Hedgehog checkpoints in medulloblastoma: the chromosome 17p deletion paradigm

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Vol.11 No.12 December 2005

Hedgehog checkpoints in medulloblastoma: the chromosome 17p deletion paradigm Elisabetta Ferretti1, Enrico De Smaele1, Lucia Di Marcotullio1, Isabella Screpanti1,2 and Alberto Gulino1,3 1

Department of Experimental Medicine and Pathology, La Sapienza University, Viale Regina Elena 324, 00161 Rome, Italy Pasteur Institute, Cenci Bolognetti Foundation, Viale Regina Elena 324, 00161 Rome, Italy 3 Neuromed Institute, Pozzilli, 86077 Isernia, Italy 2

Medulloblastomas often activate Hedgehog signaling inappropriately. The finding that mutations in components of this pathway are present only in few tumors suggests that additional genetic or epigenetic lesions can also lead to Hedgehog dysregulation. Chromosome 17p deletion, the most frequently detected genetic lesion in medulloblastoma, has recently been identified as a cause of unrestrained Hedgehog signaling. Such a deletion leads to the loss of RENKCTD11, a novel Hedgehog antagonist, thus removing a checkpoint of Hedgehog-dependent events during cerebellum development and tumorigenesis. The disruption of additional Hedgehog modulators that map to 17p suggests a rationale for a multitargeted therapeutic strategy aimed at interrupting the cooperative activation of the Hedgehog pathway.

Introduction Medulloblastoma is a highly invasive malignancy arising from cerebellar progenitor cells and accounts for 15–20% of childhood brain tumors [1]. The management of this type of tumor requires aggressive treatments, such as surgical resection, chemotherapy, or radiotherapy. Although 60% of affected children are cured in this way, there are severe long-term side-effects; risk-adapted therapies are thus needed. Medulloblastomas are genetically heterogeneous tumors [2] in which genetic alterations could define distinct clinical subsets. Their molecular classification is therefore essential because it enables improved disease risk assignment and the possibility of using targeted (and less toxic) therapies. Several genetic lesions affect the components of the signaling pathway activated by the Hedgehog family of secreted glycoproteins (Figure 1), which has a crucial role in the development of neural cells. Inappropriate activation of Hedgehog signaling occurs more frequently than previously suspected, being observed in w60% of medulloblastoma [3]. Mutations in individual components of the Hedgehog pathway are responsible for only a few (25%) Corresponding author: Gulino, A. ([email protected]).

medulloblastomas with activated Hedgehog signaling [2,4], thus raising the question of the identity of the additional signals involved in Hedgehog dysregulation. The most common medulloblastoma-associated genetic lesion (up to 50% of cases) is loss of the chromosome 17p region, frequently associated with a gain of chromosome 17q, leading to an isochromosome 17q [i(17)(q10)]. Both loss of 17p and gain of 17q are prognostically unfavorable factors [2,5,6]. Although allelic deletion in chromosome 17p is likely to affect one or more tumor suppressor genes, none of the known components of the Hedgehog pathway maps to 17p. A link between 17p deletion and Hedgehog signaling was recently suggested by the discovery of a deletion of a novel putative tumor suppressor (REN), which maps to a 17p region and functions as a repressor of the Hedgehog pathway [7], thus suggesting that REN inactivation leads to unrestrained Hedgehog signaling. Here, we review recent advances in understanding of the molecular features of medulloblastoma related to chromosome 17p and their relationships with Hedgehog signaling, in order to identify novel candidate therapeutic targets that might be useful in strategies that aim to interrupt the cooperative activation of the Hedgehog pathway.

Checkpoints of Hedgehog signaling: linking aberrant development and tumorigenesis Hedgehog signaling and cerebellum development The cerebellum is composed of different cell layers, including granule cells and Purkinje cells [8,9]. Several observations suggest that alterations of molecular events regulating the development of cerebellar granule cell progenitors (GCPs) are responsible for uncontrolled cell growth, eventually leading to medulloblastoma formation [9]. During cerebellar development, GCPs migrate from the rhombic lip, over the outer layer of the cerebellar surface [the external granule layer (EGL)] [8,9], where they initially undergo cell expansion. Subsequently, GCPs exit the cell cycle and move into the inner EGL and further inwards into the internal granule layer, where they undergo terminal differentiation [8,9]. There is now considerable evidence that factors secreted by Purkinje

