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.

Knockout and expression studies suggest that estrogen receptor ␤ (ER␤) plays a prominent role in ovarian function and pathology. Moreover, ovarian cancers are characterized by high morbidity and low responsiveness to anti-estrogens. Here we demonstrate, using quantitative PCR to measure ER␣ and ER␤ levels in 58 ovarian cancer patients, that ER␤ expression decreased in cysts and ovarian carcinomas as compared with normal ovaries and that this decrease is attributable only to a selective loss in ER␤ expression during cancer progression. To address the question of a possible involvement of ER␤ in ovarian cancers, we restored ER␣ and ER␤ expression in two human ovarian cancer cell lines PEO14 (ER␣-negative) and BG1 (ER␣-positive) using adenoviral delivery. ER␣, but not ER␤, could induce progesterone receptor and fibulin-1C. Moreover, ER␣ and ER␤ had opposite actions on cyclin D1 gene regulation, because ER␤ down-regulated cyclin D1 gene expression, whereas ER␣ increased cyclin D1 levels. Interestingly, ER␤ expression strongly inhibited PEO14 and BG1 cell proliferation and cell motility in a ligandindependent manner, whereas ER␣ had no marked effect. Induction of apoptosis by ER␤ also contributed to the decreased proliferation of ovarian cancer cells, as shown by Annexin V staining. This study shows that ER␤ is an important regulator of proliferation and motility of ovarian cancer and provides the first evidence for a proapoptotic role of ER␤. The loss of ER␤ expression may thus be an important event leading to the development of ovarian cancer.

Fibulin-1 is an extracellular matrix protein overexpressed in epithelial ovarian and breast cancers. In estrogen receptor (ER)-positive ovarian and breast cancer cell lines, fibulin-1 mRNA levels are markedly increased by estrogens. Transfection experiments using fibulin-1 promoter constructs indicate that 17beta-estradiol (E2) increases fibulin-1 gene transcription and that ERalpha is more potent than ERbeta to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. In conclusion, our data show that estrogens, via ERalpha, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.