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Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.

Background Aberrant activation of the Hedgehog pathway drives tumorigenesis of many cancers, including glioblastoma. However, the sensitization mechanism of the G protein-coupled-like receptor smoothened (SMO), a key component of Hedgehog signaling, remains largely unknown. Results In this study, we describe a novel protein SMO-193a.a. that is essential for Hedgehog signaling activation in glioblastoma. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for sonic hedgehog (Shh) induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification, and releasing SMO from the inhibition of patched transmembrane receptors. Deprivation of SMO-193a.a. in brain cancer stem cells attenuates Hedgehog signaling intensity and suppresses self-renewal, proliferation in vitro, and tumorigenicity in vivo. Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain Hedgehog signaling activation in glioblastoma. Clinically, SMO-193a.a. is more specifically expressed in glioblastoma than SMO and is relevant to Gli1 expression. Higher expression of SMO-193a.a. predicts worse overall survival of glioblastoma patients, indicating its prognostic value. Conclusions Our study reveals that SMO-193a.a., a novel protein encoded by circular SMO, is critical for Hedgehog signaling, drives glioblastoma tumorigenesis and is a novel target for glioblastoma treatment.

Objective Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. Methods We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc−/− (YFP-Ptc−/−) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. Results Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc−/− mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc−/− mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. Conclusions Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders., Highlights • Astrocytes exhibit differences in regulating the hedgehog signaling pathway. • Deletion of Ptc in Glast+ cells prevents body weight gain and insulin resistance. • Deletion of Ptc in Glast+ cells increases β oxidation. • Central hedgehog signaling participates in the regulation of whole-body metabolism.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells.

International audience; In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific mono-clonal C9C5 antibody in a subset (11-12%) of CC1 + mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendro-cyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligo-dendroglial cell body and decorates by intense puncta the processes. C9C5 + cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP + and S100β + astrocytes, and in HuC/D + neurons as shown in Ptc LacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelina-tion peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5 + CC1 + oligodendroglial cells are a source of Shh in the mouse post-natal brain.

Hyalinizing trabecular tumour (HTT) is a rare thyroid neoplasm of follicular cell origin characterised by a trabecular growth pattern and prominent intratrabecular and intertrabecular hyalinisation. These peculiar histological features allow the prompt recognition of this neoplasm in surgical specimens. However, cytological diagnosis of HTT remains elusive and misleading because of overlapping characteristics with other thyroid tumours, particularly papillary thyroid carcinoma (PTC), medullary thyroid carcinoma (MTC) and the newly described non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Nevertheless, the proper recognition of this neoplasm on preoperative cytological preparations is important to avoid unnecessary overtreatment of this indolent lesion. A thorough review of the literature has revealed that the correct diagnosis of HTT in cytological smears is achieved in only 8% of cases. In a further 6% of cases, diagnostic doubt has been indicated. Sixty percent of published cases of HTT have been misdiagnosed as suggestive, suspicious or positive for PTC. These findings underline the difficulties of a cytological-based diagnosis of such entity. In this article we review the cytomorphological features of HTT and their correlation to histological features to provide the reader with the tools to improve diagnostic performance in the identification of HTT on preoperative cytology.

Sonic hedgehog (Shh) and its patched–smoothened receptor complex control a variety of functions in the developing central nervous system, such as neural cell proliferation and differentiation. Recently, Shh signaling components have been found to be expressed at the synaptic level in the postnatal brain, suggesting a potential role in the regulation of synaptic transmission. Using in utero electroporation of constitutively active and negative-phenotype forms of the Shh signal transducer smoothened (Smo), we studied the role of Smo signaling in the development and maturation of GABAergic transmission in the somatosensory cortex. Our results show that enhancing Smo activity during development accelerates the shift from depolarizing to hyperpolarizing GABA in a manner dependent on functional expression of potassium–chloride cotransporter type 2 (KCC2, also known as SLC12A5). On the other hand, blocking Smo activity maintains the GABA response in a depolarizing state in mature cortical neurons, resulting in altered chloride homeostasis and increased seizure susceptibility. This study reveals unexpected functions of Smo signaling in the regulation of chloride homeostasis, through control of KCC2 cell-surface stability, and the timing of the GABA excitatory-to-inhibitory shift in brain maturation., Summary: The smoothened receptor controls the time course of inhibitory transmission by regulating stability of the potassium-chloride cotransporter KCC2 at the plasma membrane.