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Figure 1. Positive and negative regulation of Hedgehog signaling in vertebrates. Positive signals (red) include Shh–Ptch interactions and subsequent G-protein coupled receptor kinase 2 (GRK2)-dependent phosphorylation of Smo and interaction with b-arrestin 2, which results in Smo endocytic internalization and triggering of downstream signals [15]. Additional trafficking proteins with a positive role in Hedgehog signaling include the kinesin-like KIF3a and the intraflagellar transport proteins IFT88 and IFT172 [16]. The cytoskeleton actin-binding protein MIM/BEG4 associates with Gli to enhance its activity [17] and the kinase Dyrk1 and Iguana promote its nuclear transfer [23,24]. In vertebrates, Gli includes three different proteins: two of them undergo proteolytic processing to balance positive or negative properties (Gli2 behaves mainly as a transcriptional activator and Gli3 as a repressor), whereas Gli1 is only an activator [13]. Several negative signals (blue) switch off Hedgehog function, acting along the signaling cascade. At the ligand–receptor interaction and postreceptor levels, a negative feedback loop is triggered by Hedgehog interacting protein (HIP) and BMP, both transcriptional targets of Hedgehog: HIP binds Shh, and the BMP target Smad factor forms a complex with Gli [8,13]. Subsequently, negative regulation ranges from endocytic trafficking to transcriptional machinery.

cells regulate the proliferation of GCPs in the EGL. In particular, Purkinje cells secrete Sonic hedgehog (Shh), which is involved in the initial EGL cell population expansion and is the most potent mitogen for GCPs [9–12]. Triggering Hedgehog signals Shh, together with Indian hedgehog (Ihh) and Desert hedgehog (Dhh), belongs to a phylogenetically conserved glycoprotein family, originally described in Drosophila as a regulator of embryonic patterning and cell fate determination. Hedgehogs have a crucial role in the development of several tissues in mammals, behaving as a morphogenic, mitogenic or differentiation factor [8,9]. Although diverging at several points, Hedgehog signaling is fairly conserved between Drosophila and vertebrates [13]. Hedgehogs trigger cellular signals by interacting with the 12-pass transmembrane receptor Patched (Ptch). This interaction prevents inhibition of 7-pass transmembrane Smoothened (Smo) co-receptor by Ptch and enables it to activate downstream transcription factors belonging to the Gli family (Gli1, -2 and -3, the vertebrate homologs of Drosophila Ci); these factors translocate into the nucleus and activate the expression of target genes (including Gli1, as an automaintenance positive loop) [13,14] (Figure 1). Additional transcriptional Gli target www.sciencedirect.com

genes have been involved in the Hedgehog pathway [Hedgehog interacting protein (Hip), Ptch1 and bone morphogenetic proteins (BMPs)] and in the control of several functions such as cell proliferation and differentiation [through the target genes encoding insulin-like growth factor 2 (IGF2), platelet derived growth factor receptor a(PDGFRa), N-myc, Myc, Bmi1, cyclins D1 and D2, Wnt and Hes1] and survival (through the Bcl2 gene) in untransformed and medulloblastoma cells (Figure 1). Whereas Hedgehog-induced activation of Ci has been well characterized in Drosophila, the regulation of Gli factors in vertebrates remains to be elucidated fully, and new regulatory mechanisms are emerging. For example, Smodependent signaling is activated by several proteins involved in intracellular trafficking [including endocytosis; G-protein coupled receptor kinase 2, b-arrestin 2, kinesin family member 3a (KIF3a) and the intraflagellar transport proteins IFT88 and IFT172), cytoskeletal function [Basal cell carcinoma-enriched gene 4 (BEG4), also called Missing in metastasis (MIM)] and nuclear transfer of Gli [Dual-specificity tyrosine-phosphorylation regulated kinase 1A (Dyrk1) and the zinc-finger protein Iguana] [8,13,15–19] (Figure 1). The discovery of these mechanisms is important because defects in components of the Hedgehog signal transduction machinery are

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emerging as being crucial in tumorigenisis and these components can therefore be considered as potential therapeutic targets.

