Regulation of mineralocorticoid receptor activation by circadian protein TIMELESS.

The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.

Mineralocorticoid receptor actions in cardiovascular development and disease.

Mineralocorticoid receptors (MRs) are transcriptional regulators that mediate the diverse physiological and pathophysiological actions of corticosteroid hormones across many tissues. In the kidney aldosterone control of sodium/water resorption via DNA-binding actions of the MR is established. MRs also regulate tissues not involved in electrolyte homeostasis such as the heart, adipose tissue, brain, and inflammatory cells where the MRs can respond to both aldosterone and cortisol. The pathology of inappropriate MR activation in non-epithelial tissues are well-described, and steroidal antagonists of the MR have been clinically beneficial in the management of heart failure and blood pressure for decades. However, the role of cortisol-dependent MR activation in the physiological setting is less well defined. Like other steroid hormone receptors, the MR also regulates non-DNA-binding pathways including MAPK pathways and G protein coupled receptors to provide diversity to MR signaling. Whether nonDNA binding pathways are more relevant for MR activation in non-epithelial, versus epithelial, tissues remain unclear. This review will focus on molecular regulation of ligand-dependent MR activation and the physiology and pathophysiology of MR actions in the heart with a focus on the cardiomyocyte and provide a discussion of relevant genomic and non-genomic MR pathways and potential new transcriptional partners for the MR and their relevance for health and disease. Understanding MR actions in the heart will provide new insights into cell-selective mechanisms that underpin the therapeutic benefits of MRAs, and are a critical step towards developing next-generation tissue selective MR modulators with improved safety profiles.

A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer.

BACKGROUND: The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. METHODS: We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. RESULTS: Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARß. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. CONCLUSION: Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

Endocrine aspects of ACE2 regulation: RAAS, steroid hormones and SARS-CoV-2.

Coronavirus disease (COVID-19) is caused by a new strain of coronavirus, the severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2. At the time of writing, SARS-CoV-2 has infected over 5 million people worldwide. A key step in understanding the pathobiology of the SARS-CoV-2 was the identification of -converting enzyme 2 (ACE2) as the receptor for SARS-CoV-2 to gain entry into host cells. ACE2 is an established component of the 'protective arm' of the renin-angiotensin-aldosterone-system (RAAS) that opposes ACE/angiotensin II (ANG II) pressor and tissue remodelling actions. Identification of ACE2 as the entry point for SARS-CoV-2 into cells quickly focused attention on the use of ACE inhibitors (ACEi), angiotensin receptor blockers (ARB) and mineralocorticoid receptor antagonists (MRA) in patients with hypertension and cardiovascular disease given that these pharmacological agents upregulate ACE2 expression in target cells. ACE2 is cleaved from the cells by metalloproteases ADAM10 and ADAM17. Steroid hormone receptors regulate multiple components of the RAAS and may contribute to the observed variation in the incidence of severe COVID-19 between men and women, and in patients with pre-existing endocrine-related disease. Moreover, glucocorticoids play a critical role in the acute and chronic management of inflammatory disease, independent of any effect on RAAS activity. Dexamethasone, a synthetic glucocorticoid, has emerged as a life-saving treatment in severe COVID-19. This review will examine the endocrine mechanisms that control ACE2 and discusses the impact of therapies targeting the RAAS, glucocorticoid and other endocrine systems for their relevance to the impact of SARS-CoV-2 infection and the treatment and recovery from COVID-19-related critical illness.

Cardiomyocyte transcription is controlled by combined MR and circadian clock signalling.

We previously identified a critical pathogenic role for MR activation in cardiomyocytes that included a potential interaction between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock signalling, and vice versa. 10nM aldosterone or corticosterone regulated CRY 1, PER1, PER2 and ReverbA (NR1D1) gene expression patterns in H9c2 cells over 24hr. MR-dependent regulation of circadian gene promoters containing GREs and E-box sequences was established for CLOCK, Bmal, CRY 1 and CRY2, PER1 and PER2 and transcriptional activators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expression in hearts of mice 4hr after administration of aldosterone at 8AM versus 8PM. Our data support combined MR regulation of a subset of circadian genes and that endogenous circadian transcription factors CLOCK and Bmal modulate this response. This unsuspected relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to aldosterone as well as cortisol. These data are consistent with MR signalling in the brain where, like the heart, it preferentially responds to cortisol. Given the undisputed requirement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to the GR in the heart.

Timeless Is a Novel Estrogen Receptor Co-activator Involved in Multiple Signaling Pathways in MCF-7 Cells.

