A study to investigate the expression, structure and function of alternatively spliced progesterone receptor variants in breast cancer cells

Abstract

The progesterone receptor (PR) gene consists of eight exons and encodes the functionally distinct PR-A and B isoforms. PR alternative splicing events involving deletion of internal exons or intron retention have been reported, potentially generating proteins which lack various internal functional domains or are N-terminally truncated. PR status in breast cancer may be predictive of the efficacy of endocrine therapy and is measured using N-terminally targeted antibodies which detect both PR-A and B. In this study PR mRNA expression was assessed in breast cancer (MCF-7, T47-D and MDA-MB-231) and non-tumourigenic breast (MCF-10A) cell lines using an RT-PCR based gene walking assay. PR mRNA resulting from alternative splicing was detected in reportedly PR negative MDA-MB-231 cells using this assay. These mRNA could encode the low molecular weight nuclear and cytoplasmic PR proteins which are detected in this cell line using the C-terminal PR antibody C19. Ligand blotting, co-immunoprecipitation and DNA affinity precipitation assays demonstrated the ability of these proteins to bind progesterone, interact with the PR nuclear co-factor PSF, dimerise and bind DNA; thus potentially to function as ligand activated nuclear transcription factors. Validation studies using the gene walking assay demonstrate that alternatively spliced PR mRNA was also present in breast tumours originally characterised using N-terminal antibodies as being both PR+ and PR-. Using a novel non N-terminal PR antibody developed in this study, the nuclear PR status differed from the original status for 2 of 14 tumours examined, and cytoplasmic PR was detected. The results presented in this thesis suggest that PR undergoes extensive alternative splicing, generating potentially functional isoforms, and that expression of variant PR isoforms may affect the PR status of a tumour as determined using antibodies targeting different epitopes. PR exons 4 and 6 are flanked by consensus 5′ and 3′ splice sites and contain cryptic 3′ splice sites, as well as potential binding sites for a range of RNA binding splicing factors. siRNA knockdown of the individual SR proteins SRSF1, SRSF2, SRSF5 and SRSF6 identified potentially antagonistic roles for SRSF1 and SRSF2 in influencing the inclusion/skipping of PR exons 4 and 6, and also for SRSF6 in regulating exon 6 splicing.