Blocking signaling at the level of GLI regulates downstream gene expression and inhibits proliferation of canine osteosarcoma cells.

Joseph Alan Bauer,Mehdi Hayat Shahi,Roseline Holt,Robert B Rebhun

doi:10.1371/journal.pone.0096593

Joseph Alan Bauer, Mehdi Hayat Shahi + Show 2 more

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https://doi.org/10.1371/journal.pone.0096593

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Journal: PloS one	Publication Date: May 8, 2014
Citations: 28	License type: CC BY 4.0

Affiliation: University of California, Davis

Abstract

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.

Highlights

The Hedgehog (Hh) signaling pathway has long been known to play a critical role in early embryonic development [1]
Expression of GLI1 and GLI2 in Canine OSA We first examined the mRNA expression of the transcription factor GLI1 and found high expression of GLI1 mRNA in 2 out of 3 cell lines (Abrams and D17) relative to osteoblast cells (Fig.1A)
We investigated the mRNA expression of the patched 1 (PTCH1) transmembrane receptor and found high expression of PTCH1 mRNA in Abrams and D17 cell lines compared to canine osteoblast cells (Fig.1C)

Summary

Introduction

The Hedgehog (Hh) signaling pathway has long been known to play a critical role in early embryonic development [1]. The Hh signaling pathway is activated upon the binding of Hh ligand to its twelve transmembrane receptor patched 1 (PTCH1). This binding relieves inhibition of smoothened (SMO), a seven transmembrane receptor regulated by PTCH1. SMO enters into the cytoplasm and activates transcription factors, most notably, the GLI family of proteins. The GLI family of proteins consists of three family members, GLI1, GLI2, and GLI3 that differentially regulate downstream Hh pathways. GLI proteins are phosphorylated in the presence of fused serine/threonine kinase [8] and Costal-2 a kinase-like cytoplasmic protein (Cos2) [9]. Activated GLI proteins enter into the nucleus and bind to the promoter of various cell regulatory genes including GLI itself and PTCH1. A high level of GLI expression is often indicative of activated Hh signaling

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