Gli Activity Research Articles

GLI2 Transcription Factor Modulates CD40 Ligand Expression In Bone Marrow Stromal Cells

The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells within the host. Cytokines play a key role in mediating this crosstalk between malignant cells and the tumor microenvironment. Although this interaction is clearly established, many of the molecular mechanisms mediating these signaling events remain elusive. In previous work, we have shown that the cytokine CCL5 can regulate the expression and activity of the transcription factor GLI2 in stromal cells. GLI2 is an effector of hedgehog (HH) signaling; however, our studies reveal a role for GLI2 independent of HH. GLI2, in turn, increases the expression and secretion of IL-6 in the tumor microenvironment, highlighting a novel role for GLI2 in directly modulating cytokine genes. We therefore screened for additional cytokines regulated by this axis in HS-5 bone marrow stromal cells using expression arrays. We found that GLI2 can modulate the expression of several cytokines including CD40 ligand (CD40L), CCL2, CCL7 and CXCL11. CD40L is an immunomodulatory and proinflammatory molecule that has been shown to play a role in multiple hematologic malignancies including chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and acute myeloid leukemia (AML) pathogenesis. We first characterized the regulation of CD40L, because of the importance of CD40L in malignant cell biology. Gene expression results were validated by RT-PCR in two additional bone marrow stromal cell lines Saka and L88 where overexpression of GLI2 resulted in increased CD40L expression. Furthermore, we detected increased levels of soluble CD40L (sCD40L) in L88 and Saka cells overexpressing GLI2. Conversely, knockdown of GLI2 resulted in reduced expression and secretion of this cytokine. Biochemical mapping of the domains critical for this GLI2 regulatory mechanism reveals that N-terminal transcriptional domain is dispensable, as a mutant form of GLI2 lacking this domain (delta N-GLI2) increases CD40L gene expression by RT-PCR, protein expression by western blot and sCD40L secretion by ELISA, beyond that of the full-length GLI2 levels. Bioinformatics analysis of the CD40L promoter indicates the presence of 3 candidate binding sites for GLI proteins, suggesting the modulation of CD40L by GLI2 is mediated by direct interaction with its promoter. Ongoing experiments are aimed at determining the mechanism by which GLI2 regulates CD40L expression (either by direct binding to the CD40L promoter or through another indirect pathway). Together, our data identify a novel mechanism controlling CD40L in stromal cells in the bone marrow microenvironment. Because of the importance of CD40L in malignant cell biology, understanding the mechanism of its regulation is of great importance for the development of therapies aimed at targeting the malignant bone marrow tumor microenvironment. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Regulation Of GLI1, Potential New Therapeutic Target For Diffuse Large B-Cell Lymphoma

Hedgehog (Hh) signaling plays an important role in the pathobiology of B-cell malignancies including diffuse large B cell lymphoma (DLBCL). The GLI family members that include GLI1, GLI2 and GLI3 are Hh signaling transcription factors. While most research has focused on smoothened (SMO), Hh transducer receptor subunit, many lines of evidence indicate that other signaling pathways can activate GLI transcription factors. GLI1 is a zinc finger transcription factor and established oncogene. GLI1 expression increases in response to Hh signaling activation and potentiates the transcriptional output of Hh signaling. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and plays an important role in cell proliferation, survival and chemotolerance in DLBCL (Leukemia 2010 and Oncogene 2011). Moreover, using stable GLI1 knockdown DLBCL cell lines we found that GLI1 contributes to cell survival and proliferation of DLBCL (J Biol Chem 2013). However, our understanding of the mechanisms controlling the transcriptional activity of GLI1 is limited.To identify regulatory components that participate in the transcriptional regulation of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immuno-precipitation (IP) of endogenous GLI1. We found that the NF-κB kinase, IKKβ, is one of the proteins associated with GLI1. This finding was of interest as we found a positive correlation between NF-kB activity and Hh signaling in DLBCL (Blood 2013). To validate the functional interaction between endogenous GLI1 with IKKβ in-vivo, lysates of 293T cells were IP with GLI1 or control antibody and subjected to immunoblot analysis with antibodies recognizing IKKβ subunit and SuFu (known partner of GLI1) respectively. IKKβ and SuFu were co-immunoprecipitated with GLI1. To confirm these findings, 293T cells were transiently co-transfected with plasmids carrying GLI1 and FLAG-tagged IKKα and IKKβ wild-type or kinase dead (K44A) forms. Both forms (IKKα and IKKβ) were detected in the immunoprecipitated GLI1 protein complex, however only transfection of IKKβ resulted in abundance of GLI1 protein suggesting a functional role of IKKβ kinase in the regulation of GLI1 protein levels. The association (and nuclear co-localization) between IKKβ and GLI1 was confirmed in DLBCL cell lines and 293T cells by immunofluorescence studies.To investigate the role of IKKβ in the transcriptional activity of GLI1, we knocked down IKKβ gene in HBL1 (cells with CD79B mutation) cells using two independent IKKβ shRNAs. Silencing IKKβ resulted in a decrease of IKKβ expression associated with decreased levels of GLI1 and GLI1 downstrean targets (PTCH1, CCND1 and BCL2). To better understand the functional role of IKKβ kinase activity in the transcriptional regulation of GLI1 target genes, we explore the IKKβ dependent GLI1 phosphorylation sites. We co-transfected full length GLI1 and IKKβ plasmids in 293T cells and IKKβ-GLI1 complex were purified using anti-GLI1 antibody. Peptides resulting from digestion of GLI1 with several different proteases were analyzed by nanospray ion trap mass spectrometry. Seven phosphorylation sites in GLI1 C-terminal residues were identified during analysis of trypsin-digested GLI1 by mass spectrometry. These data suggest that IKKβ phosphorylates GLI1 and plays an important role in the regulation of GLI1 transcriptional activity. Because activation of NF-κB is a frequent event in DLBCL, our study will help to understand the cross talk between NF-κB and Hh signaling pathways and will open potential new avenues for targeted therapy in DLBCL. Disclosures:Vega:NIH: Research Funding; Leukemia & Lymphoma Society: Research Funding.

