Abstract 995: Tissue transglutaminase regulates integrin-linked kinase and beta-catenin signaling in ovarian cancer cells

Abstract Ovarian cancer (OC) is the most lethal gynecological cancer characterized by an enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and rearrangement of extra-cellular matrix (ECM) components. In OC, the interaction between fibronectin (FN) and the β1 integrins activates the adaptor protein integrin linked kinase (ILK) that acts as central hub integrating the mechanical forces with the activation of various signaling pathways, leading to cell survival, proliferation, and migration. ILK overexpression was correlated with OC progression and its functional inhibition blocked tumorigenicity by inactivation of glycogen synthase kinase-3α/β (GSK-3α/β) and the β-catenin-TCF/LEF1 transcriptional activity, thus pointing towards the direct involvement of ILK as a modulator of the crosstalk between ECM and the canonical Wnt signaling. However, the connection between integrins and β-catenin remains poorly understood. The multifunctional protein tissue transglutaminase (TG2) with enzymatic and scaffold functions is an important molecule secreted in the tumor microenvironment where it modulates oncogenic signaling by interacting with FN and integrins. Previously, we demonstrated that TG2/FN/β1 integrin clusters regulated β-catenin activation, correlating with OC progression. Here, we investigated whether TG2-FN interaction is essential to activate the integrin-ILK axis, thereby stabilizing β-catenin. First, we demonstrate that TG2-expressing OC cells plated on FN matrix increase the pool of active p-ILKSer246. Confocal microscopy shows that TG2 co-localizes with p-ILKSer246 in OC cells and that the presence of FN matrices is instrumental in the formation of active TG2/p-ILKSer246 clusters. Further, proximity ligation assay analysis reveals that each protein of the ternary TG2/FN/β1 integrin complex actively engages with p-ILKSer246 in primary ovarian tumor cells, supporting the functional relevance of TG2-mediated outside in transduction signals in vivo. Second, we show that TG2-mediated ILK activation mechanistically amplifies the response of OC cells to Wnt-3A treatment, leading to the inhibitory phosphorylation of the GSK-3α/β at Ser21/9, which in turn allows β-catenin nuclear translocation and transcriptional activity. Finally, we evaluate whether the TG2-ILK complex has functional outcomes. In the presence of an active TG2/p-ILKSer246 axis, Wnt-3a treatment efficiently increases proliferation and migration in OC cells compared to control, whereas the inhibitory anti-TG2 blocking antibody (clone 4G3) directed against the FN-binding domain of TG2 and ILK inhibition by the small molecule cpd-22 drastically reduce the Wnt-3A impact on the OC cell proliferative and migratory capacity. In sum, here we demonstrate that TG2 directly activates ILK and define a new mechanism which links ECM cues with β-catenin signaling in OC. Citation Format: Salvatore Condello, Rula Atwani, Daniela Matei. Tissue transglutaminase regulates integrin-linked kinase and beta-catenin signaling in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 995.