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Ubiquitous overactivation of Hedgehog signaling in adult pituitaries results in increased expression of proopiomelanocortin (Pomc), growth hormone (Gh) and prolactin (Prl), elevated adrenocorticotropic hormone (Acth) production and proliferation of Sox2+ cells. Moreover, ACTH, GH and PRL-expressing human pituitary adenomas strongly express the Hedgehog target GLI1. Accordingly, Hedgehog signaling seems to play an important role in pathology and probably also in homeostasis of the adult hypophysis. However, the specific Hedgehog-responsive pituitary cell type has not yet been identified. We here investigated the Hedgehog pathway activation status and the effects of deregulated Hedgehog signaling cell-specifically in endocrine and non-endocrine pituitary cells. We demonstrate that Hedgehog signaling is unimportant for the homeostasis of corticotrophs, whereas it is active in subpopulations of somatotrophs and folliculo-stellate cells in vivo. Reinforcement of Hedgehog signaling activity in folliculo-stellate cells stimulates growth hormone production/release from somatotrophs in a paracrine manner, which most likely is mediated by the neuropeptide vasoactive intestinal peptide. Overall, our data show that Hedgehog signaling affects the homeostasis of pituitary hormone production via folliculo-stellate cell-mediated regulation of growth hormone production/secretion.

PURPOSE: This study aimed to delineate the genetic basis of congenital ocular motor apraxia (COMA) in patients not otherwise classifiable. METHODS: We compiled clinical and neuroimaging data of individuals from six unrelated families with distinct clinical features of COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions associated with COMA. We used exome sequencing to identify pathogenic variants and functional studies in patient-derived fibroblasts. RESULTS: In 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as smoothened. However, analysis of expression of HH signaling target genes detected a significant increase in the general signaling activity in COMA patient-derived fibroblasts compared with control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, and Patched1. Neuroimaging revealed subtle cerebellar changes, but no full-blown molar tooth sign. CONCLUSION: Taken together, our data imply that the clinical phenotype associated with heterozygous truncating germline variants in SUFU is a forme fruste of Joubert syndrome.

Basal cell carcinoma (BCC) originate from Hedgehog/Patched signaling-activated epidermal stem cells. However, the chemically induced tumorigenesis of mice with a CD4Cre-mediated biallelic loss of the Hedgehog signaling repressor Patched also induces BCC formation. Here, we identified the cellular origin of CD4Cre-targeted BCC progenitors as rare Keratin 5+ epidermal cells and show that wildtype Patched offspring of these cells spread over the hair follicle/skin complex with increasing mouse age. Intriguingly, Patched mutant counterparts are undetectable in age-matched untreated skin but are getting traceable upon applying the chemical tumorigenesis protocol. Together, our data show that biallelic Patched depletion in rare Keratin 5+ epidermal cells is not sufficient to drive BCC development, because the spread of these cells is physiologically suppressed. However, bypassing the repression of Patched mutant cells, e.g., by exogenous stimuli, leads to an accumulation of BCC precursor cells and, finally, to tumor development.

A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS, KRAS, or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh-driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.

With nurses and midwives providing the majority of health care globally, nursing education in all countries must prepare students for broader responsibilities to move the agenda forward for equitable care and universal health coverage. Columbia University School of Nursing developed and implemented a vibrant approach to curriculum transformation that included a new didactic course followed by a program of global clinical experiences to expand students' learning environments in global health. Program planning included defining learning objectives, mobilizing support within the school, establishing new sites, recruiting and preparing students, overseeing of students with host institutions, and evaluating the program. A total of twenty-four students were placed over five sites for a six-week credit-bearing global clinical experience. Students had varied clinical experiences with new understandings of the reality of health disparities. Host sites expressed a commitment to have students return in the next year, and all students stated that they would chose a global experience again. This innovation provides a transformative addition to nursing education with a deepened understanding of health disparities and nursing roles in different health systems. It strengthens the school's network of nursing and midwifery educators and opens doors for new exchanges.

