Sonic Hedgehog Regulates Hes1 Through a Novel Mechanism That Is Independent of Canonical Notch Pathway Signalling

Oncogene (2008) 27, 1489–1500 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ONCOGENOMICS Sonic Hedgehog regulates Hes1 through a novel mechanism that is independent of canonical Notch pathway signalling

WJ Ingram1,2, KI McCue1, TH Tran1, AR Hallahan2 and BJ Wainwright1

1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia and 2Department of Paediatrics and Child Health, The University of Queensland, Royal Children’s Hospital, Brisbane, Queensland, Australia

Aberrant regulation of signalling mechanisms that normally (Smo)protein to function. Mammals have three Hedge- orchestrate embryonic development, such as the Hedgehog, hog proteins: Desert Hedgehog (Dhh), Indian Hedgehog Wnt and Notch pathways, is a common feature of tumori- (Ihh)and Sonic Hedgehog (Shh).Of these, Shh has the genesis. In order to better understand the neoplastic events most widespread expression pattern, and appears to mediated by Hedgehog signalling, we identified over 200 play the major developmental role (Jia and Jiang, 2006). genes regulated by Sonic Hedgehog in multipotent meso- Signalling through Smo causes changes in the expres- dermal cells. Widespread crosstalk with other developmen- sion, activation/repression status and cellular localiza- tal signalling pathways is evident, suggesting a complex tion of transcription factors. In vertebrates the major network of interactions that challenges the often over- transcription factors involved are members of the Gli simplistic representation of these pathways as simple linear family (reviewed by Jia and Jiang, 2006). It is thought entities. Hes1, a principal effector of the Notch pathway, that the downstream target genes regulated by such was found to be a target of Sonic Hedgehog in both factors are ultimately responsible for the biological C3H/10T1/2 mesodermal and MNS70 neural cells. Desert consequences of Hedgehog signalling, such as differ- Hedgehog also elicited a strong Hes1 response. While entiation, proliferation and tumorigenesis. Smoothened function was found necessary for upregulation While a large number of studies have used gene of Hes1 in response to Sonic Hedgehog, the mechanism expression profiling to find expression changes in various does not require c-secretase-mediated cleavage of Notch types of cancer, it is not yet clear which genes are directly receptors, and appears to involve transcription factors other influenced by aberrant Hedgehog pathway activity. than RBP-Jj. Thus, we have defined a novel mechanism for Known targets include the transcription factor Gli1 and Hes1 regulation in stem-like cells that is independent of the Patched gene itself, which are considered to be canonical Notch signalling. ‘universal markers’ of Hedgehog pathway activation in Oncogene (2008) 27, 1489–1500; doi:10.1038/sj.onc.1210767; mammalian tissues (Goodrich and Scott, 1998). Expres- published online 17 September 2007 sion profiling as a screening tool to identify large suites of transcriptional targets of Shh has shown success in Keywords: Sonic Hedgehog; Hes1; Notch; C3H/10T1/2; several model systems. Three such studies have been MNS70; gene expression profiling conducted in neural cells (Kato et al., 2001; Zhao et al., 2002; Oliver et al., 2003)Additionally, studies utilizing kidney cells and keratinocytes expressing Gli genes have also led to the identification of target genes (Yoon et al., Introduction 2002; Eichberger et al., 2006). The latter studies likely represent a subset of the genes regulated by Shh in vivo, The Hedgehog pathway was first shown to be involved since it has become clear that some Shh target genes are in tumorigenesis with the discovery of Patched as the regulated independently of Gli1. We provide evidence for a substantial number of new gene mutated in naevoid basal cell carcinoma syndrome ONCOGENOMICS (NBCCS; Hahn et al., 1996; Johnson et al., 1996). More target genes influenced at the transcriptional level by Shh recently Hedgehog pathway involvement has been stimulation, using multipotent mesodermal cells as a implicated in a range of sporadic tumour types in model system. Such cells are of particular interest, since it diverse organs