Modulation of PSMA splice variants using splice switching oligonucleotides in prostate cancer cells Public Deposited

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  • March 21, 2019
  • Williams, Tiffany L.
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
  • The Prostate Specific Membrane Antigen (PSMA), a product of the GCPII gene, is highly expressed as a largely extracellular membrane-anchored protein in malignant prostate tissues and in non-prostatic tumor neovasculature. Alternatively spliced variants of PSMA include the PSM', PSMA 6, and PSMA 18 transcripts. PSM' is produced by use of an alternate upstream 5' splice site in exon 1 and produces a cytoplasmic localized protein missing PSMA's transmembrane domain. The PSMA/PSM' splice ratio changes from predominately PSM' in normal and benign prostate tissues to predominately PSMA in prostate cancer. Variants PSMA 6 and PSMA 18 are produced by exon exclusion of exons 6 and 18, respectively, by the splicing machinery. If properly translated, PSMA 6 protein retains its membrane targeting, but lacks PSMA's enzymatic and dimerization domains; PSMA 18 lacks only the dimerization domain. Both exon exclusion products should not produce functional proteins. Extensive studies have shown the ability of antisense oligonucleotides to target specific splicing elements in pre-mRNA to alter alternative splicing patterns by interfering with splicing machinery sequence recognition. Therefore, iii antisense based splice switching oligonucleotides (SSOs) were targeted at or near the 5' splice sites of exons 1, 6, and 18 to alter PSMA splicing patterns by decreasing full length PSMA transcript levels and increasing PSM', PSMA 6, and PSMA 18 variant levels, respectively in LNCaP prostate cancer cells. We hypothesized that altering the PSMA/PSM' splicing ratio would affect specific properties of LNCaP malignancy including cellular proliferation and apoptosis levels. Also, splice switching to PSMA 6 and PSMA 18 variants while causing a decline in PSMA would aid identifying effects produced by a decline in PSMA alone. Studies in this dissertation demonstrate that treatment of LNCaP cells with SSOs did modulate splicing of PSMA pre-mRNA from the full length PSMA splice variant to three splice variants: PSM', PSMA 6, and PSMA 18 variants. Application of SSOs decreased membrane PSMA mRNA and protein levels and increased PSM', PSMA 6 and PSMA 18 transcript levels. As a result, PSM' protein was translocated to the cytoplasm while switching to PSMA 6 and PSMA 18 downregulated PSMA expression. NAALDase assays confirmed splice switching at the protein functional level and showed that PSM' retained enzymatic activity. However, we were not able to determine any specific effects of splice switching on LNCaP malignancy as detected by cellular proliferation and apoptosis.
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  • Kole, Ryszard
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  • University of North Carolina at Chapel Hill
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