Activation of estrogen receptor α (ERα) stimulates cell division and tumor growth by modulating the expression of ERα target genes. This activation involves the recruitment of specific proteins with activities that are still not fully understood. Timeless, the human homolog of the Drosophila gene involved in circadian rhythm, was previously shown to be a strong predictor of tamoxifen relapse, and is involved in genomic stability and cell cycle control. In this study, we investigated the interplay between Timeless and ERα, and showed that human Timeless is an ERα coactivator. Timeless binds to ERα and enhances its transcriptional activity. Overexpressing Timeless increases PARP1 expression and enhances ERα-induced gene regulation through the proximal LXXLL motif on Timeless protein and ERα PARylation. Finally, Timeless is recruited with ERα on the GREB1 and cMyc promoters. These data, the first to link Timeless to steroid hormone function, provide a mechanistic basis for its clinical association with tamoxifen resistance. Thus, our results identify Timeless as another key regulator of ERα in controlling ERα transactivation.

Deoxycorticosterone/Salt-Mediated Cardiac Inflammation and Fibrosis Are Dependent on Functional CLOCK Signaling in Male Mice.

Activation of the mineralocorticoid receptor (MR) promotes inflammation, fibrosis, and hypertension. Clinical and experimental studies show that MR antagonists have significant therapeutic benefit for all-cause heart failure; however, blockade of renal MRs limits their widespread use. Identification of key downstream signaling mechanisms for the MR in the cardiovascular system may enable development of targeted MR antagonists with selectivity for pathological MR signaling and lower impact on physiological renal electrolyte handling. One candidate pathway is the circadian clock, the dysregulation of which is associated with cardiovascular diseases. We have previously shown that the circadian gene Per2 is dysregulated in hearts with selective deletion of cardiomyocyte MR. We therefore investigated MR-mediated cardiac inflammation and fibrosis in mice that lack normal regulation and oscillation of the circadian clock in peripheral tissues, that is, CLOCKΔ19 mutant mice. The characteristic cardiac inflammatory/fibrotic response to a deoxycorticosterone (DOC)/salt for 8 weeks was significantly blunted in CLOCKΔ19 mice when compared with wild-type mice, despite a modest increase at "baseline" for fibrosis and macrophage number in CLOCKΔ19 mice. In contrast, cardiac hypertrophy in response to DOC/salt was significantly greater in CLOCKΔ19 vs wild-type mice. Markers for renal inflammation and fibrosis were similarly attenuated in the CLOCKΔ19 mice given DOC/salt. Moreover, increased CLOCK expression in H9c2 cardiac cells enhanced MR-mediated transactivation of Per1, suggesting cooperative signaling between these transcription factors. This study demonstrates that the full development of MR-mediated cardiac inflammation and fibrosis is dependent on intact signaling by the circadian protein CLOCK.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma.

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

Insights into Orphan Nuclear Receptors as Prognostic Markers and Novel Therapeutic Targets for Breast Cancer.

There is emerging evidence asserting the importance of orphan nuclear receptors (ONRs) in cancer initiation and progression. In breast cancer, there is a lot unknown about ONRs in terms of their expression profile and their transcriptional targets in the various stages of tumor progression. With the classification of breast tumors into distinct molecular subtypes, we assess ONR expression in the different breast cancer subtypes and with patient outcomes. Complementing this, we review evidence implicating ONR-dependent molecular pathways in breast cancer progression to identify candidate ONRs as potential prognostic markers and/or as therapeutic targets.

GEMIN4 functions as a coregulator of the mineralocorticoid receptor.

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. Coregulator proteins are known to be critical for nuclear receptor-mediated gene expression. Identification of coregulators, which mediate MR activity in a tissue-specific manner, may allow for the development of novel tissue-selective MR modulators that confer cardiac protection without adverse renal effects. Our earlier studies identified a consensus motif among MR-interacting peptides, MPxLxxLL. Gem (nuclear organelle)-associated protein 4 (GEMIN4) is one of the proteins that contain this motif. Transient transfection experiments in HEK293 and H9c2 cells demonstrated that GEMIN4 repressed agonist-induced MR transactivation in a cell-specific manner. Furthermore, overexpression of GEMIN4 significantly decreased, while knockdown of GEMIN4 increased, the mRNA expression of specific endogenous MR target genes. A physical interaction between GEMIN4 and MR is suggested by their nuclear co-localization upon agonist treatment. These findings indicate that GEMIN4 functions as a novel coregulator of the MR.

Transcriptional control of local estrogen formation by aromatase in the breast.