We analyzed the role of WIF1 in normal and acanthotic epidermis of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans-retinoic acid (ATRA)-treated and basal cell carcinoma (BCC)-bearing mice. WIF1 protein is located in the follicular infundibulum and interfollicular epidermis (IFE) in murine back skin. Within the hyperplastic epidermis of TPA- or ATRA-treated or BCC-bearing murine skin, WIF1 and Keratin 10 overlap in Ki67⁻ suprabasal layers, while basal epidermal layers expressing Ki67, and BCCs expressing Wif1 mRNA, are free of WIF1 protein. This is similar in human skin, with the exception that WIF1 protein is found in single Ki67⁻ basal epidermal cells in normal skin and additionally in Ki67+ cells in acanthotic skin. Wif1-deficiency enhances acanthosis of the murine BCC-associated epidermis, which is accompanied by an increase of Ki67+ and of Sca-1+ basal cells. WIF1 overexpression in allografted BCC-derived keratinocytes prevents growth and keratinization, involving enhanced phosphorylation of protein kinase C and extracellular signal–regulated kinase 1 and arguably factors secreted by the in vivo environment. In summary, WIF1 protein marks suprabasal layers in the normal IFE. It is also present in the epidermis overlaying BCCs where it diminishes proliferation of basal cells and production of differentiating suprabasal cells. In addition, WIF1 can prevent proliferation and keratinization of BCC-related keratinocytes.

Sonic hedgehog (SHH) is an essential morphogenetic signal dictating cell fate decisions in several developing organs in mammals. In vitro data suggest that SHH is required to specify LHX3+/LHX4+ Rathke's pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead, a critical role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 dpc (days post coitum) and total loss of pituitary tissue by 12.5 dpc. Conversely, over-activation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ve stem cell compartment by the end of gestation.

We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS. peerReviewed

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RTICC-RD12/0036/0016, /0020, /0035, /0057; and PI14/00647), Fundació A BOSCH, Fundació Amics Joan Petit, ajuts predoctorals del VHIR and RIS3CAT grants COMRDI15-1-0014 (ACCIÓ and FEDER). Altres ajuts: FEDER/COMRDI15-1-0014 Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

Objective During development of the vertebrate skeleton, chondrocytes form a cartilage template that is gradually replaced by bone. Hormones of the Hedgehog (HH) family have been implicated in the ossification process, but their exact relationship to normal or pathogenic bone formation is unclear. This study was undertaken to establish a genetic tool that allows the discrete inactivation of genes in spinal chondrocytes, and to investigate in vivo how chondrocyte-specific ablation of the inhibitory HH receptor Patched 1 (Ptch1) affects skeleton integrity. Methods A Cre-deleter mouse strain, mb1-Cre, for selective gene recombination in spinal chondrocytes was identified by in situ hybridization and histologic analysis. The mb1-Cre+/− animals were crossed with mice that harbor a loxP-flanked Ptch1 gene (Ptch1flox/flox) to abrogate the inhibition of the HH signaling pathway in chondrocytes. The skeletal integrity of F1 mice was characterized by high-resolution flat-panel–based volume computed tomography and histologic staining procedures. Results During the first weeks after birth, all mb1-Cre+/−/Ptch1flox/flox mice developed progressive spinal fusion with malformation of the vertebrae. This phenotype was caused by aberrant chondrocyte proliferation in the intervertebral discs that blocked endochondral ossification. Importantly, the disease pattern occurred in an inflammation-independent manner. Conclusion Our findings indicate that chronic activation of the HH signal pathway in spinal chondrocytes can trigger an ankylosing spine morphology without immune cell contributions. Hence, the destruction of cartilage and loss of axial joint integrity can result from chondrocyte-intrinsic defects of monogenic origin.

The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS.

