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Identification of the proteasome machinery targets in human cancer

Periodic Reporting for period 1 - Proteasome in cancer (Identification of the proteasome machinery targets in human cancer)

Reporting period: 2018-05-01 to 2020-04-30

Cancer remains one of the primary causes of death in western countries. Tendencies allow to predict that in the upcoming decade the cancers of lung, colon/rectum and pancreas will be the primary causes of neoplasia-related deaths in the EU. The proteasome machinery – consisting of 20S Proteasome, 26S Proteasome and immunoproteasome - is the major protein degradation factor in human cells. It is also strongly upregulated in most human neoplasias of various tissues, including the above-mentioned most deadly cancer types, being one of the most universal oncogenic drivers in human cancer. Proteasome inhibitors Bortezomib (1st generation) and Carflizomib (2nd generation) are clinically approved for multiple myeloma clinical treatment. Even though the proteasome has been reported as a vulnerability in multiple types of cancers, the resistance mechanisms in humans have not yet allowed the proteasome inhibitor-based solid tumor therapies to progress beyond clinical trials.
Thus, the main objective of the project was to understand the oncogenic contribution of the proteasome machinery to the reprogramming of human cancer cells and to use this knowledge to discover anti-cancer therapeutic approaches targeting the proteasome downstream effectors.
Addressing this objective is bringing a detailed knowledge of the general proteasome effect in human cells, on how these cells compensate for the lack of the proteasome activity, and how this compensation can be prevented specifically in cancer cells. This has a meaning for designing new therapeutic approaches for some of the most dangerous cancer types listed above, as well as is bringing basic, academic knowledge on the action of one of the indispensable components of human cells – the proteasome machinery.
The main objective of the project has been successfully accomplished as the proteasome machinery targets were identified using global proteomics and transcriptomics in a panel of 8 cell lines (6 cancer cell lines and 2 multiple myeloma cell lines) – and a map was made of molecular pathways regulated by the proteasome in the studied cells (see Figure 1 attached). The comparison of myeloma and cancer global proteome changes upon proteasome inhibition allowed for identification of 44 proteins/genes across 20 pathways which were candidate proteasome targets providing resistance to Carfilzomib in cancers vs. multiple myeloma. The following validation and siRNA screen allowed to focus on molecular chaperones, autophagy, endocytosis and unfolded protein response pathways - with molecular chaperones and their co-chaperones having a pivotal role in all the listed pathways. In consequence, tested specific molecular chaperone inhibitors and selected co-chaperone inhibitors provided the additive effect in killing cancer cells with Carfilzomib, allowing to bypass resistance to this inhibitor. Thus, the project, thanks to the global identification of the proteasome machinery targets, has allowed finding drug targets and preclinical protocols for treatment of colon, lung and pancreatic cancer cells, which rely on increasing the efficiency of the proteasome inhibitor Carfilzomib.

All the major points of the expected exploitation and dissemination have been successfully accomplished:
1. The project greatly helped the project’s Researcher to establish (formally in October 2018) a laboratory consisting currently of a staff of 5-10 postdocs, PhD and MSc students - Laboratory of Human Disease Multi-omics at MMRC PAS, Warsaw, Poland. The laboratory is successfully using the workflow established in this project for other studies.
2. The progress and methods were communicated during seminars in the Beneficiary institution and collaborating institutions – e.g. University of Warsaw (https://cent.uw.edu.pl/en/blog/dawid-walerych-mossakowski-medical-research-centre-warsaw/) or International Institute of Molecular and Cell Biology in Warsaw (https://www.iimcb.gov.pl/en/press-office/news/seminars?start=50).
Especially successful was the open lecture/workshop series “Cancer Fight Research Club” (https://twitter.com/FightCancerClub
https://www.facebook.com/CancerFightResearchClub/) organised at the Beneficiary institute (September 2019 – February 2020). This allowed to invite known cancer researchers from Poland and EU countries to give lectures, talk to them about the project and further collaborate in it. These meetings attracted audience from many science and education institutions, also non-professional audience – serving science popularization.
3. Two conference announcements related to the project were done: A poster during the 8th Mutant p53 Workshop in Lyon, France, 13-18 May 2019 (https://p53.iarc.fr/download/8thp53MutantWorkshopBook.pdf - the proteasome gene transcription and activity was a readout of the mutant p53 oncogenic activity) and a poster and a short talk were done at 13th Central and Eastern European Proteomic Conference, 23-25.09.2019 Ustoń, Poland (http://cd.io.gliwice.pl/ceepc13/).
4. One review manuscript was published concerning in part the proteasome machinery in cancer (https://www.mdpi.com/2072-6694/12/6/1532). The main project’s research manuscript will be submitted in the next months when the mechanism studies in compensation of the proteasome inhibition are complete, and the therapeutic tests in vivo will be completed (both are ongoing and were not listed as project tasks – they will allow to support the results better and publish them in a higher impact journal).
5. The experience on mass spectrometry and proteomics/transcriptomics data analysis gained during the project, especially during the Secondment, was indispensable for the project, extremely useful other projects and generally – for the
The project, thanks to the global identification of the proteasome machinery targets, has allowed finding drug targets and preclinical protocols for treatment of colon, lung and pancreatic cancer cells (some of the most dangerous cancer types in EU), which rely on increasing the efficiency of the proteasome inhibitor Carfilzomib.
The project is now being extended and continued by more detailed molecular mechanism studies and in vivo drug protocol testing in mouse xenografts – to complete the preclinical phase and publish the complete research data.
This extension is also a link of the project’s results with their further use. First, the new mechanisms of the compensation of the decrease in the proteasome activity in human cells have been discovered – and this will be a subject of further grant proposals and future research. Second, after the in vivo tests in mice, which are a necessary preclinical step for any new therapeutic approach, the protocol – if successful in the mice tests – can be progressed to clinical trials in human patients. This is possible thanks to collaboration with clinicians in Warsaw, Poland, including the hospital being a Partner Organization in this project, and the fact that the drugs efficiently supplementing Carfilzomib treatment discovered in the project are repositioned from other therapeutic protocols or/and are already tested in human patients for toxicity. Any new, efficient anti-cancer protocol for treatment of colon, lung or pancreatic cancers has a strong socio-economic impact and societal implications.
The workflow of the project and the map of the pathways targeted by the proteasome in cancer cells.