Hedgehog Pathway Is Deregulated In Myelodysplastic Syndrome Progenitor Bone Marrow Cells

The sonic hedgehog (Shh) pathway is a regulatory network involved in the development of cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in the inhibition of their repression on Smoothened (Smo). Smo is a seven-transmembrane protein, which inhibits Suppressor of Fused (SUFU), resulting in the stabilization and activation of the Gli family of transcription factors, followed by a transcriptional response to the “canonical Hh signaling”. SUFU is a negative regulator of the Hedgehog signaling pathway, through the restriction of Gli activity through cytoplasmic sequestration. Germline mutations in SUFU are involved in medulloblastoma. Dysregulation of Shh signaling has been reported to be involved in several malignancies, including chronic myeloid leukemia.To our knowledge, however, only one study has been carried out in myelodysplastic syndrome (MDS) and acute leukemia so far, and the role of this pathway in leukemogenesis is still unknown. Recently we showed an increased protein expression of Sonic and Dessert Hedgehog in addition to increased mRNA levels of PTCH and SMO in MDS bone marrow. Here, we investigated whether this increase was capable of activating Hedgehog pathway in total and in progenitor bone marrow cells.Bone marrow (BM) samples were collected from 39 MDS (25 low-risk, 14 high-risk, according WHO 2008) patients and 26 healthy donors. CD34+ cells were isolated from peripheral blood of 9 healthy donors and 20 (12 low-risk and 8 high risk) MDS patients. Relative expressions of PTCH, SMO, SUFU and GLI1 were obtained by Real time-PCR.Increased expression observed in ligands and receptors were not sufficient to upregulate the activity of Hedgehog pathway in MDS total bone marrow. The downstream effector GLI1 was under expressed in the disease as median [min-max] as follows: healthy donors 1.04[0.02-4.01]; low risk MDS 0.21[0.03-2.26]; high risk MDS 0.07[0.03-1.48] healthy donors vs low risk p=0.007; healthy donors vs high risk p=0.006. Furthermore, SUFU expression was similar in healthy donors 3.3[0.35-7.84] and in myelodysplastic (low risk 3.76[0.75-13, 2] and high risk 1.8[0.58-16.4]) bone marrows. Different from total bone marrow, the receptors Patched and SMO showed decreased expression in MDS CD34+ cells: Patched healthy donors 2.24[0.74-4.36]; low risk 0.6[0.1-3.34]; high risk 0.98[0.29-1.22] and SMO healthy donors 1.18[0.62-2.16]; low risk 0.79[0,11-1.87]; high risk 0.86[0.37-1.02]; healthy donors vs high risk MDS p=0.049. SUFU was overexpressed (healthy donors 1.44[0.75-2.39]; low risk 2.77[0.93-4.11]; high risk 1.68[1.07-3.43]; healthy donors vs low risk MDS p=.005). However, myelodysplastic progenitors had GLI1 overexpression (healthy donors 1.09[0.6-2.91]; low risk 2.93[0.75-14.52]; high risk 1.32[0.71-2.58]; healthy donors vs low risk p=0.03), indicating an activation of the Shh pathway in MDS progenitors.Recently, Kobune et al (Blood Cancer J. 2012 Sep 7) showed activation of Hedgehog signaling in primary CD34+ blasts from acute myeloid leukemia and MDS, as those cells expressed IHh, PTCH, SMO, GLI1 and GLI. Taken together, these results and ours led us to conclude that the Hedgehog pathway is activated in MDS. Since Shh pathway is being explored as a new opportunity for targeted therapies against tumors, our study suggests that the inhibition of this pathway could be strategic in the control of leukemic stem cell and MDS treatment. Disclosures:No relevant conflicts of interest to declare.

