Alternative promoters and splicing create multiple functionally distinct isoforms of oestrogen receptor alpha in breast cancer and healthy tissues

Abstract

Background: Oestrogen receptor alpha (ER) is involved in cell growth and proliferation and functions as a transcription factor, a transcriptional coregulator, and in cytoplasmic signalling. It affects, for example, bone, endometrium, ovaries and mammary epithelium. It is a key biomarker in clinical management of breast cancer, where it is used as a prognostic and treatment-predictive factor, and a therapeutical target. Several ER isoforms have been described, but transcript annotation in public databases is incomplete and inconsistent, and functional differences are not well understood.

Methods: We have analysed short- and long-read RNA sequencing data from breast tumours, breast cancer cell lines, and normal tissues to create a comprehensive annotation of ER transcripts and combined it with experimental studies of full-length protein and six alternative isoforms.

Results: The isoforms have varying transcription factor activity, subcellular localisation, and response to the ER-targeting drugs tamoxifen and fulvestrant. Antibodies differ in ability to detect alternative isoforms, which raises concerns for the interpretation of ER-status in routine pathology.

Conclusions: Future work should investigate the effects of alternative isoforms on patient survival and therapy response. An accurate annotation of ER isoforms will aid in interpretation of clinical data and inform functional studies to improve our understanding of the ER in health and disease.

Keywords: alternative splicing; breast cancer; isoforms; oestrogen receptor.

FIGURE 1

Heatmap illustrating the expression of splice junctions used in ESR1 transcripts among 3478 breast tumours with junctions sorted by increasing genomic coordinates from top to bottom. Only junctions with an expression of at least 2 counts per million reads (cpm) in at least 2 samples are shown. Tumours are labelled according to molecular subtype, grade and status for ER, progesterone receptor (PR), the tyrosine kinase receptor ERBB2 (also known as HER2) and positive lymph nodes at surgery (LN). ER, PR and HER2 status were defined by immunohistochemistry. Exons are marked in green if they are annotated in both GENCODE and RefSeq databases, yellow for GENCODE only, blue for RefSeq only and purple for novel exons.

FIGURE 2

Schematic illustration of the transcript structure for the most common isoforms in the long‐read sequencing data. These are isoforms that together encompass 80% of the full‐length reads for each primer pair. Annotated transcripts from GENCODE V36 and RefSeq release 109 are shown for comparison.

FIGURE 3

(A) Schematic illustration of the domain structure of the selected isoforms. (B) Isoform transcription factor activity on a dual‐luciferase reporter with firefly luciferase controlled by a 3xERE‐containing promoter. Luminescence from firefly luciferase was normalised to Renilla luciferase, and the activity was expressed relative to the baseline value for the empty pEGFP‐C1 vector with vehicle. ERfl = full‐length oestrogen receptor alpha (ER), vehicle control = 0.1% ethanol, E2 = oestradiol and 4OHT = 4‐hydroxytamoxifen. Bars indicate the mean of n = 3 replicates ± standard deviation. Two‐tailed Student's t‐test was used to test for significant differences in relative luciferase activity between each isoform and ERfl matched by treatment. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = non‐significant.

FIGURE 4

Subcellular fractionation followed by western blotting to determine the relative distribution between cytoplasm and nucleus for each isoform using (A) an N‐terminal or (B) a C‐terminal anti‐ESR1 antibody. (C) Tubulin is included as a control for the purity of the cytoplasmic fraction and (D) lamin B2 for the nuclear fraction. (E) Quantitation of nuclear fraction in vehicle control and after 30 min treatment with 10 nM E2. Bars indicate the mean of n = 3 replicates ± standard deviation. T = total lysate, C = cytoplasmic fraction and N = nuclear fraction, vehicle control = 0.1% ethanol, E2 = oestradiol, ERfl = full‐length oestrogen receptor alpha (ER). **p < 0.01 and ***p < 0.001.

FIGURE 5

(A) HepG2 cells transfected with plasmids encoding full‐length ER or one of six alternative isoforms were treated for 6 and 24 h with vehicle control (0.01% DMSO) or 1 μM fulvestrant. ER expression was assessed by western blotting using N‐terminal and C‐terminal ER antibodies, and tubulin was used as a control for equal loading. (B) Quantitation of ER expression after 24 h treatment with vehicle or 1 μM fulvestrant by western blotting with an N‐terminal ER antibody. Band intensities were normalised to tubulin and expressed relative to the vehicle control for each isoform individually. Bars indicate the mean of n = 3 replicates ±standard deviation. **p < 0.01 and ns = not significant. pEGFP‐C1 = empty vector, ERfl = full‐length oestrogen receptor alpha (ER), kDa = kilo Dalton.