Thesis presented 17 December 2015

Abstract:
The major goal of the cancer therapy is to specifically suppress malignant neoplasm without detriment to normal cells. Modern therapeutic approaches exploit characteristic hallmarks of cancer cells, which render them susceptible toward certain types of insult such as DNA damage, mitotic and oxidative stress. Recently ubiquitin-proteasome system (UPS) appeared as one of the principal cancer targets. In this work we describe a systematic approach for screening of UPS based on cascade organization. We have evaluated the effect of RNAi knockdown of individual UPS components on viability of prostate cancer (PCa) cells with major focus on TMPRSS:ERG-positive cell line, VCaP, as a model of prevalent phenotype of prostate cancer. Seven genes have been identified to be particularly important for the functioning of PCa cells.
The prevalence of the components of CRL/NEDD8 pathway in the hits (four out of seven) suggested the importance of neddylation for PCa biology. We found that partial inhibition of neddylation triggered transcriptional reprogramming of VCaP cells leading to cell quiescence and inhibition of proliferation-dependent apoptosis. This was a result of re-activation of AR program and induction of differentiation-like state. We conclude that CRL/NEDD8 pathway regulates cancer transcriptional network that underlies cancer cells plasticity. In parallel, we observed that neddylation inhibition changed membrane properties and morphology of VCaP cells. This was accompanied by dose-dependent changes in the level and the localization of several membrane-associated proteins. We thus conclude that CRL/NEDD8 pathway might be involved in sorting/trafficking of membrane proteins. This knowledge could help to find better treatments for TMPRSS2:ERG-positive cancers.
Finally, we observed that neddylation inhibition changed membrane properties and morphology of VCaP cells. This was accompanied by dose-dependent changes in the level and the localization of several membrane-associated proteins, including occludin, N-cadherin, paxillin and FAK. We thus conclude that CRL/NEDD8 pathway might be involved in sorting/trafficking of membrane proteins. This part of the work requires further investigation, as understanding of the underlying mechanisms is of general importance and may uncover a new role of CRL/NEDD8 pathway in regulation of cellular functions.

General conclusions:
1. We have obtained a comprehensive dataset on the involvement of all human E1-E2 UPS components in the regulation of viability of PCa cells, represented by five different cell lines.
2. Our work has revealed new potential drug targets for PCa treatment: UBE2U and CAND1.
3. We have demonstrated the role of CRL/NEDD8 pathway in the regulation of cancer cell plasticity and morphology.

Keywords:
Prostate cancer, cancer therapy, ubiquitin-proteasome system, UPS, RNAi.

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