and tissues (reviewed by Borzillo and appears a stem-like phenotype may frequently be the Lippa, 2005). Hedgehog is a morphogen that binds to origin or end point of oncogenic transformation. This the Patched receptor, thereby allowing the Smoothened work builds upon a previous study that demonstrated the power and validity of the approach (Ingram et al., 2002), and has now been extended to include a large portion of Correspondence: Professor BJ Wainwright, Institute for Molecular the transcriptome. The results allow a comparison of Shh Bioscience, The University of Queensland, Building 80, St Lucia target genes with those already identified from other tissue Campus, Brisbane, Queensland 4072, Australia. E-mail: [email protected] systems, and provide clues to the genes abnormally Received 3 July 2006; revised 20 June 2007; accepted 31 July 2007; expressed in tumours that may be affected by Hedgehog published online 17 September 2007 pathway activation. A number of molecules found to be Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1490 regulated by Shh stimulation are known modulators of Here, we present evidence that the regulation of Hes1 other developmental signalling pathways, suggesting a by Shh is via a novel mechanism that is independent of complex network of interactions between pathways. canonical Notch pathway signalling. This has important Of particular interest is the finding that Hairy and implications for future therapeutic approaches directed enhancer of split (Hes1)is upregulated by Shh, confirm- against these oncogenic pathways. ing the observation of Solecki et al., 2001. Hes1 encodes a basic helix–loop–helix transcription factor that functions as a classical effector of the Notch signalling Results pathway (Iso et al., 2003). Notch signalling is involved in decisions of cell fate, where it acts to maintain stem Shh regulates downstream genes with a diverse range of cell characteristics (reviewed by Hansson et al., 2004). putative functions, including many with roles in Similarly to Hedgehog signalling, Notch signalling is proliferation and development also involved in neoplasia. This was first identified from In order to elucidate transcriptional targets of Shh the finding of mutant Notch1 as an oncogene in signalling on a large scale, a multipotent mesodermal leukaemia (Ellisen et al., 1991). Abnormal Notch cell line (C3H10T1/2 clone 8, hereafter referred to as signalling is observed in a wide variety of human 10T1/2; Reznikoff et al., 1973)was employed as a model cancers (reviewed by Allenspach et al., 2002). system. This line has been widely studied in regard to its Several recent studies in mammals have suggested responsiveness to Hedgehog proteins (Kinto et al., 1997; links between Hedgehog and Notch signalling in Nakamura et al., 1997; Pathi et al., 2001)and is highly tumorigenesis. Medulloblastoma arising in heterozygous suited to downstream target gene discovery (Ingram Patched knockout mice displays elevated expression of a et al., 2002). Cells were stimulated by transfection of number of Notch pathway genes (Yokota et al., 2004; either an N-terminal Shh expression construct or a null- Dakubo et al., 2006), as do similar tumours arising in mutant control construct. mice expressing an oncogenic form of Smo (Hallahan Analysis of the data obtained from six independent et al., 2004). Similar findings are seen in human patients, experiments allowed the identification of 141 genes including the upregulation of Hes1 in a subset of showing an increase, and a further 69 genes showing medulloblastoma (Fan et al., 2004; Hallahan et al., a decrease, in transcript abundance in response to Shh 2004). Expression of Hes1 in this tumour is associated stimulation. A subset of the identified genes are with significantly shorter patient survival (Fan et al., shown in Tables 1 and 2. The full list is available as 2004). These studies indicate the importance of Hedge- Supplementary Data accompanying this paper. Genes hog–Notch pathway interactions in human cancer, and with significant signal on both channels were included, highlight the need for a greater understanding of the as were genes that only had significant signal strength relationship. for either the treatment or control cells. While the ratio

