Abstract 1408: Activation status and function of the canonical Wnt pathway in murine mammary tumor cells reexpressing Glypican-3 (GPC3)

Abstract

Abstract GPC3 is a proteoglycan downregulated in breast cancer cells. We previously demonstrated that GPC3 reexpression inhibits in vivo metastatic potential of LM3 murine mammary adenocarcinoma cells. This event was associated in vitro with a lower motility and an increased aggregation, as well as with an enhanced senescence and apoptosis. We have previous evidence indicating that the canonical Wnt pathway is inhibited in LM3-GPC3 cells. The aim of this work was to validate the inhibition of canonical Wnt signaling in GPC3 reexpressing cells and to investigate whether differences in cell behavior are due to the regulation of GPC3 on this pathway. By means of western blots (WB) we established that LM3-GPC3 cells have 5-10 folds less phosphorylated GSK3B. Using cytoplasmic protein extracts and WB we also determined that GPC3 reexpressing cells present lower levels of cytoplasmic B-Catenin. This was further studied by immunofluorescence (IF), where it was found that while LM3-vector cells show positive signal for B-catenin mainly in nucleus and cytoplasm, this staining is restricted to the membrane and cytoplasm in LM3-GPC3 cells. In addition, a gene reporter assay employing OT/OF plasmids confirmed the canonical Wnt pathway inhibition induced by GPC3. To determine whether cell properties modulated by GPC3 are mediated by the canonical Wnt pathway, we treated cells with its activator LiCl (or NaCl as control). LiCl treatment increases 10-fold the phosphorylation of GSK3B, as compared to LM3-GPC3 cells treated with NaCl, and induces an enhance of about 50% of the canonical Wnt transcriptional activity (gene reporter assay). We confirm that although LiCl partially reverted senescence induced by GPC3 reexpression (Senescent X-Gal positive cells: LM3-GPC3+NaCl= 85% vs. LM3-GPC3+LiCl= 65%, p<0.05), it had no effects on apoptosis. We established by wound healing assays that LM3-GPC3 cells +LiCl recover their ability to migrate (Coverage of the wound: LM3-GPC3+NaCl= 15% vs LM3-GPC3+LiCl= 75%, p<0.05). Finally, LiCl treatment reduced LM3-GPC3 homotypic cell aggregation, reaching similar levels to LM3-vector clones (p<0.05). In summary, we confirm by a new methodological approach that GPC3 inhibits the canonical Wnt signaling. Our results suggest that GPC3 would be modulating senescence, migration and cell aggregation of mammary tumor cells through this pathway inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1408. doi:1538-7445.AM2012-1408