Identification and Characterization of Functionally Distinct Isoforms of Estrogen Receptor Alpha in Breast Cancer

We used long- and short-read RNA sequencing of breast tumor tissue, breast cancer cell lines, and healthy tissues to create a comprehensive annotation of alternative mRNA isoforms of estrogen receptor alpha (ER, gene symbol ESR1) and combined this with functional studies of alternative protein isoforms. The ER is frequently overexpressed in breast cancer and is used in the clinic as a prognostic and treatment-predictive factor. It is also a drug target. The ER is a transcription factor and regulates gene expression in response to estrogen, a hormone that can stimulate breast cell proliferation and contribute to tumor growth. The gene has multiple promoters, and alternative mRNA isoforms have been linked to more aggressive breast tumors, suggesting that the splicing pattern of ER transcripts may influence breast cancer biology and clinical outcome. We cloned and characterized six alternative protein isoforms of ER. They were selected based on expression level in sequencing data, splice junction support, and only contained one alternative splicing event each compared to the full-length ER. Experimental characterization was done in cell lines and included transcription factor activity, alone and upon co-expression with full-length ER, response to the ER-targeting drugs tamoxifen and fulvestrant, as well as subcellular localization. Two alternative isoforms exhibited transcription factor activity, although at lower levels than the full-length ER, while isoforms with larger deletions in the DNA-binding domain or lacking parts of the ligand-binding domain were inactive. One isoform had a dominant negative effect on the activity of the full-length protein. Furthermore, full-length ER and three alternative isoforms were found in both cytoplasm and nucleus, while the remaining isoforms were only present in the cytoplasm. Isoforms capable of entering the nucleus showed increased nuclear localization upon estradiol treatment, with one isoform accumulating significantly higher and one lower than the full-length ER. Co-transfection of full-length ER and isoforms lacking transcription factor activity did not alter their distribution, suggesting a possible role of heterodimerization with full-length ER for the dominant negative isoform in the nucleus. In ongoing experiments, we are studying the regulation, expression, and function of alternative isoforms of ER in cell line models of drug resistance. These findings highlight the complexity and diversity of ER isoforms in breast cancer and suggest that specific isoforms may have distinct functional roles in tumor progression and response to therapy. Our work provides insights into the role of alternative mRNA splicing in breast cancer development and progression. Citation Format: Juliane Albrecht, Carlos Enrique Balcazar, Helena Persson, Völundur Hafstað, Cornelia Börjesson Freitag, Johan Vallon-Christersson, Jari Häkkinen. Identification and characterization of functionally distinct isoforms of estrogen receptor alpha in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A071.