The orphan nuclear receptor LRH-1 and ERα activate GREB1 expression to induce breast cancer cell proliferation

Abstract

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.

Figure 1. Modulation of LRH-1 expression in transcriptionally regulates GREB1.

(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.

Figure 2. LRH-1 binds to three ERE sites within the GREB1 promoter.

(a) Location of regulatory EREs on the distal and proximal GREB1 promoter, highlighting (bold) sequence similarity of the LRH-1 nuclear receptor half site within the ERE palindrome. (b) Chromatin immunoprecipitation (ChIP) showing occupancy of LRH-1 on the three EREs where ERα binds in the presence or absence of estradiol. Immunoprecipitation was performed with anti-LRH-1 and ERα antibodies on chromatin isolated from MCF-7 cells treated with vehicle or 10 nM 17β-estradiol for 45 mins. (c) The precipitated chromatin was analyzed by quantitative real-time PCR to demonstrate relative occupancy using the delta delta C_t method. Data is normalised to 10% of input. Data is represented from 3 or more separate treatments and separate ChIP experiments. (d) Sequential ChIP demonstrating co-localisation of ERα and LRH-1 on ERE1 of the GREB1 promoter. Figures are representative of 3 or more separate ChIP experiments.

Figure 3. LRH-1 binds to specific ERE sequences of the GREB1 and pS2 promoters.

(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 in vitro translated LRH-1 protein. In vitro 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.

Figure 4. LRH-1 acts synergistically with ERα to activate ERE containing promoters.

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<0.05, *P<0.01, ***P<0.001 compared to vehicle control unless indicated by reference line.

Figure 5. LRH-1 induces cell proliferation in 17β-estradiol and ICI 182,780 treated cells.

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<0.001 compared to control transfected cells; a,b P<0.001 compared to vehicle control.

Figure 6. Synergistic effects of LRH-1 and 17β-estradiol treatment on GREB1 expression.

Quantitation of (a) LRH-1, (b) GREB1 and (c) ERα mRNA expression in estrogen-deprived MCF-7 cells (control) or LRH-1 over-expressing (+LRH-1) MCF-7 cells treated with vehicle (veh) or 10 nM 17β-estradiol (E2) for 16 h. Data is presented as mean+SE, n = 3 separate experiments, triplicate treatments per experiment, **P<0.01, ***P<0.001 compared to vehicle control.

Figure 7. LRH-1 regulation of GREB1 expression in ER negative breast cancer cells.

MDA-MB-231 cells were transfected with empty vector (C) or expression vectors for LRH-1 alone (L), ERα alone (E) or both LRH-1 and ERα (L+E). Cells were treated with vehicle or 10 nM 17β-estradiol (E2) for 16 h. Quantitation of (a) LRH-1, (b) ERα and (c) GREB1 mRNA expression. Data is presented as mean+SE, n = 3 separate experiments, ***P<0.001 compared to vehicle control.