Expression Of Hedgehog Pathway Mediator Gli2 Represents a Clinically Negative Prognostic Marker In Acute Myeloid Leukemia and Its Inhibitor GANT61 Exerts Anti-Leukemic Effects In Vitro

▪ BackgroundLeukemic stem cells depend on signals provided by bone marrow (BM) niche cells. Due to its function in stem cell biology, we investigated expression and prognostic impact of the Hedgehog pathway in acute myeloid leukemia (AML). Methods and resultsPre-treatment samples from 104 patients with newly diagnosed AML (AMLSG 07-04 trial) were analyzed by quantitative PCR. Expression of receptors Smoothened and Patched and downstream transcription factors Gli1, Gli2 and Gli3 was found in 69%, 41%, 73%, 20% and 26% of cases, respectively. However, no expression of Hedgehog ligands was observed. Gli2 expression had a significant influence on event-free, relapse-free and overall survival (p=0.037, p=0.026 and p=0.013, respectively) and was furthermore correlated to FLT3 mutational status (p<0.001).To verify our findings in a second, independent patient cohort, microarray-based gene expression data of patients published by Valk et.al.(N Engl J Med. 2004 Apr 15;350(16):1617-28) was used. Clinical data of 291 AML patients from this cohort were available and the negative prognostic impact of Gli2 expression on overall and event-free survival could be confirmed (p=0.003 for overall and p=0.007 for event-free survival, respectively; no data for relapse-free survival available). Additionally, the correlation of Gli2 expression to FLT3 mutational status was also observed in this patient cohort (p=0.003).Although Hedgehog ligands were not expressed in AML bone marrow aspirates, Desert Hedgehog (DHH) plasma levels were significantly increased in AML patients compared to healthy subjects (p=0.002). Provision of Desert Hedgehog could be ascribed to BM niche cells including endothelial cells and osteoblasts, since DHH expression was found in outgrowth endothelial cells (OECs) and osteoblasts in vitro and on immunohistochemistry of leukemic bone marrow samples in patients.Next, we investigated the effect of Gli inhibition in vitro. AML cell lines KG-1, OCI-AML5 and UKE-1 as well as freshly isolated primary AML cells were treated with the specific Gli inhibitor GANT61. GANT61 dose-dependently induced apoptosis in all three cell lines investigated. Furthermore, GANT61 treatment resulted in significantly increased apoptosis in primary AML cells upon treatment with 10 µM, 30 µM and 90 µM GANT61 (n=10 pts). Additionally, a pronounced growth inhibitory effect on primary AML cells was observed with a significant reduction in cell count to 82 ± 13%, to 54 ± 11% and to 47 ± 13% upon treatment with 10 µM, 30 µM and 90 µM of GANT61, respectively (n=9 pts). Incubation of KG-1, UKE-1 and OCI-AML5 cells for 7 days with 30 µM GANT61 resulted in reductions in cell numbers compared to the control to 14 ± 2%, to 44 ± 6% and to 31 ± 0%, respectively. Moreover, GANT61 mediated a strong and significant inhibitory effect on the colony formation capacity of AML cell lines and primary AML cells. The average number of colonies was reduced to 44 ± 16% and 4 ± 4% upon treatment with 10 µM and 30 µM GANT61, respectively, while no colony formation was observed with a concentration of 90 µM GANT61 in primary AML cells (n=4 pts). ConclusionIn conclusion, Gli2 expression is a negative prognostic factor in AML that mirrors activated Hedgehog signaling induced by paracrine stimulation through BM niche cells. Inhibition of Hedgehog signaling by blocking Gli activity might represent a promising therapeutic approach for AML treatment with special regard to patients carrying a mutated FLT3. Disclosures:Fiedler:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Titration of GLI3 Repressor Activity by Sonic Hedgehog Signaling Is Critical for Maintaining Multiple Adult Neural Stem Cell and Astrocyte Functions

Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.

Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.

Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis

The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components.

Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma

Abstract Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p&amp;lt;0.05, FC &amp;gt;2 or &amp;lt;-2). Unsupervised clustering revealed two distinct subgroups with differential ShH activity (GLI1 z-score -2.000 vs 2.000) and expression of GLI1, GLIPR1, PTCHD1 and SMO. Protein profiling revealed high expression of TLN1, CLU, EEF2 (FC &amp;gt;2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p&amp;lt;0.05, FC &amp;gt;2 or &amp;lt;-2). Concordant differences in DNA methylation were detected in related genes, including GLI1, FOXF1, SMO, SHH, and SUFU (ANOVA, p&amp;lt;0.05, FC&amp;lt;-2 or &amp;gt;2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.

Menin Directly Represses Gli1 Expression Independent of Canonical Hedgehog Signaling

Multiple endocrine neoplasia type 1 (MEN-1), is a familial tumor syndrome resulting from mutations in the tumor suppressor gene menin (MEN1). Menin plays an essential role in both repressing and activating gene expression. However, it is not well understood how menin represses expression of multiple genes. Upon MEN1 excision, the transcription factor Gli1 and its target genes, including Ptch1 and c-Myc, were shown to be elevated in the absence of an apparent Hedgehog) pathway-activating ligand or when Smoothened (SMO), a key component of the pathway, is inhibited. Menin binds to the GLI1 promoter and recruits PRMT5, a histone arginine methyltransferase associated with transcriptional repression. Both PRMT5 binding and histone H4 arginine 3 methylation (H4R3m2s) are decreased at the GLI1 promoter in MEN1-excised cells. Moreover, MEN1 ablation resulted in increased binding of transcriptionally active Gli1 at the GLI1 promoter in a manner not influenced by the canonical Hedgehog signaling pathway. Inhibition of Gli1 by the small-molecule inhibitor GANT-61 led to decreased expression of Gli1 and its target genes in MEN1-depeleted cells. Furthermore, GANT-61 potently suppressed proliferation of MEN1-excised cells as compared with control cells. These findings uncover a novel epigenetic link whereby menin directly represses Gli1 expression, independent of the canonical Hedgehog signaling pathway, via PRMT5 and its repressive H4R3m2s mark. Inhibition of GLI1 suppresses neuroendocrine tumors harboring mutations in the MEN1 gene.

Fem1b promotes ubiquitylation and suppresses transcriptional activity of Gli1

The mammalian Fem1b gene encodes a homolog of FEM-1, a protein in the sex-determination pathway of the nematode Caenorhabditis elegans. Fem1b and FEM-1 proteins each contain a VHL-box motif that mediates their interaction with certain E3 ubiquitin ligase complexes. In C. elegans, FEM-1 negatively regulates the transcription factor TRA-1, and functions as an E3 ubiquitin ligase substrate recognition subunit to target TRA-1 for ubiquitylation. TRA-1 is homologous to the mammalian Gli1 protein, a transcription factor that mediates Hedgehog signaling as well as having Hedgehog-independent functions. Whether the interaction between nematode FEM-1 and TRA-1 proteins is conserved, between corresponding mammalian homologs, has not been reported. Herein, we show that Fem1b interacts with Gli1 within cells, and directly binds Gli1. Fem1b also promotes ubiquitylation of Gli1, suppresses transcriptional activation by Gli1, and attenuates an oncogenic Gli1 autoregulatory loop in cancer cells, all dependent on the VHL-box of Fem1b. These findings have implications for understanding the cellular functions of Fem1b, and the regulation of Gli1 oncoprotein activity.

Nonclassical hedgehog‐gli signaling and its clinical implications

Hedgehog (Hh) signaling regulates embryonic patterning and organ morphogenesis. It is also involved in regeneration and repair of tissues. Aberrant Hh pathway activation is a feature of many human malignancies. Classical Hh signaling is activated by Hh ligands that can signal in an autocrine or paracrine manner generating a tumor-stromal crosstalk. In contrast to canonical Hh signaling that culminates in the activation of GLI transcription factors, "noncanonical" Hh signaling does not involve GLI transcriptional activity. Several Hh pathway inhibitors have progressed to clinical trials, where the outcomes have not been very encouraging in many solid tumors. Here we discuss the likely role of "nonclassical" Hh-GLI signaling that is activated by growth factors and cytokines from the tumor and/or its microenvironment; these uncouple Hh signaling from the vital regulatory protein Smoothened, and result in the activation of GLI. While efforts are being made to target tumor-intrinsic Hh targets, it is imperative to acknowledge the role of the complex molecular networks and crosstalk between different components of the tumor microenvironment that can result in the emergence of resistance to conventional Hh therapy. These considerations have an important bearing on appreciating the need to mitigate the effects of tumor microenvironment to combat resistance to Hh inhibitors.

