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Inhibitory effects of estrogen receptor beta on specific hormone-responsive gene expression and association with disease outcome in primary breast cancer.

ABSTRACT: INTRODUCTION: The impact of interactions between the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, on gene expression in breast cancer biology is not clear. The goal of this study was to examine transcriptomic alterations in cancer cells co-expressing both receptors and the association of gene expression signatures with disease outcome. METHODS: Transcriptional effects of ERbeta overexpression were determined in a stably transfected cell line derived from ERalpha-positive T-47D cells. Microarray analysis was carried out to identify differential gene expression in the cell line, and expression of key genes was validated by quantitative polymerase chain reaction. Microarray and clinical data from patient samples were then assessed to determine the in vivo relevance of the expression profiles observed in the cell line. RESULTS: A subset of 14 DNA replication and cell cycle-related genes was found to be specifically downregulated by ERbeta. Expression profiles of four genes, CDC2, CDC6, CKS2, and DNA2L, were significantly inversely correlated with ERbeta transcript levels in patient samples, consistent with in vitro observations. Kaplan-Meier analysis revealed better disease outcome for the patient group with an expression signature linked to higher ERbeta expression as compared to the lower ERbeta-expressing group for both disease-free survival (p = 0.00165) and disease-specific survival (p = 0.0268). These findings were further validated in an independent cohort. CONCLUSION: Our findings revealed a transcriptionally regulated mechanism for the previously described growth inhibitory effects of ERbeta in ERalpha-positive breast tumor cells and provide evidence for a functional and beneficial impact of ERbeta in primary breast tumors.

SUBMITTER: Lin CY

PROVIDER: S-EPMC1868918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Estrogens play an important role in breast cancer (BC) development and progression; when the two isoforms of the estrogen receptor (ER? and ER?) are co-expressed each of them mediate specific effects of these hormones in BC cells. ER? has been suggested to exert an antagonist role toward the oncogenic activities of ER?, and for this reason it is considered an oncosuppressor. As clinical evidence regarding a prognostic role for this receptor subtype in hormone-responsive BC is still limited and conflicting, more knowledge is required on the biological functions of ER? in cancer cells. We have previously described the ER? and ER? interactomes from BC cells, identifying specific and distinct patterns of protein interactions for the two receptors. In particular, we identified factors involved in mRNA splicing and maturation as important components of both ER? and ER? pathways. Guided by these findings, here we performed RNA sequencing to investigate in depth the differences in the early transcriptional events and RNA splicing patterns induced by estradiol in cells expressing ER? alone or ER? and ER?.Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We identified several splicing events induced by ER? alone and by ER?+ER?, demonstrating for the first time that ER? significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ER?-dependent splicing by ER?, as well as by the presence of splicing isoforms only in ER?+cells. In particular, we observed that ER?+BC cell lines exhibited around 2-fold more splicing events than the ER?- cells. Interestingly, we identified putative direct targets of ER?-mediated alternative splicing by correlating the genomic locations of ER? and ER? binding sites with estradiol-induced differential splicing in the corresponding genes.Taken together, these results demonstrate that ER? significantly affects estrogen-induced early transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of ER? in these tumors.