Objective In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. Design The authors used transgenic mice in which the Hh receptor Patched1 ( Ptch1 ) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. Results Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. Conclusion Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

Activation of the Hedgehog (Hh)-signaling pathway due to deficiency in the Hh receptor Patched1 (Ptch) is the pivotal defect leading to formation of basal cell carcinoma (BCC). Recent reports provided evidence of Ptch-dependent secretion of vitamin D3-related compound, which functions as an endogenous inhibitor of Hh signaling by repressing the activity of the signal transduction partner of Ptch, Smoothened (Smo). This suggests that Ptch-deficient tumor cells are devoid of this substance, which in turn results in activation of Hh-signaling. Here, we show that the application of the physiologically active form of vitamin D3, calcitriol, inhibits proliferation and growth of BCC of Ptch mutant mice in vitro and in vivo. This is accompanied by the activation of the vitamin D receptor (Vdr) and induction of BCC differentiation. In addition, calcitriol inhibits Hh signaling at the level of Smo in a Vdr-independent manner. The concomitant antiproliferative effects on BCC growth are stronger than those of the Hh-specific inhibitor cyclopamine, even though the latter more efficiently inhibits Hh signaling. Taken together, we show that exogenous supply of calcitriol controls the activity of 2 independent pathways, Hh and Vdr signaling, which are relevant to tumorigenesis and tumor treatment. These data suggest that calcitriol could be a therapeutic option in the treatment of BCC, the most common tumor in humans. Mol Cancer Ther; 10(11); 2179–88. ©2011 AACR.

Background & Aims Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation. Methods Hedgehog activity was modulated genetically in both cell type–specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence. Results Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells. Conclusions We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria., Graphical abstract

Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptchflox/floxERT2+/− knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca2+ signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling. Cancer Res; 70(7); 2739–48

Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2′deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model. [Cancer Res 2009;69(3):887–95]

Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice, we show that this differentiation step requires Patched (Ptch), the cell surface–bound receptor for Hedgehog (Hh). In the absence of Ptch, the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently, the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally, adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism.

Rapid detection of genetic contamination is critical in mouse studies involving inbred strains. During a Quantitative Trait Locus (QTL) study using simple sequence length polymorphism (SSLP) markers, we noticed heterozygosity at some loci of a commercially available inbred C57BL/6N mouse strain, suggesting a contamination by another mouse strain. A panel of 100 single-nucleotide polymorphism (SNP) markers was used to confirm and specify the genetic contamination suspected. Retrospective analyses demonstrated that the contamination took place as early as autumn 2003 and has persisted ever since at a fairly constant level. Contaminating alleles most probably originated from a DBA strain. Our data demonstrate the suitability of SNP markers for rapid detection and identification of the source of genetic contamination. Further, our results show the importance of a state-of-theart genetic monitoring of the authenticity of murine inbred strains.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is divided into two major histological subgroups, i.e., embryonal (ERMS) and alveolar RMS (ARMS). RMS can show HEDGEHOG/SMOOTHENED (HH/SMO) signaling activity and several clinical trials using HH inhibitors for therapy of RMS have been launched. We here compared the antitumoral effects of the SMO inhibitors GDC-0449, LDE225, HhA, and cyclopamine in two ERMS (RD, RUCH-2) and two ARMS (RMS-13, Rh41) cell lines. Our data show that the antitumoral effects of these SMO inhibitors are highly divers and do not necessarily correlate with inhibition of HH signaling. In addition, the responsiveness of the RMS cell lines to the drugs is highly heterogeneous. Whereas some SMO inhibitors (i.e., LDE225 and HhA) induce strong proapoptotic and antiproliferative effects in some RMS cell lines, others paradoxically induce cellular proliferation at certain concentrations (e.g., 10 μM GDC-0449 or 5 μM cyclopamine in RUCH-2 and Rh41 cells) or can increase HH signaling activity as judged by GLI1 expression (i.e., LDE225, HhA, and cyclopamine). Similarly, some drugs (e.g., HhA) inhibit PI3K/AKT signaling or induce autophagy (e.g., LDE225) in some cell lines, whereas others cannot (e.g., GDC-0449). In addition, the effects of SMO inhibitors are concentration-dependent (e.g., 1 and 10 μM GDC-0449 decrease GLI1 expression in RD cells whereas 30 μM GDC-0449 does not). Together these data show that some SMO inhibitors can induce strong antitumoral effects in some, but not all, RMS cell lines. Due to the highly heterogeneous response, we propose to conduct thorough pretesting of SMO inhibitors in patient-derived short-term RMS cultures or patient-derived xenograft mouse models before applying these drugs to RMS patients.

Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system [Formula: see text] that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.

Mutations in the human homologue of Drosophila Patched1 (PTCH1) have been found in several common tumours including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (RMS). Medulloblastoma and RMS are also present in the murine model for Ptch1 deficiency. Tumours in heterozygous Ptch1(neo67/+) mice consistently exhibit elevated transcript levels of the proto-oncogene Gli1, of Ptch1 itself, and of the insulin-like growth factor 2 (Igf2). The present study has investigated additional molecular changes in RMSs of Ptch1 mutant mice by means of microarray analysis and protein expression analysis. The data show activation of the cell survival-promoting Akt/protein kinase B (Pkb). Furthermore, RMSs express increased levels of the anti-apoptotic protein Bcl-2 and of genes and proteins known to inhibit cell proliferation, including Gadd45a and p27kip1. Taken together, the data suggest that the formation of RMSs in Ptch1 mutants is associated with the ability of tumour cells to resist apoptosis.

Summary In the adult brain, self-renewal is essential for the persistence of neural stem cells (NSCs) throughout life, but its regulation is still poorly understood. One NSC can give birth to two NSCs or one NSC and one transient progenitor. A correct balance is necessary for the maintenance of germinal areas, and understanding the molecular mechanisms underlying NSC division mode is clearly important. Here, we report a function of the Sonic Hedgehog (SHH) receptor Patched in the direct control of long-term NSC self-renewal in the subependymal zone. We show that genetic conditional activation of SHH signaling in adult NSCs leads to their expansion and the depletion of their direct progeny. These phenotypes are associated in vitro with an increase in NSC symmetric division in a process involving NOTCH signaling. Together, our results demonstrate a tight control of adult neurogenesis and NSC renewal driven by Patched., Graphical Abstract, Highlights Ptc inactivation in the adult NSCs expands the NSC pool and depletes their progeny Neurogenesis blockade is related to the increase of NSC symmetric division PTC exerts a central role via NOTCH, in the regulation of adult NSC self-renewal, In the adult brain, self-renewal is essential for the persistence of neural stem cells throughout life. Traiffort, Ruat, and colleagues report that the Sonic Hedgehog receptor Patched controls long-term self-renewal of these cells in the subependymal zone by increasing their symmetric division in a process involving NOTCH signaling.

Basai Cell Carcinomas (BCCs) are the most commonly diagnosed tumors among people in the western world. Most BCCs are caused by mutational inactivation of the tumor suppressor Patched (PTCH), which results in activation of Smoothened (SMO) and of the Hedgehog (HH) signaling pathway. Recent studies indicate that BCC progression involves a crosstalk between Hh signaling, vitamin D derivatives and the vitamin D receptor (Vdr) signaling pathway. This has been demonstrated in BCC-bearing Ptch mutant mice and BCC cell lines, in which both vitamin D3 and its active metabolite calcitriol ( 1 a-25(OH)2D3) exert antitumor effects. Interestingly, the antitumor effects are mainly ascribed to an inhibition of Hh signaling. Furthermore, as evident from studies in Vdr deflcient mice, calcitriol may also repress the activity of Hh signaling in a Vdr-dependent fashion thereby establishing an additional inhibitory feedback on Hh signaling activity. In this chapter, we discuss the current understanding and controversial findings of the inhibition of Hh signaling by vitamin D derivatives and the implication of these findings for BCC carcinogenesis.

