Canonical Hedgehog Signaling Research Articles

Deciphering the role of hedgehog signaling in pancreatic cancer

Pancreatic cancer, mostly pancreatic ductal adenocarcinoma (PDAC), is a leading cause of cancer-related death in the US, with a dismal median survival of 6 months. Thus, there is an urgent unmet need to identify ways to diagnose and to treat this deadly cancer. Although a number of genetic changes have been identified in pancreatic cancer, their mechanisms of action in tumor development, progression and metastasis are not completely understood. Hedgehog signaling, which plays a major role in embryonic development and stem cell regulation, is known to be activated in pancreatic cancer; however, specific inhibitors targeting the smoothened molecule failed to improve the condition of pancreatic cancer patients in clinical trials. Furthermore, results regarding the role of Hh signaling in pancreatic cancer are controversial with some reporting tumor promoting activities whereas others tumor suppressive actions. In this review, we will summarize what we know about hedgehog signaling in pancreatic cancer, and try to explain the contradicting roles of hedgehog signaling as well as the reason(s) behind the failed clinical trials. In addition to the canonical hedgehog signaling, we will also discuss several non-canonical hedgehog signaling mechanisms.

Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination.

SummaryEnhancing repair of myelin is an important, but still elusive therapeutic goal in many neurological disorders1. In Multiple Sclerosis (MS), an inflammatory demyelinating disease, endogenous remyelination does occur but is frequently insufficient to restore function. Both parenchymal oligodendrocyte progenitor cells (OPCs) and endogenous adult neural stem cells (NSCs) resident within the subventricular zone (SVZ) are known sources of remyelinating cells2. Here, we characterize the contribution to remyelination of a subset of adult NSCs, identified by their expression of Gli1, a transcriptional effector of the Sonic Hedgehog (Shh) pathway. We show that these cells are recruited from the SVZ to populate demyelinated lesions in the forebrain but never enter healthy, white matter tracts. Unexpectedly, recruitment of this pool of NSCs, and their differentiation into oligodendrocytes, is significantly enhanced by genetic or pharmacological inhibition of Gli1. Importantly, complete inhibition of canonical hedgehog signaling was ineffective indicating that Gli1’s role in both augmenting hedgehog signaling and retarding myelination is specialized. Indeed, inhibition of Gli1 improves the functional outcome in a relapsing/remitting model of experimental autoimmune encephalomyelitis (RR-EAE) and is neuroprotective. Thus, endogenous NSCs can be mobilized for the repair of demyelinated lesions by inhibiting Gli1, identifying a new therapeutic avenue for the treatment of demyelinating disorders.

Abstract 5061: Astrocytes promote medulloblastoma tumorigenesis via sonic hedgehog secretion

Abstract Tumor development is a complex process that involves interactions between tumor cells and the surrounding cells (the microenvironment). However, in medulloblastoma (MB), the most common malignant pediatric brain tumor, the contribution of the tumor microenvironment in tumorigenesis still remains elusive. Using MB mouse models, we have recently found that astrocytes represent the major component of the MB microenvironment. Astrocytes significantly enhance MB cell proliferation in vitro and genetic ablation of astrocytes dramatically prevents MB cell proliferation and inhibits MB growth in tumor-bearing mice. These results indicate the critical role of astrocytes during MB development. Our studies have further revealed that astrocytes stimulate proliferation of MB cells by secretion of sonic hedgehog (Shh), a mitogen required for neuronal proliferation during normal development. It is well established that Shh triggers canonical hedgehog signaling via activation of the Gli1 transcription factor. Interestingly, astrocyte-derived Shh stimulates MB cell proliferation independent of Gli1 activation, suggesting that astrocytes promote MB tumorigenesis through a novel Shh signaling pathway. Together, our studies shed light on the important function of the tumor microenvironment in MB initiation and progression, and pave the road to develop therapeutic approaches to treat MB by targeting Shh secretion in astrocytes. Note: This abstract was not presented at the meeting. Citation Format: Larra Walling Yuelling, Fang Du, Eric Lee, Renata Muradimova, Peng Li, Zeng-jie Yang. Astrocytes promote medulloblastoma tumorigenesis via sonic hedgehog secretion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5061. doi:10.1158/1538-7445.AM2015-5061

Abstract 4689: Targeting constitutive GLI activation in colon carcinomas and cancers with oncogenic KRAS signaling