Summary Identifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury., Highlights • Hh shortens the cell cycle of activated neural stem cells (NSCs) in culture • Hh activation depletes the pool of proliferating activated NSCs at long term • Quiescent NSCs accumulates after a long period of Hh activation in NSCs, Hedgehog (Hh) signaling is implicated in tissue regeneration. Ruat, Boussin, and colleagues show that short-term Hh pathway activation in adult neural stem cells (NSCs) results in increases in activated NSCs. Surprisingly, long-term pathway activation causes a large accumulation of quiescent NSCs and impaired neurogenesis. Thus, the Hh pathway has a novel role in balancing quiescent and activated adult NSC numbers.

The SHH signaling pathway is critical for gastrointestinal development and organic patterning, and dysregulation of SHH pathway molecules has been detected in multiple gastrointestinal neoplasms. This study investigated the role of the SHH signaling pathway in PJS. Expression of SHH, PTCH, and GLI1 was examined by real-time PCR and immunohistochemistry in 20 normal tissues and 75 colorectal lesions (25 PJPs, 25 adenomas, and 25 adenocarcinomas). Expression of SHH, PTCH, and GLI1 mRNA was higher in PJPs than in normal tissue (P < .05) and gradually increased along the PJP-adenoma-adenocarcinoma sequence (P < .05). Immunostaining indicated that SHH expression was present in 60% of PJPs, 72% of adenomas, and 84% of carcinomas, whereas 68% of PJPs, 72% of adenomas, and 88% of carcinomas exhibited cytoplasmic expression of PTCH. Moreover, high GLI1 expression was detected in 56% of PJPs, 64% of adenomas, and 80% of carcinomas; and high nuclear expression of GLI1 was observed in 8 adenomas with atypia and 15 carcinomas. Increased SHH, PTCH, and GLI1 protein correlated positively with tumor grade (P = .012, P = .003, and P = .007, respectively), tumor depth (P = .024, P = .007, and P = .01), and lymph node metastasis (P = .05, P = .015, and P = .005). This study identified aberrant expression of SHH pathway molecules in PJS, and the findings may supply a novel mechanism for the development of PJ polyps.

Klein et al. show that Ptch2 loss in either the niche or in hematopoietic cells drives myeloproliferation and accelerates JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias., JAK2V617F+ myeloproliferative neoplasms (MPNs) frequently progress into leukemias, but the factors driving this process are not understood. Here, we find excess Hedgehog (HH) ligand secretion and loss of PTCH2 in myeloproliferative disease, which drives canonical and noncanonical HH-signaling. Interestingly, Ptch2−/− mice mimic dual pathway activation and develop a MPN-phenotype with leukocytosis (neutrophils and monocytes), strong progenitor and LKS mobilization, splenomegaly, anemia, and loss of lymphoid lineages. HSCs exhibit increased cell cycling with improved stress hematopoiesis after 5-FU treatment, and this results in HSC exhaustion over time. Cytopenias, LKS loss, and mobilization are all caused by loss of Ptch2 in the niche, whereas hematopoietic loss of Ptch2 drives leukocytosis and promotes LKS maintenance and replating capacity in vitro. Ptch2−/− niche cells show hyperactive noncanonical HH signaling, resulting in reduced production of essential HSC regulators (Scf, Cxcl12, and Jag1) and depletion of osteoblasts. Interestingly, Ptch2 loss in either the niche or in hematopoietic cells dramatically accelerated human JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias with >30% blasts in the peripheral blood. Our findings suggest HH ligand inhibitors as possible drug candidates that act on hematopoiesis and the niche to prevent transformation of MPNs into leukemias.

Cardiac fibroma is an extremely rare benign tumor that remains poorly characterized genetically. Somatic copy number alterations are common in tumors and have been defined as a crucial factor leading to tumors. In this study, we present a child diagnosed with cardiac fibroma with somatic copy number losses of a total of three discontinuous segments from 9q21.33 to 9q22.33, including a mosaic deletion of PTCH1 . PTCH1 has been associated with sporadic cardiac fibroma. Sequencing analysis of the PTCH1 gene has not revealed any causative mutation. Quantitative PCR analysis of PTCH1 further confirms somatic copy number losses. Our data narrow down the critical causative deletions for sporadic cardiac fibroma to a region more precise than any other previously reported one. Our results suggest important roles of somatic copy number losses on chromosome 9q21.33q22.33 in the development of sporadic cardiac fibroma; these findings may provide a better understanding of sporadic cardiac fibroma pathogenesis and contribute to the identification of novel diagnostic biomarkers of this neoplasm.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC -mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC -signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal ( CBX4 and CBX8 . In our combined cohort, we identified frequent inactivating mutations in AJUBA , ZNF750 , and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1 , in addition to known mutations. Functional analyses suggest roles for several genes ( CBX4 , CBX8 , AJUBA , and ZNF750 ) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.

