Replication-selective oncolytic adenoviruses represent a promising anticancer approach with proven efficacy in cancer cell lines and tumour xenografts in vivo. Anti-tumour efficacy, both in preclinical studies and clinical trials, was significantly improved in combination with chemotherapeutics in numerous cancers, including prostate cancer. It has been established that expression of the viral E1A gene is essential for the enhancement of cell killing in combination with cytotoxic drugs. The overall goal of this project is to identify specific E1A gene regions involved in the sensitization to the cytotoxic drugs mitoxantrone and docetaxel, the current standard of care for late stage prostate cancers, to enable the development of improved anti-cancer therapies. Specific regions in the E1A proteins bind to numerous cellular factors to regulate the host cell function and the viral life cycle, including the p300, p400 and pRb family proteins. This work was aimed at determining the mechanisms involved in the synergistic cell killing in prostate cancer cells in response to the combination of the replication-selective (oncolytic) mutant AdΔΔ with cytotoxic drugs. Previous findings suggested an enhancement of drug-induced apoptosis. I found that the small E1A12S protein, unable to induce viral replication, is sufficient to sensitize the prostate cancer cells, 22Rv-1 (AR+), and PC-3 and DU145 (AR-), to drugs. The non-replicating AdE1A12S-mutant AdE1A1104 (defective in p300-binding) could not sensitize the cells while mutants with intact E1A-p300 binding (AdE1A12S, AdE1A1102, AdE1A1108) and defective in p400- (AdE1A1102) or pRb-binding (AdE1A1108) potently sensitized all tested cell lines. In fact, all mutants except AdE1A1104 potently synergised with mitoxantrone and docetaxel to kill the prostate cancer cells. When comparing the non-replicating E1A12S mutants with the corresponding replicating E1A-deletion mutants (expressing E1A12S and 13S) synergy was demonstrated with all replicating mutants except dl1104, which caused an additive effect with mitoxantrone. We hypothesised that the synergistic cell killing is the result of pathway convergence through E1A-p300 and mitoxantrone-activated DNA-damage/apoptosis events. To address this I employed an extensive miRNA array screen to identify potential pathways. Several miRNAs were found to be differentially regulated in response to the combination of AdE1A12S with mitoxantrone compared to each single agent treatment. The majority of these miRNAs are reported to be part of cell death and survival pathways (e.g. apoptosis and autophagy) and to be differentially regulated in prostate cancer. To further investigate the role of these pathways, I determined changes in expression levels of key proteins that had previously been suggested to be targeted by the identified miRNAs, thereby preventing translation of the respective mRNAs. The greatest changes in protein levels in response to AdE1A12S and mitoxantrone were observed for Bcl-2, p-Akt, LC3BII and p62. Finally, I verified similar mechanisms of action when the oncolytic AdΔΔ was combined with mitoxantrone under synergistic conditions. These findings will direct future investigations aimed at dissecting the mechanisms of action for virus-induced sensitization to cytotoxic drugs and may aid in the development of improved therapies for prostate cancer by design of novel oncolytic mutants and combination strategies and/or identification of targets for small molecules inhibitors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:667111 |
| Date | January 2013 |
| Creators | Maya-Pineda, Héctor Rubén |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/8482 |
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