Aromatase is the critical enzyme that converts androgens to estrogens. It is frequently highly expressed in the tumour bearing breast of women diagnosed with estrogen receptor positive tumours, resulting in dramatically increased local estrogen production to drive tumour progression. Expression of aromatase is regulated primarily at the transcriptional level of its encoding gene CYP19A1, located on chromosome 15 of the human genome. A characteristic feature of CYP19A1 expression is its use of alternative promoters to regulate transcription in a tissue-specific manner. In breast cancer, the increase in aromatase expression is mediated via higher expression of the distal adipose-specific promoter I.4 and a switch to the preferential use of proximal promoters I.3 and II. This results in a net increase of CYP19A1 transcripts in tumour-bearing breast up to 3-4-fold higher than normal breast. Current aromatase inhibitors - whilst efficacious - exhibit significant side effects that reduce patient compliance. Understanding the transcription factors and signalling pathways that control aromatase expression will lead to opportunities to develop breast-specific inhibitors with an improved side-effects profile. This article is part of a Special Issue entitled 'Essential role of DHEA'.

Estradiol regulates Tumor Necrosis Factor-α expression and secretion in Estrogen Receptor positive breast cancer cells.

The cytokine Tumor Necrosis Factor-α is critical to Estrogen Receptor positive breast cancer pathology, stimulating estrogen-biosynthesis pathways and preventing the differentiation of estrogen-producing fibroblasts. High concentrations of TNFα are detected in the tumor microenvironment, and infiltrating immune cells are thought to be a major source. This study identifies that TNFα is also a tumor-derived factor, expressed in ER+ tumour epithelial cells and regulated by 17-ß-estradiol (E2). Treatment of MCF-7, T47D and ZR-75 breast cancer cells with E2 increased TNFα mRNA and protein expression and secretion. This effect was mitigated with the use of ERα inhibitors 4-hydroy-tamoxifen and ICI-182780, indicating that E2-mediated TNFα induction was via the actions of ERα. Chromatin immunoprecipitation reveals ERα binding to the TNFα promoter upon stimulation with E2. This study demonstrates for the first time a positive feedback loop between estradiol and TNFα, critical in maintaining high levels of the hormone within the ER+ breast tumour microenvironment.

Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.

The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.

Conditional overexpression of liver receptor homolog-1 in female mouse mammary epithelium results in altered mammary morphogenesis via the induction of TGF-ß.

Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation, and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signaling to promote breast cancer cell proliferation. We used small interfering RNA knockdown strategies to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1-dependent mechanisms. We identified key genes involved in TGF-ß signaling to be highly responsive to LRH-1 knockdown. This relationship was validated in 2 breast cancer cell lines overexpressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 overexpression in mammary epithelial cells. Notably, TGF-ß signaling was activated in LRH-1-overexpressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 overexpression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-ß expression. The regulation of TGF-ß isoforms and SMAD2/3-mediated downstream signaling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively, these data demonstrate that LRH-1 regulates TGF-ß expression and downstream signaling in mouse mammary glands.

Endocrine disruption of the epigenome: a breast cancer link.

The heritable component of breast cancer accounts for only a small proportion of total incidences. Environmental and lifestyle factors are therefore considered to among the major influencing components increasing breast cancer risk. Endocrine-disrupting chemicals (EDCs) are ubiquitous in the environment. The estrogenic property of EDCs has thus shown many associations between ongoing exposures and the development of endocrine-related diseases, including breast cancer. The environment consists of a heterogenous population of EDCs and despite many identified modes of action, including that of altering the epigenome, drawing definitive correlations regarding breast cancer has been a point of much discussion. In this review, we describe in detail well-characterized EDCs and their actions in the environment, their ability to disrupt mammary gland formation in animal and human experimental models and their associations with exposure and breast cancer risk. We also highlight the susceptibility of early-life exposure to each EDC to mediate epigenetic alterations, and where possible describe how these epigenome changes influence breast cancer risk.

Hepatic glucose intolerance precedes hepatic steatosis in the male aromatase knockout (ArKO) mouse.

Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17ß-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.

Distinct nuclear receptor expression in stroma adjacent to breast tumors.

The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor growth. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription factors that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated fibroblasts (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose fibroblasts (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-ß); and RAR-related orphan receptor beta (ROR-ß)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-ß (TR-ß); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-ß, and PPAR-γ proteins in stromal fibroblasts from an independent panel of breast cancers (ER-positive (n = 15), ER-negative (n = 15), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.

Oestradiol reduces liver receptor homolog-1 mRNA transcript stability in breast cancer cell lines.

The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER- cells. However, the presence of LRH-1 protein in ER- cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER- breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER- compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER- versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER- cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER- cells as well as ER- tumors suggests a possible role in the development of ER- tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER- and ER+ breast cancer.