Scientific results:
Modern methods of the study of the molecular mechanisms of carcinogenesis in the lungs allowed to determine new target proteins for targeted pharmaceutical therapy, among which of special interest are the proteins contributing to the metastasis and activation of anti-apoptotic cascades, one of which is transglutaminase 2 (TG2).
TG2 is a highly conserved ferment that catalyzes Ca2+-dependent transamidation of proteins. Conformational changes in the protein structure give TG2 the capability to also manifest GTP-ase, serine/threonine-specific kinase and protein disulfide isomerase activity. It is known that changes in the activity or regulation of TG2 can lead to onset of a number of neoplastic and fibroproliferative diseases, including malignant tumors and kidney, lung and heart fibrosis. Apart from the intracellular functions, TG2 mediates the interaction of integrins with fibronectin and binds extracellular matrix proteins in the intercellular space, which, in turn, influences the metastasis of tumor cells. According to literature data, it is known that a high level of TG2 in hepatocellular carcinoma (HCC) cells correlates with a low survival rate of patients and an increased resistance against chemotherapy agents, while TG2 inhibition suppresses the growth of renal cancer tumors by the р53-mediated induction of apoptosis. However, the role of TG2 in the modulation of the activity of the р53 oncosuppressor in the context of lung cancer remains unexplained.
To research a potential mechanism of the interaction of TG2 and р53, we have produced the isogenic cell lines А549 (wild-type of р53) and H1975 (mutant р53) with a knockout and an overexpression of TG2 respectively. Further analysis of the nature of changes in р53 and its target proteins was conducted using the western blot method and RT-PCR. The influence of TG2 on changes in the resistance against genotoxic drugs and the proliferation potential of cells was assessed using the clonogenic and MTT assays as well as using the RTCA xCelligence eSight cell analyzer. Collectively the obtained data allow to suggest that TG2-mediated degradation only of wild-type р53 can contribute to an increase of the survival rate of cancer cells in the presence of anti-tumor drugs. Further work will be focused on the comprehensive study of the mechanism of the interaction of TG2 with р53 and the possible participation of MDM2 ubiquitin ligase in this process.
Therefore, we managed to prove that the TG2 fragment participates in the degradation of wild-type р53 but does not affect the oncogenic functions of mutant р53 in lung adenocarcinoma cells, which leads to an increase in the resistance of cancer cells with TG2 expression against standard chemotherapy drugs.
Transglutaminase 2 (TG2) participates in the regulation of proteostasis in the conditions of cell stress by the post-translational regulation of the heat shock factor 1 (HSF1; Rossin and others, 2018). HSF1 is the main factor of transcription that regulates the reaction to proteotoxic stress, controlling the transcription of multiple genes sensitive to stress, including heat shack proteins. We have identified a new molecular mechanism of the control of HSF1 activity. In particular, we have demonstrated that TG2 via its disulfide-isomerase activity launches the trimerization and activation of HSF1, regulating the adaptation of a cell to stress and proteostasis disorders. More specifically, we have found that the loss of TG2 function correlates with the disorder of nuclear translocation of HSF1 and its capability to bind with the promoter of the HSP70 gene.
One more result of of the conducted research uncovers a possible role of TG2 in the development of hepatocellular carcinoma (HCC), for instance, the participation of TG2 in nonalcoholic fatty liver disease (NAFLD). We have demonstrated that the influence of TG2 on the progression of NAFLD induced by a high-fat diet, both on wild-type mice, as well as on TG2 knockout mice. The animals were fed a standard diet or a high-fat (42 per cent of energy from fat) diet throughout 16 weeks. The results of the experiemnts demonstrated that the absence of TG2 functionality causes disruption of autophagy/mitophagy and lead to the progression of the disease. The data was confirmed by the pharmacological inhibition of TG2 in wild-type animals.
To determine the role of TG2 in the development of hepatocellular carcinoma, which is the main goal of the grant, three-month-old wild-type TG2-knowckout (TG2 KO) C57BL/6 mice were subjected to the procedure of induction of liver carcinogenesis diethylnitrosamine (DEN) in combination with a high-fat diet (HFD). This model completely reproduces all the clinical features of HCC, such as insulin resistance, inflammation, fibrosis and carcinogenesis. As a result, we have found that in wild-type mice a large number of tumors can develop that have a much more pronounced size than in TG2 KO mice. These results have high significance since they demonstrate that the absence of TG2 prevents the progression of HCC. In fact, in mice deprived of TG2 small pre-neoplastic nodules (1-2 mm) developed that very rarely reach sizes comparable to tumors detected in wild-type mice. It is worth noting that in the livers of TG2 KO mice we found a much lower level of pro-inflammatory cytokines after the induction of carcinogenesis.
This data suggest that the inhibition of TG2 can become a new method of battling the development of HCC (a patent application has been submitted for the use of the inhibition of HSF1 and TG2 for the treatment of HCC). According to these results, a search in the TCGA database has demonstrated that TG2 expression in patients with HCC is a negative prognostic marker. This confirms that the oncogenic role of TG2 provides grounds for the potential clinical use of its genetic or pharmacological inhibition. It is also worth mentioning that results similar to genetic knockout were obtained by treatment of mice with cysteamine, an FDA-approved TG2 inhibitor, which confirms the potential possibility of its use in treatment of patients. We are currently studying TG2 expression in vivo in about 50 HCC bioptates to confirm the results collected using the above-mentioned mouse model.
Education and career development:
- Within the Laboratory, postgraduate students of the Institute of Cytology of the Russian Academy of Sciences and undergraduate students of Saint Petersburg universities perform their research.
- In 2018-2020, students participating in the project defended two bachelor’s degree theses 4 master’s degree theses .
- In 2019, a postgraduate student working in our Laboratory completed an internship in modern methods of proteomics at the laboratory of the leading scientist Mauro Piacentini at Tor Vergata University of Rome (Italy).
- Two employees of the Laboratory have completed the additional training course «Modern methods of the use of laboratory rodents in translational biomedical research» at the Moscow State University (November 2019).
- Employees of the Laboratory organized and staged the international conference «Regulation of proteostasis in cancer» (Saint Petersburg, 2019).
Organizational and structural changes:
We have built a vivarium to keep mice with immune deficiency. The vivarium is designed to keep about 200 animals.
Other results:
- With support from a Russian Foundation for Basic Research, we have found and demonstrated an important role of the adhesion receptor conjugate with G proteins, GPR123, as a new marker of human pluripotent cells and an effector of the process of reprogramming.
- With support from a Russian Science Foundation grant, using mathematical modeling we have demonstrated the feasibility of conducting the selection of the best clones of induced pluripotent stem cells (iPSC) according to morphological features as a non-invasive method of assessment. Using neural networks, we have built a classifier that has demonstrated the capability to determine the phenotype of an iPSC colony from the morphologucal criteria present in phase-contrast images with a high degree of precision (89 per cent detection precision).
- With support from a grant from the Ministry of Science and Higher Education of Russia (pursuant to an agreement signed on 28 September 2021), we demonstrated the role of mutations in the CaSR gene in the induction of osteodifferentiation. It has been found that patient-specific induced pluripotent stem cells (iPSCs) carrying a mutation in the CaSR gene are incapable of full osteodifferentation in comparison with iPSCs acquired from healthy donors, which can point at a correlation between this gene and the osteoporosis pathogen.
Collaborations:
Tor Vergata University of Rome (Italy): joint research and publications.