BackgroundBasal cell nevus syndrome (BCNS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), maxillary keratocysts, and cerebral calcifications. BCNS most commonly is caused by a germline mutation in the patched-1 (PTCH1) gene. PTCH1 mutations are also described in patients with holoprosencephaly.MethodsWe have established a locus-specific database for the PTCH1 gene using the Leiden Open Variation Database (LOVD). We included 117 new PTCH1 variations, in addition to 331 previously published unique PTCH1 mutations. These new mutations were found in 141 patients who had a positive PTCH1 mutation analysis in either the VU University Medical Centre (VUMC) or Maastricht University Medical Centre (MUMC) between 1995 and 2015.ResultsThe database contains 331 previously published unique PTCH1 mutations and 117 new PTCH1 variations.ConclusionWe have established a locus-specific database for the PTCH1 gene using the Leiden Open Variation Database (LOVD). The database provides an open collection for both clinicians and researchers and is accessible online at http://www.lovd.nl/PTCH1..

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. In children, the 2 major RMS subtypes are alveolar and embryonal RMS. Aberrant Hedgehog/Patched1 (Hh/Ptch) signaling is a hallmark of embryonal RMS. We demonstrate that mice carrying a Ptch mutation in mesodermal Delta1-expressing cells develop embryonal-like RMS at a similar rate as mice harboring a Ptch mutation in the germline or the brachury-expressing mesoderm. The tumor incidence decreases dramatically when Ptch is mutated in Myf5- or Pax3-expressing cells. No RMS develop from Myogenin/Mef2c-expressing cells. This suggests that Hh/Ptch-associated RMS are derived from Delta1-positive, Myf5-negative, Myogenin-negative and Pax3-negative mesodermal progenitors that can undergo myogenic differentiation but lack stable lineage commitment. Additional preliminary genetic data and data on mesodermal progenitors further imply an interplay of Hh/Ptch and Delta/Notch signaling activity during RMS initiation. In contrast, Wnt signals supposedly suppress RMS formation because RMS multiplicity decreases after inactivation of the Wnt-inhibitor Wif1. Finally, our results strongly suggest that the tumor-initiating event determines the lineage of RMS origin.

Intrinsic molecular determinants of neurodevelopmental outcomes assume new, albeit related roles during adult neural regeneration. Here we studied and identified a facilitatory role for Sonic hedgehog protein (Shh), a morphogen that influences motor neuron floor plate architecture, during adult peripheral neuron regeneration. Shh and its receptors were expressed in adult dorsal root ganglia (DRG) neurons, axons and glia and trended toward higher levels following axotomy injury. Knockdown of Shh in adult sensory neurons resulted in decreased outgrowth and branching in vitro, identifying a role for Shh in facilitating outgrowth. The findings argued for an intrinsic action to support neuron regeneration. Support of advancement and turning however, were not identified in adult sensory neuron growth cones in response to local extrinsic gradients of Shh. That intrinsic Shh supported the regrowth of peripheral nerves after injury was confirmed by the analysis of axon regrowth from the proximal stumps of transected sciatic nerves. By exposing regenerating axons to local infusions of Shh siRNA in vivo within a conduit bridging the transected proximal and distal stumps, we achieved local knockdown of Shh. In response, there was attenuated axonal and Schwann cell outgrowth beyond the transection zone. Unlike its role during neurodevelopment, Shh facilitates but does not confer regenerative outgrowth properties to adult neurons alone. Exploring the differing properties of morphogens and related proteins in the adult nervous system identifies new and important roles for them.

The Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D3, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane domain or the cysteine-rich domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition, which did not result from elevated calcitriol bioavailability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers.

The evolution of imaginal cells, or stem cell-like cells, contributed to the spectacular diversification of holometabolous insects, which undergo complete metamorphosis. The proliferation and differentiation of these imaginal cells is under the control of juvenile hormone (JH), but which patterning genes respond to JH is currently unknown. Here, the role of Hedgehog (Hh) signaling in the development of imaginal cells was investigated. RNA interference-mediated knockdown of the components of the Hh signaling pathway showed that Hh is required for the proliferation of polymorphic and imaginal cells in Tribolium castaneum. Hh was also necessary for the regeneration of larval appendages. In contrast, knockdown of Hh signaling antagonists, patched and costal 2 led to the overgrowth and precocious maturation of structures derived from imaginal cells and the occasional appearance of ectopic appendages from the head epidermis. In addition, JH suppressed the expression of hh both in vivo and in vitro. Our findings suggest that imaginal cells are created and maintained by modulating Hh signaling. Thus, Hh signaling may have played a critical role during the evolution of complete metamorphosis.