Abstract The GLI genes, GLI1 and GLI2, encode transcription factors that regulate target genes at the distal end of the canonical Hedgehog signaling (HH) pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation. In cancers, both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated. Oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer, circumvent the HH-GLI axis to further drive GLI activation, which serves as a nodal channel for oncogenic signals, and provides a pivotal role for GLI in cell survival. GANT61, a small molecule inhibitor of GLI-dependent transcription, terminates oncogenic HH-GLI and KRAS/BRAF-GLI signaling, inducing DNA damage and extensive cell death in seven human colon carcinoma cell line models examined. The overall goal is to understand how GANT61 induces cell death by inhibiting GLI-dependent transcription mechanisms. We have demonstrated that GANT61 is: a) specific for binding to GLI, and does not bind to DNA or to other transcription factors; b) selective for cancer cells vs normal cells that lack constitutive GLI activation. Inhibition of GLI1 and GLI2 by GANT61 rapidly reduces GLI binding to consensus sequences in target gene promoters and inhibits GLI-dependent transcription. Stalling of Pol II occurs adjacent to GLI binding regions at target gene promoters (FOXM1, BCL-2), associated with inhibition of GLI binding, enrichment of Pol II, and modification of promoter-bound pause (DSIF, NELF) or pause-release (P-TEFb) factors within a 300bp region of the FOXM1 promoter. Single-stranded DNA and RNA:DNA hybrids determining R-loop formation (distributed throughout the nucleoplasm) occur at sites of stalled Pol II, inhibited by RNAseH. Consistent with transcriptional inhibition, GANT61 induced DNA damage (γH2AX foci) in S-phase and non-S-phase cells, recognized at the initiation of S-phase (G1/S), prior to the onset of cell death. This may be due to one of two mechanisms: 1) The GLI1 target gene FOXM1, and its transcriptional target CDC6, downregulated in GANT61-treated cells, regulate Pre-Replication Complex assembly at G1/S thereby inhibiting the initiation of DNA replication; this controls outcome of the GANT61 response, dependent on GLI; 2) Transcriptional R-loop formation can conflict with DNA replication in GANT61-treated cells. These events inhibit initiation of DNA replication from unfired origins at early replicons and inhibit further progression through S-phase. GLI1 and/or GLI2 are constitutively activated in epithelial cancers of the GI tract, brain tumors, melanoma, pediatric solid tumors, liver, lung, breast, pancreatic and prostate cancers. Further, KRAS is mutated in one third of all human cancers and in 50% of colon carcinomas. Targeting GLI therefore has the potential for high impact in therapeutics, and is anticipated to lead to new approaches and therapeutic strategies for treating cancer. Citation Format: Tapati Mazumdar, Babal K. Jha, Akwasi Agyeman, Janet A. Houghton. Targeting constitutive GLI activation in colon carcinomas and cancers with oncogenic KRAS signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2015-4689

The presence of primary cilia in cancer cells does not predict responsiveness to modulation of smoothened activity.

Primary cilia are microtubule-based organelles that regulate smoothened-dependent activation of the GLI transcription factors in canonical hedgehog signaling. In many cancers, primary cilia are markedly decreased or absent. The lack of primary cilia may inhibit or alter canonical hedgehog signaling and, thereby, interfere in the cellular responsiveness to modulators of smoothened activity. Clinical trials of smoothened antagonists for cancer treatment have shown the best response in basal cell carcinomas, with limited response in other solid tumors. To determine whether the presence or absence of primary cilia in cancer cells will predict their responsiveness to modulation of smoothened activity, we compared the ability of an agonist and/or inhibitor of smoothened (SAG and SANT1, respectively) to modulate GLI-mediated transcription, as measured by GLI1 mRNA level or GLI-luciferase reporter activity, in non-cancer cells with primary cilia (ovarian surface epithelial cells and breast fibroblasts), in cancer cells that cannot assemble primary cilia (MCF7, MDA-MB-231cell lines), and in cancer cells with primary cilia (SKOV3, PANC1 cell lines). As expected, SAG and SANT1 resulted in appropriate modulation of GLI transcriptional activity in ciliated non-cancer cells, and failed to modulate GLI transcriptional activity in cancer cells without primary cilia. However, there was also no modulation of GLI transcriptional activity in either ciliated cancer cell line. SAG treatment of SKOV3 induced localization of smoothened to primary cilia, as assessed by immunofluorescence, even though there was no increase in GLI transcriptional activity, suggesting a defect in activation of SMO in the primary cilia or in steps later in the hedgehog pathway. In contrast to SKOV3, SAG treatment of PANC1 did not cause the localization of smoothened to primary cilia. Our data demonstrate that the presence of primary cilia in the cancer epithelial cells lines tested does not indicate their responsiveness to smoothened activation or inhibition.

