Characterisation of Estrogen Responses in Breast Cancer Cells Highlights LRH-1 as a New Target for Breast Cancer Treatment.

Abstract

Abstract Background: Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). It is expressed in endodermal derived tissues and has been linked to a number of developmental, metabolic and proliferative processes. LRH-1 plays an important role in cholesterol and lipid homeostasis. Recent findings have shown that intestinal production of glucocorticoids is regulated by LRH-1, a deficiency of which sensitizes mice to colitis. Uncontrolled LRH-1 activity participates in the induction of intestinal tumours. LRH-1 protein expression has also been detected by immunohistochemistry in tumour cells of human mammary ductal carcinomas.Materials and Methods: Using a panel of breast cancer cell lines and human tissues we have studied the expression of LRH-1 together with another Ftz-F1 subfamily member steroidogenic factor 1 (SF-1) and the potent LRH-1 co-repressors small heterodimer partner (SHP) and DAX-1/Dax-1 (dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome 1). RNA ligase Mediated Rapid amplification of 5' complementary DNA ends (5'RLM-RACE) was performed in order to map the estrogen regulated transcription start sites for the LRH-1 gene. No natural ligand for LRH-1 has been described. Using fluorescence resonance energy transfer (FRET)-based assays a series of unusual bicyclic compounds have been defined as potent LRH-1 ligands. The effects of these compounds on breast cancer cell growth and gene expression has been investigated using SRB growth assays and quantitative RT-PCR.Results & Discussion: LRH-1 expression was found to be highest in estrogen receptor positive cell lines and breast, colon,small intestine and liver tissue. Treatment with 17β-estradiol increased LRH-1 levels in estrogen receptor positive cell lines, while treatment with anti-estrogens Tamoxifen and ICI 182,780 (Fulvestrant) reduced LRH-1 levels. siRNA mediated RNA interference against LRH-1 significantly reduced the estrogen-dependent growth of estrogen receptor positive breast cancer cell lines. We have shown that LRH-1 regulates the expression of the LRH-1 target gene Inhibin-α (a member of the transforming growth factor-β superfamily) in breast cancer cell lines. 5' RACE identified two new variant forms of LRH-1, which we have named LRH-1 289 and LRH-1 325 respectively. These arise from a novel promoter region lying between exon 2 and exon 3. RT- PCR for the LRH-1 289 variant showed E2 regulation in MCF7 cells and was seen to account for the majority of LRH-1 expression in this line. SRB growth assays demonstrated that these bicyclical compounds stimulate the growth of estrogen receptor/LRH-1 positive breast cancer cell lines in a dose dependent manner. Luciferase assays have demonstrated a titratable increase in LRH-1 reporter activity following the addition of these compounds.Our studies show that LRH-1 is a potently estrogen regulated gene in breast cancer cells, which acts to regulate cell growth and gene expression. These results therefore help validate LRH-1 as a potential new target for breast cancer treatment Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4143.