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Table 1. Molecular genetics and epigenetics of human medulloblastoma Gene product Chromosome 17p loss REN

How are Hedgehog signals switched off? Shh activity is interrupted in the cerebellar inner EGL. In keeping with this observation, the expression of Gli1 is restricted to proliferating outer EGL GCPs, whereas it is silenced in the inner EGL [20]. Failure to withdraw Shh signals leads to uncontrolled proliferation, and constitutive activation of the Hedgehog pathway is responsible for medulloblastoma development. Several checkpoints are responsible for the negative regulation of Hedgehog signaling (Figure 1). In the absence of Shh, the activity of Gli factors is limited, at the Smo level, by the kinesin Kif7 (the vertebrate homolog of Drosophila Cos2) [21], which retains the Gli factors in the cytoplasm as inactive multicomponent complexes. The endocytic trafficking of Hedgehog signals is negatively regulated by Rab23 [13]. Post-translational regulation of Gli proteins occurs by their protein kinase A-mediated processing to transcriptional repressors (i.e. Gli3) [13]. Hedgehog signaling is also switched off by the FK506binding protein FBP8, belonging to the immunophilin family of proteins that bind immunosuppressant drugs [22]. A powerful antagonist of Gli signaling is also suppressor of fused (SUFU), which binds Gli proteins, thereby inhibiting nuclear transfer and interfering with its transcriptional activity [23–26]. The Gli1 nuclear transfer step is also targeted by REN, encoded by a gene frequently deleted in medulloblastoma [7]. Deregulation of Hedgehog pathway in medulloblastoma Disruption of several signaling pathways occurs in medulloblastoma, as a consequence of various genetic and epigenetic changes [2,9,27] (Table 1); here, we focus on the Hedgehog pathway. Failure to switch off growthpromoting Hedgehog signals during GCP development is a primary event in medulloblastoma development. Indeed, germline and somatic gain-of-function (Smo) or loss-offunction (Ptch1 and -2 and SUFU) mutations in components of the Hedgehog signaling, all leading to activation of ligand-independent signals, are observed in human medulloblastomas [2,4] (Table 1). The idea that uncontrolled activation of the Hedgehog pathway sustains the development of medulloblastoma is confirmed by mouse models in which heterozygous deletion of Ptch1 or activatory mutations of Smo genes result in tumor development [3,28]. The status of the wild-type Ptch1 allele in these tumors is controversial because its expression has been reported in tumor tissue [29,30], whereas others have suggested that the wild-type allele is epigenetically silenced [31]. However, loss of the wild-type Ptch1 allele and disruption of GCP development have been reported recently in preneoplastic cerebellar lesions of Ptch1C/K mice, suggesting that loss of Ptch1 is an early event in tumorigenesis [32]. Conversely, specific antagonists of Smo inhibit the growth of medulloblastoma primary cultures and of tumors in vivo in Ptch1-deficient mice [29,31,33,34]. Gli1 appears to be an important effector of Hedgehog-induced tumorigenesis because inactivation of

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Genetically determined medulloblastoma mouse models: Mutant mice: p53K/K and Ptc1K/K, Kip1K/K, Ink4cK/K, Ink4dK/KKip1C/K, Ink4dC /K Ink4cK/K, RbK/K, PARP1K/K, DNA Lig4K/K. b Adenocarcinomas in mammary gland-targeted conditional MntK/K mice. c Hic1K/K mice develop various tumors but no medulloblastomas. d Ptc1K/K (14% penetrance) and Ptc1K/K p53K/K (100% penetrance) mice. e Mutated SmoA1 transgenic (48% penetrance). f Undetectable in 40% of cases [63]. g Overexpressed and amplified. h Overexpressed in 90% of anaplastic medullobastomas. i Only in metastatic, no overexpression in nonmetastatic. For references, see text. a

both Gli1 alleles in Ptch1C/K mice significantly reduces medulloblastoma formation [35]. Likewise, switching off inappropriately activated Gli1 by small interfering RNA or a dominant-negative Gli1 mutant suppresses bromodeoxyuridine (BrdU) incorporation in a medulloblastoma cell line in vitro [7]. The observation that, although !25% of medulloblastomas carry mutations of components of the Hedgehog pathway, inappropriate activation of Hedgehog signaling occurs in w60% of tumors [3] suggests that additional alterations in other pathways cooperate synergistically with Hedgehog signaling for tumorigenesis. The chromosome 17p deletion is a good example of

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how distinct dysregulated pathways might be reconciled into a common network.

antagonized by p53 via upregulation of the cyclindependent kinase inhibitor p21 and the proapoptotic protein Bax (Figure 2a). Furthermore, in mouse medulloblastoma models carrying disruptions of p53 together with other checkpoints of the cell cycle (Kip1K/Kp53K/K, Ink4cK/Kp53K/K, Ink4dK/KKip1C/Kp53K/K, Ink4dC/K Ink4cK/Kp53K/K, PARP-1K/Kp53K/K or Lig4K/Kp53K/K mice), tumors display increased levels of Gli1 [40,41], suggesting that the different models converge, in some way, upon upregulation of Hedgehog signaling.

