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Hedgehog signaling plays pivotal roles in embryonic development, adult homeostasis and tumorigenesis. However, its engagement in the pituitary gland has been long underestimated although Hedgehog signaling and pituitary embryogenic development are closely linked. Thus, deregulation of this signaling pathway during pituitary development results in malformation of the gland. Research of the last years further implicates a regulatory role of Hedgehog signaling in the function of the adult pituitary, because its activity is also interlinked with homeostasis, hormone production, and most likely also formation of neoplasms of the gland. The fact that this pathway can be efficiently targeted by validated therapeutic strategies makes it a promising candidate for treating pituitary diseases. We here summarize the current knowledge about the importance of Hedgehog signaling during pituitary development and review recent data that highlight the impact of Hedgehog signaling in the healthy and the diseased adult pituitary gland.

It is unclear how epithelial tissues adjust cell division rates to cell density. Here, the authors show that Plexin-B1 and Plexin-B2 sense mechanical compression (crowding) of epidermal stem cells, resulting in inactivation of YAP and suppression of cell proliferation.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch+/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC.

In the Ptch+/- mouse model for embryonal rhabdomyosarcoma (ERMS), we recently showed that oncogenic (onc) H-, K- or NRAS mutations do not influence tumor growth when induced at the advanced, full-blown tumor stage. However, when induced at the invisible ERMS precursor stage at 4 weeks of age, tumor development was enforced upon oncHRAS and oncKRAS but not by oncNRAS, which instead initiated tumor differentiation. These data indicate that oncRAS-associated processes differ from each other in dependency on the isoform and their occurrence during tumor development. Here, we investigated the outcome of oncNRAS induction at an earlier ERMS precursor stage at 2 weeks of age. In this setting, oncNRAS accelerates tumor growth because it significantly shortens the ERMS-free survival and increases the ERMS incidence. However, it does not seem to alter the differentiation of the tumors. It is also not involved in tumor initiation. Together, these data show that oncNRAS mutations can accelerate tumor growth when targeting immature ERMS precursors within a specific time window, in which the precursors are permissive to the mutation and show that oncNRAS-associated processes differ from each other in dependency on their occurrence during tumor development.

The development of skeletal muscle from immature precursors is partially driven by canonical WNT/β-catenin signaling. Rhabdomyosarcomas (RMS) are immature skeletal muscle-like, highly lethal cancers with a variably pronounced blockade of muscle differentiation. To investigate whether canonical β-catenin signaling in RMS is involved in differentiation and aggressiveness of RMS, we analyzed the effects of WNT3A and of a siRNA-mediated or pharmacologically induced β-catenin knock-down on proliferation, apoptosis and differentiation of embryonal and alveolar RMS cell lines. While the canonical WNT pathway was maintained in all cell lines as shown by WNT3A induced AXIN expression, more distal steps including transcriptional activation of its key target genes were consistently impaired. In addition, activation or inhibition of canonical WNT/β-catenin only moderately affected proliferation, apoptosis or myodifferentiation of the RMS tumor cells and a conditional knockout of β-catenin in RMS of Ptchdel/+ mice did not alter RMS incidence or multiplicity. Together our data indicates a subordinary role of the canonical WNT/β-catenin signaling for RMS proliferation, apoptosis or differentiation and thus aggressiveness of this malignant childhood tumor.

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.

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.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

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.

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.

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.

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.

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.

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 to Ptch mutations 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 conditional Ptch knockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditional Ptchflox/flox mice and the K5-Cre-ERT+/− driver.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Aberrant Hedgehog (Hh) signaling is characteristic of the embryonal subtype (ERMS) and of fusion-negative alveolar RMS. In the mouse, ERMS-like tumors can be induced by mutations in the Hh receptor Patched1 (Ptch). As in humans these tumors show increased Hh pathway activity. Here we demonstrate that the treatment with the active form of vitamin D3, calcitriol, inhibits Hh signaling and proliferation of murine ERMS in vivo and in vitro. Concomitantly, calcitriol activates vitamin D receptor (Vdr) signaling and induces tumor differentiation. In addition, calcitriol inhibits ERMS growth in Ptch-mutant mice, which is, however, a rather late response. Taken together, our results suggest that exogenous supply of calcitriol could be beneficial in the treatment of RMS, especially in those which are associated with aberrant Hh signaling activity.

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 Ptch1neo67/+ 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. Copyright © 2003 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]