Hedgehog Ligand Research Articles

Sonic hedgehog connects podocyte injury to mesangial activation and glomerulosclerosis.

Glomerular disease is characterized by proteinuria and glomerulosclerosis, two pathologic features caused by podocyte injury and mesangial cell activation, respectively. However, whether these two events are linked remains elusive. Here, we report that sonic hedgehog (Shh) is the mediator that connects podocyte damage to mesangial activation and glomerulosclerosis. Shh was induced in glomerular podocytes in various models of proteinuric chronic kidney diseases (CKD). However, mesangial cells in the glomeruli, but not podocytes, responded to hedgehog ligand. In vitro, Shh was induced in podocytes after injury and selectively promoted mesangial cell activation and proliferation. In a miniorgan culture of isolated glomeruli, Shh promoted mesangial activation but did not affect the integrity of podocytes. Podocyte-specific ablation of Shh in vivo exhibited no effect on proteinuria after adriamycin injection but hampered mesangial activation and glomerulosclerosis. Consistently, pharmacologic blockade of Shh signaling decoupled proteinuria from glomerulosclerosis. In humans, Shh was upregulated in glomerular podocytes in patients with CKD and its circulating level was associated with glomerulosclerosis but not proteinuria. These studies demonstrate that Shh mechanistically links podocyte injury to mesangial activation in the pathogenesis of glomerular diseases. Our findings also illustrate a crucial role for podocyte-mesangial communication in connecting proteinuria to glomerulosclerosis.

The Mechanism of Cholesterol Modification of Hedgehog Ligand.

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing. © 2019 Wiley Periodicals, Inc.

Leukotriene Synthesis Is Critical for Medulloblastoma Progression.

Here, we examined the role of leukotrienes, well-known inflammatory mediators, in the tumorigenesis of hedgehog pathway-associated medulloblastoma, and tested the efficacies of antagonists of leukotriene biosynthesis in medulloblastoma treatment.Experimental Design: We examined the leukotriene levels in medulloblastoma cells by ELISA. We next tested whether leukotriene synthesis in medulloblastoma cells relied on activation of hedgehog pathway, or the presence of hedgehog ligand secreted by astrocytes. We then investigated whether leukotriene mediated hedgehog-induced Nestin expression in tumor cells. The functions of leukotriene in tumor cell proliferation and tumor growth in medulloblastoma were determined through knocking down 5-lipoxygenase (a critical enzyme for leukotriene synthesis) by shRNAs, or using 5-lipoxygenase-deficient mice. Finally, the efficacies of antagonists of leukotriene synthesis in medulloblastoma treatment were tested in vivo and in vitro. Leukotriene was significantly upregulated in medulloblastoma cells. Increased leukotriene synthesis relied on hedgehog ligand secreted by astrocytes, a major component of medulloblastoma microenvironment. Leukotriene stimulated tumor cells to express Nestin, a cytoskeletal protein essential for medulloblastoma growth. Genetic blockage of leukotriene synthesis dramatically suppressed medulloblastoma cell proliferation and tumor growth in vivo. Pharmaceutical inhibition of leukotriene synthesis markedly repressed medulloblastoma cell proliferation, but had no effect on proliferation of normal neuronal progenitors. Moreover, antagonists of leukotriene synthesis exhibited promising tumor inhibitory efficacies on drug-resistant medulloblastoma. Our findings reveal a novel signaling pathway that is critical for medulloblastoma cell proliferation and tumor progression, and that leukotriene biosynthesis represents a promising therapeutic target for medulloblastoma treatment.

Cholesterol accessibility at the ciliary membrane controls hedgehog signaling.

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-- a palmitoyl group at the N-terminus and a cholesteroyl group at the C-terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with the re-assessment of prior cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic Hedgehog (SHH) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signalling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, SHH inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.

Sonic hedgehog selectively promotes lymphangiogenesis after kidney injury through noncanonical pathway.