Basal cell carcinoma (BCC) is the most common human tumor. Mutations in the hedgehog (HH) receptor Patched (PTCH) are the main cause of BCC. Due to their high and increasing incidence, BCC are becoming all the more important for the health care system. Adequate animal models are required for the improvement of current treatment strategies. A good model should reflect the situation in humans (i.e., BCC initiation due toPtchmutations on an immunocompetent background) and should allow for (i) BCC induction at a defined time point, (ii) analysis of defined BCC stages, and (iii) induction of BCC in 100% of animals. In addition, it should be easy to handle. Here, we compare several currently existing conventional and conditionalPtchknockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditionalPtchflox/floxmice and theK5-Cre-ERT+/−driver.

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines. peerReviewed

Embryonal rhabdomyosarcoma (ERMS) is a tumor of the skeletal muscle in children and is frequently initiated by heterozygous germline mutations in the Hedgehog (Hh) receptor Patched1 (Ptch), both in humans and mice. Using a conditional knock-out strategy in Ptch(flox/+) mice, we demonstrate that early embryonic stages are more susceptible to ERMS development than later stages and that cells normally not committed to undergo myogenesis at this stage represent the major source of ERMS. We found that deletion of a single copy of the Ptch allele at E9.5 using the ubiquitously active Rosa26CreERT2 resulted in a tumor incidence of 88% but reached only 44% and 12% when the Ptch allele was inactivated at E11.5 and E13.5, respectively. Induction of the Ptch mutation at E9.5 did also significantly shorten ERMS-free survival and increased tumor multiplicity compared with tumor induction at E11.5 and E13.5. Interestingly, we observed a more that 10-fold reduction of ERMS incidence when the Ptch mutation was specifically introduced in Myf5-expressing cells, which is the myogenic factor expressed in all muscle cells at E9.5. We conclude that Myf5-negative cells are more susceptible to ERMS development than Myf5-positive embryonic precursors. As the propensity to undergo tumorigenic transformation declined with age, concomitant with the increase of stably committed muscle cells, it seems likely that the Ptch mutation favors tumor formation in progenitor cells, which have not yet acquired a muscle cell fate.

We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type–specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Seventy-five percent of ARMS harbor reciprocal chromosomal translocations leading to fusion genes of the forkhead transcription factor FOXO1 and PAX3 or PAX7. The hedgehog (Hh) pathway has been implied in tumor formation and progression of various cancers including RMS. However, whether Hh pathway activation presents a general feature of RMS or whether it is restricted to specific subgroups has not yet been addressed. Here, we report that marker genes of active Hh signaling, that is, Patched1 (Ptch1), Gli1, Gli3 and Myf5, are expressed at significantly higher levels in ERMS and fusion gene-negative ARMS compared with fusion gene-positive ARMS in two distinct cohorts of RMS patients. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene-negative ARMS, but not in fusion gene-positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene-negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation. Moreover, Myf5 is identified as a novel excellent class predictor for RMS by receiver operating characteristic analysis. Importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene-negative RMS underscoring the clinical relevance of these findings. By showing that Hh signaling is preferentially activated in specific subgroups of RMS, our study has important implications for molecular targeted therapies, such as small molecule Hh inhibitors, in RMS.

Background & Aims The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. Methods We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. Results Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. Conclusions Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse.

Mutations in the Hedgehog (Hh) receptor Patched (Ptch) are responsible for a variety of tumors, which show ligand-independent stimulation of the Hh/Ptch signaling cascade. Cyclopamine is an alkaloid of the corn lily Veratrum californicum, which blocks activity of the pathway by inhibition of Smoothened (Smo), the signal transduction partner of Ptch. This results in growth inhibition of Hh/Ptch-dependent tumor cells in vitro, of subcutaneous xenografts as well as of precancerous lesions in Ptch+/− mice. However, the evidence that treatment with cyclopamine is an effective anti-cancer therapy against full-blown tumors is sparse. Here, we have investigated the responsiveness of full-blown Hh/Ptch-associated rhabdomyosarcoma (RMS) to this drug. Hh pathway activity and proliferation of cultured primary RMS cells was inhibited by cyclopamine. Hh signaling was also partially suppressed by the drug in RMS in vivo, but cyclopamine treatment did not result in stable disease or tumor regression. It also did not affect proliferation, apoptosis or the differentiation status of RMS. This was in contrast to anti-proliferative effects on tumor growth caused by doxorubicin, an anthracycline routinely used in therapy of human RMS. In summary, our data indicate that there must be additional factors that render full-blown Hh/Ptch-associated RMS insensitive against anti-proliferative effects of cyclopamine in vivo. © 2007 Wiley-Liss, Inc.

