Aromatase Is a Direct Target of FOXL2: C134W in Granulosa Cell Tumors via a Single Highly Conserved Binding Site in the Ovarian Specific Promoter

BACKGROUND: Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells. METHODOLOGY/PRINCIPAL FINDINGS: In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (-516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (-1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W. CONCLUSIONS/SIGNIFICANCE: These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation

BACKGROUND: Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. LRH-1 plays important roles in embryonic development, steroidogenesis and cholesterol homeostasis. In tumor cells, LRH-1 induces proliferation and cell cycle progression. High LRH-1 expression is demonstrated in breast cancers, positively correlating with ERα status and aromatase activity. LRH-1 dependent cellular mechanisms in breast cancer epithelial cells are poorly defined. Hence in the present study we investigated the actions of LRH-1 in estrogen receptor α (ERα) positive breast cancer cells. RESULTS: The study aimed to investigate LRH-1 dependent mechanisms that promote breast cancer proliferation. We identified that LRH-1 regulated the expression of Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in MCF-7 and MDA-MB-231 cells. Over-expression of LRH-1 increased GREB1 mRNA levels while knockdown of LRH-1 reduced its expression. GREB1 is a well characterised ERα target gene, with three estrogen response elements (ERE) located on its promoter. Chromatin immunoprecipitation studies provided evidence of the co-localisation of LRH-1 and ERα at all three EREs. With electrophoretic mobility shift assays, we demonstrated direct binding of LRH-1 to EREs located on GREB1 and Trefoil Factor 1 (TFF1, pS2) promoters. LRH-1 and ERα co-operatively activated transcription of ERE luciferase reporter constructs suggesting an overlap in regulation of target genes in breast cancer cells. Over-expression of LRH-1 resulted in an increase in cell proliferation. This effect was more pronounced with estradiol treatment. In the presence of ICI 182,780, an ERα antagonist, LRH-1 still induced proliferation. CONCLUSIONS: We conclude that in ER-positive breast cancer cells, LRH-1 promotes cell proliferation by enhancing ERα mediated transcription of target genes such as GREB-1. Collectively these findings indicate the importance of LRH-1 in the progression of hormone-dependent breast cancer and implicate LRH-1 as a potential avenue for drug development

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

In human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.We show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.We demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription

Modulation of LRH-1 expression in transcriptionally regulates GREB1.

<p>(a) Changes in LRH-1 mRNA and (b) protein levels in MCF-7 cells transfected with siRNA for LRH-1 (−LRH-1) or control; with pcDNA only or LRH-1-pcDNA (+LRH-1) constructs 24 h post transfection. (c) The expression levels of GREB1 in response to LRH-1 knockdown (siRNA) and over-expression (+LRH-1). Data were presented as % fold change compared to controls of the normalized expression levels, as mean ± SD, n = 3 separate experiments.</p

LRH-1 acts synergistically with ERα to activate ERE containing promoters.

<p>Transcriptional activation of (a) 2×ERE and (b) GREB-ERE2 luciferase reporters by ERα and LRH-1 with vehicle (veh) or 10 nM 17β-estradiol (E2). Estrogen-deprived MCF-7 cells were over expressed with LRH-1 or ERα alone, or in combination with the appropriate reporter construct. Cells were treated with 17β-estradiol for 16 h prior to luciferase assays. Data is presented as mean+SE, n = 3 separate experiments, treatments in triplicate per experiment. *P&lt;0.05, *P&lt;0.01, ***P&lt;0.001 compared to vehicle control unless indicated by reference line.</p

LRH-1 binds to specific ERE sequences of the GREB1 and pS2 promoters.

<p>(a) EMSA showing binding of LRH-1 to the EREs present in the GREB1 promoter. Radiolabeled ERE1-GREB1, ERE2-GREB1 and ERE3-GREB1 probes were incubated with <i>in vitro</i> translated LRH-1 protein. <i>In vitro</i> translation of the empty vector was used as a negative control. Anti-LRH-1 antibody was added in addition to the probe and the LRH-1 protein to indicate specificity of protein binding. (b) EMSA showing binding of LRH-1 to the EREs present in the GREB1 and pS2 promoters. Radiolabeled LRHRE probe (containing the LRH-1 response element derived from the aromatase promoter), whole cell nuclear extracts infected with a LRH-1 viral construct were incubated with various oligonucleotides (as listed in the figure) including unlabeled LRHRE, mutated LRHRE, ERE1-GREB1, ERE2-GREB1, ERE3-GREB1 and ERE-pS2 which were added in 200 fold excess. Anti-LRH-1 antibody and IgG were also added in addition to the probe and the nuclear extract to indicate specificity of protein binding.</p

LRH-1 induces cell proliferation in 17β-estradiol and ICI 182,780 treated cells.

<p>Cell proliferation was measured in pcDNA alone transfected, estrogen-deprived MCF-7 cells (control) or LRH-1 over-expressing (+LRH-1) MCF-7 cells treated with vehicle, 10 nM 17β-estradiol (E2) or 10 nM 17β-estradiol and 1 nM ICI 182,780, an ERα antagonist for 5 days. Data is presented as mean+SEM, n = 3 separate experiments, triplicate treatments per experiment, ***P&lt;0.001 compared to control transfected cells; a,b P&lt;0.001 compared to vehicle control.</p