The Transcription Factor GLI1 Modulates the Inflammatory Response during Pancreatic Tissue Remodeling

Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process.

Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor β1 in mice

Hepatic encephalopathy (HE) is a neurologic disorder that develops during liver failure. Few studies exist investigating systemic-central signalling during HE outside of inflammatory signalling. The transcription factor Gli1, which can be modulated by hedgehog signalling or transforming growth factor β1 (TGFβ1) signalling, has been shown to be protective in various neuropathies. We measured Gli1 expression in brain tissues from mice and evaluated how circulating TGFβ1 and canonical hedgehog signalling regulate its activation. Mice were injected with azoxymethane (AOM) to induce liver failure and HE in the presence of Gli1 vivo-morpholinos, the hedgehog inhibitor cyclopamine, Smoothened vivo-morpholinos, a Smoothened agonist, or TGFβ-neutralizing antibodies. Molecular analyses were used to assess Gli1, hedgehog signalling, and TGFβ1 signalling in the liver and brain of AOM mice and HE patients. Gli1 expression was increased in brains of AOM mice and in HE patients. Intra-cortical infusion of Gli1 vivo-morpholinos exacerbated the neurologic deficits of AOM mice. Measures to modulate hedgehog signalling had no effect on HE neurological decline. Levels of TGFβ1 increased in the liver and serum of mice following AOM administration. TGFβ neutralizing antibodies slowed neurologic decline following AOM administration without significantly affecting liver damage. TGFβ1 inhibited Gli1 expression via a SMAD3-dependent mechanism. Conversely, inhibiting TGFβ1 increased Gli1 expression. Cortical activation of Gli1 protects mice from induction of HE. TGFβ1 suppresses Gli1 in neurons via SMAD3 and promotes the neurologic decline. Strategies to activate Gli1 or inhibit TGFβ1 signalling might be developed to treat patients with HE.

Hedgehog signalling in liver regeneration

Regeneration of adult livers requires Hh-dependent modulation of epithelial-mesenchymal transitions in multipotent progenitors. Data generated by studying models of acute and chronic liver injury reveal that robust epithelial to mesenchymal transitions (EMT) normally occur in injured livers, and show that conditionally abrogating canonical Hedgehog signaling in multi-potent aSMA-expressing cells blocks these EMT, instead triggering a cascade of mesenchymal-to-epithelial transitions (MET). This has global consequences for liver wound healing. Regeneration of different types of liver cells becomes unbalanced, leading to excessive accumulation of quiescent HSC but reduced outgrowth of hepatocytic and ductular progenitors and their progeny, as well as depletion of MF populations. Thus, de-regulating Hh-sensitive EMT/MET responses disrupts both scarring and regeneration of injured livers. These findings, in turn, suggest that the liver is uniquely suited for regeneration because it harbors large populations of multi-potent progenitors and is generally able to constrain signals, such as Hedgehog, that modulate the differentiation of these cells towards less epithelial (and more mesenchymal) phenotypes.

Mode and specificity of binding of the small molecule GANT61 to GLI determines inhibition of GLI-DNA binding.

The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation. Both GLI1 and GLI2 are oncogenes, constitutively activated in many types of human cancers. In colon cancer cells oncogenic KRAS-GLI signaling circumvents the HH-SMO-GLI axis to channel through and activate GLI in the transcriptional regulation of target genes. We have observed extensive cell death in a panel of 7 human colon carcinoma cell lines using the small molecule GLI inhibitor GANT61. Using computational docking and experimental confirmation by Surface Plasmon Resonance, GANT61 binds to the 5-zinc finger GLI1 protein between zinc fingers 2 and 3 at sites E119 and E167, independent of the GLI-DNA binding region, and conserved between GLI1 and GLI2. GANT61 does not bind to other zinc finger transcription factors (KLF4, TFIIβ). Mutating the predicted GANT61 binding sites in GLI1 significantly inhibits GANT61-GLI binding and GLI-luciferase activity. Data establish the specificity of GANT61 for targeting GLI, and substantiate the critical role of GLI in cancer cell survival. Thus, targeting GLI in cancer therapeutics may be of high impact.