Project description:BACKGROUND: Oestrogen receptor beta (ERbeta) modulates ERalpha activity; wild type ERbeta (ERbeta1) and its splice variants may therefore impact on hormone responsiveness of breast cancer. ERbeta2/ERbetacx acts as a dominant negative inhibitor of ERalpha and expression of ERbeta2 mRNA has been proposed as a candidate marker for outcome in primary breast cancer following adjuvant endocrine therapy. We therefore now assess ERbeta2 protein by immunostaining and mRNA by quantitative RT-PCR in relation to treatment outcome. METHODS: ERbeta2-specific immunostaining was quantified in 141 primary breast cancer cases receiving adjuvant endocrine therapy, but no neoadjuvant therapy or adjuvant chemotherapy. The expression of mRNA for ERbeta2/ERbetacx was measured in 100 cases by quantitative RT-PCR. Statistical analysis of breast cancer relapse and breast cancer survival was performed using Kaplan Meier log-rank tests and Cox's univariate and multivariate survival analysis. RESULTS: High ERbeta2 immunostaining (Allred score >5) and high ERbeta2 mRNA levels were independently associated with significantly better outcome across the whole cohort, including both ERalpha positive and negative cases (Log-Rank P < 0.05). However, only ERbeta2 mRNA levels were significantly associated with better outcome in the ERalpha + subgroup (Log-Rank P = 0.01) and this was independent of grade, size, nodal status and progesterone receptor status (Cox hazard ratio 0.31 P = 0.02 for relapse; 0.17 P = 0.01 for survival). High ERbeta2 mRNA was also associated with better outcome in node negative cases (Log Rank P < 0.001).ERbeta2 protein levels were greater in ERalpha positive cases (T-test P = 0.00001), possibly explaining the association with better outcome. Levels of ERbeta2 protein did not correlate ERbeta2 mRNA levels, but 34% of cases had both high mRNA and protein and had a significantly better outcome (Log-Rank relapse P < 0.005). CONCLUSION: High ERbeta2 protein levels were associated with ERalpha expression. Although most cases with high ERbeta2 mRNA had strong ERbeta2 immunostaining, mRNA levels but not protein levels were independently predictive of outcome in tamoxifen-treated ERalpha + tumours. Post-transcriptional control needs to be considered when assessing the biological or clinical importance of ERbeta proteins.

Project description:IntroductionOxidative stress can modify estrogen receptor (ER) structure and function, including induction of progesterone receptor (PR), altering the biology and clinical behavior of endocrine responsive (ER-positive) breast cancer.MethodsTo investigate the impact of oxidative stress on estrogen/ER-regulated gene expression, RNA was extracted from ER-positive/PR-positive MCF7 breast cancer cells after 72 hours of estrogen deprivation, small-interfering RNA knockdown of ER-alpha, short-term (8 hours) exposure to various oxidant stresses (diamide, hydrogen peroxide, and menadione), or simultaneous ER-alpha knockdown and oxidant stress. RNA samples were analyzed by high-throughput expression microarray (Affymetrix), and significance analysis of microarrays was used to define gene signatures responsive to estrogen/ER regulation and oxidative stress. To explore the association of these signatures with breast cancer biology, microarray data were analyzed from 394 ER-positive primary human breast cancers pooled from three independent studies. In particular, an oxidant-sensitive estrogen/ER-responsive gene signature (Ox-E/ER) was correlated with breast cancer clinical parameters and disease-specific patient survival (DSS).ResultsFrom 891 estrogen/ER-regulated probes, a core set of 75 probes (62 unique genes) responsive to all three oxidants were selected (Ox-E/ER signature). Ingenuity pathway analysis of this signature highlighted networks involved in development, cancer, and cell motility, with intersecting nodes at growth factors (platelet-derived growth factor-BB, transforming growth factor-beta), a proinflammatory cytokine (tumor necrosis factor), and matrix metalloproteinase-2. Evaluation of the 394 ER-positive primary breast cancers demonstrated that Ox-E/ER index values correlated negatively with PR mRNA levels (rp = -0.2; P = 0.00011) and positively with tumor grade (rp = 0.2; P = 9.741 x e-5), and were significantly higher in ER-positive/PR-negative versus ER-positive/PR-positive breast cancers (t-test, P = 0.0008). Regardless of PR status, the Ox-E/ER index associated with reduced DSS (n = 201; univariate Cox, P = 0.078) and, using the optimized cut-point, separated ER-positive cases into two significantly different DSS groups (log rank, P = 0.0009).ConclusionAn oxidant-sensitive subset of estrogen/ER-responsive breast cancer genes linked to cell growth and invasion pathways was identified and associated with loss of PR and earlier disease-specific mortality, suggesting that oxidative stress contributes to the development of an aggressive subset of primary ER-positive breast cancers.

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Inhibitory effects of estrogen receptor beta on specific hormone-responsive gene expression and association with disease outcome in primary breast cancer.

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