Registration Number
0218U001147,
0112U003291
, R & D reports
Title
Potential oncoproteins and suppressor proteins of brain tumours and their participation with cellular signaling pathways
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Head
Areshkov Pavlo Oleksandrovych, Avdieiev Stanislav Sergeevich,
Registration Date
24-01-2018
Organization
Institute of Molecular Biology and Genetics of NASU
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The object of study: tumor-associated proteins CHI3L1 and CHI3L2 as signaling cascade activators. The goal of the project is to find out the role of interaction of the tumor-associated proteins CHI3L1 and CHI3L2 with MAPK/ERK1/2 and PI3K/AKT1 signaling cascades in the acquisition of proliferative and invasive properties of tumorous and non-tumorous cells, as well as the searching of specific inhibitors of these processes. Methods of investigation – reverse transcription polymerase chain reaction (RT-PCR), mammalian cell culture and transfection, molecular cloning, Western blot analysis, laser scanning confocal microscopy, analysis of cell’s colony-formation capacity in the soft agar, cell viability analysis, cells implantation in the brain of adult immunocompetent rats. The project is aimed at solving one of the fundamental problems of molecular biology - the specification and characterization of malignant cell transformation mechanisms. To understand the complex biology of oncogenesis, it is important to identify genes that change their expression during tumor development. Earlier by the methods of expression genetics, we discovered a group of genes with increased expression in gliomas and investigated the oncogenic properties of CHI3L1 and CHI3L2 as the genes with the most pronounced change in expression from this list. A further comparative characterization of the corresponding human pseudohitinases CHI3L1 and CHI3L2 indicated that these related proteins have opposite effects on the growth of tumor and non-tumor cell lines. For the first time, it has been shown that, unlike mitogen-stimulating action of CHI3L1, the addition of recombinant CHI3L2 to the serum-free medium of U-251 and 293 cell lines results in suspension of their growth. Antitumor therapy, aimed at one target, has only limited efficacy primarily due to the branching and convergence of the signaling pathways associated with highly invasive and angiogenic properties of malignant neoplasias. Detection and characterization of tumor-associated proteins with growth-modulating properties is an urgent task for modern cancer biology through the possibility of their use in new combined treatment approaches. In this work, we found that the presence of both the glycoprotein CHI3L1 and the recombinant CHI3L2 in a nutritious serum-free environment leads to stimulation of phosphorylation of the key units of the signaling cascades MARK and RI-3K protein kinase ERK1/2 and ACT1, respectively, in the cells of 239 and U-251 cell lines. However, the growth-inhibiting effect of CHI3L2 is mediated by long-term phosphorylation of ERK1/2, whereas addition of mitogen-stimulating glycoprotein CHI3L1 results in short-term activation of these kinases. Analysis of the localization of activated ERK1/2 in cells of line 293 after treatment with CHI3L1 and CHI3L2 showed the accumulation of most phospho-ERK1/2 in the nucleus, whereas in the stimulated by the proteins cells U-251 activated forms of protein kinases were localized in the cytoplasm. This indicates that the intracellular localization of phosphorylated ERK1/2 by the action of chitinase-like proteins depends on the cell type. In addition, it has been shown that the characteristic feature of cell signaling induced by CHI3L1 and CHI3L2 is the interaction between the MAPK and PI-3K cascades, which may be involved in the realization of the functional properties of these proteins. It has also been demonstrated that the epidermal growth factor receptor (EGFR) and apoptotic signal kinase 1 (ASK1) may be the components of signaling cascades that mediate the effect of CHI3L1 and CHI3L2 on cells. Since our previous results of the analysis of the malignant transformation of cells by cDNA CHI3L1 indicated that this gene could be considered as a new potential oncogene, the characterization of the 293 cell line, with stable and ectopic production of the corresponding protein, was continued. It has been shown that intracranial implantation of 293_CHI3L1 cells leads to the formation of tumors in the brain of rats in the absence of systemic immunosuppression. Based on these data, we have proposed a new brain tumor model that can be used to test antitumor drugs. Owing to massive research developments, drugs based on oligonucleotides are a promising direction of modern oncology. Many of them are already in use at the clinic and their quality is enhanced during the final stages of clinical trials. An important tool for studying the biological effects of genes and their corresponding proteins is to manipulate the level of their expression in cells using short-interfering RNA molecules (siRNAs). For the purpose of verifying the possibility of using the aberrant expression of CHI3L1 as a prognostic factor or therapeutic target for complex therapy of CHI3L1-associated tumors, we used two siRNAs in the course of our research which correspond to the CHI3L1 mRNA sequence at the boundary of the 6th and 7th exons and 10 th exon, respectively. Experimental data indicate that the inhibition of the production of glycoprotein CHI3L1 by specific short interfering RNAs (siRNAs) leads to suppression of features of malignant transformation of cells 293_CHI3L1. Moreover, the use of siRNA-CHI3L1 in combination with the treatment of cells with antagonists of bradykinin or derivatives of 4-thiazolidinones (the potent antitumor drugs with a wide range of biological properties) has the consequence of enhancing the cytotoxic effect. The results substantially broaden the existing picture of the biological properties of the CHI3L1 and CHI3L2 genes and their protein products, primarily in the processes of initiation and progression of malignant tumors, in particular glioms, and also allow them to be considered as targets in the development of new therapeutic approaches to the treatment of diseases associated with them.
Product Description
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Єршов А.В.
Бельтюкова Л.В.
Губернаторова А.О.
Кашуба В.В.
Чаусовський Т.І.
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2020-04-02
