Abstract 842: Breast cancer: Relationship between GPC3 and EMT process
Authors: Lilian F. Castillo, Gisela V. Novack, Elisa Bal de Kier Joffé, María G. Peters
Journal: Cancer Research (2017)

Abstract Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells acquire mesenchymal fibroblast-like properties and display reduced intracellular adhesion and increased motility. Glypican-3 (GPC3) is a heparan sulfate proteoglycan associated with cancer. We have established that GPC3 regulates the EMT process underwent by breast cancer cells. However, cellular and molecular events underlying this modulation are not completely described. At the present work we analyzed the effect of GPC3 on cell adhesion and on the expression of molecules associated with this property. In addition, molecular regulators of EMT were also studied. We employed a murine breast cancer cell line (LM3: GPC3-) as well as human lines (MDA-MB231: GPC3- and MCF-7: GPC3+). We have blocked GPC3 expression in MCF-7 cells, while it was overexpressed in MDA-MB231 and LM3 ones. It is know that EMT depends on a reduction in cell adhesion. We determined that GPC3 overexpression induced an increase in the homotypic adhesion of MDA-MB231 and LM3 cells, although GPC3 silencing did not lead significant changes in the organization of MCF-7 spheroids. GPC3 was also able to modulate the adhesion to extracellular matrix components. GPC3 overexpressing cells were more adherent to fibronectin than controls, but no changes were observed in their adhesion to laminin. E-Cadherin, a molecule involved in homotypic adhesion, is considered a suppressor of invasion and growth of many epithelial cancers. Here, we have corroborated by WB that GPC3 overexpression induces the E-Cadherin upregulation in LM3 and MDA-MB231 cells, whereas GPC3 silencing inhibits the expression of this epithelial marker in MCF-7 cells. We also confirmed the called "cadherin switch", since N-Cadherin was downregulated in GPC3 overexpressing cells and upregulated in GPC3 silenced ones. β-Catenin is a key downstream effector in the Wnt pathway. We confirmed that GPC3 expressing breast cancer cells exhibit a canonical Wnt signaling inhibition. β-Catenin also activates Slug. Similar to Snail, Slug binds to the E-Cadherin promoter to repress transcription. By WB and qPCR we demonstrated that GPC3 induces a decrease in Slug and Snail levels. Surprisingly, Twist expression was higher in GPC3 expressing cells. It was reported that Slug is able to represses integrins expression. However, β1-Integrin levels were decreased in GPC3 overexpressing MDA-MB231 cells, while they were increased in GPC3 blocked MCF-7 sublines. Our results suggest the key role of GPC3 in the EMT regulation. GPC3 would modulate cell adhesion and expression of E-Cadherin, N-Cadherin and β1-Integrin. In addition, GPC3 would regulate the expression of different EMT molecules (as Slug, Snail and Twist) as well as signaling pathways involved in that process (like canonical Wnt). The set and balance of these regulations would result in the reversal of EMT, reinforcing the function of GPC3 as an inhibitor of mammary tumor progression. Citation Format: Lilian F. Castillo, Gisela V. Novack, Elisa Bal de Kier Joffé, María G. Peters. Breast cancer: Relationship between GPC3 and EMT process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2017-842