Table 1 Genes showing increased transcript abundance in response to Sonic Hedgehog Accession Gene name Symbol FCMolecule type Function

Qualitative change: basal mRNA level below threshold, increasing to signiﬁcant level after Shh stimulation NM_008343 Insulin-like growth factor Igfbp3 8.5a Extracellular Cell growth/proliferation binding protein 3 AK020715 Patched homolog 1 Ptch1, Ptc1 5.3a Membrane Developmental processes, Hedgehog pathway NM_011896 Sprouty homolog 1 Spry1, sprouty1 4.1a Membrane Developmental processes NM_009349 Indolethylamine Inmt, Temt 3.8a Metabolic enzyme Catecholamine metabolism N-methyltransferase NM_008235 Hairy and enhancer of split 1 Hes1, Hry 3.4a Transcription factor Transcription regulation, cell cycle, Notch pathway NM_010231 Flavin containing Fmo1 3.0a Metabolic enzyme Amino acid metabolism monooxygenase 1 AB046537 Protease inhibitor 16 Pi16 2.6a Extracellular Pathogenesis NM_020259 Hedgehog-interacting protein Hip1 2.2a Extracellular, Developmental processes, membrane Hedgehog, Wnt and Fgf pathways AJ277212 Mustang Mustn1 2.1a Nuclear protein Musculoskeletal development NM_020612 Cell matrix adhesion regulator Cmar, PNG, SPG7 1.9a Metalloprotease Cell adhesion NM_008462 Killer cell lectin-like receptor A2 Klra2, Ly49b 1.9a Membrane Cell adhesion NM_009651 A kinase (PRKA)anchor protein 4 Akap4, Fsc1, p82 1.9 a Membrane Serine/threonine kinase pathway AK007287 RIKEN cDNA 1700126A01 gene — 1.9a —— AK017269 Microﬁbrillar-associated protein Mfap3 l, NYD-sp9 1.9a Membrane — 3-like AK009996 RAD51-like 1 Rad51l1 1.8a Nuclear DNA repair NM_019487 Heme binding protein 2 Hebp2, SOUL 1.8a Cytoplasmic Circadian rhythm NM_011280 Tripartite motif protein 10 Trim10, Rnf9 1.8a Intracellular Mesoderm determination NM_019734 N-acylsphingosine amidohydrolase Asah1, AC, Asah 1.7a Metabolic enzyme cGMP biosynthesis 1 NM_010296 GLI-Kruppel family member GLI1 Gli1 1.7a Transcription factor Developmental processes, Hedgehog pathway

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1491 Table 1 (continued ) Accession Gene name Symbol FCMolecule type Function

NM_013690 Endothelial-speciﬁc receptor Tek, Tie2 1.7a Kinase Cell adhesion/migration, tyrosine kinase angiogenesis NM_007855 Twist homolog 2 Twist2, Dermo1 1.6a Transcription factor Transcription regulation NM_016719 Growth factor receptor bound Grb14 1.6a Cytoplasmic Insulin pathway protein 14 NM_008813 Ectonucleotide pyrophosphatase/ Enpp1 1.6a Enzyme Skeletal development phosphodiesterase 1 NM_021504 N-glycanase 1 Ngly1, PNGase 1.6a Enzyme Degradation of misfolded proteins

