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Towards the Understanding a Metal-Tumour-Metabolism

Periodic Reporting for period 3 - ToMeTuM (Towards the Understanding a Metal-Tumour-Metabolism)

Okres sprawozdawczy: 2021-01-01 do 2022-06-30

Recent advances in understanding the human genome have been made possible due to intensive multidisciplinary cooperation involving the life sciences and technology. Genomics has succeeded in producing complete genome DNA sequences of a number of living organisms, but we are still some way from understanding the difference between the normal and pathological functions of cells and organisms. Currently, attention is directed towards proteomics providing information about protein localization, structure and function, and most importantly, interactions with other proteins.
Metalloproteins are one of the most diverse classes of proteins, with the intrinsic metal atoms providing a catalytic, regulatory and structural role crucial to protein functioning. One of the interesting proteins known as a marker of heavy metal poisoning with potential to be considered as a marker of tumour diseases is metallothionein (MT). In spite of the fact that metal-binding ability of metallothionein has been known for decades, we are for the first time suggesting that MTs are protecting cancer cells and helping them to grow and invade to the surrounding tissue, which makes a tumour more aggressive.
Exploring the role of MT in a mechanism of tumour defence against treatment strategies based on platinum drugs is very challenging task, where there exists a presumption of the fact that MT plays a crucial role in the formation of platinum-based-cytostatics resistance; however, there is still no clear evidence on this. Therefore, multi-instrumental approach covering electrophoretic, mass spectrometric, electrochemical and immunochemical methods will be utilized to test the hypotheses in vitro and then in vivo. In addition, the obtained multi-instrumental data will be further employed as a basis for development and preclinical testing of bio-compatible nanovehicles with smart tuneable properties that could be efficient in management of hard-to-treat tumours with frequently occurring intrinsic chemoresistance to platinum cytostatics.
We have been developing several methods and protocols to detect MTs with special attention paid to distinguishing MTs isoforms and oligomers. The most attention and effort has been paid to mass spectrometric analysis and measurements as well as biological methods including immunological and nucleic acids based protocols. We developed new selective recognition elements based on molecularly imprinted polymers enabling effective MT detection in complex samples. Such approach would enable discrimination between various MT oligomers. New approach of antibody labelling by metal nanoparticles was also introduced leading to significantly simplified and shortened conjugation procedure compared to EDC/NHS strategy. Besides, we succeeded in the first in silico modelling and developed a unique methodology for precise construction of high-resolution protein models with improved rotamer classification, which is of utmost importance for molecular dynamics/mechanics simulations. Moreover, we have been optimizing electrochemical and capillary electrophoretic methods for studying of MTs. This includes advanced methodologies of detection of protein oligomers by using FRET-mediated distance detection. We were also successful in gathering comprehensive proteomics and transcriptomics data that describe a set of cellular properties involved in a chemoresistance of cells to CDDP (including cells with transient MTs up-regulation) and constructed comprehensive molecular maps of possible interactions of MTs with a plethora of biological processes and druggable targets. We also described a participative role of MT-3 on chemoresistance to CDDP through induction of oncogene-induced senescence/cell cycle and apoptosis. The obtained data were validated by functional analyses and published in two publications (others are in preparation). Additionally, we were successful in constructing high precise homology models of MTs, which are currently utilized in homology modelling experiments focused on energetic requirements during MTs oligomerization under different pathophysiological conditions. Preliminary optimization data and findings that are crucial for such models and also for the above mentioned achievements were successfully published in 19 publications.
Suggestion of new and challenging treatment strategy based on silencing of genes for MT to suppress a tumour growth and its protection mechanism is unique. The first part of the project solution succeeded in a development of cutting-edge methodologies suitable for investigation of different aspects of MTs behaviour, including their oligomerization, classification and metal saturation. The computational part of the project succeeded in suggesting and validating novel pipeline for production of high-resolution protein models with precisely optimized rotamers, which will be extensively utilized in the following part of the project, in particular for in silico studies of MTs homo- and heterooligomerization. In addition, pilot molecular biology experiments revealed a highly complex biology of MTs and underpinned their importance of cancer cells and particularly for development of their chemoresistant phenotypes. The published findings will serve as a solid basis for follow-up experiments that could bring novel insights into the cancer biology and could also resulted in development of novel class of highly selective anticancer drugs. We plan to utilize the obtain data to design and develop smart bio-compatible nanovehicles that will be able to systematically inhibit activity of MTs with a parallel production of cytotoxic effect. In future, such modalities could be of utmost interest for a treatment of highly chemoresistant and inoperable tumours.
Vojtěch Adam & his team participate in Researchers Night in 2018
Vojtěch Adam as guest of Science Café in Brussels on February 28,2019