Distinct Effects of the mesenchymal dysplasia Gene Variant of Murine Patched-1 Protein on Canonical and Non-canonical Hedgehog Signaling Pathways

Hedgehog (Hh) signaling requires regulation of the receptor Patched-1 (Ptch1), which, in turn, regulates Smoothened activity (canonical Hh signaling) as well as other non-canonical signaling pathways. The mutant Ptch1 allele mesenchymal dysplasia (mes), which truncates the Ptch1 C terminus, produces a limited spectrum of developmental defects in mice as well as deregulation of canonical Hh signaling in some, but not all, affected tissues. Paradoxically, mes suppresses canonical Hh signaling and binds to Hh ligands with an affinity similar to wild-type mouse Ptch1 (mPtch1). We characterized the distinct activities of the mes variant of mPtch1 mediating Hh signaling through both canonical and non-canonical pathways. We demonstrated that mPtch1 bound c-src in an Hh-regulated manner. Stimulation with Sonic Hedgehog (Shh) of primary mammary mesenchymal cells from wild-type and mes animals activated Erk1/2. Although Shh activated c-src in wild-type cells, c-src was constitutively activated in mes mesenchymal cells. Transient assays showed that wild-type mPtch1, mes, or mPtch1 lacking the C terminus repressed Hh signaling in Ptch1-deficient mouse embryo fibroblasts and that repression was reversed by Shh, revealing that the C terminus was dispensable for mPtch1-dependent regulation of canonical Hh signaling. In contrast to these transient assays, constitutively high levels of mGli1 but not mPtch1 were present in primary mammary mesenchymal cells from mes mice, whereas the expression of mPtch1 was similarly induced in both mes and wild-type cells. These data define a novel signal transduction pathway involving c-src that is activated by the Hh ligands and reveals the requirement for the C terminus of Ptch in regulation of canonical and non-canonical Hh signaling pathways.

Molecular modeling study on the dynamical structural features of human smoothened receptor and binding mechanism of antagonist LY2940680 by metadynamics simulation and free energy calculation

BackgroundThe smoothened (SMO) receptor, one of the Class F G protein coupled receptors (GPCRs), is an essential component of the canonical hedgehog signaling pathway which plays a key role in the regulation of embryonic development in animals. The function of the SMO receptor can be modulated by small-molecule agonists and antagonists, some of which are potential antitumour agents. Understanding the binding mode of an antagonist in the SMO receptor is crucial for the rational design of new antitumour agents. MethodsMolecular dynamics (MD) simulation and dynamical network analysis are used to study the dynamical structural features of SMO receptor. Metadynamics simulation and free energy calculation are employed to explore the binding mechanism between the antagonist and SMO receptor. ResultsThe MD simulation results and dynamical network analysis show that the conserved KTXXXW motif in helix VIII has strong interaction with helix I. The α-helical extension of transmembrane 6 (TM6) is detected as part of the ligand-binding pocket and dissociation pathway of the antagonist. The metadynamics simulation results illustrate the binding mechanism of the antagonist in the pocket of SMO receptor, and free energy calculation shows the antagonist needs to overcome about 38kcal/mol of energy barrier to leave the binding pocket of SMO receptor. ConclusionsThe unusually long TM6 plays an important role on the binding behavior of the antagonist in the pocket of SMO receptor. General significanceThe results can not only profile the binding mechanism between the antagonist and Class F GPCRs, but also supply the useful information for the rational design of a more potential small molecule antagonist bound to SMO receptor.

Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma

The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells. Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system. Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive. Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

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.

Hedgehog and Gli Signaling in Embryonic Mammary Gland Development

The first mouse mutation associated with a heritable defect in embryonic mammary gland development was Extratoes. It represents a functional null-mutation of the gene encoding Gli3, which is best known as a transcription factor mediating canonical Hedgehog (Hh) signaling. Here we review the roles of Hh and Gli proteins in murine embryonic mammary development. We propose that an off-state for Hh signaling, mediated by Gli3-repressor, is determinant for induction of a mammary instead of hair follicle fate in the trunk surface ectoderm.

Structure of the human smoothened receptor bound to an antitumour agent

The smoothened (SMO) receptor, a key signal transducer in the hedgehog signalling pathway, is responsible for the maintenance of normal embryonic development and is implicated in carcinogenesis. It is classified as a class frizzled (class F) G-protein-coupled receptor (GPCR), although the canonical hedgehog signalling pathway involves the GLI transcription factors and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure of the transmembrane domain of the human SMO receptor bound to the small-molecule antagonist LY2940680 at 2.5 Å resolution. Although the SMO receptor shares the seven-transmembrane helical fold, most of the conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulphide bonds. The ligand binds at the extracellular end of the seven-transmembrane-helix bundle and forms extensive contacts with the loops.