Patched1 (PTCH1/Ptch1) is a member of the SHH/Shh signaling pathway, where it serves as a receptor for SHH/Shh. Inactivating mutations in the PTCH1/Ptch1 gene result in a pathological activation of the pathway, which may lead to several forms of familial and sporadic cancers. Although PTCH1/Ptch1 is generally supposed to be a tumor suppressor gene, mutational inactivation of one allele seems to be sufficient for tumor formation. In this chapter the current knowledge about the mode of tumor formation caused by PTCH1/Ptch1 mutations is presented. Most of the insights in these processes are derived from Ptch1 mutant mice. The impact of the genetic background on tumor susceptibility, the involvement of additional molecular pathways and epigenetic mechanisms leading to tumor formation, are discussed.

Hemizygous Ptc1 mice have many features of Gorlin syndrome, including predisposition to medulloblastoma development. Ionizing radiation synergize with Ptc1 mutation to induce medulloblastoma only in neonatally exposed mice. To explore the mechanisms underlying age-dependent susceptibility, we irradiated Ptc(neo67/+) mice at postnatal day 1 (P1) or 10 (P10). We observed a dramatic difference in medulloblastoma incidence, which ranged from 81% in the cerebellum irradiated at P1 to 3% in the cerebellum irradiated at P10. A striking difference was also detected in the frequency of cerebellar preneoplastic lesions (100 versus 14%). Our data also show significantly lower induction of apoptosis in the cerebellum of medulloblastoma-susceptible (P1) compared to -resistant (P10) mice, strongly suggesting that medulloblastoma formation in Ptc1 mutants may be associated with resistance to radiation-induced cell killing. Furthermore, in marked contrast with P10 mice, cerebellum at P1 displays substantially increased activation of the cell survival-promoting Akt/Pkb protein, and markedly decreased p53 levels in response to radiation-induced genotoxic stress. Overall, these results show that developing cerebellar granule neuron precursors' (CGNPs) radiosensitivity to radiation-induced cell death increases with progressing development and inversely correlates with their ability to neoplastically transform.

Rhabdomyosarcoma (RMS) is a highly malignant tumor that is histologically related to skeletal muscle, yet genetic and molecular lesions underlying its genesis and progression remain largely unknown. In this study we have compared the molecular profiles of two different mouse models of RMS, each associated with a defined primary genetic defect known to play a role in rhabdomyosarcomagenesis in man. We report that RMS of heterozygous Patched1 (Ptch1) mice show less aggressive growth and a greater degree of differentiation than RMS of heterozygous p53 mice. By means of cDNA microarray analysis we demonstrate that RMS in Ptch1 mutants predominantly express a number of myogenic markers, including myogenic differentiation 1, myosin heavy chain, actin, troponin and tropomyosin, as well as genes associated with Hedgehog/Patched signaling like insulin-like growth factor 2, forkhead box gene Foxf1 and the growth arrest and DNA-damage-inducible gene Gadd45a. In sharp contrast, RMS in p53 mutants display higher expression levels of cell cycle-associated genes like cyclin B1, cyclin-dependent kinase 4 and the proliferation marker Ki-67. These results demonstrate that different causative mutations lead to distinct gene expression profiles in RMS, which appear to reflect their different biological characteristics. Our results provide a first step towards a molecular classification of different forms of RMS. If the described differences can be confirmed in human RMS our results will contribute to a new molecular taxonomy of this cancer, which will be critical for gene mutation- and expression-specific therapy.

