LRH-1 as a Key Regulator of Estrogen Responses in Breast Cancer Cells

Abstract

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). LRH- 1 has been linked to a number of key developmental, metabolic and proliferative processes and is known to play an important role in the regulation of cholesterol biosynthesis, lipid homeostasis and the control of steroid aromatisation. In this respect, LRH-1 is recognised to play an important role in breast cancer, where it acts to regulate aromatase activity, leading to the paracrine production of estrogen. Recent findings have also suggested a direct role for LRH-1 in cancer, where LRH-1 is found to be involved in the induction of intestinal tumours and LRH-1 has been found by immunohistochemistry in tumour cells of human mammary ductal carcinomas. Recently, a gene expression microarray analysis of estrogen responses in an engineered breast cancer cell line, where Estrogen Receptor-α (ERα) activity can be conditionally repressed, provisionally identified LRH-1 as an estrogen responsive gene that may be important in the estrogen-regulated growth of breast cancer cells. Based on this initial observation, I have gone on to study the role of LRH-1 in the estrogen response in breast cancer cells. Using ERα-positive breast cancer cell lines, I have confirmed that LRH-1 levels increase in response to estrogen and are inhibited by anti-estrogens (tamoxifen and ICI 182,780). Using 5`RNA Ligase Mediated Rapid Amplification of cDNA Ends (5`RLM-RACE) to characterise the 5’ end of the LRH-1 mRNA, I have found that the estrogen regulation of LRH-1 is mediated through a previously undescribed gene promoter, which results in the production of a variant LRH-1 mRNA species that initiates transcription just upstream of exon 2 of the LRH-1 gene. Further, reverse transcriptase polymerase chain reaction (RT-PCR) showed that the newly identified variant LRH-1 transcript is expressed in tissues in which LRH-1 expression has previously been described. Moreover, this variant was seen to be the major form of LRH-1 expressed in breast cancer cell lines. Having established the estrogen regulation of LRH-1, studies were carried out to investigate the role of LRH-1 in growth and gene expression in breast cancer cells. siRNA-mediated inhibition of LRH-1 expression inhibited proliferation of MCF-7, ZR-75-1 and T47-D breast cancer cell lines but did not inhibit BT474 and MDAMB- 231 breast cancer cells, in which LRH-1 is not expressed. Further, a group of recently described synthetic agonists for LRH-1 stimulated the growth of breast cancer cell lines expressing LRH-1 in a dose dependent manner, but did not stimulate growth in those breast cancer cell lines which do not express LRH-1. Finally, RNA interference experiments directed against LRH-1 identified ERα as an important LRH-1 regulated gene. These results show that LRH-1 potentially plays a key role in regulating estrogen responses in ERα–positive breast cancer cells, primarily through the direct regulation of ERα gene expression. These new findings, taken together with the previously described role for LRH-1 in regulating aromatase gene expression identify LRH-1 as a potentially important target for the development of new therapies for the treatment of breast cancer.