Basal cell carcinoma (BCC), the most common type of skin cancer, is occasionally aggressive with deep invasion, destruction of adjacent structures, recurrence and, on very rare occasions, regional and distant metastases. Mutations that occur in BCC in hedgehog (Hh) pathway genes primarily involve the genes encoding patched homolog (PTCH) and smoothened homolog (SMO). Several animal models have demonstrated the functional relevance of genetic alterations in the Hh pathway during tumorigenesis. Recently, targeted therapy has become available both commercially and in the context of human clinical trials. Interestingly, Hh pathway inhibitors not only suppress BCC progression but also promote acquired immune responses. Since immune responses are crucial for long-term tumor control, new clinical trials, such as those involving a combination of Hh inhibitors with immune modifiers, are needed to supplement standard methods of tumor control.

Down Syndrome (DS) is a highly complex developmental genetic disorder caused by trisomy for human chromosome 21 (Hsa21). All individuals with DS exhibit some degree of brain structural changes and cognitive impairment; mouse models such as Ts65Dn have been instrumental in understanding the underlying mechanisms. Several phenotypes of DS might arise from a reduced response of trisomic cells to the Sonic Hedgehog (SHH) growth factor. If all trisomic cells show a similar reduced response to SHH, then up-regulation of the pathway in trisomic cells might ameliorate multiple DS phenotypes. We crossed Ptch1tm1Mps/+ mice, in which the canonical SHH pathway is expected to be up-regulated in every SHH-responsive cell due to the loss of function of one allele of the pathway suppressor, Ptch1, to the Ts65Dn DS model and assessed the progeny for possible rescue of multiple DS-related phenotypes. Down-regulation of Ptch produced several previously unreported effects on development by itself, complicating interpretation of some phenotypes, and a number of structural or behavioral effects of trisomy were not compensated by SHH signaling. However, a deficit in a nest-building task was partially restored in Ts;Ptch+/- mice, as were the structural anomalies of the cerebellum seen in Ts65Dn mice. These results extend the body of evidence indicating that reduced response to SHH in trisomic cells and tissues contributes to various aspects of the trisomic phenotype.

The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation.

Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Background/Aims: There is interest in drugs and rehabilitation methods to enhance neurogenesis and improve neurological function after brain injury or degeneration. Resveratrol may enhance hippocampal neurogenesis and improve hippocampal atrophy in chronic fatigue mice and prenatally stressed rats. However, its effect and mechanism of neurogenesis after stroke is less well understood. Sonic hedgehog (Shh) signaling is crucial for neurogenesis in the embryonic and adult brain, but relatively little is known about the role of Shh signaling in resveratrol-enhanced neurogenesis after stroke. Methods: Neural stem cells (NSCs) before oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were pretreated with resveratrol with or without cyclopamine. Survival and proliferation of NSCs was assessed by the CCK8 assay and BrdU immunocytochemical staining. The expressions and activity of signaling proteins and mRNAs were detected by immunocytochemistry, Western blotting, and RT-PCR analysis. Results: Resveratrol significantly increased NSCs survival and proliferation in a concentration-dependent manner after OGD/R injury in vitro. At the same time, the expression of Patched-1, Smoothened (Smo), and Gli-1 proteins and mRNAs was upregulated, and Gli-1 entered the nucleus, which was inhibited by cyclopamine, a Smo inhibitor. Conclusion: Shh signaling mediates resveratrol to increase NSCs proliferation after OGD/R injury in vitro.

Purpose Heterozygous germline PTCH1 mutations are causative of Gorlin syndrome (naevoid basal cell carcinoma), but detection rates > 70% have rarely been reported. We aimed to define the causative mutations in individuals with Gorlin syndrome without PTCH1 mutations. Methods We undertook exome sequencing on lymphocyte DNA from four unrelated individuals from families with Gorlin syndrome with no PTCH1 mutations found by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), or RNA analysis. Results A germline heterozygous nonsense mutation in SUFU was identified in one of four exomes. Sanger sequencing of SUFU in 23 additional PTCH1-negative Gorlin syndrome families identified a SUFU mutation in a second family. Copy-number analysis of SUFU by MLPA revealed a large heterozygous deletion in a third family. All three SUFU-positive families fulfilled diagnostic criteria for Gorlin syndrome, although none had odontogenic jaw keratocysts. Each SUFU-positive family included a single case of medulloblastoma, whereas only two (1.7%) of 115 individuals with Gorlin syndrome and a PTCH1 mutation developed medulloblastoma. Conclusion We demonstrate convincing evidence that SUFU mutations can cause classical Gorlin syndrome. Our study redefines the risk of medulloblastoma in Gorlin syndrome, dependent on the underlying causative gene. Previous reports have found a 5% risk of medulloblastoma in Gorlin syndrome. We found a < 2% risk in PTCH1 mutation–positive individuals, with a risk up to 20× higher in SUFU mutation–positive individuals. Our data suggest childhood brain magnetic resonance imaging surveillance is justified in SUFU-related, but not PTCH1-related, Gorlin syndrome.

Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 μm in 2 hours) and high-throughput (150 μm in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm3. Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.

Objectives: To examine Hedgehog signaling in cutaneous fibrosing disorders for which effective approved therapies are lacking, expand our knowledge of pathophysiology, and explore the rationale for targeted inhibition. Methods: Stain intensity and percentage of cells staining for Sonic hedgehog (Shh), Indian hedgehog (Ihh), Patched (Ptch), glycogen synthase kinase 3 β (GSK3-β), β-catenin, and Snail were evaluated in human skin biopsy specimens of keloid, hypertrophic scar (Hscar), scleroderma, nephrogenic systemic fibrosis (NSF), scar, and normal skin using a tissue microarray. Results: Ihh, but not Shh, was detected in a significantly larger proportion of cells for all case types. Ptch, GSK3-β, and β-catenin showed a gradient of expression: highest in NSF and keloid; moderate in normal skin, scar, and Hscar; and lowest in scleroderma. Snail expression was binary: low in normal skin but high in all fibrosing conditions studied. Conclusions: Differential overexpression of Hedgehog and Snail in cutaneous fibrosing disorders demonstrates a role for targeted inhibition. Ptch, GSK3-β, and β-catenin can help differentiate scleroderma from NSF in histologically subtle cases. Differences in expression between keloid and hypertrophic scar support the concept that they are pathophysiologically distinct disorders. Our findings implicate Snail as a target for the prevention of fibrogenesis or fibrosis progression and may offer a means to assess response to therapy.

Anaplastic thyroid carcinoma is the least common form of thyroid cancer; however, it accounts for the majority of deaths associated with this family of malignancies. A number of genetically engineered immunocompetent mouse models recapitulating the genetic and histological features of anaplastic thyroid cancer have been very recently generated and represent an invaluable tool to dissect the mechanisms involved in the progression from indolent, well differentiated tumors to aggressive, undifferentiated carcinomas, and to identify novel therapeutic targets. In this review, we focus on the relevant characteristics associated with these models and on what we have learned in terms of anaplastic thyroid cancer biology, genetics, and response to targeted therapy.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter ( K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Objective Hedgehog signaling proteins were assessed in patients with cervical carcinoma receiving chemoradiation. Associations between five Hedgehog proteins and prognosis were studied. Methods In all, 131 cases of cervical carcinomas (FIGO stages I–IV) were immunohistochemically (IHC) analyzed for Patched (PTCH), Smoothened (SMO), and GLI1, GLI2 and GLI3 protein expression. Associations between Hedgehog protein expressions, clinicopathological factors, and clinical outcome data were examined. Results Positive IHC staining for the five Hedgehog proteins was recorded in 8% to 37% of the tumor cells. The highest frequency was noted for SMO and the lowest for GLI1. There was a significant association between low SMO- and GLI2-expression and KRAS-mutation. Tumors with overexpressed SMO had a higher frequency of residual tumor or local recurrences than tumors with low SMO expression. Patients with tumors expressing PTCH in more than 75% of the cells had significantly (P=0.023) better recurrence-free survival than patients with tumors with low expression. The opposite situation was true for SMO. For GLI2, there was a statistically significant difference with regard to overall (P=0.004) and distant (P=0.015) relapse rate for groups with expression of GLI2 in the range of 5–25% compared to higher rates. Conclusions A predictive and prognostic value was found for PTCH, SMO, and GLI2 with regard to residual carcinoma, local recurrences, and for GLI2 distant relapses. The Hedgehog signaling pathway also seems to play an important role in cervical carcinogenesis together with HPV16-infection and KRAS-mutation.