Kidney fibrosis is associated with an increased lymphangiogenesis, characterized by the formation and expansion of new lymphatic vessels. However, the trigger and underlying mechanism responsible for the growth of lymphatic vessels in diseased kidney remain poorly defined. Here, we report that tubule-derived sonic hedgehog (Shh) ligand is a novel lymphangiogenic factor that plays a crucial role in mediating lymphatic endothelial cell proliferation and expansion. Shh was induced in renal tubular epithelium in various models of fibrotic chronic kidney disease, and this was accompanied by an expansion of lymphatic vessels in adjacent areas. In vitro, Shh selectively promoted the proliferation of human dermal lymphatic endothelial cells (HDLECs) but not human umbilical vein endothelial cells, as assessed by cell counting, MTT assay, and bromodeoxyuridine incorporation. Shh also induced the expression of vascular endothelial growth factor receptor-3, cyclin D1, and proliferating cell nuclear antigen in HDLECs. Shh did not affect the expression of Gli1, the downstream target and readout of canonical hedgehog signaling, but activated ERK-1/2 in HDLECs. Inhibition of Smoothened with small-molecule inhibitor or blockade of ERK-1/2 activation abolished the lymphatic endothelial cell proliferation induced by Shh. In vivo, inhibition of Smoothened also repressed lymphangiogenesis and attenuated renal fibrosis. This study identifies Shh as a novel mitogen that selectively promotes lymphatic, but not vascular, endothelial cell proliferation and suggests that tubule-derived Shh plays an essential role in mediating lymphangiogenesis after kidney injury.

Abstract 3083: A novel hedgehog signaling inhibitor for targeting pancreatic ductal adenocarcinoma

Abstract Sonic Hedgehog (Shh) signaling is deregulated in pancreatic cancer and can drive tumor onset and progression. Previous attempts to block Shh signaling used Smoothened (Smo) inhibitors, which target canonical Shh signaling. These approaches met with limited success, due to development of resistance, and contributions from non-canonical, Smo-independent signaling pathways that operate within tumor epithelial cells. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for palmitoylation of Shh, is a novel target for inhibition of Shh signaling in human and mouse pancreatic cancer cells and tumors. TDI-003410, a small molecule inhibitor of Hhat recently developed by our group, reduced proliferation of AsPC1 cells, a human pancreatic cancer cell line, in vitroand in subcutaneous xenografts in mice in vivo. The mechanism is via induction of a G2/M cell cycle arrest leading to apoptosis. We next used cell lines isolated from tumors formed in KPC (K-RasG12D/p53R172H) mice, a genetically engineered mouse model of pancreatic cancer.TDI-003410 inhibited the growth of KPC cell linesand also reduced Gli1 levelsin vitro. Neither LDE225, a Smo inhibitor, nor SAG, a Smo activator, had any effect on KPC cell proliferation or Gli1 levels in vitro. In orthotopic xenografts using KPC cells implanted into the pancreas, TDI-3410 treatment significantly reducedin vivotumor burden. No significant changes in CD31 or Trichrome (collagen) stainingwith TDI-3410 were detected in KPC orthotopic tumor tissue. However, Gli-1 levels in tumor tissue from TDI-3410 treated mice were significantly reduced compared to the vehicle controls. These findings provide proof of concept for the potential therapeutic efficacy of Hhat inhibition in pancreatic cancer. The concept of using an Hhat inhibitor is unique: Hhat is a novel target that hits upstream, directly at hedgehog ligands. This approach can inhibit both canonical and non-canonical (Smo-independent) as well as autocrine and paracrine Shh signaling. Citation Format: Atul Khire, Yi Zhong, Kalpana Rajanala, Christine Iacobuzio-Donahue, Marilyn D. Resh. A novel hedgehog signaling inhibitor for targeting pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3083.