The chromosome 17p deletion paradigm: it is not just one hit Deletion of chromosome 17p, occurring in up to 50% of medulloblastomas and frequently restricted to the common 17p13.2–13.3 region [2,5], leads to the loss of several putative tumor suppressor genes (Figure 2a). Below, we describe the tumor-associated changes and the relationships with the Hedgehog pathway of these genes.

Hic1 A p53 target also mapping to 17p13.3 is Hypermethylated in cancer (Hic1), which is deleted and specifically silenced by hypermethylation in medulloblastoma [42,43]. Loss of Hic1 accentuates p53-dependent tumorigenesis [44]. However, its role is still controversial because tumors developed in Hic1-deficient mice do not include medulloblastomas [44].

p53 The tumor suppressor gene p53, encoding a transcription factor that promotes growth arrest and apoptosis, maps to 17p13.2–13.3 and displays allelic deletion in most medulloblastomas carrying 17p loss. Although !10% of tumors have p53 mutations [2], p53 hemizygosity is sufficient per se to increase susceptibility to malignancy [36], and, most interestingly, p53 loss cooperates with Hedgehog signaling by increasing significantly the frequency of medulloblastomas occurring in Ptc1C/Kp53K/K mice [37]. Indeed, p53 is expressed in EGL GCPs [38], implying a role for p53 in counteracting the mitogenic and antiapoptotic activity sustained by Shh. Shh targets cyclins D1 and D2 promote cell cycle progression and the target Bcl2 promotes cell survival [27,39] and, conversely, they all are strongly

Mnt The Myc inhibitor Mnt is an additional tumor suppressor gene mapping to 17p13.3 that is frequently deleted and downregulated in medulloblastoma [45] (Figure 2a). Loss of this gene leads to disrupted cell cycle control and tumorigenesis [46]. The Mnt protein heterodimerizes with Max and suppresses Myc-dependent cell transformation (b)

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Figure 2. (a) Hedgehog antagonists encoding genes mapping to chromosome 17p13.2.13.3. 17p-associated Hedgehog antagonist gene products suppress Gli nuclear transfer (REN) or the function of Hedgehog target genes (functions dependent on cyclins D1 and D2 and Bcl2 by p53; Myc function by Mnt). Star symbols indicate genetic lesions observed in human medulloblastoma and affecting the function of encoded proteins, as described in the text. Abbreviations: CDK, cyclin-dependent kinase; Hic1, hypermethylated in cancer 1; p21, p21WAF1; p27, p27Kip1; Rb, retinoblastoma. (b) Role for REN in cerebellar GCP development and tumorigenesis. REN expression (blue) increases (in association with p27, green) along the transition from outer to inner EGL GCPs, thus lowering the Hedgehog–Gli1 (red) signaling, activated in the outer EGL. In this way, REN promotes the growth arrest of inner EGL GCPs and enables granule cells to differentiate and migrate into the internal granule layer (IGL). The number of developing granule cells is also limited by REN-induced apoptosis. Chromosome 17p deletion-dependent loss of REN function removes the constraints from Hedgehoginduced immature cell expansion, thus favoring tumorigenesis. Modified, with permission, from Ref. [20]). Copyright 2005 by the Society for Neuroscience. www.sciencedirect.com