Quantitative change: signiﬁcant mRNA levels before and after Shh stimulation (fold change >2) M55181 Preproenkephalin 1 Penk1, ENK, PPA 4.8 Neuropeptide Neuropeptide signaling NM_008048 Insulin-like growth factor binding Igfbp7, mac25 4.5 Extracellular Cell growth/proliferation protein 7 NM_025312 Sclerostin domain containing 1 Sostdc1, ectodin 4.1 Extracellular Wnt and BMP pathways NM_009378 Thrombomodulin Thbd, CD141, TM 3.9 Extracellular Blood coagulation, developmental processes NM_010637 Kruppel-like factor 4 (gut)Klf4, EZF, Gklf 3.5 Transcription factor Transcription regulation AK018379 PTK7 protein tyrosine kinase 7 Ptk7, Cck4 3.4 Membrane protein Developmental processes, cell adhesion U01136 RIKEN cDNA 2310032D16 gene — 3.3 — — NM_008895 Pro-opiomelanocortin-alpha Pomc1, ACTH 3.2 Extracellular Neuropeptide signalling NM_008242 Brain factor 2/Forkhead box D1 BF-2, Foxd1 2.9 Transcription factor Transcription regulation NM_010217 Insulin-like growth factor binding Igfbp8, Ctgf 2.7 Extracellular Cell growth/proliferation, protein 8 adhesion, migration NM_007833 Decorin Dcn, DC, DSPG2 2.7 Extracellular Cell adhesion AK020379 Solute carrier family 35, Slc35e3 2.6 Mitochondrion Transport member E3 BC005558 Phosphatidic acid phosphatase Ppap2b 2.5 Membrane protein Developmental processes, type 2B Wnt pathway NM_010286 Glucocorticoid-induced leucine Gilz, TSC-22R 2.4 Transcription factor Transcription regulation, zipper antiapoptosis NM_009320 Solute carrier family 6, member 6 Slc6a6 2.3 Membrane protein Neurotransmitter transport NM_008597 Matrix Gla protein MGP, Mglap 2.3 Extracellular Developmental processes, bone mineralization, TGF-beta pathway NM_008700 NK2 transcription factor related, Nkx2-5, Csx 2.3 Transcription factor Transcription regulation locus 5 NM_013642 Dual speciﬁcity phosphatase 1 Dusp1, mkp-1 2.2 Phosphatase Cell cycle AK017223 RIKEN cDNA 5133401H06 gene — 2.1 Membrane protein Metabolism NM_010902 Nuclear factor, erythroid derived 2, Nfe2l2 2.1 Transcription factor Transcription regulation like 2 AF072758 Solute carrier family 27, member 3 Slc27a3 2.1 Mitochondrion Lipid transport NM_007555 Bone morphogenetic protein 5 Bmp5 2.0 Extracellular Developmental processes, TGF-beta pathway NM_010517 Insulin-like growth factor binding Igfbp4 2.0 Extracellular Cell growth/proliferation protein 4 AK003535 RIKEN cDNA 1110007F12 gene — 2.0 — — NM_007442 Aristaless 4 Alx4, lst 2.0 Transcription factor Transcription regulation NM_019571 Tetraspanin 5 Tm4sf9, NET-4 2.0 Membrane Cell growth/proliferation, adhesion, motility

Quantitative change: selected genes of interest (fold change o2) NM_009155 Selenoprotein P, plasma, 1 Sepp1, Se-P 1.7 Extracellular Cell growth and/or maintenance NM_007752 Ceruloplasmin Cp 1.6 Extracellular Copper ion transport NM_008885 Peripheral myelin protein Pmp22, Gas-3 1.6 Extracellular, Neural cell insulation, cell membrane growth/proliferation, cell cycle NM_007836 Growth arrest and Gadd45a 1.6 Nuclear Response to DNA damage, DNA-damage-inducible 45 alpha cell cycle NM_010917 Nidogen 1 Nid1, entactin 1.6 Extracellular Adhesion

Abbreviations: FC, fold change, Shh, Sonic Hedgehog. FC shown is the geometric mean of the change in expression between Shh-treated and pair- matched control cells (n ¼ 6). Information was assigned to genes based on information from the Compugen, Gene Ontology, LocusLink, Unigene and PubMed databases. aFC obtained from microarray probably underestimates true value for this gene (refer text). obtained from the latter is not quantitative, the fact that the noise threshold, but rises to a significant signal after such genes have reproducibly gone from having no Shh treatment. Genes displaying such behaviour (‘qua- significant signal to a high signal is clearly informative. litative’ targets)have been included in the target lists Genes such as Gli1 (Table 1)fit into this category, where separately from genes having strong signals both before the microarray signal strength in control cells is below and after treatment (‘quantitative’ targets).

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1492 Table 2 Genes showing decreased transcript abundance in response to Sonic Hedgehog Accession Gene name Symbol FCMolecule Type Function

Qualitative change: signiﬁcant mRNA level before stimulation, decreasing below threshold after Shh stimulation NM_016910 Protein phosphatase 1D magnesium- Ppm1d, Wip1 À1.7a Nuclear Cell cycle dependent, delta isoform AK019341 DIRAS family, GTP-binding RAS-like 2 Diras2 À1.7a GTPase — U50955 — — À1.7a —— AK004552 RIKEN cDNA 1200002N14 gene — À1.7a —— NM_026481 RIKEN cDNA 2700055K07 gene CGI-38 À1.6a —— AF153440 BMP and activin membrane-bound Bambi À1.6a Membrane TGF-b pathway inhibitor