Abstract 3661: GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells

Abstract Ewing sarcoma (EWS) is characterized by TET-ETS fusions, most commonly the EWSR1-FLI1 fusion. GLI1, a transcription factor in the Hedgehog (HH) signal transduction pathway and target of canonical HH signaling, is a direct transcriptional target of EWSR1-FLI1. A role for the HH signaling pathway and GLI1 in acquisition of a multidrug resistance phenotype has been previously observed in several human cancers. The role of the HH signal transduction pathway and GLI1 in the biology of EWS remains incompletely understood. We hypothesize that the majority of EWS cell lines express GLI1 based on non-canonical up-regulation by EWSR1-FLI1 and that further up-regulation of GLI1 may be associated with the development of drug resistance. Here, we show that EWS cell lines (CHLA-9, CHLA-10, CHLA-258, TC-32 and TC-71) express canonical HH pathway components (PTCH1, SMO, GLI1 and GLI3) and that cells lines established at the time of recurrence have higher GLI1 expression (CHLA-258 and TC-71) than those established at the time of diagnosis (CHLA-9, CHLA-10 and TC-32). Exposure to Sonic HH ligand in order to activate canonical HH signaling did not affect cell viability (MTT assay), proliferation (BrdU assay), or apoptosis (caspase 3/7 assay). However, inhibition of GLI1 activity with GANT61 reduced GLI1 expression (qRT PCR), cell viability (MTT assay) and cell proliferation (BrdU assay) but increased apoptosis (caspase 3/7 assay) and sensitivity of the cells to vincristine (VCR) (MTT assay), suggesting GLI1 plays roles in each of these processes. We then established VCR-resistant EWS cell lines (TC-71 [45-fold increase in the IC-50] and TC-32 [27-fold increase in the IC-50]) by exposing cells to serially increasing concentrations of VCR. We used an 86-gene cancer drug resistance PCR array (Qiagen) to characterize gene expression differences between untreated vs. recurrent EWS cell lines and between parental vs. VCR-resistant EWS cell lines. Among the changes in gene expression, we showed higher GLI1 expression in the recurrent EWS cell lines (CHLA-258 cells [4.8-fold] and TC-71 [3.4-fold]) vs. untreated CHLA-9 cells, and in VCR-resistant TC-71 cells (2.2-fold) vs. parental TC-71 cells. Down-regulation of GLI1 by GANT61 or GLI1 siRNA enhanced sensitivity of the cells to VCR. Our results suggest that GLI1 expression contributes to proliferation, survival and drug-resistance of EWS cell lines. Strategies to inhibit GLI1 may have therapeutic benefit in EWS. Citation Format: Joon Won Yoon, Marilyn Lamm, Philip Iannaccone, David Walterhouse. GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3661.

Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancer cells

Aberrant activity of the hedgehog (Hh) pathway is prevalent in pathologies such as cancer. Improved understanding of Hh activity in the aggressive tumor cell phenotype is being pursued for development of targeted therapies. Recently, we described a link between Hh activity and carbonic anhydrase XII (CAXII) expression. Extracellular facing CAs (IX/XII) are highly expressed in hypoxia, contribute to tumor pH regulation and are thus of clinical interest. Here we have extended the investigation of potential interactions between Hh activity and CAXII utilizing genomic disruption/knockout of either GLI1 (the main transcriptional factor induced with Hh activity) or CAXII in the triple negative breast cancer cell lines MDA-MB-231 and BT-549. Knockout of GLI1 and CAXII significantly decreased hallmarks of tumor aggressiveness including proliferation and migration. Most intriguingly, CAXII knockout caused a massive induction of the Sonic hedgehog (Shh) ligand expression (gene and protein). This novel finding indicates that CAXII plays a potential role in suppression of Shh and may act in a feedback loop to regulate overall Hh activity. Enhanced knowledge of these CA-Hh interactions in future studies may be of value in understanding this currently ‘incurable’ subclass of breast cancer.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

Downregulation of hedgehog ligands in human simple steatosis may protect against nonalcoholic steatohepatitis: Is TAZ a crucial regulator?

The conversion of simple steatosis into nonalcoholic steatohepatitis (NASH) in patients with nonalcoholic fatty liver disease (NAFLD) has attracted many attentions in recent years. The role of the hedgehog (HH) pathway in the regulation of lipogenesis has been addressed in the literature. This study aimed to investigate the levels of the sonic hedgehog (SHH) and Indian hedgehog (IHH) ligands and the correlation of these ligands with levels of proteins involved in the transforming growth factor-β1 (TGF-β1) pathway, as well as the evaluation of the transcriptional coactivator with PDZ binding motif (TAZ) expression in human simple steatosis, NASH cirrhosis, and controls. Patients were divided into two groups: the first group consisted of patients diagnosed with simple steatosis (n = 16) and the second group included those diagnosed with NASH cirrhosis (n = 15). As a control group, 18 histologically normal liver tissues were collected in this study. The expression of the TGF-β1pathway components and SHH and IHH ligands were analyzed by means of the quantitative real-time polymerase chain reaction and western blot analyses. A significant decrease was found in the hepatic expression of the SHH, IHH, and TGF-β1 pathways along with the expression of TAZ in tissue specimens with simple steatosis in comparison with patients affected by NASH cirrhosis and controls. Also, the levels of SHH and IHH proteins were significantly correlated with the expression of proteins involved in the TGF-β1 pathway. Moreover, the expression of the HH pathway ligands was positively associated with the expression of TAZ, supporting the notion that TAZ may play a role in the activation of the HH pathway thereby regulating the expression of its ligands. It seems that in patients with NAFLD, the downregulation of the HH pathway ligands may stem from steatosis; however, at the same time, it may prevent the conversion of simple steatosis into NASH in patients with liver diseases. © 2019 IUBMB Life, 71(9):1382-1390, 2019.

