Abstract 508: Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland.

Abstract

Abstract GPC3 was proposed as a negative regulator of cell proliferation in the mammary gland. Whereas normal breast tissues present high GPC3 levels, its expression is reduced in breast tumors. We have transfected LM3 murine mammary tumor cells with the GPC3 gene. We found that GPC3 reexpression inhibited metastasis in vivo. Although GPC3 signaling pathway is unclear, we reported that this glypican modulates Wnt, p38 and Akt pathways. However, regulation hierarchy has not been determined and we have no data in human cells. We believe it possible to generate an anti-metastasis therapy targeting GPC3 pathway. To test it, we developed a pre-clinical human model. First, we showed by qPCR that GPC3 expression levels are inversely proportional to the metastatic ability of a panel of human breast cancer cells. The metastatic Hs578T and MDA-MB231 cells expressed less GPC3 than the poorly-metastatic ZR-75-1 and MCF7 lines. We selected MDA-MB231 and MCF7 to be modified by genetic engineering. GPC3 expression was blocked by RNA interference technology. We transfected four shRNA-GPC3 and one shRNA scramble (shNC) into MCF7. After selection and cloning, we chose clone 2 that expressed about 75% less GPC3 for further characterization. On the other hand, GPC3 was reexpressed in MDA-MB231 cells by transduction of pLV-GPC3-GFP (empty vector was used as control). GPC3 mRNA increased up to 200-fold in pLV-GPC3-GFP transduced cells. Then we evaluated cell properties in the generated models. Surprisingly, MDA-MB231-GPC3 cells showed a higher proliferation rate than controls, that became significant from 48 h of exponential growth (Population doubling time= 45 h for control vs. 24 h for GPC3 reexpressing cells). On the other side, MCF7-shGPC3 bulk and clone 2 proliferated faster than -shNC. This suggests that, in this cell line, GPC3 is inhibiting proliferation. GPC3 reexpression induced a 30% inhibition on MDA-MB231 viability when cells were serum starved for 96 h, while parental and -GFP remained 100% viable. No differences were detected among MCF7 engineered cells. So, the effect of GPC3 on cell viability would depend on the cellular context. We performed a wound healing assay to evaluate migration, determining that MDA-MB231-GPC3 cells were significantly less migrant than their controls (Wound coverage: MDA-MB231-GPC3 10% vs. parental and -GFP 90%). In sum, we have developed a human cell model to study the role of GPC3 on tumor progression. Our preliminary in vitro results reveal the importance of GPC3 in the biology of breast cancer cells. Citation Format: Lilian F. Castillo, Rocio Tascon, María Amparo Lago Huvelle1, Ancély Ferreira dos Santos, Letícia Ferreira Terra, Rosangela A. m. Wailemann, Talita C. Oliveira, Mari Sogayar, Leticia Labriola, Elisa Bal de Kier Joffe, Maria Giselle Peters. Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2013-508