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Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

ABSTRACT: 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.

SUBMITTER: Dago DN

PROVIDER: S-EPMC4424892 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

Dago Dougba Noel DN Scafoglio Claudio C Rinaldi Antonio A Memoli Domenico D Giurato Giorgio G Nassa Giovanni G Ravo Maria M Rizzo Francesca F Tarallo Roberta R Weisz Alessandro A

BMC genomics 20150509

<h4>Background</h4>Estrogens play an important role in breast cancer (BC) development and progression; when the two isoforms of the estrogen receptor (ERα and ERβ) are co-expressed each of them mediate specific effects of these hormones in BC cells. ERβ has been suggested to exert an antagonist role toward the oncogenic activities of ERα, 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 ...[more]

PMID: 25956916

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Project description:BackgroundAlternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ER?) and their ligands promote alternative splicing. The endogenous targets of ER?:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ER? on E2-induced splicing are unknown.MethodsMCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ER? binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.ResultsWe identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ER? cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ER? binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ER? and splicing factors, influenced ER?:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ER?-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ER?-dependent isoform switching, which correlated with altered response to KGF.ConclusionE2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

Project description:Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERbeta-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

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Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

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Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

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