The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis. Hh-induced tumors arise from various tissues and they may be indolent or aggressive, as is the case with skin basal cell carcinoma (BCC) or cerebellar medulloblastoma, respectively. Little is known about common cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of hematopoietic and embryonic stem cells, as well as for the propagation of acute myeloid and T-lymphoblastic leukemias. We report here that Zfx facilitates the development of experimental BCC and medulloblastoma in mice initiated by deletion of the Hh inhibitory receptor Ptch1. Simultaneous deletion of Zfx along with Ptch1 prevented BCC formation and delayed medulloblastoma development. In contrast, Zfx was dispensable for tumorigenesis in a mouse model of glioblastoma. We used genome-wide expression and chromatin-binding analysis in a human medulloblastoma cell line to characterize direct, evolutionarily conserved targets of Zfx, identifying Dis3L and Ube2j1 as two targets required for the growth of the human medulloblastoma cells. Our results establish Zfx as a common cell-intrinsic regulator of diverse Hh-induced tumors, with implications for the definition of new therapeutic targets in these malignancies. Cancer Res; 74(20); 5914–24. ©2014 AACR.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Normally, sonic hedgehog (Shh) signaling induces high levels of Patched 1 (Ptc1) and its associated transcription factor Gli1 with genesis of specific neuronal progeny. But their roles in the neural stem cells (NSCs), including glial precursor cells (GPCs), of Alzheimer's disease (AD) are unclear. Here, we show that Ptc1 and Gli1 are significantly deficits in the hippocampus of an aged AD transgenic mouse mode, whereas these two molecules are highly elevated at young ages. Our similar findings in autopsied AD brains validate the discovery in AD mouse models. To examine whether Aβ peptides, which are a main component of the amyloid plaques in AD brains, affected Ptc1-Gli1 signaling, we treated GPCs with Aβ peptides, we found that high dose of Aβ1-42 but not Aβ1-40 significantly decreased Ptc1-Gli1, while Shh itself was elevated in hippocampal NSCs/GPCs. Furthermore, we found that deficits of Ptc1-Gli1 signaling induced NSCs/GPCs into asymmetric division, which results in an increase in the number of dividing cells including transit-amplifying cells and neuroblasts. These precursor cells commit to apoptosis-like death under the toxic conditions. By this way, adult neural precursor cell pool is exhausted and defective neurogenesis happens in AD brains. Our findings suggest that Ptc1-Gli1 signaling deregulation resulting abnormal loss of GPCs may contribute to a cognition decline in AD brains. The novel findings elucidate a new molecular mechanism of adult NSCs/GPCs on neurogenesis and demonstrate a regulatory role for Ptc1-Gli1 in adult neural circuit integrity of the brain.

Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-srcAct mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty.

Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.

ATAD2 is associated with many cellular progresses such as cell growth, differentiation and apoptosis. Some studies suggest ATAD2 is highly expressed in cancer cells. In our previous studies, we found that ATAD2 is highly expressed in HCC tissues, compared with adjacent normal tissues, and patients with high expression of ATAD2 had a poorer prognosis. Moreover, we found mir-372 can regulate the expression of ATAD2 in HCC cell lines. We also detected a relationship between the mRNA expression of ATAD2 and Ptch1 by gene microarray. Here, we completed the function studies of ATAD2 in vivo and in vitro, and tested whether ATAD2 could regulate the Hh pathway. ATAD2 and Hh pathway protein expressions in 80 HCC specimens were examined by immunohistochemistry (IHC). The mRNA expression of ATAD2 and Hh pathway members in paired-HCC tissues and cell lines were, respectively, analyzed using quantitative PCR. ATAD2‑RNAi was transduced into HCCLM3 and Huh7 cells, using a lentiviral vector. The effect of ATAD2 in HCC cell lines on cell cycle and apoptosis were evaluated by flow cytometry. Tumorigenicity experiments in nude mice were performed to test the function of ATAD2 in vivo. Pharmacological regulation of Hh signaling was performed to test the relation between the ATAD2 and Hh pathways and C-myc. We found that ATAD2 and Ptch1 were both highly expressed in HCC tissues, compared with paired normal hepatic tissues. In addition, we found that ATAD2 could affect the expression of the Hh pathway by PCR and western blot anaysis in HCC cell lines, by observing the outcome before and after transfection. We speculate that ATAD2 cooperates with the MYC gene to regulate the expression of SMO and Gli, activating the Hh pathway and inducing an active feedback of the Hh pathway.

