Constitutive expression of the AR corepressor, Hey1, from a nonreplicating adenovirus, sensitises prostate cancer cells to chemotherapeutic agents through multiple pathways.

Abstract

Androgen receptor (AR) cell signalling is active in most castration-resistant prostate cancer (PCa) tumours and suppression is hypothesized to impede cell proliferation. Hey1, a corepressor of AR is being investigated as a therapeutic transgene for late-stage PCa. A replication-defective recombinant adenovirus deleted for E1 and E3 and expressing Hey1 under a CMV promoter was constructed (Ad5Hey1). A dual luciferase reporter system demonstrated that Ad5Hey1 repressed AR activity in a dose dependent manner in miboleronestimulated 22Rv1 cells. Ad5Hey1 was cytotoxic in both AR-positive 22Rv1 and LNCaP and AR-negative DU145 cells. The doses required to kill 50% of cells (EC50) were comparable to those of AdE1A12S expressing the cytotoxic E1A12S gene from an identical vector. The mechanisms of Ad5Hey1-induced cell killing were investigated in 22Rv1 and DU145 cells. Using RNA interference towards AR or p53 in 22Rv1 cells we concluded both proteins were required for optimal cell killing by Ad5Hey1. In DU145 cells, with non-functional p53, Ad5Hey1 decreased levels of phospho- STAT3 and total STAT3 suggesting Ad5Hey1 might inhibit STAT3 signalling while the JAK1/2 inhibitor, AZD1480 was ineffective at sensitising DU145 cells to Ad5Hey1. Preliminary data therefore suggests Ad5Hey1 may interfere with JAK/STAT signalling in these cells. Cell-killing efficacy with Ad5Hey1 in combination with cytotoxic drugs currently used in the clinic for the treatment of late-stage PCa, mitoxantrone and docetaxel, resulted in a synergistic enhancement of cell death in 22Rv1 and DU145 cells. LNCaP cells were also sensitised to the drugs. Characterisation of the mode of cell killing demonstrated augmented mitochondrial membrane depolarisation and caspase-3 activation when combined with docetaxel in all cell lines and with mitoxantrone in 22Rv1 and LNCaP cells, typical of apoptotic death. In DU145 cells, the combination of Ad5Hey1 with mitoxantrone decreased the proportion of apoptotic cells suggesting cells are dying by alternative cell death mechanisms. In this thesis I have demonstrated that Ad5Hey1 potently eliminates PCa cells both in the presence and absence of functional AR or p53, and that cell killing is 6 improved in combination with cytotoxic drugs. I demonstrate that the mechanisms by which Ad5Hey1 acts as a cell death enhancer is mainly through cooperation with drugs on apoptotic pathways while other factors such as inhibition of survival are also involved. In conclusion, these data suggest that it is feasible to develop a future replication-selective adenovirus expressing Hey1 as a cytotoxic transgene to improve antitumour efficacy in vitro and in vivo, especially in combination with apoptosis-inducing drugs