The Role of Transglutaminase 2 isoforms in Breast Carcinogenesis

Abstract

Background: The formation of cancer stem cells is an important factor for breast cancer growth, metastasis, and response to treatment, hence prognosis. Elevated levels of the Ca2+ dependent enzyme transglutaminase 2 (TG2) have been identified in cancer cells displaying resistance to anti-cancer treatment or those isolated from metastatic sites, implying TG2’s involvement in these processes. TG2 is a multifunctional enzyme with different activities, including transamidase, GTPase, protein disulfide isomerase, and protein kinase. The various enzymatic activities and functions of TG2 may be referred to alternative splicing of TG2 mRNA. This study aims to investigate the role of the TG2 different isoforms in breast carcinogenesis by assessing their contribution to various carcinogenic processes, including transformation to cancer stem cells and acquisition of the EMT phenotype. Methods: The protein levels of the different TG2 isoforms have been investigated in the ERα positive MCF-7 and T47D and the triple negative MDA-MB-231 and MDA-MB-468 breast cancer cell lines. The transamidase activity was measured by TG2-CovTest. The ability of the different TG2 isoforms to confer stemness to these cells has been evaluated following mammosphere formation and expression level of the cancer stem cell (CSC) markers CD44 and CD24 in the presence or absence of the different TG2 isoforms. EMT has been evaluated by following the protein expression of the epithelial marker E-cadherin and the mesenchymal markers N-cadherin and vimentin. Results: MDA-MB-231, expresses both the TG2-L and TG2-S isoforms. MCF-7 and T47D also express both isoforms but to a very lower extent compared to MDA-MB-231, whereas MDA-MB-468 expresses only the TG2-S isoform. ZDON increased the CD44+/CD24-/low phenotype and ALDH activity. NC9 induced the CD44+/CD24-/low phenotype and increased ALDH activity to a higher extent than ZDON. In MDA-MB-231, silencing of the TG2 gene expression by siRNA significantly reduced CD44+/ CD24-/low phenotype, ALDH activity and mesenchymal marker vimentin. Conclusion: Human breast cancer cell lines express two TG2 isoforms, which exhibit differential roles in CSC and EMT. The CD44+/CD24-/low phenotype is linked to high basal expression of TG2-L in MDA-MB-231. The GTP-binding domain (GTPase activity) of TG2 plays an important role in promoting its carcinogenic effects, whereas its transamidase activity is not essential. The catalytic-binding domain (transamidase activity) reduces the oncogenic effect of TG2, reduces stemness traits, and EMT phenotype.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sally Freeman (Supervisor) & Costas Demonacos (Supervisor)