Projects / Programmes
The role of cysteine protease inhibitors in NK cell mediated lysis of tumour cells
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 3.04 |
Medical and Health Sciences |
Medical biotechnology |
proteolysis, cancer,NK cells, cystatin F,cathepsins, granzyme, cellular therapy
Researchers (16)
Organisations (2)
Abstract
Natural killer (NK) cells are large granular lymphocytes that mediate natural immunity against a variety of tumour and virally infected cells. They have been shown to lose cytotoxic function following their interaction with target cells. This inactivation of the cytotoxicity of NK cells, but not their proliferative function and secretion of cytokines, is termed ˝split anergy˝. Many factors responsible for the tumour associated suppression of NK cell cytotoxicity have been identified, however, the mechanisms by which tumour cells contribute to the induction of NK suppression are complex and have not been fully elucidated. Cysteine protease cathepsin C has been identified as a key regulator of the cytotoxicity of NK cells activating granule serine peptidases granzymes A and B. Granule exocytosis includes the release of a pore-forming protein, perforin, and activated granzymes into the synapse formed between the killer and the target cell. Although cathepsin C generates the majority of granzyme activity, recent studies support alternative mechanisms for granzyme processing, including cathepsin H as an alternative pro-granzyme convertase.
The activity of cysteine cathepsins is controlled by their endogenous inhibitors, cystatins. Cystatins are considered as emergency inhibitors, trapping proteases escaped from the endosomes/lysosomes or cells in stable, proteolytically inactive complexes and are thus not considered to regulate protease activity within the endosomal/lysosomal pathway. However, cystatin F is a suitable candidate for regulating the proteolytic activity of cysteine proteases within the cells. Unlike other cystatins it is localized in endosomal/lysosomal vesicles and may thus directly inactivate lysosomal enzymes. Cystatin F is produced in cells as a disulphide-linked dimer inactive as an inhibitor of cathepsins. In vitro, unusually strong reducing conditions are needed to dissociate dimer to monomer. However, truncation of the N-terminal region significantly enhances the monomerization and also changes the inhibitory properties of the monomer. By N-terminal processing, cystatin F becomes a strong inhibitor of cathepsins C and H, main pro-granzyme convertases in NK cells.
In our preliminary study we demonstrated that the activity of cathepsin C in NK cells is related to the expression of cystatin F and its translocation to endosomal/lysosomal vesicles. We hypothesise that the changed inhibitory profile of cystatin F, that results from its N-terminal processing in endosomal/lysosomal vesicles, impairs the function of cathepsins C and H as pro-granzyme convertases and, consequently, granzyme-related cytotoxic function. Furthermore, the regulation of cystatin F function is a part of the mechanism activated by targeted tumour cells to induce split anergy in NK cells. We expect that cystatin F can be transferred as an inactive dimer from tumour cells and adjacent monocytes and macrophages to NK cells and, after internalisation, reaches vesicular cathepsins C and H and impairs their enzymatic activities. Our hypothesis will be tested on cell models utilising stable cell lines and primary NK cells, monocytes, tumour cells and tumour stem cells obtained from cell collections and patients treated in UCLA. The expression and location of the enzymes, cystatin F, receptors and cytokines will be followed and related to the cytotoxycity of NK cells and the status of the cancer patients. The main goal of this project is to confirm that cysteine proteases cathepsins C and H and their inhibitor cystatin F are the main regulators of split anergy of NK cells and that they can be used as therapeutic targets to improve cell therapy of cancer patients. Additionally, we aim to demonstrate that the regulation of cystatin F transcription and translation by genetic tools and its final processing into an active form by protease inhibitors constitute an applicable method to strengthen the response of NK cells to cancer.
Significance for science
Immunosuppression and tumour escape from immune recognition are major factors responsible for the development and progression of cancer. However, neither the underlying physiological significance nor the exact mechanisms by which immunosuppression occurs are well understood. Nowadays it is well accepted that effectors of adaptive and innate immunity need to cooperate in order to reject tumours, however, activation of the adaptive immune response plays a predominant role in eradicationg malignant cells. The understanding of molecular mechanisms of T cell and NK cell cytotoxicity and the processes triggered by tumour cells to eliminate their cytotoxic action, can provide new tools to develop more effective anticancer therapies. Proteolytic enzymes are believed to be key players in these processes and the regulation of their activity by endogenous inhibitors is obviously one of the mechanisms that tumour cells utilize to avoid their destruction. New knowledge, obtained through this project will enrich scientific field and open up new investigations toward understanding immunosuppression. Furthermore, it can foster the development of new approaches to bypassing the inactivation of cytotoxicity. Our project group used the most recent techniques and methods, which have already been introduced into our laboratory practice. Collaboration with leading researchers in Slovenia and worldwide will guarantee top rank science, rapid dissemination of knowledge, and successful implementation of the project.
Significance for the country
The results of the proposed project will make possible the development of new technologies and products, which will be economically interesting for pharmaceutical and biotechnological companies. They can also serve a a basis for establishing new start-up companies. In Slovenia and the European Union, biotechnology, biomedicine and pharmacy constitute a leading scientific and economic priority. The pharmaceutical industry has exhibited outstanding growth in recent years and the implementation of new products, either original or generic, constitutes the main driving force. The scientific knowledge arising from top class research in this field, to which our proposed project has contributed, is essential for further development of the pharmaceutical industry. Besides potential new drugs, the project employed the most recent methods and techniques, which can easily be adopted by industrial laboratories. Furthermore, the proposed project also contributed to all levels of university education. It enabled students to acquire new knowledge and the project’s implementation provided opportunities for diploma and doctoral training in the fields of pharmaceutical biochemistry, biotechnology and tumour biology. The results of this project will also foster our collaboration with excellent research groups, increasing knowledge exchange through organising international scientific conferences and the opportunity to obtain new European and other funds for financing our science.
Most important scientific results
Annual report
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2014,
2015,
final report
