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

Abstract

Background: 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 still limited and conflicting, more knowledge is required on the biological functions of ERβ in cancer cells. We have previously described the ERβ and ERα 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 ERα and ERβ 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 ERα alone or ERα and ERβ.

Results: Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We identified several splicing events induced by ERα alone and by ERα+ERβ, demonstrating for the first time that ERβ significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ERα-dependent splicing by ERβ, as well as by the presence of splicing isoforms only in ERβ+cells. In particular, we observed that ERβ+BC cell lines exhibited around 2-fold more splicing events than the ERβ- cells. Interestingly, we identified putative direct targets of ERβ-mediated alternative splicing by correlating the genomic locations of ERβ and ERα binding sites with estradiol-induced differential splicing in the corresponding genes.

Conclusions: Taken together, these results demonstrate that ERβ significantly affects estrogen-induced early transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of ERβ in these tumors.

Figure 1

Venn diagram of expressed genes in ERβ + and wt breast cancer cell lines. The Venn diagram shows the number of genes regulated by estradiol in the three cell lines, as indicated: wt (parental MCF-7 cells); Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the N-terminus). The pie charts in the lower panels specify the direction of regulation by estradiol (induction or repression) of the genes whose regulation is present in wt cells but is lost in both ERβ + cell lines (left panel), or of the genes whose regulation by estradiol is not present in the wt cells but appears in both ERβ + lines (right panel).

Figure 2

Annotation of splice events in ERα + and ERα + ERβ + BC cell lines. (A) The bar plot shows the number of all alternative splicing events occurring in the cell lines analyzed. Inclusion and exclusion behavior for each event are shown (FDR ≤ 0.05; c ≤ |0.1|). (B) Genes whose regulation has opposite direction in the ERβ + lines compared to the wt MCF-7. The heat map on the right side shows the gene expression fold changes induced by estradiol. The matrix on the left side shows in black those genes for which a splicing event was detected in at least one of the cell lines. The nomenclature for the cell lines is the following: wt (parental MCF-7 cells); Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the N-terminus).

Figure 3

Selected isoform switches affected by the expression of ERβ. The ERβ-dependent differential splicing events that affect most prominently the balance of different isoforms for each gene were identified by using the following parameters: (i) isoform ratio (FPKM_isoform/FPKM_gene %) changing of at least 10% after estradiol stimulation, either in the wt cells or in both the ERβ + cells in at least one of the isoforms of the gene; (ii) at least one isoform of the gene significantly regulated by estradiol (p-value ≤ 0.05; |FC| ≥ 1.3) either in the wt cells or in both the ERβ + cells; (iii) isoform ratio changing in opposite direction in the wt cells compared to the ERβ + cells; (iv) at least one splicing event identified by MATS analysis. Thirty-five genes satisfied all the selection requirements; of these, only 2 to 3 isoforms were selected for presentation, according to the expression levels and the regulation by estradiol. Left panel: heat map of regulation of the selected genes by estradiol in: Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the N-terminus); wt (parental MCF-7 cells). Right panel: heat map with the change in isoform ratio (FPKM_isoform/FPKM_gene %) between E2-treated and non-treated cells in the indicated cell lines; for each gene, at least two different isoforms are presented, to show estrogen-induced switch from one isoform to the other.

Figure 4

Examples of ERβ-specific splicing events. A) Example of alternative splicing in the SGK1 gene. The upper panel shows a schematic representation of two differentially regulated isoforms of the gene (same as those shown in Figure 3 for the same gene), differing in the transcription start site, in the inclusion of the first intron (the gene is encoded by the reverse strand) and in the transcription stop site. The lower panels show a representation of the RNA-Seq reads and junction reads associated with the gene in the different conditions: Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus) without or with E2 stimulation, and wt (parental MCF-7 line) without or with E2 stimulation. B) Example of ERβ-specific alternative promoter usage in the gene PSD3. The left panel shows a heat map with the fold change of all the primary transcripts associated with the gene in: Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the N-terminus); wt (parental MCF-7 cells). The right panels show the logarithm base 10 (log₁₀) FPKM for each of the different transcript start sites (from TSS01 to TSS09) corresponding with the different gene isoforms in the above described cell lines, without (left) or with (right) E2 stimulation.

Figure 5

Correlation between ERβ-specific splicing and ER binding. Circos plot of differential spliced genes (DSGs) common to both ERβ + cell lines which contain at least one ER binding event within a window of 10 kB around the gene. The outer ring shows chromosome ideograms with the relative genes located in their respective chromosomal locations, with the following color code: the genes that have both ERβ and ERα binding are in black; the genes which have only ERβ binding are in red; the genes which have only ERα binding are in blue. The two internal rings represent ERα and ERβ binding events in blue and red, respectively.