Medulloblastoma (MB) is a highly malignant embryonal tumor of the cerebellum with a preferential manifestation in children. Although the majority of MBs occur sporadically, this tumor is also associated with familial cancer syndromes including the nevoid basal cell carcinoma or Gorlin syndrome. Mutations in the tumor suppressor gene PATCHED 1 (PTCH1) have been described in both familial and sporadic cases and inactivation of one Patched 1 (Ptch1) allele in mice promotes development of MB. In order to determine candidate genes involved in tumorigenesis of MB, we have screened tumors of heterozygous Ptch1 mice for differentially expressed genes by means of cDNA microarray technology. Our data show that genes involved in cell cycle, signal transduction and metastasis are transcriptionally up-regulated in MB compared to normal cerebellum. Gene ontology analysis reveals cell cycle regulators to be the predominant functional gene class altered in MB of Ptch1 mutants, including D-type cyclins and cyclin-dependent kinase 4. We furthermore describe that overexpression of the growth arrest and DNA-damage-inducible gene Gadd45a is common in Ptch1-associated tumors and Ptch1 null embryos. These results suggest that cDNA microarray technology is a useful tool to discover genes involved in the development of MB that arise in response to a persistent activation of sonic hedgehog (Shh) signaling. This approach may provide novel data for diagnosis, treatment and prevention of human PTCH1-related malignancies.

Mutations in the Patched (Ptch1) gene are responsible for various familial and sporadic cancers. Ptch1 neo67/+ mice, in which exons 6 and7 are deleted, show genetic background-dependent susceptibility to the development of muscle tumors resembling humanrhabdomyosarcoma (RMS); BALB/c (BALB) is a susceptible strain whereas C57BL/6 (B6) shows resistance. A genome-wide linkageanalysis was carried out using Ptch1 neo67/+ mice produced from B6 (BALB B6) backcrosses to identify loci involved in the control ofRMS susceptibility. Quantitative trait locus mapping with the censored tumor latency time as the quantitative parameter was used to detect asignificant RMS susceptibility modifier locus, Parms1 (Patched-Associated RMS 1), on chromosome 2 between D2Mit37 and D2Mit102(LRS = 10). A Kaplan-Meier survival curve revealed that mice with the B6/BALB genotype develop tumors more frequently and much fasteras compared to mice homozygous for the B6 allele (P = 0.02). Additional loci not reaching linkage significance were also detected formedulloblastoma resistance.D 2004 Elsevier Inc. All rights reserved.

Cultivation of retrovirus packaging cells at 32 degrees C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 degrees C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 degrees C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 degrees C, production of MLV-A and the Shh signalling pathway.

The gene coding for the human homologue of the Drosophila segment polarity gene patched (PTCH1) is mutated in several common human tumors. In mice, haplodeficiency at the Ptch1 locus results in severe histologic defects in mammary ductal structure. We found no mutations within the coding region of PTCH1 in 17 human primary breast carcinomas. However, the biallelic Pro1315Leu (C3944T) polymorphism of PTCH1 was significantly associated with breast cancer in 41 Bavarian patients compared to 85 healthy controls. We investigated whether this variant influences susceptibility for breast cancer in 611 breast cancer patients diagnosed by age 50 years and 1,057 controls matched by age and study region in Germany and in 1,093 breast cancer patients from the United Kingdom. Allele and genotype frequencies were not different between cases and controls. However, multivariate logistic regression analysis revealed an effect modification of oral contraceptive use (OC) on breast cancer risk by Leu-carrier status. Compared to women who have Pro/Pro and never used OC, Pro/Pro OC users had an increased odds ratio for breast cancer of 1.7. The odds ratio was also 1.7 for Leu-carriers who never used OC, but this was attenuated among Leu-carriers who ever used OC by 20%. The gene-environmental interaction was confirmed in case-only analysis of the German and British studies, yielding an interaction odds ratio of 0.7 for premenopausal women (p = 0.06). Longer duration of pill use was associated with a significantly greater risk reduction (p for trend = 0.015). Our novel observation of a differential effect of OC use on breast cancer risk by PTCH1 1315Leu-carrier status suggests the interesting possibility of the Sonic hedgehog/Patched (SHH/PTCH1) signaling pathway being involved in hormone-induced development of breast carcinoma. © 2002 Wiley-Liss, Inc.

