Inhibiting androgen receptor using decoy binding sites as a new treatment option for prostate cancer

The androgen receptor (AR) is a key driver of prostate cancer progression. Although current treatments target AR signalling, resistance frequently develops while AR activity persists, highlighting the need for alternative therapeutic approaches. This study investigated the use of androgen response element (ARE)-based decoy binding sites to inhibit AR signalling using a mutated TAT-STOP plasmid containing indirect palindromic ARE repeats. It was hypothesised that ARE decoys could competitively sequester AR and reduce AR-driven gene expression irrespective of resistance mechanisms, including AR mutations and splice variants. Site-directed mutagenesis was used to introduce a premature stop codon into the TAT-GRE-E1B-LUC plasmid, generating the TAT-STOP construct. Reporter assays in COS1 cells demonstrated dose-dependent inhibition of AR activity following co-transfection with TAT-STOP, reducing reporter activity to approximately 20% of MIB-treated controls at 1000 ng. Western blot analysis showed that reduced reporter output was not solely explained by changes in AR protein levels. To assess therapeutic relevance, TAT-STOP was evaluated in 22RV1 cells, a model of therapy-resistant prostate cancer. qPCR analysis demonstrated reduced expression of the AR-regulated genes NDRG1 and KLK2, while IncuCyte imaging showed suppression of androgen-induced cell proliferation. DNA nanoflowers were also explored as a potential delivery strategy, although further optimisation is required. Overall, these findings demonstrate that TAT-STOP acts as an effective decoy to inhibit AR signalling and AR-regulated gene expression, supporting ARE-based decoy strategies as a potential therapeutic approach for AR-positive prostate cancer.