Transcriptional Regulation of Serine/Threonine Protein Kinase (AKT) Genes by Glioma-associated Oncogene Homolog 1

Aberrant activation of Hedgehog signaling has been described in a growing number of cancers, including malignant lymphomas. Here, we report that canonical Hedgehog signaling modulates the transcriptional expression of AKT genes and that AKT1 is a direct transcriptional target of GLI1. We identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter, and site-directed mutagenesis assays. Moreover, we provide evidence that GLI1 contributes to the survival of diffuse large B-cell lymphoma (DLBCL) cells and that this effect occurs in part through promotion of the transcription of AKT genes. This finding is of interest as constitutive activation of AKT has been described in DLBCL, but causative factors that explain AKT expression in this lymphoma type are not completely known. In summary, we demonstrated the existence of a novel cross-talk at the transcriptional level between Hedgehog signaling and AKT with biological significance in DLBCL.

Abstract 669: A unique acquired resistance mechanism to agents that target GLI (GANT61) in the Hedgehog (HH) signaling pathway in human colon cancer cells.

Abstract In normal cellular processes, the HH signaling pathway has critical roles in embryonic development, tissue patterning, stem cell function, and differentiation. Canonical HH signaling engages the transmembrane receptor PTCH, the intermediary signaling molecule SMO, and the transcriptional regulators of target genes and the HH signaling response, GLI1 and GLI2. In colon cancers, GLI is aberrantly regulated by oncogenic signaling pathways, driving cell survival and genomic instability involved in oncogenesis, progression and metastasis. Using the small molecule inhibitor of GLI1/GLI2 transcription, GANT61, we have demonstrated that inhibition of HH signaling at the level of GLI induces extensive cell death in human colon carcinoma cell line models in contrast to targeting SMO. Cells with acquired resistance to SMO inhibitors also remain highly sensitive to GANT61, underscoring GLI as a critical target in colon cancer. We have now identified a previously undescribed, novel mechanism by which cancer cells evade cell death following protracted, targeted exposure of GLI by GANT61. The classic pharmacologic approach of stepwise increasing drug selection did not produce a proliferative colon cancer cell line resistant to GANT61, confirming the critical importance of GLI genes to cell survival. In contrast, prolonged exposure (1-3 weeks) of HT29 cells to semi-lethal concentrations of GANT61 (10 μM; HT29-G10), induced GLI1/GLI2 inhibition, reduced proliferation, decreased appearance of γH2AX nuclear foci, and increased methylation of histone H3K9 (H3K9me1-3) within the GLI1 and GLI2 promoters. Methylation of H3K9 lysine residues is correlated with either an inactive or silent state of gene expression, and can exist in mono-, di-, or tri- methylated states. It has been demonstrated by others that methylation at H3K9 in the promoters of p53, p21Waf1 or DHFR genes, can regulate cell proliferation or function at the G1/S transition. In addition to GLI1 and GLI2, H3K9 methylation was also observed in HT29-G10 cells in the promoter regions of FOXM1 and CDC6, G1/S regulatory genes. These are GLI target genes, which regulate the G1/S transition, or PreReplication Complex assembly at initiation of DNA replication, respectively. Data suggest that the GLI2->GLI1->FOXM1->CDC6 promoters are coordinately regulated by H3K9 methylation in a pathway to promote gene silencing, reduce gene transcription, and reduce cell cycle transition at G1/S, constituting a unique mechanism of acquired resistance to drugs that target GLI. It is anticipated that these approaches will provide new insight into critical targets that determine HH-dependent survival, and lead to the translation of agents that target or regulate GLI function. Citation Format: Akwasi Agyeman, Tapati Mazumder, Janet Houghton. A unique acquired resistance mechanism to agents that target GLI (GANT61) in the Hedgehog (HH) signaling pathway in human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2013-669

A unique acquired resistance mechanism to agents that target GLI (GANT61) in the Hedgehog (HH) signaling pathway

