The role of tissue transglutaminase active site mutants in renal cell carcinoma and epithelial to mesenchymal transition

Multifunctionality of tissue transglutaminase 2 (TG2) allowed protein to act as a crosslinking enzyme, GTPase/ATPase, protein disulfide isomerase and as a protein kinase. TG2 is involved in several cellular processes such as adhesion, migration, invasion, cell growth, and epithelial mesenchymal transition (EMT). Evidence suggests that EMT might be crucial in the cellular processes including metastasis and drug resistance in many different tumor types. Renal cell carcinoma (RCC) is known as the subtype of kidney cancer and it accounts three per cent of the malignancies in adults. Our previous findings suggested that the upregulation in the expression level of TG2 in RCC was associated with tumor metastasis and with a significant decrease in disease- and cancer-specific survival outcome. Given the importance of the pro-metastatic function of TG2 in RCC, we aimed to investigate the possible allosteric contribution of TG2 enzyme in cell migration, invasion, EMT, and cancer stemness for RCC with a special emphasis on the TG2- NFκB axis. For that purpose, RenCa TG2 variants were generated by transducing mouse RCC cell line RenCa using wt-TG2, GTP-binding deficient form TG2-R580A, transaminase-deficient form with a low GTP-binding affinity TG2-C277S and transaminase-inactive form TG2-W241A. Results from CD marker analysis and spheroid assay suggested that GTP-binding function was important in the maintenance of mesenchymal phenotype and cancer stemness profile. The expression level analysis of EMT transcriptional factors and matrix metalloproteinases (MMPs) together with scattering and Matrigel invasion assays in RenCa TG2 variants suggested that GTPase activity of TG2 was indispensable for the cell migration and invasion of RCC. In addition, EMSA experiments performed on control and TG2-expressing mutant RenCa cells indicated that GTP-binding function of TG2 was essential for the activation of NFκB through the canonical pathway. Taken together, these findings supported that pro-metastatic role of TG2 in RCC was mainly dependent on the GTP-binding function of the enzyme. This project was funded by FP-7 TRANSPATH 289964 grant.