIP140 gene. We identified several noncoding exons with alternative splicing and localized the promoter region more than 100 kilobases upstream from the coding exon. Although we mapped a perfect consensus estrogen response element able to bind ER␣ in gel shift and in chromatin immunoprecipitation experiments, the effect of E2 on RIP140 gene transcription was very modest. This might result at least in part from the presence of an overlapping aryl hydrocarbon receptor (AhR) binding site, which interfered with the E2 response on both the transiently transfected reporter construct and the accumulation of the endogenous RIP140 mRNA. Altogether, our data indicate that the RIP140 gene exhibits a complex structure with several noncoding exons and supports transcriptional cross-talk and feedback involving the ER␣ and AhR nuclear receptors.Estrogens are steroid hormones that regulate proliferation and differentiation of target tissues such as mammary glands, reproductive organs, and skeletal, cardiac, and neural cells. They act mainly by controlling the expression of a number of specific genes through binding to two distinct nuclear estrogen receptors, ER␣ and ER␤. These receptors are ligand-activated transcription factors that subsequently bind as homo-or heterodimers to estrogen responsive elements (ERE) located in the regulatory region of target gene promoters. ERs, like other nuclear receptors, stimulate transcription using both a constitutive amino-terminal and a ligand-dependent carboxy-terminal activation function (AF1 and AF2, respectively), the latter being associated with the ligand-binding domain. These activation functions act independently or synergistically, depending on the cell type and promoter context, by recruiting a number of cofactors that are able either to stabilize the transcription preinitiation complex or to alter chromatin structure through histonemodifying enzymes, thus regulating transcription factor accessibility and binding.RIP140 was one of the first cofactors to be isolated through its recruitment by ER␣ AF2 in the presence of ligand (Cavailles et al., 1995). It has been shown to interact with many nuclear receptors such as ER␣, thyroid hormone receptor, retinoic acid receptor, and retinoid X receptor (L'Horset et al., 1996), adrenergic receptor (Ikonen et al., 1997),...