The oncolytic adenoviral AdΔΔ mutant sensitizes prostate cancer cells to mitoxantrone by promoting apoptosis and attenuating autophagy
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Prostate cancer (PCa) is the second most common cause of cancer-related deaths in men in the Western world. Advanced PCa is initially managed by anti-androgen therapy however, resistance frequently develops resulting in progressive metastatic disease. The current standard of care for hormone-insensitive PCa includes the cytotoxic drugs docetaxel and mitoxantrone although resistance rapidly develops to all available therapies. We demonstrated that the replication-selective oncolytic adenoviral mutant AdΔΔ enhanced drug-induced cell killing in several preclinical cancer models. AdΔΔ is deleted in the viral E1ACR2 and E1B19K, to prevent pRb-binding and enhance drug-mediated apoptotic cell killing, respectively. In drug-insensitive PCa tumour-xenografts, in vivo administration of AdΔΔ greatly enhanced drug-mediated tumour regression. The aim of my thesis project was to investigate the role of apoptosis and pro-survival pathways, including drug-induced autophagy, in AdΔΔ-mediated drug-sensitisation. I have demonstrated that autophagy was activated in a dose-dependent manner in response to mitoxantrone in the human PCa cell lines PC3, PC3M and 22Rv1. Low doses of mitoxantrone (<EC50-values) caused initiation of autophagy, determined as increased conversion of LC3I to LC3II and increased number of acidic vesicles, indicating autophagosome formation. At higher doses degradation of p62 was also observed, suggesting autophagosome fusion with the lysosome. AdΔΔ attenuated the drug-induced activation of autophagy by restoring basal LC3II/I ratios, and increasing apoptosis, determined as increased PARP-cleavage and mitochondrial depolarization. The autophagyinducer rapamycin prevented AdΔΔ-mediated sensitization in PC3 cells increasing mitoxantrone EC50-values 3-fold and attenuating apoptosis induction. In contrast, the autophagy-inhibitor chloroquine further sensitized PC3 and 22Rv1 cells to the combinationtreatment, decreasing mitoxantrone EC50-values by 40% and increasing apoptotic cell death. Atg7 is a key-factor in the autophagy pathway and siRNA-mediated knockdown prevented increases in LC3II/I ratios. In siAtg7-transfected PC3 cells mitochondrial depolarization was further promoted in combination-treated cells, similar to the results with chloroquine. The cellular Bcl-2-protein has important roles as a mediator of both anti-apoptotic and antiautophagic functions. In cells transfected with siBcl-2 the LC3II/I ratios increased and AdΔΔ- mediated sensitization to mitoxantrone was prevented, indicating initiation of autophagy. In addition, mitoxantrone-induced degradation of Bcl-2 was attenuated by AdΔΔ infection, suggesting stabilization of the protein. The mechanisms for the AdΔΔ-mediated increases in cell killing were also demonstrated in 3-dimensional co-culture models of PC3 or 22Rv1 in combination with prostate stromal cells and extracellular matrix proteins using confocal microscopy. These data demonstrate that AdΔΔ attenuates drug-induced cell survival/rescue and promotes elimination of cancer cells through apoptosis and viral lysis, and that Bcl-2 was essential for the sensitisation.
AuthorsAguirre, Hernandez, Carmen
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