Hairless mice carrying homozygous mutations in hairless gene manifest rudimentary hair follicles (HFs), epidermal cysts, hairless phenotype, and enhanced susceptibility to squamous cell carcinomas. However, their susceptibility to basal cell carcinomas (BCCs), a neoplasm considered originated from HF-localized stem cells, is unknown. To demonstrate the role of HFs in BCC development, we bred Ptch(+/-)/C57BL6 with SKH-1 hairless mice, followed by brother-sister cross to get F2 homozygous mutant (hairless) or wild-type (haired) mice. UVB-induced inflammation was less pronounced in shaved haired than in hairless mice. In hairless mice, inflammatory infiltrate was found around the rudimentary HFs and epidermal cysts. Expression of epidermal IL1f6, S100a8, vitamin D receptor, repetin, and major histocompatibility complex II, biomarkers depicting susceptibility to cutaneous inflammation, was also higher. In these animals, HF disruption altered susceptibility to UVB-induced BCCs. Tumor onset in hairless mice was 10 weeks earlier than in haired littermates. The incidence of BCCs was significantly higher in hairless than in haired animals; however, the magnitude of sonic hedgehog signaling did not differ significantly. Overall, 100% of hairless mice developed12 tumors per mouse after 32 weeks of UVB therapy, whereas haired mice developed fewer than three tumors per mouse after 44 weeks of long-term UVB irradiation. Tumors in hairless mice were more aggressive than in haired littermates and manifested decreased E-cadherin and enhanced mesenchymal proteins. These data provide novel evidence that disruption of HFs in Ptch(+/-) mice enhances cutaneous susceptibility to inflammation and BCCs.

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60–90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog ( Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by—and possibly attributable to—altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

Basal cell carcinosarcoma is a rare biphenotypic malignant skin tumor, in which one tumor component has light microscopic features of basal cell carcinoma, whereas the other has features of sarcoma. Clinical experience with this tumor is limited, and associated molecular genetic alterations are unknown. Herein, we report a unique case of metastatic basal cell carcinosarcoma, in which we analyzed the 2 components of the primary tumor as well as the metastasis by next-generation sequencing. The patient was a 72-year-old man who presented with a 7-year history of a large tumor of the left forearm. The tumor showed mixed features of basal cell carcinoma and undifferentiated sarcoma. The patient underwent a wide local excision and sentinel lymph node biopsy, which revealed microscopic subcapsular deposits of metastatic sarcomatoid tumor. One year later, intra-abdominal metastatic tumor was detected and resected. It had sarcomatoid features by light microscopy and failed to stain for epithelial markers by immunohistochemistry. DNA was extracted separately from the epithelial and sarcomatoid component of the primary tumor, intra-abdominal metastasis, and normal tissue. All exons of 230 cancer-associated genes were sequenced to an average read depth of >500-fold. This revealed multiple identical mutations in epithelial and sarcomatoid tumor compartments. Both compartments harbored 2 identical mutations, a truncating and a missense mutation, in the patched gene (PTCH1). This finding is not only of interest for a shared heritage of different subpopulations in a biphenotypic tumor, but also relevant clinically. It provides a rationale for the clinical use of hedgehog pathway inhibitors for treatment of patients affected by this tumor. Unfortunately, the patient reported herein died of metastatic disease before targeted therapy could be initiated.

Recent advances in sequencing technology allow genome-scale approaches to cancer mutation discovery. Such data-intensive methods have been applied to cutaneous squamous cell carcinomas (SCCs) and melanomas but have not, to our knowledge, been applied to basal cell carcinomas (BCCs). We used whole-exome sequencing to characterize the mutational landscape of sporadic BCCs. We show that BCCs are the most mutated type of human cancer. Tumors from anatomical regions with chronic UV exposure were associated with higher mutation rates than those with intermittent exposure. The majority of all mutations (75.7%) were UV signature. Using a conventional binomial probability model, several genes were found mutated significantly. However, this model assumes a uniform distribution of mutations throughout the genome. We also used a more stringent approach called InVEx that uses a permutation-based framework to pick drivers from passengers. After correction for multiple hypothesis testing, InVEx identified only PTCH1 (Patched 1) as having a significant functional mutation burden. We also found three genes, STAT5B, CRNKL1, and NEBL, with mutational hot spots at a single base in 3 of 12 tumors sequenced. Our findings support the central role of PTCH1 mutations in BCC genesis. Moreover, our discovery of the uniquely high number of mutations in this tumor may lend insight into its biological behavior.

Cerebellar granule neuronal progenitors (GNPs) are the precursors of cerebellar granule cells (CGCs) and are believed to be the cell of origin for medulloblastoma (MB), yet the molecular mechanisms governing GNP neurogenesis are poorly elucidated. Here, we demonstrate that storkhead box 1 (Stox1), a forkhead transcriptional factor, has a pivotal role in cerebellar granule neurogenesis and MB suppression. Expression of Stox1 is upregulated along with GNP differentiation and repressed by activation of sonic hedgehog (SHH) signaling. Stox1 exerts its neurogenic and oncosuppressing effect via direct transcriptional repression of Math1, a basic helix-loop-helix transcription activator essential for CGC genesis. This study illustrates a SHH-Stox1-Math1 regulatory axis in normal cerebellar development and MB formation.

