Canonical Notch Signaling Is Not Required for the Growth of Hedgehog Pathway-Induced Medulloblastoma

Oncogene (2010) 29, 3465–3476 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Canonical Notch signaling is not required for the growth of Hedgehog pathway-induced medulloblastoma E Julian1, RK Dave1, JP Robson1, AR Hallahan2 and BJ Wainwright1 1Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia and 2Department of Paediatrics and Child Health, University of Queensland, Brisbane, Australia Current treatment for medulloblastoma is successful in deficiencies, renal failure, hearing loss and psychiatric more than half of all cases but results in substantial and social difficulties. The elucidation of signaling disability in survivors. Accordingly, there is considerable pathways involved in medulloblastoma pathogenesis interest in drugs that may target specific signaling could substantially improve the clinical management of pathways activated in the tumors, with inhibitors of both these neoplasms, a more accurate prediction of the the Hedgehog and Notch pathways currently proposed as disease risk could be achieved and new targeted possible therapeutics. Here, we tested the hypothesis that treatments developed. Notch pathway inhibition in vivo may block the formation A number of studies have identified candidate- of Hedgehog-dependent medulloblastoma. We took the signaling pathways, which may have a role in the general approach of using a cre recombinase under the genesis of medulloblastoma (Marino, 2005). The poten- control of the GFAP promoter to generate medulloblas- tial complexity of the interaction of signaling pathways toma in mice carrying a conditional Ptc1 allele and that give rise to medulloblastoma is reflected in the introduced a conditional RBP-J allele to ablate canonical diversity of knockout and transgenic mouse models that Notch signaling. Loss of RBP-J from the developing have been found to develop medulloblastoma. These cerebellum led to a modest loss of stem cells and an overall include cell cycle regulators such as Ink4c (Zindy et al., developmental delay. These phenotypes could be partially 2007), p53/Rb (Marino et al., 2000; Wetmore et al., compensated by activation of the Hedgehog pathway. 2001), Bmi1 (Leung et al., 2004), RenKCTD11 (Di Hedgehog-dependent medulloblastoma were not blocked Marcotullio et al., 2004), Brca2 (Frappart et al., 2007) by loss of RBP-J, indicating that canonical Notch signaling and Parp (Tanori et al., 2008), chemokine receptor is not required for tumor initiation and growth in this model. Cxcr6 (Sasai et al., 2007), growth factors Hgf (Binning Oncogene (2010) 29, 3465–3476; doi:10.1038/onc.2010.101; et al., 2008), Igf1 (Rao et al., 2004) and Ifna (Wang published online 26 April 2010 et al., 2003), a regulator of PKA activity, Pacap (Lelievre et al., 2008), and the transcription factor Keywords: medulloblastoma; Notch; Hedgehog; therapy; Hic1 (Briggs et al., 2008). In the mouse at least most of RBP-J these manipulations appear to either directly modulate the Hedgehog-signaling pathway or cooperate with it to generate the medulloblastoma. Insight into the molecular basis of medulloblastoma Introduction has come from studies of the Hedgehog-signaling pathway. Sonic hedgehog (Shh) is a secreted protein Medulloblastoma are tumors of the cerebellum that that has a critical role in patterning of the nervous most commonly occur within the first few years of life. system, the limbs, the skin and other tissues (Wicking Children with the disease face a 3-year event-free et al., 1999). Patched1 (Ptc1) is a transmembrane protein survival rate of less than 35% if the tumor is metastatic that can bind Shh and appears to serve as a receptor. or recurrent or if the child is o3 years old (Packer, The first evidence that Hedgehog signaling might be 1994). The approach of clinical trial cooperative groups involved in medulloblastoma came from studies of the toward these patients has been the intensification of human PATCHED (PTCH1) gene in an inherited post-surgical chemotherapeutic and brain radiation cancer disposition syndrome, Naevoid Basal Cell regimens, but recent advances have been limited. Carcinoma Syndrome (NBCCS), which is characterized Even when successful, current therapies cause major toxi- by widespread skin tumors, craniofacial and skeletal cities, including severe cognitive impairment, endocrine abnormalities, and an increased incidence of medulloblastoma. PTCH1 was found to be mutated in both the Correspondence: Professor BJ Wainwright, Institute for Molecular inherited and sporadic form of basal cell carcinoma Bioscience, The University of Queensland, Building 80, St Lucia (Hahn et al., 1996) and subsequently it was discovered Campus, Brisbane, Queensland 4072, Australia. that in some studies up