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[47]. Myc and N-myc are Gli targets [27,48] and are overexpressed in medulloblastoma (Table 1), N-myc being crucially required for the expansion of proliferating EGL progenitor cells [2,48,49]. Accordingly, Myc has been shown to cooperate with Shh to enhance in vivo medulloblastoma formation from neural progenitors in mice [50]. Therefore, loss of Mnt could be an additional link between 17p deletion and Hedgehog signaling. Interestingly, it has recently been observed that the transcription factor developmentally regulated high mobility group AT-hook 1 (HMGA1), which is overexpressed in medulloblastoma [51], is a direct target of N-myc in neuroblastic tumors [52]. Furthermore, enhanced N-myc signaling might impair p53 function because upregulation of HDM2, the human homolog of murine double minute chromosome gene 2 and an inhibitor of p53, has been reported to be induced by N-myc [53], thus suggesting an autoregulatory circuitry between genes located in the 17p region. REN REN (also called KCTD11) was identified as a novel putative tumor suppressor gene, mapping to 17p13.2, whose expression is enhanced by retinoic acid, epidermal growth factor and nerve growth factor [54]. REN has a role in neurogenesis, being expressed initially in the neural fold epithelium during gastrulation and, subsequently, throughout the ventral neural tube and in postmitotic neuroblasts of the outer layer of the ventricular encephalic neuroepithelium [47]. REN promotes neural cell growth arrest and p27kip1 expression, and its abrogation in embryonic stem cells prevents the retinoic acid-induced upregulation of the proneural basic helix–loop–helix transcription factors neurogenin1 and NeuroD. NeuroD and the cell cycle inhibitor p27Kip1 promote differentiation of inner EGL GCPs [54,55]. Therefore, REN might be recruited by differentiating signals (e.g. retinoic acid or nerve growth factor) during the proliferative phases of neurogenesis, and might contribute to terminating epidermal growth factor-derived growth-enhancing signals, thereby enabling progenitor cells to undergo growth arrest and postmitotic differentiation. Accordingly, REN could have a role in neonatal cerebellum development, as suggested by its pattern of expression, which is restricted to low proliferating inner EGL GCPs and internal granule layer neurons [20]. Likewise, REN expression increases during in vitro differentiation of cultured GCPs and REN overexpression consistently promotes GCP growth arrest, differentiation and apoptosis. Interestingly, REN antagonizes Hedgehog signaling by affecting Gli1 nuclear transfer. Conversely, REN functional knockdown enhances Hedgehog signaling and proliferation of GCPs and impairs neuronal differentiation, thus supporting a role for REN as an inhibitory signal required for withdrawing GCP expansion at the outer to inner EGL transition [20]. The loss of such an inhibitory signal might therefore lead to the Hedgehog-dependent uncontrolled GCP overgrowth linked to medulloblastoma development (Figure 2b). REN loss of function (owing to hemizygosity and methylation-dependent silencing) is observed in all 17p-deleted medulloblastomas, and downregulated expression is also www.sciencedirect.com

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observed in biallelic tumors [7]. Restoring high REN expression suppresses medulloblastoma growth in vitro and in vivo by negatively regulating Hedgehog signaling [7] because inactivation of Gli1 by small interfering RNA or a dominant-negative Gli1 mutant abrogates inhibition of cell proliferation induced by REN. Therefore, REN is suggested to be a missing checkpoint in the Hedgehog pathway in medulloblastoma development (Figure 2). In summary, 17p carries several genes whose dysregulation interferes with the signaling cascade of the Hedgehog pathway, ranging from Gli1 itself (REN) to target genes of Gli proteins (cyclin-dependent cell cycle machinery genes, Bcl2 and Myc). Therefore, 17p deletion might lead to the loss of Hedgehog checkpoints by integrating multiple haploinsufficient conditions until they reach the threshold necessary to unmask cooperation with the Hedgehog master oncogenic pathway. Cooperating with Hedgehog for cancer stem cell maintenance The ability of the above-described genes located in the 17p region to cooperate with the Hedgehog pathway in disrupting developmental processes raises the question of their influence on cancer stem cells. Indeed, human medulloblastomas contain cancer stem cells, which have the ability to differentiate but also to retain the property of self-renewal, thus representing a never-ending reservoir for the maintenance of the tumor mass [56]. Hedgehog might have a crucial role, as it sustains stemness in both adult and embryonic neural progenitors [57,58]. Inappropriate activation of Hedgehog signaling might confer to tumor cells features potentially related to either stem cell identity and/or self-renewal. We can speculate that deletion of Hedgehog-antagonist genes from chromosome 17p (i.e. REN) might favor stemness of cancer cells by leading to the loss of checkpoints operating directly upon the Hedgehog pathway. REN has frequently been described as being expressed during the early stages of embryogenesis and to be linked to differentiation of neural progenitors [20,47] . Moreover, loss of REN function has been reported to maintain undifferentiated developing cerebellar GCP [20] and is therefore suggested to influence undifferentiated medulloblastoma cells in a similar way. By contrast, an indirect regulation (i.e. disruption of cell cycle machinery by defective p53) might affect Hedgehog function as a mitogenic and stem cell-maintaining factor. Interestingly, p53 has a novel role in promoting differentiation of embryonic stem cells by suppressing the expression of Nanog, a gene required for stem cell self-renewal [59]. p53 deletion, by maintaining stem cell properties, might also keep Hedgehog activity high by hampering the developmentally regulated activation of still-unknown inhibitors (that is, by fostering secondary genetic hits). The 17p deletion might additionally lead to the maintenance of malignant stem cells by operating at the effector level downstream of Hedgehog. By having a crucial role in the maintenance of progenitor cell features, N-myc and Myc are such Hedgehog effectors [49,60]. An additional Hedgehog effector, although unrelated to