Quantitative change: signiﬁcant mRNA levels before and after Shh stimulation (fold change >2) NM_009144 Secreted frizzled-related protein 2 Sfrp2, Sdf5 À3.2 Extracellular Apoptosis, Wnt pathway L29479 Polo-like kinase 4 Plk4, Sak, À3.0 Kinase Cell cycle Stk18 AF013576 IG kappa chain complex variable region Igk-V À2.9 Immunoglobulin Immune response NM_018820 SERTA domain containing 1 Sertad1, Sei1 À2.7 Transcription factor Cell cycle NM_009369 Transforming growth factor beta induced Tgfbi À2.6 Extracellular Cell growth/proliferation, adhesion AK008943 Secreted frizzled-related protein 1 Sfrp1 À2.5 Extracellular Apoptosis, Wnt pathway D90225 Pleiotrophin Ptn, Osf1 À2.4 Extracellular Cell cycle, ossiﬁcation X94418 IG heavy chain (V7183 family)Igh-V7183 À2.4 Immunoglobulin Immune response X17164 IG heavy chain 4 (serum IgG1)Igh-6 À2.4 Immunoglobulin Immune response AF093558 — — À2.3 — — AK018071 Leucine-rich repeat containing 17 Lrrc17, À2.3 — — P37nb AK016598 RIKEN cDNA 4933401B06 gene — À2.2 — — AK005783 — — À2.1 — — NM_009525 Wingless-related MMTV integration Wnt5b À2.1 Extracellular Developmental processes, Wnt site 5B pathway NM_010268 Ganglioside-induced differentiation- Gdap10 À2.1 — — associated-protein 10 NM_007633 Cyclin E1 Ccne1 À2.1 Nuclear Cell cycle X16223 — — À2.1 — — NM_024283 RIKEN cDNA 1500015O10 gene Ecrg4 À2.0 — —

Abbreviations: Bambi, BMP and activin membrane-bound inhibitor; FC, Fold change, Shh, Sonic Hedgehog. FC shown is the geometric mean of the change in expression between Shh-treated and pair-matched control cells (n ¼ 6). A preceding ‘—’ indicates downregulation. Information was assigned to genes based on information from the Compugen, Gene Ontology, LocusLink, Unigene and PubMed databases. aFC obtained from microarray probably underestimates true value for this gene (refer text).

A sample of 25 putative targets, representing both IGF (Igbbp3, Igfbp4, Igfbp7, Igfbp8),TGF-b (Bmp5, highly and weakly expressed genes, were validated by Sostdc1)and Notch ( Hes1)pathways. northern blotting using RNA from independent experiments. Examples are shown in Figures 1 and 2. Greater Shh regulates Hes1 via a mechanism independent than 90% of the genes investigated were confirmed as of Notch receptor cleavage targets in this way in multiple experiments. These results, As Hes1 is regulated by Shh in both mesodermal and along with the suite of already known Hedgehog pathway neural precursor cells (Figures 2a and b), the link between targets in the gene lists, indicated the data obtained Hedgehog and Notch signalling provided by Hes1 were of high quality. During the validation procedure was investigated in detail. Comparative studies showed genes were also investigated as to their response in an that Hes1 is strongly regulated by both Shh and Dhh, independent cell type—the multipotent rat neural line and more modestly upregulated in response to Ihh MNS70 (Nakagawa et al., 1996). Three of the genes (Figure 2c). In these experiments, transfection constructs tested were found to be regulated by Shh in MNS70 cells: encoded full-length Shh, Dhh and Ihh, revealing that Cyclin E1, Ceruloplasmin and Hes1 (Figure 2b). naturally processed Hedgehog proteins, with attached Some of the newly identified genes have been lipid moieties, can induce the Hes1 response. Like many previously reported as Shh targets in other cell types, genes, Hes1 basal mRNA level is slightly increased after while the involvement of the majority of genes has not protein synthesis inhibition; however, the large increase in previously been described. Genes showing changes in Hes1 mRNA seen after Shh stimulation was inhibited by mRNA abundance in response to Shh encode a diverse cycloheximide (Figure 2a). The appropriate concentra- array of molecules involved in a wide range of cellular tion of cycloheximide to use in 10T1/2 cells was functions. Of particular interest, crosstalk between the determined via a novel method assaying translation of Hedgehog pathway and other developmental signalling luciferase mRNA (refer materials and methods). mechanisms appears to be widespread. Notable Hedge- Given that Hes1 represents a common intersection of hog target genes identified include known modulators of two major developmental/tumourigenic signalling path- the Wnt (Wnt5b, Ppap2b, Sfrp1, Sfrp2, Sostdc1, Bambi), ways, we examined whether Shh acts to increase Hes1 by

