Background: Metastatic melanoma remains a life-threatening disease because most tumours develop resistance in response to conventional treatment. Even though targeted drugs represent promising therapeutics, the clinical outcome remains poor, with high relapse rates coinciding with pronounced metastatic outgrowth. Therefore, successful therapy is still challenging, and alternative treatment options are demanded as first or second line therapy to overcome acquired resistance. In this context, cell death induction by the tumour-selective death ligand TRAIL (Tumour necrosis factor-Related Apoptosis-Inducing Ligand) might serve as an alternative single or co-treatment option. Unfortunately, melanoma cells were shown to stay largely resistant against conventional TRAIL treatment and the first-generation TRAIL-based therapeutics failed in clinical trials due to the limited therapeutic activity in patients. To overcome this therapeutic limitation, a second-generation hexavalent TRAIL receptor agonist IZI1551 has been developed showing increased bioactivity thereby enhancing the cytotoxic effects on cancer cells. Questions and Hypothesis. We hypothesized that cells that do not receive the full but only a sublethal drug-dose may not only be responsible for drug resistance but may also confer tumour relapse and metastatic outgrowth. By switching the signal transduction from pro-apoptotic to anti-apoptotic and pro-survival, these cells may foster an aggressive phenotype with enhanced proliferation and migration. Therefore, an in depth understanding of the underlying mechanisms of emerging drug resistance that may additionally trigger secondary metastasis formation is required to identify new therapeutic targets and alternative treatment options. Methodology. Melanoma cell lines were conditioned to the TRAIL receptor agonist IZI1551 and the expression of members of the anti-apoptotic NFκB and MAPK pathways as well as of the TRAIL receptor-driven apoptotic pathway were investigated by semi-quantitative Western-blot analysis. Protein expression/activation data of parental IZI1551-sensitive versus conditioned IZI1551-resistant melanoma cells were implemented into a network topology derived from literature. A Dynamic Bayesian Network (DBN) model was combined with a sophisticated regularisation strategy resulting in sparse and context-sensitive networks to identify cell line-specific deregulations within the signalling network. Predictions of the model were confirmed by siRNA-mediated knock down. Enhanced proliferation and migration of resistant-cells were investigated by proliferation, clonogenic, and scratch assays. Following the 2D studies, migration and invasion were monitored by confocal microscopy in 3D migration/invasion assays and 3D spheroids models. Expression of pro-metastatic cell adhesion molecules was evaluated by flow cytometry. In order to identify potential regulators of the aggressive phenotype, quantitative transcriptome analysis (RNA-seq) was performed. The therapeutic outcome of the new identified treatment options with IZI1551 alone or in combination with Smac mimetics or bortezomib was evaluated by cell death detection ELISA. Results. In this thesis, IZI1551 was shown to induce pronounced apoptotic cell death in melanoma cells compared to mutation specific targeted kinase inhibitors, as being used in the clinic. Comparing IZI1551-sensitive to IZI1551-resistant melanoma cells, the DBN model accurately predicted activation of NFκB in concert with upregulation of the anti-apoptotic protein XIAP to be the key mediator of IZI1551 resistance. Moreover, XIAP was identified to serve as a potential biomarker for TRAIL responsiveness. According to these findings, human melanoma cell lines were re-sensitised to TRAIL in vitro by co-application of the IAP antagonists Smac mimetics as well as bortezomib, a proteasome inhibitor currently used in cancer treatment. In addition, by triggering survival instead of apoptotic signalling pathways, resistant cells caused an aggressive phenotype with enhanced proliferation and migration/invasion into 3D collagen matrices, coinciding with upregulation of the cell adhesion molecules MelCAM and αVβ3 integrin, which are known to promote tumour progression and metastasis. Combining in silico studies of RNA-seq and protein expression data, YAP, an intracellular transducer of mechanical stimuli, and its upstream regulator FAK, a component of the focal adhesion complex, were identified as key promoters of proliferation and migration. Conclusions. Identification of new biomolecular markers or targets combining experimental and computational approaches is a promising avenue to assess the effects of drug combinations and to identify responders to selected combination therapies. In this thesis, IZI1551 was identified as an alternative treatment option for metastatic melanoma. Our data also suggest that XIAP expression may serve as a potential predictive marker for the sensitivity of tumour cells to TRAIL-induced apoptosis. Above this, we demonstrated that three alternative treatment options with IZI1551 in combination with Smac mimetics, bortezomib or FAK inhibitors may represent a promising approach for the treatment of TRAIL-resistant melanomas and to prevent undesired metastatic outgrowth.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:77977 |
| Date | 10 February 2022 |
| Creators | Del Mistro, Greta |
| Contributors | Kulms, Dagmar, Mansfeld, Jörg, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1038/s41540-018-0075-y |
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