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Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells

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Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells Mehdi Hayat Shahi, Roseline Holt, Robert B. Rebhun* The Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, California, United States of America 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. Citation: Shahi MH, Holt R, Rebhun RB (2014) Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells. PLoS ONE 9(5): e96593. doi:10.1371/journal.pone.0096593 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received August 8, 2013; Accepted April 9, 2014; Published May 8, 2014 Copyright: ß 2014 Shahi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Bernice Barbour Foundation, the Center for Companion Animal Health-School of Veterinary Medicine, and NCRR K01 RR031272 (currently supported by the Office of Research Infrastructure Programs 8K01OD011111-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction [6,7,11]. This pathway mediates cell growth and survival through activation of pathways associated with transcription factors GLI1 The Hedgehog (Hh) signaling pathway has long been known to and GLI2 [12,13]. However, GLI2 often acts as a main play a critical role in early embryonic development [1]. It is now transcription factor in the absence of GLI1 [14,15]. Recent known that Hh signaling contributes to tumor growth and studies have found that human OSA cells have low expression of chemoresistance in a variety of human tumors including osteo- GLI1 compared to GLI2 and that GLI2 appears to serve as the sarcoma (OSA) [2–7]. The Hh signaling pathway is activated upon driving transcription factor of Hh signaling thus contributing to the binding of Hh ligand to its twelve transmembrane receptor the growth of OSA [7,16]. A separate study also identified GLI2 as patched 1 (PTCH1). This binding relieves inhibition of smooth- the main transcription factor of Hh signaling in human OSA ened (SMO), a seven transmembrane receptor regulated by growth, and further found that expression of GLI2 correlated with PTCH1. Upon activation, SMO enters into the cytoplasm and poor outcome in human patients with OSA [7]. Additional studies activates transcription factors, most notably, the GLI family of have found that inhibition of SMO in human OSA cell lines and proteins. In mammals, the GLI family of proteins consists of three mouse xenografts can inhibit canonical Hh signaling and may be family members, GLI1, GLI2, and GLI3 that differentially an effective therapeutic approach for the treatment of human regulate downstream Hh pathways. GLI1 and GLI2 are typically OSA [6]. found to induce distinct and overlapping target genes, whereas As an alternative to targeting SMO, compounds such as arsenic GLI3 can additionally serve as a repressor. GLI proteins are trioxide have been found to inhibit GLI transcription factors and phosphorylated in the presence of fused serine/threonine kinase can inhibit growth of sarcoma or OSA cells that express GLI [8] and Costal-2 a kinase-like cytoplasmic protein (Cos2) [9]. Activated GLI proteins enter into the nucleus and bind to the [17,18]. Recently, two small molecules have been identified, promoter of various cell regulatory genes including GLI itself and GANT61 and GANT58, which specifically inhibit GLI proteins PTCH1. Therefore, a high level of GLI expression is often and serve to prevent their interaction with DNA. Subsequently it indicative of activated Hh signaling. has been shown that GLI inhibitors can effectively block Hh While canonical Hh signaling is mainly mediated by PTCH1 signaling and inhibit cancer growth in vitro and in vivo [7,19]. and SMO, recent data has clearly demonstrated a parallel Targeting the Hh signaling pathway at the level of GLI, instead of existence of non-canonical Hh signaling pathways [10]. The Hh at the level of SMO, may represent an attractive approach since it signaling pathway has been found to play a critical role in many could potentially inhibit both canonical and non-canonical human cancers including basal cell carcinoma, medulloblastoma, mediated up-regulation of this pathway [20]. glioma, colon, breast, lung, pancreas and recently in OSA PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e96593 Inhibition of GLI in Canine Osteosarcoma Cells Spontaneously occurring OSA in the dog very closely models GLI1 mRNA expression compared to vehicle DMSO treated and the human disease with striking similarities at the clinical and untreated cells (Fig.4A). We also observed a significant decrease in molecular levels [21–23]. While very little is known about the GLI2 mRNA expression after the same treatment with GANT61 contribution of Hh-GLI signaling in spontaneously arising canine when compared with vehicle DMSO treated and untreated cells cancers, alterations in this pathway have been implicated in both (Fig.4B). Importantly, changes in GLI mRNA expression were canine appendicular and extra-skeletal mammary OSA [24,25]. found to correlate with changes at the protein level as determined Based on the importance of Hh-GLI signaling pathways in human by Western blot analyses (Fig. 5). cancers including OSA, we set out to determine whether GLI signaling is present and active in canine OSA cell lines. We found Expression of GLI downstream target genes after that GLI signaling is indeed present and active in multiple canine GANT61 treatment in canine OSA cell OSA cell lines and that GANT61 inhibition at the level of GLI We next wanted to examine whether GANT61 inhibition of modulates expression of downstream target genes and reduces GLI expression led to alterations in downstream target gene proliferation of canine OSA cells. mRNA and protein expression. We found that GANT61 decreased levels of PTCH1 mRNA expression by approximately Results 60% in D17 cells compared to vehicle DMSO treated and Expression of GLI1 and GLI2 in Canine OSA untreated cells (Fig.4C). Furthermore, expression of one of the putative downstream target genes of the GLI signaling pathway, We first examined the mRNA expression of the transcription PAX6, showed approximately a 40% decrease in mRNA factor GLI1 and found high expression of GLI1 mRNA (transcript) expression in canine D17 cells after GANT61 treatment compared in 2 out of 3 cell lines (Abrams and D17) relative to osteoblast cells to vehicle DMSO treated and untreated cells (Fig.4D). mRNA (Fig.1A). We then checked the expression of GLI2 at the mRNA changes correlated with protein expression changes for GLI1, level and also found high expression of GLI2 mRNA in both GLI2, PAX6, and PTCH1 (Fig. 5). Taken together, these data Abrams and D17 cell lines (Fig.1B). In contrast, Moresco cells expressed relatively low levels of GLI1 and GLI2 mRNA when demonstrate that GLI signaling is active and indeed regulates its compared to osteoblasts or Abrams and D17 OSA cell lines. target genes in canine OSA cells. Western blot results confirmed varying expression of GLI1 and GLI2 proteins in all canine OSA cell lines. (Fig. 2) Expression of GLI1 and GLI2 in Moresco cells after GANT61 treatment Constitutive Expression of GLI target genes in Canine Because we observed inhibition of proliferation and colony OSA formation in Moresco cells despite relatively low constitutive We next investigated the mRNA expression of the PTCH1 expression of GLI1 and GLI2, we wanted to confirm the effect of transmembrane receptor and found high expression of PTCH1 GANT61 on the mRNA expression of GLI1 and GLI2 in Moresco mRNA in Abrams and D17 cell lines compared to canine cells. Despite the lower relative expression of GLI in Moresco cells, osteoblast cells (Fig.1C). As would be expected based on GLI after 96 hrs of exposure to 12 mM of GANT61, we indeed expression, Moresco cells expressed lower constitutive levels of observed a significant reduction of GLI1 and GLI2 mRNA PTCH1 than osteoblast, D17, or Abrams cell lines. While little is expression in Moresco cells (Fig. 6), confirming that even relatively known about PAX6 in OSA, it is a homeodomain-transcription low constitutive levels of GLI signaling can be inhibited. factor I and one of the putative downstream targets of the Hh-GLI signaling pathway [26]. We therefore examined PAX6 gene Discussion expression and found that PAX6 was highly expressed in Abrams Dogs with spontaneously arising OSA have been found to be an and D17 OSA cell lines that expressed high constitutive levels of excellent model for the study of human OSA and this disease GLI (Fig.1D).
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