to 20% of sporadic medullo- E-mail: [email protected] Received 27 September 2009; revised 15 February 2010; accepted blastoma have causative mutations at the PTCH1 locus 4 March 2010; published online 26 April 2010 (Pietsch et al., 1997; Vorechovsky et al., 1997; Dong RBP-J is not required for Hedgehog-induced medulloblastoma E Julian et al 3466 et al., 2000). Mutation of the Hedgehog pathway media- accepted and relevant model for human medullo- tor suppressor of fused has also been observed in blastoma (Pazzaglia et al., 2006; Yang et al., 2008; medulloblastoma and provides a potential mechanistic Thomas et al., 2009), and here we addressed the link between Hedgehog signaling and the Wnt signaling intersection of Hedgehog and Notch signaling in vivo pathway in this tumor (Taylor et al., 2002). Although using a genetic approach to ablate Notch signaling in mutation of Hedgehog pathway components is present this model. The developing cerebellum expresses multi- in a minority of medulloblastoma (approximately 25%), ple components of the Notch-signaling pathway, in- the pathway is detected as perturbed by unknown cluding at least 4 ligands, 4 receptors and multiple mechanisms in most tumors (Hallahan et al., 2004; putative downstream effectors, and the rhombic lip/ Thompson et al., 2006). Small molecule inhibitors of the EGL itself expresses receptors Notch1, Notch2, Notch4 Hedgehog pathway show significant activity in Ptc1 and ligands Jag1, Dll1, at least. Although the combina- medulloblastoma animal models and on that basis are torial complexity of Notch ligand/receptor is high, all currently in clinical trials (Romer and Curran, 2005; ‘canonical’ Notch signaling requires the CSL/Rbpj Rudin et al., 2009). (RBP-J) transcription co-factor so in practice, inhibition The Notch receptor-signaling pathway has a key role of RBP-J function will negate Notch receptor signaling in many different cell fate decisions and has a particular (Komine et al., 2007). There is some emerging genetic prominence due to its function as a key determinant of evidence to suggest the existence of an RBP-J-indepen- asymmetric/symmetric cell division (Corbin et al., 2008). dent (‘non-canonical’) pathway through which Notch Notch signaling uses a membrane bound ligand that receptor signaling may exert some influence (Talora contacts an appropriate receptor leading to the release et al., 2008). However, the key effectors implicated in of a cytoplasmic fragment of the receptor, which then medulloblastoma growth, Hes1 and Hes5 are clearly binds to a multi-unit complex capable of transcriptional both ‘canonical’ Notch targets (Jarriault et al., 1995, regulation, transforming it from a repressor to an 1998; Sweeney et al., 2004; Ong et al., 2006). Given the activator complex. In mammals, ligands include mem- number of ligands and receptors potentially having a bers of the Delta-like (Dll1, Dll3, Dll4) and Jagged role in the cerebellum, here we took the approach of (Jag1, Jag2) families, and receptors include Notch1–4. blocking Notch signaling by deleting the common Given the array of Notch ligands and receptors it is not pathway effector RBP-J, which has proved to be a surprising that this pathway forms a complex network, highly effective strategy in analyses of Notch signaling which interacts with many other pathways such as (Fujikura et al., 2006; Komine et al., 2007). Our results Hedgehog, Wnt/wingless and TGFb (Hurlbut et al., indicate that although loss of RBP-J function results in a 2007). The Notch pathway likely has a significant role in developmental delay both within the stem cell niche and the genesis of medulloblastoma and it clearly has a role consequently in granule neuron precursor development, in normal cerebellar development (Tanaka et al., 1999; this leaves cerebellar development largely intact and Klein et al., 2004; Komine et al., 2007; Koo et al., 2007). these effects can be moderated by Hedgehog pathway Perturbation of Notch1 receptor signaling negatively activation. Medulloblastoma formed at high frequency regulates growth of the external germinal layer (EGL) following Hedgehog pathway activation and loss of (Lutolf et al., 2002) whereas activation of Notch2 Notch signaling failed to prevent either expansion of receptor promotes granule cell proliferation (Solecki granule neuron precursors (GNPs) or the subsequent et al., 2001). Other ligands, receptors and targets (Jag1, development of medulloblastoma. Jag2, Dll1, Dll3, Notch3, Notch4, Hes1, Hes5) are also expressed in the developing cerebellum (Stump et al., 2002; Gazit et al., 2004; Irvin et al., 2004; Weller et al., 2006). A number of studies have noted dysregulation of Results Notch pathway components in both human and murine medulloblastoma (Berman et al.,

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