Periodic Reporting for period 4 - ToMeTuM (Towards the Understanding a Metal-Tumour-Metabolism)
Okres sprawozdawczy: 2022-07-01 do 2023-02-28
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 toward spatial and structural 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 intrinsic metal atoms providing a catalytic, regulatory and structural role crucial to protein functioning. One of these interesting proteins, metallothionein (MT), is known as a marker of heavy metal poisoning and could be considered a marker of tumor diseases. Despite the fact that the metal-binding abilities of MTs have been known for decades, we are the first to suggest that MTs not only protect cancer cells but also help them grow and invade the surrounding tissue, which makes a tumor more aggressive.
Exploring the role of MTs in a mechanism of tumor defense against treatment strategies based on platinum drugs is a very challenging task. There is a presumption that MT plays a crucial role in the formation of platinum-based cytostatic resistance; however, there is still no clear evidence on this phenomenon. Therefore, a multi-instrumental approach covering electrophoretic, mass spectrometric, electrochemical and immunochemical methods was utilized to test the proposed hypotheses in vitro and then in vivo. In addition, the obtained multi-instrumental data were further employed as a basis for the development and preclinical testing of biocompatible nanovehicles with smart tunable properties that could be efficient in the management of hard-to-treat tumors with frequently occurring intrinsic chemoresistance to platinum cytostatics. With this consideration in mind, the project ToMeTuM yielded a plethora of exceptional results covering novel insights into the interactions of MTs with metals, in silico tools to analyze these interactions and the construction of high-resolution 3D models of proteins that, with the advent of computational biology and chemistry, will help the community better understand the role of MTs in healthy vs. cancer cells. Furthermore, a variety of biological experiments conducted during the project revealed the importance of different isoforms of MTs for cancer aggressiveness, angiogenesis and chemoresistance to chemotherapeutics, which is of utmost importance for cutting-edge precision medicine.
Metalloproteins are one of the most diverse classes of proteins, with intrinsic metal atoms providing a catalytic, regulatory and structural role crucial to protein functioning. One of these interesting proteins, metallothionein (MT), is known as a marker of heavy metal poisoning and could be considered a marker of tumor diseases. Despite the fact that the metal-binding abilities of MTs have been known for decades, we are the first to suggest that MTs not only protect cancer cells but also help them grow and invade the surrounding tissue, which makes a tumor more aggressive.
Exploring the role of MTs in a mechanism of tumor defense against treatment strategies based on platinum drugs is a very challenging task. There is a presumption that MT plays a crucial role in the formation of platinum-based cytostatic resistance; however, there is still no clear evidence on this phenomenon. Therefore, a multi-instrumental approach covering electrophoretic, mass spectrometric, electrochemical and immunochemical methods was utilized to test the proposed hypotheses in vitro and then in vivo. In addition, the obtained multi-instrumental data were further employed as a basis for the development and preclinical testing of biocompatible nanovehicles with smart tunable properties that could be efficient in the management of hard-to-treat tumors with frequently occurring intrinsic chemoresistance to platinum cytostatics. With this consideration in mind, the project ToMeTuM yielded a plethora of exceptional results covering novel insights into the interactions of MTs with metals, in silico tools to analyze these interactions and the construction of high-resolution 3D models of proteins that, with the advent of computational biology and chemistry, will help the community better understand the role of MTs in healthy vs. cancer cells. Furthermore, a variety of biological experiments conducted during the project revealed the importance of different isoforms of MTs for cancer aggressiveness, angiogenesis and chemoresistance to chemotherapeutics, which is of utmost importance for cutting-edge precision medicine.
We have been developing several methods and protocols to detect MTs, with special attention given to distinguishing MT isoforms and oligomers. Most attention and effort has been paid to mass spectrometric analysis and measurements as well as biological methods, including immunological and nucleic acid-based protocols. We developed new selective recognition elements based on molecularly imprinted polymers, enabling effective MT detection in complex samples. Such an approach would make it possible to discriminate between various MT oligomers. A new approach involving antibody labeling by metal nanoparticles was also introduced, leading to a significantly simplified and shortened conjugation procedure compared to the EDC/NHS strategy. In addition, the first in silico modeling was successful, allowing us to develop a unique methodology for the precise construction of high-resolution protein models with improved rotamer classification, which is of utmost importance for molecular dynamics/mechanics simulations. Moreover, we optimized electrochemical and capillary electrophoretic methods for studying MTs, including 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 the chemoresistance of cells to CDDP and sorafenib (including cells with transient MT upregulation) and constructed comprehensive molecular maps of possible interactions of MTs with a large number of biological processes and druggable targets. We also described a participative role of MT-3 in chemoresistance to CDDP through the induction of oncogene-induced senescence/cell cycle and apoptosis processes. The obtained data were validated by functional analyses and published in several publications (some of which are under review or in preparation). Importantly, beyond the scope of the project, we have revealed that MTs are important factors of energetic metabolism and can affect the development and progression of severe liver diseases, such as nonalcoholic fatty liver disease. Additionally, we were successful in constructing highly precise homology models of MTs, which are currently utilized in homology modeling experiments focused on energetic requirements during MT oligomerization under different pathophysiological conditions. Preliminary optimization data and findings that are crucial for such models and for the abovementioned achievements were successfully published.
A new and challenging treatment strategy based on the silencing of genes for MT to suppress tumor growth and its protection mechanism is suggested. The first part of the project solution succeeded in the development of cutting-edge methodologies suitable for the investigation of different aspects of MT behavior, including oligomerization, classification and metal saturation. The computational part of the project succeeded in suggesting and validating a novel pipeline for the production of high-resolution protein models with precisely optimized rotamers, which will be extensively utilized in the subsequent parts of the project, particularly for in silico studies of MT homo- and heterooligomerization. In addition, pilot molecular biology experiments revealed the highly complex biology of MTs and underpinned their importance in cancer cells, particularly for the development of their chemoresistant phenotypes. During the project, we synthesized a variety of nanomaterials based on ZnO coordination compounds and confirmed their applicability for the selective and efficient killing of triple-negative breast cancer cells. Our CRISPR‒Cas9 experiments revealed that in prostate cancer cells, MT-3 acts as an inhibitor of cancer progression, and its knockout triggers a highly aggressive phenotype with extensive angiogenesis. Considering these results, we were the first team to employ propulsive metal-based nanorobots with swarming properties to directly deliver MT into the intracellular region of hard-to-transfect prostate cells.