Sonic Hedgehog Ligand: A Role in Formation of a Mesenchymal Niche in Human Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterised by desmoplasia, thought to support progression and chemotherapeutic resistance. The Hedgehog pathway is known to play an important role in this cancer. While the upregulation of Sonic hedgehog (Shh) in the epithelium of PDAC is known, we investigated its expression in the tumour microenvironment in order to find new targets for new chemotherapeutical approaches. Immunohistochemistry was used for the investigation of Shh and Vimentin in primary human pancreatic tissues. Gene (qRT-PCR) and protein (immunofluorescence) expression of Shh, αSMA (a marker of the mesenchymal phenotype) and periostin (a marker of mesenchymal cells within a mixed population) were investigated in in vitro cell models. Shh expression was significantly upregulated in the stromal and epithelial compartments of poorly-differentiated PDAC samples, with a strong correlation with the amount of stroma present. Characterisation of stromal cells showed that there was expression of Shh ligand in a mixed population comprising αSMA+ myofibroblasts and αSMA− mesenchymal stem cells. Moreover, we demonstrated the interaction between these cell lines by showing a higher rate of mesenchymal cell proliferation and the upregulation of periostin. Therefore, targeting stromal Shh could affect the equilibrium of the tumour microenvironment and its contribution to tumour growth.

Sonic hedgehog (Shh) and CC chemokine ligand 2 signaling pathways in asthma.

Asthma is a chronic inflammatory disease of the airways in which many cells are involved, including mast cells, eosinophils, T lymphocytes, and so on. During the process, many chemokines and mediators are released to engage in recruiting and activating eosinophils and other inflammatory cells. Also, some signaling pathways are involved in the pathobiology of asthma. Sonic hedgehog (Shh) is one of the members of hedgehog gene families. Shh signaling plays a critical role in the embryonic development, including the lung. Previous findings from our team reveal that Shh is involved in the asthma pathogenesis. Recombinant Shh could induce the CC chemokine ligand 2 (CCL2) overexpressing and Smo inhibitor GDC-O449 could inhibit CCL2 expression in airway epithelial cells, monocytes, or macrophages. Hence, we reviewed the effects of Shh and CCL2 signaling pathways, and the interaction between signaling pathways in asthma.

Hedgehog Signaling and Embryonic Craniofacial Disorders.

Since its initial discovery in a Drosophila mutagenesis screen, the Hedgehog pathway has been revealed to be instrumental in the proper development of the vertebrate face. Vertebrates possess three hedgehog paralogs: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Of the three, Shh has the broadest range of functions both in the face and elsewhere in the embryo, while Ihh and Dhh play more limited roles. The Hedgehog pathway is instrumental from the period of prechordal plate formation early in the embryo, until the fusion of the lip and secondary palate, which complete the major patterning events of the face. Disruption of Hedgehog signaling results in an array of developmental disorders in the face, ranging from minor alterations in the distance between the eyes to more serious conditions such as severe clefting of the lip and palate. Despite its critical role, Hedgehog signaling seems to be disrupted through a number of mechanisms that may either be direct, as in mutation of a downstream target of the Hedgehog ligand, or indirect, such as mutation in a ciliary protein that is otherwise seemingly unrelated to the Hedgehog pathway. A number of teratogens such as alcohol, statins and steroidal alkaloids also disrupt key aspects of Hedgehog signal transduction, leading to developmental defects that are similar, if not identical, to those of Hedgehog pathway mutations. The aim of this review is to highlight the variety of roles that Hedgehog signaling plays in developmental disorders of the vertebrate face.

Investigating Novel Roles for Hedgehog Co‐Receptors During Craniofacial Development

