Laboratory of Growth Regulators
Palacký University Olomouc 
RECAMO – Top center for cancer research, based Masaryk Oncological Institute in Brno offers several themes of doctoral theses..
Defining new signalling pathways and therapeutic potential of the receptor signalling protein IFITM1 and TSPAN1 in human cervical cancer
Supervisor: RNDr. Bořivoj Vojtěšek, DrSc.
Drug resistant and invasive cervical cancer is a disease with a high unmet clinical need. Novel strategies to improve target selection, patient stratification, and to develop new therapeutics are required to improve outcomes in advanced disease. Emerging strategies in cancer target discovery involve the interrogation of patient-derived cancer biopsies using cutting-edge proteomics and RNA expression technologies to identify expressed cancer-specific targets. We have used these methodologies to define two highly expressed pro-oncogenic receptors in cervical cancer from patient biopsies using a dual isobaric labeling proteomic and transcriptomic platform. We have developed a suite of recombinant monoclonal antibodies (MABs) to these targets (IFITM1 and TSPAN1) and confirm the MABs detect target protein expression in cervical cancers. We propose to (i) validate these two receptors in cervical cancer cell lines and cervical biopsies from patients undergoing late stage chemotherapy treatment as potential therapeutic targets for use in metastatic disease; (ii) develop companion biomarkers from cancer biopsies and cervical smears that can be used to access target inhibition; (iii) identify new interacting proteins for IFITM1 and TSPAN1 using mass spectrometry; and (iv) determine whether drugging either receptor alone or in combination using recombinant multivalent MAbs has the most therapeutic potential. These approaches will validate a set of clinically relevant, highly-penetrant, pro-metastatic receptors in cervical cancer with the aim of providing new strategies for improved stratification and personalization in the treatment of cervical cancer. This project will be realized in cooperation with Edinburgh Cancer Research Centre (Prof. Ted Hupp).
Extracellular activity of AGR proteins and its significance for tumour tissue growth
Supervisor: RNDr. Bořivoj Vojtěšek, DrSc.
AGR2 and AGR3 proteins belong to the protein-disulphide isomerase (PDI) family of proteins that have been associated with various cancers recently. Preceding studies have localised AGRs into specific subcellular compartments which may indicate proteins essential intracellular functions. Immunofluorescence microscopy and cytometric studies have confirmed AGR2 localisation into endoplasmic reticulum (ER) where it apparently participates on protein folding as protein chaperone and takes part in ER stress reaction. Further but still without any specific function, AGR2 has been detected in other subcellular locations such as membrane and also in extracellular space, including extracellular body fluids such as gastrointestinal mucus, blood serum and urine. The AGR3 homologue was detected in rather distinct locations compared to AGR2. It has been detected in endosomes of T47D cells identified among a set of breast cancer associated membrane proteins so far.
It is assumed, that extracellular effect of human AGRs is principally similar to that documented in salamanders, where it was shown that secreted nAG binds to Prod-1 receptor and stimulates blastemal cells growth during wound healing processes. Although there is not known a human homologue of Prod-1, structural studies using recombinant monomeric AGR2 protein revealed that AGR2 has putative cell surface binding sites. Indeed, former work has identified C4.4A (LYPD3) as a functional cell surface receptor for the extracellular AGR2 in pancreatic cancer cells.
Autocrine and paracrine extracellular signalling represent substantial field of cancer research in terms of identification of molecules that may provide us information on prognostic markers and targets of cancer therapy. AGR proteins have been validated as prognostic markers of breast, ovarian and lung cancer detectable in extracellular fluids however the exact mechanism and significance of extracellular AGRs has to be elucidated. The aim of the thesis will particularly consist of i) revealing of molecular mechanisms controling and participating on AGRs secretion; ii) identification of molecules participating on AGRs – cell surface interaction; iii) functional effects of extracellular AGRs on cancer cell viability and invasive properties.
The molecular mechanism of MDM2’s switch from negative to positive regulator of p53 tumour suppressor activity
Supervisor: RNDr. Bořivoj Vojtěšek, DrSc.
The main regulators of p53 tumour suppressor activity are the MDM2 and its homologue MDMX. Both proteins bind to the N-terminus of p53 and prevent p53 activity by blocking its trans activity and to target it for degradation via the 26S proteasome pathway under normal conditions. These interactions are the subjects of industrial and academic efforts aimed at manipulating p53 activity in cancers. During DNA damage, the cells activate p53 in order to either arrest the cell cycle and repair the cells or to induce irreversible apoptosis. This is regulated by the ATM kinase and involves preventing MDM2/MDMX from binding the p53 protein and to instead bind the p53 mRNA in order to stimulate p53 synthesis. The highly conserved BOX-I of p53 encodes both the RNA sequence and the peptide sequence that bind MDM2 which allows MDM2 to switch from negative to positive regulator of p53, depending on cellular conditions.
In order to exploit MDM2’s capacity to activate p53 for therapeutic purposes we are now interested to further understand the molecular mechanism that underlie MDM2’s positive activity towards p53. We know that phosphorylation of MDM2 at serine 395 by ATM opens up an RNA binding pocket in the MDM2 C-terminal RING domain. We now i) want to know the allosteric effects of this phosphorylation event of MDM2 and of the RNA ligand binding using deuterium exchange followed by mass spectrometry analysis. We also ii) want to know how these allosteric changes affect MDM2’s interaction with other known cellular factors by using IP of MDM2 phosphomimetic mutations in the presence of p53 mRNA followed by MS analysis. Finally, we will iii) address the molecular mechanism of MDM2’s capacity to stimulate translation initiation of the p53 mRNA and identify the pathway involved.
