Investigation of circular RNA and androgen receptor signalling in cancer: RSK crosstalk and androgen response element variation

Cancer incidence rates continue to increase, and hence there is a need to better understand the molecular mechanisms that regulate disease development and progression. This project focussed on three aspects of cancer biology: circRNA-mediated regulatory networks in oral and prostate cancer, the impact of regulatory single nucleotide polymorphisms (rSNPs) within androgen response elements (AREs) on androgen receptor (AR) activity, and the role of ribosomal S6 kinases (RSKs 1-4) in modulating AR signalling. Computational analysis of publicly available RNA sequencing datasets identified dysregulated circRNAs in oral squamous cell carcinoma (OSCC) and prostate cancer. Differential expression and predicted circRNA-miRNA-mRNA interactions suggested potential miRNA sponging mechanisms involved in cancer-related pathways. In OSCC, hsa_circ_0056440 was strongly upregulated, while hsa_circ_0064977 and hsa_circ_0010794 were identified as candidate regulatory circRNAs in prostate cancer. Functional growth assays in 22Rv1 prostate cancer cells provided evidence that these circRNAs may influence cell proliferation. Reporter assays investigating rSNP-containing AREs demonstrated androgen-dependent transcriptional activation following mibolerone treatment, consistent with AR-mediated regulation. The MAF SNP-containing construct showed reduced androgen-dependent reporter activity, suggesting that this variant may impair AR-mediated transcription. To further investigate potential effects on AR-DNA interactions, bacterial protein production optimisation was performed for the AR DNA-binding domain (DBD), enabling recovery of soluble GST-AR DBD to support future DNA-binding studies. In parallel, the potential crosstalk between AR signalling and RSKs was explored. RSK2 showed the strongest suppressive effect on reporter activity, whereas RSK1 produced the strongest increase in cell proliferation, highlighting potentially isoform-specific effects of RSKs on AR signalling and prostate cancer cell behaviour. Overall, this study used computational and experimental approaches to investigate circRNA regulatory networks, androgen receptor signalling, and associated regulatory variants in cancer, identifying candidates for further functional investigation.