Hedgehog (Hh) signaling is largely suppressed in the normal differentiated tissues of the adult but activated in many cancers. The Hh pathway can either be activated by the expression of Hh ligands, or by mutations that cause constitutive, ligand-independent signaling. Colorectal cancer cells frequently express Hh ligands that are believed to exert paracrine effects on the stromal component of the tumor. Evidence for a more direct role of Hh signaling on the growth and evolution of colorectal cancer cell clones has been lacking. Here, we report a loss-of-function mutation of PTCH1, a tumor suppressor in the Hh pathway, in a colorectal cancer that exhibits transcriptional upregulation of the downstream Hh gene GLI1. This finding demonstrates that autocrine Hh signaling can provide a selective advantage to evolving tumors that arise in the colorectal epithelia, and suggests a definable group of colorectal cancer patients that could derive enhanced benefit from Hh pathway inhibitors.

In mice, activated Hedgehog (Hh) signaling induces tumors with myogenic differentiation. In humans, hyperactive Hh signaling due to germline PATCHED1 (PTCH1) mutations has been linked to nevoid basal cell carcinoma syndrome (NBCCS). We report an embryonal rhabdomyosarcoma in a 16-month-old girl with NBCCS and review the literature on myogenic neoplasms in NBCCS, including 8 fetal rhabdomyomas and 3 rhabdomyosarcomas. Of note, 3 population studies, including 255 individuals with NBCCS aged 4 months to 87 years, did not identify any myogenic tumors. Thus, myogenic tumors in NBCCS are rare and include both rhabdomyosarcomas and fetal rhabdomyomas.

Basal cell nevus syndrome (BCNS) has existed at least since Dynastic Egyptian times. In 1960, Gorlin and Goltz first described the classic clinical triad: multiple basal cell carcinomas (BCCs), jaw keratocysts, and bifid ribs. As an autosomal-dominant disorder, it is characterized by tumorigenesis and developmental defects.To review the current literature on BCNS, including reports on epidemiology, pathogenesis, clinical presentation, diagnostic criteria, management, treatment, and prognosis.A literature review of currently available articles related to BCNS.Individuals with a mutation in the tumor suppressor gene PTCH1 are predisposed to tumorigenesis and developmental defects. Clinical features include BCCs, often with onset in adolescence, jaw keratocysts, bifid ribs, craniofacial defects, palmar-plantar pits, and ectopic intracranial calcification. Despite high cure rates for individual lesions and various treatment modalities including excision, Mohs micrographic surgery, photodynamic therapy, and topical imiquimod, management of BCCs is challenging. The development of an oral hedgehog pathway inhibitor, vismodegib, has added a new dimension to current treatment algorithms.Adolescents and young adults with BCC should be evaluated for BCNS. Early diagnosis of BCNS is critical for possible prevention of the devastating effects of BCCs and establishment of multidisciplinary care.

Gorlin syndrome or nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant condition mainly characterized by the development of mandibular keratocysts which often have their onset during the second decade of life and/or multiple basal cell carcinoma (BCC) normally arising during the third decade. Cardiac and ovarian fibromas can be found. Patients with NBCCS develop the childhood brain malignancy medulloblastoma (now often called primitive neuro-ectodermal tumor [PNET]) in 5% of cases. The risk of other malignant neoplasms is not clearly increased, although lymphoma and meningioma can occur in this condition. Wilms tumor has been mentioned in the literature four times. We describe a patient with a 10.9 Mb 9q22.3 deletion spanning 9q22.2 through 9q31.1 that includes the entire codifying sequence of the gene PTCH1, with Wilms tumor, multiple neoplasms (lung, liver, mesenteric, gastric and renal leiomyomas, lung typical carcinoid tumor, adenomatoid tumor of the pleura) and a severe clinical presentation. We propose including leiomyomas among minor criteria of the NBCCS. © 2013 Wiley Periodicals, Inc.

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to α v β 3 , α v β 5 , and α 5 β 1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α 5 β 1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F–Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F–Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5–Fc as targeted molecular probes for brain tumor imaging.

Objective— To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. Approach and Results— In vitro, human coronary arterial smooth muscle cells were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression while concomitantly decreasing human coronary arterial smooth muscle cell growth to that similar under normal flow conditions. In vivo, ligation injury–induced low flow increased vascular smooth muscle cell growth and vascular remodeling, while increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow–induced increases in Ptc-1/Notch expression and markedly reduced the subsequent vascular remodeling. Conclusions— These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.