The effect of hypoxia on alternative splicing in prostate cancer cell lines

Hypoxia is defined as the state in which the availability or delivery of oxygen is insufficient to meet tissue demand. It occurs particularly in aggressive, fast-growing tumours in which the rate of new blood vessel formation (angiogenesis) cannot match the growth rate of tumour cells. Cellular stresses such as hypoxia can cause cells to undergo apoptosis; however some tumour cells adapt to hypoxic conditions and evade apoptosis. Tumour hypoxia has been linked to poor prognosis and to greater resistance to existing cancer therapies. This thesis provides evidence that alterations in alternative splicing patterns of key genes is one method tumour cells adapt to hypoxia. This study confirms a hypoxic-induced change in the alternative splicing of carbonic anhydrase IX (CA IX) following 1% oxygen treatment. CA IX is one of the best studied hypoxia markers, involved in maintaining an intracellular pH that favours tumour cell growth. Furthermore, evidence is provided here that in PC3 cells the regulation of CA IX splicing involves the SAFB1 and PRPF8 splice factors. Additionally, SAFB1 expression is shown to decrease in hypoxia. This study further demonstrates that alternative splicing patterns of previously documented cancer-associated genes are altered in hypoxia. PCR analysis showed that hypoxia significantly altered the alternative splicing of apoptotic-associated genes: caspase-9; Mcl-1; Bcl-x; survivin. The expression of the pro-apoptotic isoforms of the first two genes, and the anti-apoptotic isoforms of the latter two genes were favoured by hypoxia. Furthermore, high-throughput PCR analysis provided evidence of significant changes in the alternative splicing of several other cancer-associated genes in hypoxia: APAF1; BTN2A2; CDC42BPA; FGFR1OP; MBP; PTPN13; PUF60; RAP1GDS1; TTC23; UTRN. Most notably, the pro-oncogenic isoforms of APAF1, BTN2A2 and RAP1GDS1 were favoured in hypoxia. The majority of alternative splicing changes were found in the PC3 cell line. However changes in alternative splicing patterns that mirrored those in the PC3 cell line were also found in the VCaP (CDC42BPA, RAP1GDS1 and UTRN) and PNT2 (BTN2A2, CDC42BPA, FGFR1OP and TTC23) cell lines. The mRNA expression of splice factors (SRSF1, SRSF2, SRSF3, SAM68, HuR and hnRNP A1) and splice factor kinases (CLK1 and SRPK1) were shown to significantly increase in hypoxia. Subsequent experiments provided evidence that CLK1 and SRSF1 protein expression also increased in hypoxia. The phosphorylation of SRSF4 and SRSF5 were demonstrated to increase in hypoxia. However, the phosphorylation of SRSF6 was not. In addition, siRNAs and chemical inhibitors of CLK1 (TG003) and SRPK1 (SPHINX) were used to assess the effect of these splice factor kinases on the subsequent splicing of cancer-associated genes. There were no significant changes to splicing found with SRPK1 siRNA knockdown or SPHINX treatment. However CLK1 siRNA knockdown and TG003 treatment demonstrated a shift in FGFR1OP splicing that mirrored the effect of hypoxia on FGFR1OP splicing. This suggests that CLK1 activity is inhibited in hypoxia. Furthermore, in contrast to previous research CLK1 was found to be localised to the cytoplasm in both normoxia and hypoxia in the PC3 cell line. This work has uncovered factors and provided an insight into mechanisms that are involved in alternative splicing changes in hypoxia in mammalian cell lines. It is hoped that these novel research findings will aid in the understanding of how cells adapt to hypoxia especially in regards to alternative splicing, and may offer future therapeutic targets in hypoxic tumours.