Abstract B56: Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells
Authors: Condello Salvatore , Sima Livia , Ivan Cristina , Cardenas Horacio , Schiltz Gary , Mishra Rama , Matei Daniela 
Published: September 2020

Ovarian cancer (OC) is the most lethal gynecologic cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The metastatic progression and post-chemotherapy recurrence of OC is linked to a small population of cancer stem cells (CSCs) detectable as ALDH+/CD133+ phenotype. In OC, the tumor microenvironment (TME) provides a favorable milieu that protects quiescent CSCs during chemotherapy and supports their tumorigenic functions. The multifunctional protein tissue transglutaminase (TG2), with enzymatic and scaffold functions, is an important molecule secreted in the TME where it modulates oncogenic signaling by interacting with extracellular matrix (ECM) components, such as fibronectin (FN) and integrins. Its aberrant expression is correlated with OC progression and with the tumorigenic OCSC phenotype. Here, we hypothesized that the formation of the TG2/FN/integrins ternary complex at the cell membrane promotes survival of CSCs and supports OC spheroid formation by regulating the Wnt/beta-catenin pathway. Our data demonstrate that TG2, FN, and integrin β1 mRNA expression is markedly increased in OCSCs (ALDH+/CD133+) compared with non-CSC (ALDH-/CD133-) and highly enriched in OC cells grown as spheroids compared with monolayers (p&lt;0.01). Immunofluorescence (IF) staining showed abundant complexes of TG2 with integrin β1 and FN in OC cells grown as spheroids, and the protein-protein interactions were detected in human tumors by proximity ligation assay. TG2 expression was strongly correlated with ITGB1 (R=0.23, P&lt;0.0001) and with FN1 (R=0.39, P&lt;0.0001) in the TCGA ovarian cancer database. Use of function-inhibiting antibody against the FN-binding domain of TG2 (4G3) dramatically suppressed spheroid proliferation, TG2/FN/integrin complex formation, and tumor growth in a xenograft model. Disruption of TG2/FN complex altered key oncogenic signaling pathways, and in particular blocked the canonical Wnt/β-catenin signaling pathway essential to sustaining cancer cell stemness. The Wnt receptor Frizzled genes (Fzd1, Fzd7 and Fzd8) showed the greatest difference in gene expression, indicating a direct correlation between TG2/FN complex and Wnt pathway activation. Co-immunoprecipitation and IF staining demonstrated that TG2 co-localizes and directly binds to Fzd7. Furthermore, protein docking and peptide inhibition demonstrated that the interaction between TG2 and Fzd7 overlapped with the FN-binding domain of TG2. Finally, blockade of TG2/FN complex and shRNA-mediated Fzd7 knockdown attenuated spheroids proliferation and targeted gene expression induced by Wnt3a/7a. This study provides strong evidence supporting a key function of TG2/FN complexes in OCSCs. By demonstrating that TG2 directly binds Fzd7, we identified a novel function of TG2 and a new mechanism promoting CSCs’ proliferation and tumorigenicity. These results point to TG2/FN clusters or the newly discovered TG2/Fzd7 complex as potential new therapeutic CSC targets.

Citation Format: Salvatore Condello, Livia Sima, Cristina Ivan, Horacio Cardenas, Gary Schiltz, Rama Mishra, Daniela Matei. Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B56.