Patching the gaps in Hedgehog signalling

The Hedgehog (Hh) pathway plays central roles in animal development and stem-cell function. Defects in Hh signalling lead to birth defects and cancer in humans. The first and often genetically damaged step in this pathway is the interaction between two membrane proteins - Patched (Ptc), encoded by a tumour suppressor gene, and Smoothened (Smo), encoded by a proto-oncogene. Recent work linking Hh signalling to sterol metabolites and protein-trafficking events at the primary cilium promises to shed light on the biochemical basis of how Patched inhibits Smoothened, and to provide new avenues for cancer treatment.     

Chromosome 17p Deletion in Human Medulloblastoma: A Missing Checkpoint in the Hedgehog Pathway

Although deregulation of Hedgehog signalling is considered to play a crucial oncogenic role and commonly occurrs in medulloblastoma, genetic lesions in components of this pathway are observed in a minority of cases. The recent identification of a novel putative tumor suppressor (RENKCTD11) on chromosome 17p13.2, a region most frequently lost in human medulloblastoma, highlights the role of allelic deletion of the gene in this brain malignancy, leading to the loss of growth inhibitory activity via suppression of Gli-dependent activation of Hedgehog target genes. The presence on 17p13 of another tumor suppressor gene (p53) whose inactivation cooperates with Hedgehog pathway for medulloblastoma formation, suggests that 17p deletion unveils haploinsufficiency conditions leading to abrogation of either direct and indirect checkpoints of Hedgehog signalling in cancer.     

A PTCH1 Homolog Transcriptionally Activated by p53 Suppresses Hedgehog Signaling

The p53-mediated responses to DNA damage and the Hedgehog (Hh) signaling pathway are each recurrently dysregulated in many types of human cancer. Here we describe PTCH53, a p53 target gene that is homologous to the tumor suppressor gene PTCH1 and can function as a repressor of Hh pathway activation. PTCH53 (previously designated PTCHD4) was highly responsive to p53 in vitro and was among a small number of genes that were consistently expressed at reduced levels in diverse TP53 mutant cell lines and human tumors. Increased expression of PTCH53 inhibited canonical Hh signaling by the G protein-coupled receptor SMO. PTCH53 thus delineates a novel, inducible pathway by which p53 can repress tumorigenic Hh signals.     

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling

During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.     

Polymorphisms in p53 and the p53 pathway: roles in cancer susceptibility and response to treatment

The p53 tumour suppressor protein lies at the crossroads of multiple cellular response pathways that control the fate of the cell in response to endogenous or exogenous stresses and inactivation of the p53 tumour suppressor signalling pathway is seen in most human cancers. Such aberrant p53 activity may be caused by mutations in the TP53 gene sequence producing truncated or inactive mutant proteins, or by aberrant production of other proteins that regulate p53 activity, such as gene amplification and overexpression of MDM2 or viral proteins that inhibit or degrade p53. Recent studies have also suggested that inherited genetic polymorphisms in the p53 pathway influence tumour formation, progression and/or response to therapy. In some cases, these variants are clearly associated with clinico-pathological variables or prognosis of cancer, whereas in other cases the evidence is less conclusive. Here, we review the evidence that common polymorphisms in various aspects of p53 biology have important consequences for overall tumour susceptibility, clinico-pathology and prognosis. We also suggest reasons for some of the reported discrepancies in the effects of common polymorphisms on tumourigenesis, which relate to the complexity of effects on tumour formation in combination with other oncogenic changes and other polymorphisms. It is likely that future studies of combinations of polymorphisms in the p53 pathway will be useful for predicting tumour susceptibility in the human population and may serve as predictive biomarkers of tumour response to standard therapies.     

In vivo inhibition of endogenous brain tumors through systemic interference of Hedgehog signaling in mice

The full spectrum of developmental potential includes normal as well as abnormal and disease states. We therefore subscribe to the idea that tumors derive from the operation of paradevelopmental programs that yield consistent and recognizable morphologies. Work in frogs and mice shows that Hedgehog (Hh)-Gli signaling controls stem cell lineages and that its deregulation leads to tumor formation. Moreover, human tumor cells require sustained Hh-Gli signaling for proliferation as cyclopamine, an alkaloid of the lily Veratrum californicum that blocks the Hh pathway, inhibits the growth of different tumor cells in vitro as well as in subcutaneous xenografts. However, the evidence that systemic treatment is an effective anti-cancer therapy is missing. Here we have used Ptc1(+/-); p53(-/-) mice which develop medulloblastoma to test the ability of cyclopamine to inhibit endogenous tumor growth in vivo after tumor initiation through intraperitoneal delivery, which avoids the brain damage associated with direct injection. We find that systemic cyclopamine administration improves the health of Ptc1(+/-);p53(-/-) animals. Analyses of the cerebella of cyclopamine-treated animals show a severe reduction in tumor size and a large decrease in the number of Ptc1-expressing cells, as a readout of cells with an active Hu-Gli pathway, as well as an impairment of their proliferative capacity, always in comparison with vehicle treated mice. Our data demonstrate that systemic treatment with cyclopamine inhibits tumor growth in the brain supporting its therapeutical value for human HH-dependent tumors. They also demonstrate that even the complete loss of the well-known tumor suppressor p53 does not render the tumor independent of Hh pathway function.     

Dynamic phosphorylation of the kinesin Costal-2 in vivo reveals requirement of fused kinase activity for all levels of hedgehog signalling

Hedgehog (Hh) proteins are secreted molecules that play an essential role in development and tumorigenesis. In Drosophila cultured cells, phosphorylation of the kinesin-like Costal2 (Cos2) protein at Ser572 is triggered by the kinase fused (Fu) upon Hh pathway activation. Here, we validate the first phospho-antibody for one of the Hh pathway components, Cos2, as a universal in situ readout of Hh signal transduction. For the first time, this tool allows the visualisation of a gradient of signalling activity and therefore the range of the activating Hh ligand in different tissues. We also show that, in vivo, Fu kinase is activated by and necessary to transduce all levels of intracellular Hh signalling. Our study fills a gap in the understanding of the Hh pathway by showing that the molecular cascade leading to Cos2 phosphorylation is conserved in all cells activated by Hh. Therefore, we propose that the extracellular Hh information is conveyed to an intracellular signal through graded Fu kinase activity.     

Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome

Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a, -1b and -2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a, even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.     

Multiple roles for lipids in the Hedgehog signalling pathway

The identification of endogenous sterol derivatives that modulate the Hedgehog (Hh) signalling pathway has begun to suggest testable hypotheses for the cellular biological functions of Patched, and for the lipoprotein association of Hh. Progress in the field of intracellular sterol trafficking has emphasized how tightly the distribution of intracellular sterol is controlled, and suggests that the synthesis of sterol derivatives can be influenced by specific sterol-delivery pathways. The combination of this field with Hh studies will rapidly give us a more sophisticated understanding of both the Hh signal-transduction pathway and the cell biology of sterol metabolism.     

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. Here we show that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. Our observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated.