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

Dougba Noel Dago, Claudio Scafoglio, Antonio Rinaldi, Domenico Memoli, Giorgio Giurato, Giovanni Nassa, Maria Ravo, Francesca Rizzo, Roberta Tarallo, Alessandro Weisz

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. 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. 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.