Inherited mutations of the human tumor suppressor gene Patched (PTCH) lead to an autosomal dominant disorder known as Nevoid Basal Cell Carcinoma Syndrome (NBCCS). The syndrome is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Tumors in patients with NBCCS include basal cell carcinoma, medulloblastoma, fibroma and rhabdomyosarcoma (RMS). RMS are also present in 15 % of mice haplodeficient for Ptch. To investigate whether mutations in PTCH are a general feature in rhabdomyosarcomagenesis we sequenced the protein-coding region in sporadic human cases of these tumors. For this purpose we first determined the distribution and frequency of polymorphisms in 23 exons of PTCH in 48 healthy caucasians. Ten new polymorphisms were identified (IVS11 + 15-17del AAA; IVS14 + 25T>C; 2485G>A; IVS15 + 9G>C; IVS17 + 21A>G; 3033T>C; 3149T>C; 3387T>C; 3617G>A; 4080C>T). Next, the PTCH coding region in 14 RMS was sequenced. Whereas one case with LOH at the PTCH locus was detected, none of the cases showed nonsense or missense mutations in the coding region of PTCH. These data do not support the existence of frequent mutations in the protein-coding region of PTCH in RMS.

Individuals affected with the Gorlin syndrome inherit a germ-line mutation of the patched (Ptc1) developmental gene and, analogously to Ptc1 heterozygous mice, show an increased susceptibility to spontaneous tumor development. Human and mouse Ptc1 heterozygotes (Ptc1(+/-)) are also hypersensitive to ionizing radiation (IR)-induced tumorigenesis in terms of basal cell carcinoma (BCC) induction. We have analysed the involvement of Ptc1 in the tumorigenic response to a single dose of 3 Gy X-rays in neonatal and adult Ptc1 heterozygous and wild type mice. We report that irradiation dramatically increased the incidence of medulloblastoma development (51%) over the spontaneous rate (7%) in neonatal but not adult Ptc1 heterozygotes, indicating that medulloblastoma induction by IR is subjected to temporal restriction. Analysis of Ptc1 allele status in the tumors revealed loss of the wild type allele in 17 of 18 medulloblastomas from irradiated mice and in two of three spontaneous medulloblastomas. To our knowledge, irradiated newborn Ptc1(+/-) heterozygous mice constitute the first mouse model of IR-induced medulloblastoma tumorigenesis, providing a useful tool to elucidate the molecular basis of medulloblastoma development.

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and Treg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo. peerReviewed

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

Inherited mutations of Patched (PTCH) in the nevoid basal cell carcinoma syndrome (NBCCS) lead to several developmental defects and contribute to tumor formation in a variety of tissues. PTCH mutations have been also identified in sporadic tumors associated with NBCCS including basal cell carcinoma (BCC) and medulloblastoma. Mice heterozygous for Ptch recapitulate the typical developmental symptoms of NBCCS and develop rhabdomyosarcoma (RMS) and medulloblastoma. PTCH is assumed to act as a tumor suppressor gene although inactivation of both alleles has been demonstrated only in a fraction of tumors. We have investigated the status of Ptch in RMS of heterozygous Ptch(neo67/+) mice. Although the wild-type Ptch allele was retained in tumor tissue, the high levels of Ptch mRNA in these tumors result from overexpression of the mutant Ptch transcript. Our results suggest that the wild-type Ptch allele might be selectively silenced in RMS tissue or, alternatively, that haploinsufficiency of Ptch is sufficient to promote RMS formation in mice.