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17p, is Bmi1, a repressor of the p16Ink4a and p19Arf cell cycle inhibitors and a promoter of stem cell maintenance, which is required for cerebellar development and is also overexpressed in most human medulloblastomas [61]. Additional pathways, such as Notch and Wnt signaling, which are known to favor the maintenance of stem cells and progenitor cells [62], have also been reported to crosstalk with Hedgehog in a dysregulated way in medulloblastoma. Inappropriate activation of Notch signaling is observed in human medulloblastoma [3,63]. Interestingly, Shh and Notch cooperate for maintaining undifferentiated cerebellar GCPs via upregulation of the shared target gene Hes1 [64]. Medulloblastoma is also associated with dysregulated Wnt signaling [2]. Wnt and Hedgehog share common regulators (SUFU and glycogen synthase kinase 3b) which inhibit Gli proteins and the Wnt target b-catenin [65,66]. Reciprocal regulation of components of both pathways is described (enhancement of Wnt expression by Shh and b-catenin-induced upregulation of Shh) [67]. However, crosstalk between Hedgehog and Wnt in cerebellar and medulloblastoma cells remains to be elucidated. In summary, loss of genes located on chromosome 17p might also sustain the maintenance of cancer stem cells (besides the known regulators Notch and Wnt), possibly by cooperating with Hedgehog signaling.

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Hedgehog checkpoints and beyond: hunting for therapeutic targets We have proposed here that the dysregulation of developmental networks has a central role in medulloblastoma formation, with the Hedgehog pathway mostly involved as an orchestrating player. The chromosome 17p deletion feature might represent a model by which several genetic and epigenetic lesions either affect the crosstalk of Hedgehog machinery with other signals (REN) or lie downstream of the Hedgehog signaling cascade at the level of Gli targets involved in cell cycle progression, differentiation and survival (p53, Mnt, N-myc and Myc). These events occur in addition to other alterations, unrelated to 17p deletion, affecting gene products which are components of the Hedgehog signaling cascade (Bmi1, N-myc, Myc, HMGA1, IGF2, PDGFRa, Notch with the effector Hes1, and Wnt). This scenario has profound implications for novel therapeutic strategies for medulloblastoma (Figure 3). In this regard, several questions now need to be addressed (Box 1). Drug-mediated blocking of Hedgehog signaling has been shown to be successful for medulloblastoma therapy: mice carrying Hedgehog-dependent tumors were cured when treated with Hedgehog antagonists (cyclopamine and small-molecule Smo antagonists) [29,34]. Novel therapeutic targets are suggested by the alterations in genes located on chromosome 17p and their roles

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Figure 3. Schematic representation of the Hedgehog pathway and interacting networks controlling medulloblastoma cells, as potential therapeutic targets. Gene products altered in medulloblastoma are indicated by green symbols. The red arrows indicate the activation pathways potentially involved in tumorigenesis. Smo-dependent Gli activation of the target genes encoding Bmi1, cyclins D1 and D2, Myc and HMGA1, Bcl2, PDGFRa, IGF2, Wnt and Hes1 and intersection with surrounding pathways are indicated. Glycogen synthase kinase 3b(GSK3b) inhibits both Gli and Wnt-dependent b-catenin, which is also repressed by the APC–Axin complex. SUFU suppresses both Gli and b-catenin. g-secretase-cleaved Notch activates Hes1. Potential therapeutic agents are also indicated in blue. Abbreviations: APC, adenomatous polyposis coli; b-cat, bcatenin; CDK, cyclin-dependent kinase; Frz, Frizzled; g-sec, g-secretase; TCF, T cell factor. www.sciencedirect.com

