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Regulation of ETS1 Expression in Prostate Cancer Cells

Hope, Danika (2014) Regulation of ETS1 Expression in Prostate Cancer Cells. Honours thesis, Murdoch University.

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ETS transcription factors are widely expressed during development and in adult tissues and regulate a variety of cellular processes including cell proliferation, migration and invasion. In prostate cancers, ETS factors are commonly abnormally expressed, with up to 70% of prostate tumours containing chromosomal rearrangements that disrupt genes encoding the ETS factors, ERG, ETV1, ETV4 and ETV5. The prototypical ETS transcription factor, ETS1 is overexpressed in ~70% of prostate cancers, and this is correlated with higher grade tumours, more rapid progression to castrate-resistant disease and poor patient prognosis. ETS1 overexpression in prostate tumours is not associated with chromosomal rearrangements and mechanisms underlying its elevated expression, including sequence alterations, abnormal turnover or altered transcriptional regulation are unknown.

To identify mutations in the ETS1 coding region, RNA extracted from 45 prostate tumour specimens was reverse transcribed, PCR-amplified in 4 overlapping fragments and sequenced by Sanger sequencing. Although no mutations were identified, 32 specimens were heterozygous or homozygous for the common c.1011A>G polymorphism and single specimens had the rarer c.789C>T and c.816A>G polymorphisms. These findings indicated that if present in prostate tumours, ETS1 mutations are rare or occur in a low proportion of cells. ETS1 mRNA levels in the prostate cancer cell lines LNCaP, DU145 and PC-3 were determined using RT-qPCR, with ETS1 expression ~129 fold higher in DU145 cells and ~17 fold higher in PC-3 cells compared to LNCaP cells. Consistent with these findings, ETS1 protein levels investigated by western blotting were found to be higher in DU145 and PC-3 compared to LNCaP cells. ETS1 protein turnover in the cell lines was estimated by western blotting following 0-24 hours of treatment with the protein synthesis inhibitor, cycloheximide, from which the half-life of ETS1 protein in DU145 and PC-3 cells was estimated to be 4-6 hours. In contrast, the half-life of ETS1 protein in LNCaP cells was >24 hours, indicating that although ETS1 expression was low, the protein was very stable.

Bioinformatics analysis of the ETS1 3’untranslated region (3’UTR) identified two putative androgen responsive elements at 931-949 and 2911-2929 in the 3’UTR and 22 miRNA potential binding sites including miR-125b and miR-221/222 which are up regulated in prostate cancer and miR-101, miR-145 and miR-200bc, which are downregulated in prostate cancer. The predicted mRNA folding structure of the ETS1 3’UTR identified that the putative transcription factor and miRNA binding sites were located in stem-loop regions, which would facilitate access by regulatory factors. Analysis of the 5’ETS1 gene promoter identified 11 putative androgen responsive elements and a large CpG island flanking the transcription start site suggesting that DNA methylation may contribute to the regulation of ETS1 expression. Initial studies using the methyl transferase inhibitor, 5-Aza-2’-deoxycytidine and the histone deacetylate inhibitor, Trichostatin A did not indicate contributions of DNA hypermethylation or histone modification to the low ETS1 protein levels in LNCaP cells, however further investigation may identify ETS1 promoter methylation in human prostate or prostate tumours. Findings of this study indicate complex mechanisms of regulation of ETS1 expression including transcriptional control and modification of mRNA and protein turnover which may each contribute to the upregulation of ETS1 levels in human prostate tumours.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Bentel, Jacky, Thomas, Marc and Mead, Robert
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