Hedgehog (HH) signaling is essential for correct patterning of vertebrate embryos, including during craniofacial development. HH ligands signal through the canonical receptor Patched1 (PTCH1), and three co‐receptors–growth arrest specific 1 (GAS1), cell adhesion molecule‐related/down‐regulated by oncogenes (CDON), and brother of CDON (BOC). Together, these co‐receptors are required during embryogenesis to mediate proper HH signaling. However, recent genetic studies suggest tissue‐specific, antagonistic roles for these proteins. For example, Gas1 restricts the HH pathway during mouse tooth development, while Boc antagonizes HH signaling during zebrafish jaw formation. Here I explored possible antagonistic roles for GAS1, CDON and BOC in HH‐dependent mammalian craniofacial development. Genetic deletion of these co‐receptors results in variable holoprosencephaly (HPE) phenotypes (failure to divide the forebrain into two cerebral hemispheres). Gas1 mutant mice display a spectrum of HPE phenotypes, and significant patterning defects in the neural tube and limb. In contrast, genetic deletion of Boc does not perturb patterning in any of these tissues. Strikingly, the combined deletion of Boc and Gas1 results in more severe neural tube and limb patterning defects than those observed in Gas1 single mutants. However, the Gas1 craniofacial phenotype is partially ameliorated in Gas1;Boc double mutant embryos as determined by the levels of NKX2.1, Gli1, Ptch1 and internasal distance quantitation. Micro‐computed tomography and skeletal analyses reveal that the rescue of the craniofacial defects in Gas1;Boc double mutants is restricted to the nasal bone and nasal capsule; other craniofacial bones are more severely affected, similar to what is observed in the neural tube and the limb. This effect does not appear to be due to differential GAS1 and BOC function in the forebrain neuroepithelium as overexpression of these co‐receptors in the developing chicken forebrain both promote HH signaling. Instead, I hypothesize that these co‐receptors have distinct, tissue‐specific functions in the craniofacial mesenchyme or the surface ectoderm. To further explore an antagonistic role for BOC during craniofacial development, I am currently deleting Boc in a sensitized HH genetic background through breeding with Ptch1 and Shh heterozygous mutant animals. These findings suggest that GAS1 and BOC do not function simply as redundant HH co‐receptors, but instead display distinct, tissue‐specific functions, particularly during craniofacial development.Support or Funding InformationNational Sciences Foundation Graduate Research Fellowship Program (DGE 1256260), Rackham Pre‐Candidate Graduate Student Research Grant, Bradley Merill Patten Fellowship and Organogenesis Training Grant (NIH T32HD007505)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Protein Kinases in Hedgehog Pathway Control and Implications for Cancer Therapy.

Hedgehog (HH) signaling is an evolutionarily conserved pathway that is crucial for growth and tissue patterning during embryonic development. It is mostly quiescent in the adult, where it regulates tissue homeostasis and stem cell behavior. Aberrant reactivation of HH signaling has been associated to several types of cancer, including those in the skin, brain, prostate, breast and hematological malignancies. Activation of the canonical HH signaling is triggered by binding of HH ligand to the twelve-transmembrane protein PATCHED. The binding releases the inhibition of the seven-transmembrane protein SMOOTHENED (SMO), leading to its phosphorylation and activation. Hence, SMO activates the transcriptional effectors of the HH signaling, that belong to the GLI family of transcription factors, acting through a not completely elucidated intracellular signaling cascade. Work from the last few years has shown that protein kinases phosphorylate several core components of the HH signaling, including SMO and the three GLI proteins, acting as powerful regulatory mechanisms to fine tune HH signaling activities. In this review, we will focus on the mechanistic influence of protein kinases on HH signaling transduction. We will also discuss the functional consequences of this regulation and the possible implications for cancer therapy.

3373 Modulation of Hedgehog Signaling Alters Immune Infiltration in Pancreatic Cancer

OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.

Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

Hedgehog Signaling Modulates Interleukin-33-Dependent Extrahepatic Bile Duct Cell Proliferation in Mice.

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH (pCMV‐Shh), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 (Gli1lacZ/lacZ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.

How Bad Is the Hedgehog? GLI-Dependent, Hedgehog-Independent Cancers on the Importance of Biomarkers for Proper Patients Selection.

Hedgehog (HH) signaling plays an important role both during embryonic development and adult life. It is involved in the regulation of cell differentiation, cell proliferation and tissue polarity, as well as in the maintenance of stem cells, tissue repair, and regeneration (Briscoe and Therond, 2013; Jiang and Hui, 2008). Three ligands, Indian, Sonic, and Desert HH, can activate this pathway. Binding of HH ligands to their receptor, PTCH1 (Figure1) lift its inhibition on SMO, resulting in activation and nuclear translocation of GLI transcription factors (Javelaud etal., 2012). The vertebrate GLI gene family is composed of three distinct genes GLI1, GLI2, and GLI3, encoding Krüppel-like transcription factors. GLI proteins exhibit distinct regulations, biochemical properties, and target genes. GLI3 acts as the main repressor of the pathway in the absence of HH ligands, whereas, in their presence, GLI2 is the main HH effector that drives the expression of GLI1 (Briscoe and Therond, 2013).