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Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secon...

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Autores principales: Bowler, Elizabeth, Porazinski, Sean, Uzor, Simon, Thibault, Philippe, Durand, Mathieu, Lapointe, Elvy, Rouschop, Kasper M. A., Hancock, John, Wilson, Ian, Ladomery, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879922/
https://www.ncbi.nlm.nih.gov/pubmed/29606096
http://dx.doi.org/10.1186/s12885-018-4227-7
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author Bowler, Elizabeth
Porazinski, Sean
Uzor, Simon
Thibault, Philippe
Durand, Mathieu
Lapointe, Elvy
Rouschop, Kasper M. A.
Hancock, John
Wilson, Ian
Ladomery, Michael
author_facet Bowler, Elizabeth
Porazinski, Sean
Uzor, Simon
Thibault, Philippe
Durand, Mathieu
Lapointe, Elvy
Rouschop, Kasper M. A.
Hancock, John
Wilson, Ian
Ladomery, Michael
author_sort Bowler, Elizabeth
collection PubMed
description BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. METHODS: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. RESULTS: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. CONCLUSIONS: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12885-018-4227-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-58799222018-04-04 Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells Bowler, Elizabeth Porazinski, Sean Uzor, Simon Thibault, Philippe Durand, Mathieu Lapointe, Elvy Rouschop, Kasper M. A. Hancock, John Wilson, Ian Ladomery, Michael BMC Cancer Research Article BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. METHODS: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. RESULTS: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. CONCLUSIONS: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12885-018-4227-7) contains supplementary material, which is available to authorized users. BioMed Central 2018-04-02 /pmc/articles/PMC5879922/ /pubmed/29606096 http://dx.doi.org/10.1186/s12885-018-4227-7 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Bowler, Elizabeth
Porazinski, Sean
Uzor, Simon
Thibault, Philippe
Durand, Mathieu
Lapointe, Elvy
Rouschop, Kasper M. A.
Hancock, John
Wilson, Ian
Ladomery, Michael
Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title_full Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title_fullStr Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title_full_unstemmed Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title_short Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells
title_sort hypoxia leads to significant changes in alternative splicing and elevated expression of clk splice factor kinases in pc3 prostate cancer cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879922/
https://www.ncbi.nlm.nih.gov/pubmed/29606096
http://dx.doi.org/10.1186/s12885-018-4227-7
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