Abstract 133: Role of Glypican-3 (GPC3) on tumor progression of the human mammary gland
Authors: Castillo Lilian F. , Tascon Rocio S. , Joffé Elisa Bal de Kier , Peters Maria G. 
Published: May 2015

We have previously shown that the reexpression of Glypican-3 (GPC3), a proteoglycan downregulated in breast cancer, leads to the impairment of the in vivo metastatic capacity of the murine LM3 mammary adenocarcinoma cells. On the basis of clinical and translational potential of GPC3, the aim of this work was to assess whether GPC3 acts as a metastasis suppressor in human cells. So, we generated a preclinical breast cancer cell model. We chose MCF-7 cell line (poorly-metastatic, GPC3 +) and MDA-MB231 cell line (metastatic, GPC3 -) to be genetically modified.

GPC3 expression was blocked in MCF-7 cells by siRNA. We demonstrated that MCF-7-shGPC3 cells proliferate faster than controls (Population doubling time (h): 72 -shGPC3 vs 87 -shNC). In agreement, silencing of GPC3 increased clonogenic capacity (Colony number: 130 -shGPC3 vs 37 -shNC). No differences were found in viability. By wound healing assay we determined that -shGPC3 cells are significantly more motile than controls (Wound coverage (%): 15 -shGPC3 vs 2 -shNC). GPC3 blocking induced an increase in homotypic MCF-7 cell adhesion (Cellular aggregation (%): 56 -shGPC3 vs 21 -shNC). We performed an in vivo tumor growth assay, by sc inoculation of MCF-7 engineered cells into nude mice. MCF-7-shGPC3 tumor-bearing mice showed shorter tumor latency than controls (Md [Rg] (days): 30 [30-44] -shGPC3 vs 49 [30-71] -shNC), and an increase in tumorigenic ability (Tumorigenicity (%): 80 -shGPC3 vs 60 -shNC), as well as in the tumor growth rate (mm3/day: 26±2.2 -shGPC3 vs 0.2±0.02). Spontaneous metastases were not detected in any of the MCF-7 tumor-bearing mice.

On the other hand, GPC3 was reexpressed in MDA-MB231 cells by lentiviral infection. Surprisingly, GPC3 reexpressing cells showed a higher proliferation rate (Population doubling time (h): 24 -GPC3 vs 45 -vector), but their clonogenic capacity was less (Colony number: 8 -GPC3 vs 40 -vector). While GPC3 reexpression induced a 30% inhibition on MDA-MB231 viability, controls remained 100% viable. We also determined that -GPC3 cells are less migrant than controls (Wound coverage (%): 10 -GPC3 vs 90 -vector), and also they exhibit a reduction in their homotypic adhesion (Cellular aggregation (%): 33 -GPC3 vs 81 -vector). In relation to in vivo behavior, MDA-MB231-GPC3 tumor-bearing mice showed shorter tumor latency than controls (Md (days): 14 -GPC3 vs 97 -vector). However, GPC3 reexpression induced a reduction in tumorigenicity: 40% -GPC3 vs 60% -vector. The tumor growth rate was higher for GPC3 reexpressing cells (mm3/day: 72.73±9.46 -GPC3 vs 1.9±0.21). Spontaneous metastases were detected in lungs, showing an incidence of 20% for -GPC3 vs 40% -vector cells.

In sum, we generated a preclinical human breast cancer cell model with differential GPC3 expression. Our in vitro and in vivo results reveal the key role of GPC3 in the biology of breast cancer cells.

Citation Format: Lilian F. Castillo, Rocio S. Tascon, Elisa Bal de Kier Joffé, Maria G. Peters. Role of Glypican-3 (GPC3) on tumor progression of the human mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 133. doi:10.1158/1538-7445.AM2014-133