Abstract
Purpose
We have shown that GPC3 overexpression in breast cancer cells inhibits in vivo tumor progression, by acting as a metastatic suppressor. GPC3-overexpressing cells are less clonogenic, viable and motile, while their homotypic adhesion is increased. We have presented evidences indicating that GPC3 inhibits canonical Wnt and Akt pathways, while non-canonical Wnt and p38MAPK cascades are activated. In this study, we aimed to investigate whether GPC3-induced Wnt signaling inhibition modulates breast cancer cell properties as well as to describe the interactions among pathways modulated by GPC3.
Methods
Fluorescence microscopy, qRT-PCR microarray, gene reporter assay and Western blotting were performed to determine gene expression levels, signaling pathway activities and molecule localization. Lithium was employed to activate canonical Wnt pathway and treated LM3-GPC3 cell viability, migration, cytoskeleton organization and homotypic adhesion were assessed using MTS, wound healing, phalloidin staining and suspension growth assays, respectively.
Results
We provide new data demonstrating that GPC3 blocks—also at a transcriptional level—both autocrine and paracrine canonical Wnt activities, and that this inhibition is required for GPC3 to modulate migration and homotypic adhesion. Our results indicate that GPC3 is secreted into the extracellular media, suggesting that secreted GPC3 competes with Wnt factors or interacts with them and thus prevents Wnt binding to Fz receptors. We also describe the complex network of interactions among GPC3-modulated signaling pathways.
Conclusion
GPC3 is operating through an intricate molecular signaling network. From the balance of these interactions, the inhibition of breast metastatic spread induced by GPC3 emerges.
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Acknowledgements
This work was supported by Argentine grants from FONCyT (PICT 2013-1337 Préstamo BID)—Ministry of Science and Technical, CONICET (PIP 2013-112200120100100CO) and Fundación Florencio Fiorini. We would like to thank Dr. Osvaldo Rey for assistance with immunofluorescence and Dr. Mariana Salatino for help with conditional medium concentration.
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432_2018_2751_MOESM1_ESM.tif
Fig. 1 GPC3 expression levels. A RT-PCR. Total RNA from LM3-GPC3 and LM3-vector cells was retrotranscribed and used as template in PCR reactions. Agarose gel representative images are shown. B WB. Total protein extracts from LM3-GPC3 and LM3-vector cells were analyzed by WB to determine GPC3 levels. The arrows indicate GPC3 full-length core protein and N-terminal subunit. Actin was employed as seeding control (TIF 1033 KB)
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Fernández, D., Guereño, M., Lago Huvelle, M.A. et al. Signaling network involved in the GPC3-induced inhibition of breast cancer progression: role of canonical Wnt pathway. J Cancer Res Clin Oncol 144, 2399–2418 (2018). https://doi.org/10.1007/s00432-018-2751-0
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DOI: https://doi.org/10.1007/s00432-018-2751-0