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Box 1. Outstanding questions † How can the dysregulated molecular checkpoints of Hedgehog signaling be targeted to interrupt its tumor-promoting activity? † How can medulloblastoma-associated mutations and their relationships with Hedgehog signaling be exploited to design a multitargeted therapeutic strategy directed at cancer stem cells? † Are there additional checkpoints in the Hedgehog pathway in 17p or in other common chromosomal lesions? † How do the different chromosome 17p-mapping checkpoints operate either directly or indirectly upon Hedgehog signaling and how can they be targeted by therapeutic agents? † Do these dysregulated checkpoints operate in additional types of neoplasias in which a wider role for inappropriate Hedgehog signaling is emerging?

in cooperating with Hedgehog. Rescue of the function of the tumor suppressor p53 is induced by small molecules protecting it from the inhibitory effects of HDM2 [68,69]. Rescue of loss of Mnt function or activation of myc activity might be targeted by small inhibitors of Max–myc heterodimerization or myc mRNA antisense oligonucleotides [70]. Furthermore, the ability of retinoids to inhibit medulloblastoma cell growth [71], possibly by modulating the expression of several 17p-related (REN) or -unrelated (Otx2, N-myc, HMGA1) genes dysregulated in medulloblastoma [47,51,70,71,], suggests that these drugs are worth considering as potential therapy for this tumor. Finally, additional signals that crosstalk with Hedgehog are proposed as potential therapeutic targets (i.e. suppression of both Notch–Hes1 function and medulloblastoma growth by g-secretase inhibitors or Wnt–TCF–bcatenin antagonists) [3,63,72]. Tyrosine kinase receptors such as PDGFRa might also be targeted by specific inhibitors (such as STI-571), although the limitations of such inhibitors might include the development of acquired resistance [73]. The redundancy of medulloblastoma-associated mutations and their relationships with Hedgehog signaling might have implications for designing a multitargeted therapeutic strategy directed at the stemness feature of cancer cells. Indeed, medulloblastoma generation is suggested to aberrantly recapitulate normal ontogeny, which follows an orchestrated transition from stem cell identity determination, self-renewal and expansion, cellfate specification and differentiation, in which each signal has a sequential role. Although it is known that Hedgehog signaling and related pathways occur during the early ontogenetic phases (stem cells), it will be crucial to identify the mechanism for controlling cancer stem cell identity determination and self-renewal. Indeed, if stem cell identity or stem cell niches cannot be targeted by pathway antagonists, which might only affect self-renewal, endogenous cancer stem cells might remain in a quiescent condition during pathway blockade and their expansion might occur once therapy is withdrawn. The targeting of the above-described molecular events, specifically characterizing such orchestrated sequential steps, might thus interrupt the tumorigenic process at the earliest phase. This awaits the elucidation of the reciprocal relationships that exist between the Hedgehog master oncogenic signaling and the surrounding pathways. www.sciencedirect.com

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How this might apply to additional types of neoplasia in which a wider role for deregulated Hedgehog signaling is emerging (basal cell carcinoma, prostate, lung and upper gastrointestinal cancer, including pancreas) [27,74–78], is still an open question. Therefore, taking the medulloblastoma model as a paradigm, the recently discovered molecular events that cooperate with Hedgehog signaling might also be potential candidates for validation and targeting by therapeutic agents in a wide range of tumors. Acknowledgements We thank Edoardo Alesse, Rita Gallo, Francesca Zazzeroni, Beatrice Argenti, Marella Maroder and other members of Alberto Gulino’s laboratory, whose work and discussions have contributed to the realization of this review. We thank the Associazione Italiana Ricerca sul Cancro, Telethon #GGP04168, the National Research Council, the Ministry of University and Research and the Ministry of Health, the Center of Excellence for Biology and Molecular Medicine (BEMM) and the Rome Oncogenomic Center for financial support. E. De Smaele was supported by the FIRC fellowship. We apologize to investigators whose research was not cited in this review, as a result of space constraints.

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