Canonical HH signaling engages the transmembrane receptor PTCH, the intermediary signaling molecule SMO, and the HH signaling response molecules, GLI1 and GLI2. In colon cancers, GLI is aberrantly regulated, driving cell survival and genomic instability in oncogenesis, progression and metastasis. Following inhibition of GLI by a protracted exposure of HT29 to GANT61, we have identified a previously undescribed, novel mechanism by which cancer cells evade cell death. Stepwise selection did not produce a proliferative colon cancer cell line resistant to GANT61, confirming the critical importance of GLI genes to cell survival. In contrast, in HT29 cells, by inhibiting GLI1/GLI2 by prolonged exposure to semi‐lethal concentrations of GANT61 (10 μM; G10), we observed reduced proliferation, decreased appearance of γH2AX nuclear foci, and increased methylation of histone H3K9 (H3K9me1–3) within the promoters of the GLI1 and GLI2 genes. Increased H3K9 methylation was also demonstrated in HT29(G10) cells in the promoter regions of FoxM1 and CDC6, a G1/S and PreReplication Complex assembly regulatory genes, respectively. Data suggest that the GLI2‐>;GLI1‐>;FOXM1‐>;CDC6 promoters are coordinately regulated by H3K9 methylation in a pathway to promote gene silencing, reduce gene transcription, and reduce cell cycle transition at G1/S, constituting a unique mechanism of acquired resistance to drugs that target GLI.This work is supported by NCI awards RO1 CA 32613 (J.A. Houghton) and NCI RO1 108929 (J.A. Houghton)

Altered canonical hedgehog-gli signalling axis in pesticide-induced bone marrow aplasia mouse model

The mechanistic interplay between pesticide exposure and development of marrow aplasia is not yet well established but there are indices that chronic pesticide exposure in some instances causes marrow aplasia like haematopoietic degenerative condition in human beings. Canonical Hedgehog (Hh) signalling has multiple roles in a wide range of developmental processes, including haematopoiesis. The present study was designed to explore the status of four important components of the canonical Hedgehog signalling cascade, the Sonic Hedgehog (Shh), Ptch1, Smo, and Gli1, in a mouse model of chronic pesticide-induced bone marrow aplasia. We used 5 % aqueous mixture of pesticides (chlorpyriphos, prophenophos, cypermethrin, alpha-methrin, and hexaconazole) for inhalation and dermal exposure of 6 hours per day and 5 days a week up to 90 days. Murine bone marrow aplasia related to chronic pesticide treatment was confirmed primarily by haemogram, bone marrow cellularity, short term bone marrow explant culture for cellular kinetics, bone marrow smear, and fl ow cytometric Lin-Sca-1+C-kit+ extracellular receptor expression pattern. Later, components of hedgehog signalling were analysed in the bone marrow of both control and pesticide-treated aplastic groups of animals. The results depicted pancytopenic feature of peripheral blood, developmental anomaly of neutrophils, depression of primitive stem and progenitor population along with Shh, Ptch1, Smo and Gli1 expression in aplasia group. This investigation suggests that pesticide-induced downregulation of two critically important proteins--Ptch1 and Gli1--inside the haematopoietic stem and progenitor cell population impairs haematopoietic homeostasis and regeneration mechanism in vivo concurrent with bone marrow aplasia.

Detection of Canonical Hedgehog Signaling in Breast Cancer by 131-Iodine-Labeled Derivatives of the Sonic Hedgehog Protein

Activation of hedgehog (HH) pathway signaling is observed in many tumors. Due to a feedback loop, the HH receptor Patched (PTCH-1) is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH-1 ligand sonic (SHH) for detection of cancer cells with canonical HH activity. Receptor binding of 131I-SHH was increased in cell lines with high HH pathway activation. Our findings also show that PTCH-1 receptor expression is decreased upon treatment with HH signaling inhibitors, and receptor binding of 131I-SHH is significantly decreased following treatment with cyclopamine. In vivo imaging and biodistribution studies revealed significant accumulation of 131I-SHH within tumor tissue as compared to normal organs. Tumor-to-muscle ratios were approximately 8 : 1 at 5 hours, while tumor to blood and tumor to bone were 2 : 1 and 5 : 1, respectively. Significant uptake was also observed in liver and gastrointestinal tissue. These studies show that 131I-SHH is capable of in vivo detection of breast tumors with high HH signaling. We further demonstrate that the hedgehog receptor PTCH-1 is downregulated upon treatment with hedgehog inhibitors. Our data suggests that radiolabeled SHH derivatives may provide a method to determine response to SHH-targeted therapies.