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress

The Hedgehog (Hh) signaling regulates tissue development, and its aberrant activation is a leading cause of malignancies, including medulloblastoma (Mb). Hh-dependent tumorigenesis often occurs in synergy with other mechanisms, such as loss of p53, the master regulator of the DNA damage response. To date, little is known about mechanisms connecting DNA-damaging events to morphogen-dependent processes. Here, we show that genotoxic stress triggers a cascade of signals, culminating with inhibition of the activity of Gli1, the final transcriptional effector of Hh signaling. This inhibition is dependent on the p53-mediated elevation of the acetyltransferase p300/CBP-associated factor (PCAF). Notably, we identify PCAF as a novel E3 ubiquitin ligase of Gli1. Indeed PCAF, but not a mutant with a deletion of its ubiquitination domain, represses Hh signaling in response to DNA damage by promoting Gli1 ubiquitination and its proteasome-dependent degradation. Restoring Gli1 levels rescues the growth arrest and apoptosis effect triggered by genotoxic drugs. Consistently, DNA-damaging agents fail to inhibit Gli1 activity in the absence of either p53 or PCAF. Finally, Mb samples from p53-null mice display low levels of PCAF and upregulation of Gli1 in vivo, suggesting PCAF as potential therapeutic target in Hh-dependent tumors. Together, our data define a mechanism of inactivation of a morphogenic signaling in response to genotoxic stress and unveil a p53/PCAF/Gli1 circuitry centered on PCAF that limits Gli1-enhanced mitogenic and prosurvival response.

Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation

Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling.

The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury

In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro. In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses. In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.

Interaction of smoothened with integrin‐linked kinase in primary cilia mediates Hedgehog signalling

Here we report that ILK localizes in the mouse primary cilium, a sensory organelle required for signalling by the Hedgehog (Hh) pathway. Genetic or pharmacological inhibition of ILK blocks ciliary accumulation of the Hh pathway effector smoothened (Smo) and suppresses the induction of Gli transcription factor mRNAs by SHh. Conditional deletion of ILK or Smo also inhibits SHh-driven activation of Gli2 in the embryonic mouse cerebellum. ILK regulation of Hh signalling probably requires the physical interaction of ILK and Smo in the cilium, and we also show selective cilia-associated interaction of ILK with β-arrestin, a known mediator of Smo-dependent signalling.

Activation of the Transcription Factor GLI1 by WNT Signaling Underlies the Role of SULFATASE 2 as a Regulator of Tissue Regeneration

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced β-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and β-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

Mutational Analysis of Hedgehog Signaling Pathway Genes in Human Malignant Mesothelioma

BackgroundThe Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown.Methodology/Principal FindingsReal-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour.Conclusions/SignificanceWe identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

Noncanonical Regulation of the Hedgehog Mediator GLI1 by c-MYC in Burkitt Lymphoma

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5'-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box-dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers.

Specific Requirement of Gli Transcription Factors in Hedgehog-mediated Intestinal Development

Hedgehog (Hh) signaling is involved in multiple aspects of embryonic gut development, including mesenchymal growth and smooth muscle differentiation. The Gli family transcription factors is thought to collectively mediate Hh signaling in mammals. However, the function of different Gli proteins in gut development remains uncharacterized. Here, we genetically dissect the contribution of Gli transcriptional activation and de-repression in intestinal growth and patterning. We find that removal of the Gli3 repressor is dispensable for intestinal development and does not play a major role in Hh-controlled gut development. However, Gli2 activation is able to fully rescue the Smoothened (Smo)-null intestinal phenotype, suggesting that the Gli2 transcription factor is the main effector for Hh signaling in the intestine. To understand further the molecular mechanism underlying Hh/Gli function in the developing gut, we identify a subset of small leucine-rich glycoproteins (SLRPs) that may function downstream of Hh signaling in the mesenchyme. We show that osteoglycin, a SLRP, inhibits Hh-induced differentiation toward the smooth muscle lineage in C3H10T1/2 pluripotent mesenchymal cells. Taken together, our study reveals, for the first time, the distinct roles of Gli proteins in intestine development and suggests SLRPs as novel regulators of smooth muscle cell differentiation.

Gli1 Mediates Lung Cancer Cell Proliferation and Sonic Hedgehog-Dependent Mesenchymal Cell Activation

Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options.