- Proteases in the tumor microenvironment, possibilities of their targeting
- Role of membrane proteases in gliomagenesis, namely in the regulation of stemness, cell proliferation, differentiation, invasion and tumour progression
- Regulation of cancer cell membrane proteases, namely "Dipeptidyl peptidase IV activity and/or structure homologues“ (DASH) in cells of neuroectodermal origin
- Role of membrane proteases in cell signaling. Prereceptor proteolytic modification of biologically active peptides by DASH molecules and its role in signal transduction diversification
Our work demonstrated existence of cell specific expression patterns of DASH molecules in transformed neuroectodermal cells of differing degree of malignity and under varying growth conditions. The results of other authors also support the importance of the "DASH system" (comprising DASH molecules, namely those localized in the plasma membrane, their biologically active substrates and corresponding receptors) in the processes of cancer development (Wesley et al. 2004, Chen and Kelly 2003). Our results, obtained using human bioptic material, cell clones with regulated expression of DASH molecules and their mutated variants and animal studies suggest the coregulation of DASH molecules and receptors of their biologically active substrates. As an example, we described codistribution of DPP-IV and FAP as well as DPP-IV and chemokine receptor CXCR4 in human glioblastoma (Stremenova et al. 2007).
A phylogenetic tree of DPP-IV family protein sequence relationships. Distances represent relationship predicted after alignment using the CLUSTAL2 algorithm followed by PHYLIP analysis (Genbank accession numbers: DPP4: NP_001926;DPP6.1: NP_570629; DPP6.2: NP_001927; DPP7/DPPII/QPP: NP_037511; DPP8.1: NP_569118; DPP8.2: NP_060213; DPP8.3: NP_932064;DPP8.4: NP_932065; DPP9: NP_631898; DPP10-S: NP_001004360; DPP10-L: NP_065919; FAP-alpha/seprase: NP_004451). The blue boxes indicate enzymatically active proteins and the brown boxes indicate members with no dipeptidyl peptidase-IV enzymatic activity. All proteins are members of the SC clan representing enzymes with an alpha/beta-hydrolase fold; family S9B represents the dipeptidyl peptidase-IV family, whereas peptidase family S28 contains the DPP-IV-sequence divergent DPP7/DPPII/QPP. DPP6 and DPP10 are classified as S9 homologs with no peptidase activity. The Family descriptor is followed by the unique family identifier under which the general substrate specificity is indicated (http://merops.sanger.ac.uk/cgi-bin/name_index?id = P;action = A). To the right of each colored box are listed known interactions/functionalities believed to be independent of enzymatic activity (Sedo et al, 2008).
Current research projects focus on the role of FAP in the progression of glioblastoma and its potential targeting. In cooperation with the Institute of Organic Chemistry and Biochemistry of the Academy of Science of Czech Republic, we demonstrated the applicability of a FAP-specific iBody for the isolation of FAP from various biological sources as well as for its detection by ELISA, Western blot, flow cytometry, and confocal microscopy. Our results show the iBody is a useful tool for FAP targeting in vitro and potentially also for specific anticancer drug delivery (Dvorakova et al 2017).
A complex portfolio of biochemical, immunochemical, cell and molecular biology and advanced preclinical imaging methods is available. It comprises inducible expression of transgenic products, use of Crispr-Cas9, isolation of primary cell cultures including glioma stem-like cells, mouse cancer models (genetically engineered mouse models, orthotopic xenotransplantations under stereotactic control etc.), confocal microscopy, flow cytometry, in vivo PET/SPECT/CT imaging etc.
|In vivo imaging of orthotopically implanted glioma cells transfected with a fluorescent protein mKate2. Images were taken at 1 (figure A) and 5 weeks (figure B) after implantation
Localization of DPP-IV and FAP in U87 (A) and U138 cell lines (B).
PET imaging of glioblastoma tumor in mouse model
- Novel Concepts for the terapeutic targeting of tumor microenvironment in human glioblastomas. (2015, Czech health research council - AZV ČR, project 15-31379A)
- Study of PIWI-interacting RNAs in glioblastoma stem cells and their potential clinical implications (2019, Czech health research council - AZV ČR, project NV19-03-00501)
- Complex oncological program II. (2017, Charles University Research Development Schemes, project Progres Q28/LF1/1)
- EATRIS project LM2015064
- Center for Tumor Ecology – Research of the Cancer Microenvironment Supporting Cancer Growth and Spread (reg. n. CZ.02.1.01/0.0/0.0/16_019/0000785) is supported by the Operational Programme Research, Development and Education