Glypican-3 (Gpc3) Inhibits Metastatic Dissemination In A Preclinical Human Breast Cancer Model

Although GPC3 has been linked to cancer, its role during mammary tumor progression is barely known. With the aim to develop a pre-clinical breast cancer model, we genetically modified MCF-7 (poorly-metastatic, GPC3 +) and MDA-MB231 (metastatic, GPC3 -) cell lines. GPC3 expression was blocked in MCF-7 by siRNA (generating MCF-7-shGPC3 sublines), while it was overexpressed in MDA-MB231 by viral infection (producing MDA-MB231-GPC3 sublines). First, we analyzed the in vivo tumor growth by s.c. inoculation of MCF-7 and MDA-MB231 engineered cells into nude mice. Silencing GPC3 stimulated tumorigenicity, while its overexpression inhibited it. The histological analysis of MCF-7-shGPC3 tumors revealed extensive invasion of the muscle and the dermis, while control tumors did not invade. In addition, MDA-MB231-GPC3 tumors were less invasive than MDA-MB231-vector ones. We also evaluated spontaneous metastasis capacity by lung histological analysis. While no metastases were found in MCF-7-sh scramble tumor-bearing mice, lung nodules were detected in mice inoculated with GPC3 silenced cells. On the other hand, mice bearing MDA-MB231-GPC3 tumors showed a decrease in the incidence of metastasis. Next, we did a panel of in vitro tests. Although MCF-7-shGPC3 and control cells were morphologically similar, an increase in the number of actin stress fibers was found in GPC3 silenced cells. In association with this mesenchymal characteristic, this subline exhibited a decrease in E-Cadherin expression. While MDA-MB231-vector cells presented a fibroblastic appearance, GPC3 overexpression induced a drastic change in cell morphology turning to an epithelial phenotype. In addition, although control cells showed large stress fibers, GPC3 overexpressing cells localized their actin in cortical position. Moreover, GPC3 induced a reexpression of the epithelial marker E-Cadherin. We tested anchorage-independent growth, finding that GPC3 silencing disorganized MCF-7 spheroids. MDA-MB231-GPC3 cells formed larger spheroids than those generated by controls. Blocking E-cadherin, employing a neutralizing antibody, reversed the 3-D growth ability of GPC3 overexpressing cells. The sensitivity to nutrient depletion was evaluated. We found, through propidium iodide/Hoescht staining, a reduction in starved MCF-7-shGPC3 cell death. While MCF-7-sh scramble cells showed morphological alterations characteristic of apoptotic death as detected by orange acridine/ethidium bromide staining, no apoptosis was found in MCF-7-shGPC3 cells. GPC3 overexpression increased MDA-MB-231 cell susceptibility to death. Although there was no apoptosis in MDA-MB231 control cells, the overexpression of GPC3 promoted an increase in the number of cells with nuclear and cytoplasmic manifestations of apoptosis. We demonstrate a central role of GPC3 in human breast cancer biology. Our results indicate that it acts a metastasis suppressor in human mammary cancer. Citation Format: Lilian F. Castillo, Rocio Tascon, Elisa Bal de Kier Joffe, Maria G. Peters. Glypican-3 (GPC3) inhibits metastatic dissemination in a preclinical human breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1419. doi:10.1158/1538-7445.AM2015-1419