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1493 Sprouty1 Igfbp8 Twist2 AK003535 Shh: – + – + – + – + 4.5 3.1 14.6 4.1 4.4 4.4 2.4 2.4 2.4 2.4 1.4 1.4 1.4 1.4

Fbn2 BambiWnt5b Ptn Shh: – + – + – + – + 10.2 0.4 0.4 0.4

2.4 2.4 4.4

1.4 9.5 2.4 1.4 7.5

Figure 1 Examples of novel Sonic Hedgehog (Shh)target genes identified by microarray and validated by northern blotting. 10T1/2 cells treated for 3 days with Shh-N conditioned media ( þ )show changes in mRNA abundance compared to control media treated cells (À). Size markers in kb. Sprouty, Igf-binding protein 8 (Igfbp8), Twist2, Fibrillin 2 (Fbn2)and uncharacterized gene AK003535 show upregulation, while BMP and activin membrane-bound inhibitor (Bambi), Wnt5b and Pleiotrophin (Ptn)show downregulation in response to Shh. Numbers above blots correspond to fold change in response to Shh, and are normalized to corresponding GAPDH hybridizations (G). simply activating the canonical Notch pathway, or 12 RBP-Jk binding motifs driving luciferase expression whether the interaction is via an independent mechanism. (pGA981-6; Minoguchi et al., 1997; Das et al., 2004). To address this, an inhibitor of Notch pathway signalling, The dose–response curve shows that DAPT is a potent N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine inhibitor of canonical Notch signalling under our t-butyl ester (DAPT; Geling et al., 2002), was used to treat experimental conditions (Figure 3a). cells during stimulation with Shh. When Notch signalling was inhibited in MNS70 cells, In canonical Notch signalling receptors on the surface a dramatic change in cell morphology was observed, as of a receiving cell are activated when ligands (members shown by phase contrast microscopy and staining of of the Jagged and Delta families)are presented on the nuclei (Figure 4). DAPT treatment led to large cells surface of adjacent cells. The Notch receptor is then dominating the dish surface, and to an increase in the cleaved, allowing an intracellular portion of the protein number of smaller cells growing on top. The latter cells (Notch-IC)to enter the nucleus and control transcrip- occurred in clusters and were poorly adherent. Surpris- tion of downstream genes (reviewed by Mumm and ingly, when DAPT was added to MNS70 cells that were Kopan, 2000). Recombining binding protein suppressor stimulated with Shh, the cells did not undergo this of hairless J kappa (RBP-Jk, also known as RBPSUH/ morphology change (Figures 4d and h). CSL/CBF-1)is a transcriptional activator for primary Notch pathway targets, such as Hes1, after it has formed Regulation of Hes1 by Shh appears to be independent a complex with Notch-IC (Jarriault et al., 1995). DAPT of the transcription factor RBP-Jk acts by inhibiting cleavage of activated Notch receptors Activity of the RBP-Jk reporter construct was investi- by g-secretase, thereby preventing signalling. In 10T1/2 gated during Shh stimulation of 10T1/2 cells to determine and MNS70 cells, DAPT did not prevent the induction whether Shh-mediated regulation of Hes1 is via the of Hes1 mRNA in response to Shh (Figures 3b and c). transcription factor RBP-Jk. The human Notch-IC Although DAPT caused a drop in basal Hes1 levels in construct pcDNA3-ICTX (Weng et al., 2003)was used both cell types, particularly in MNS70, there was no as a positive control for Notch pathway activation, and decrease in the fold change in Hes1 mRNA caused by led to a significant increase in reporter activity. No Shh stimulation. increase in RBP-Jk activity was seen with Shh, regardless DAPT suppressed canonical Notch signalling, as of the stimulation method (Figure 5a), and the same shown in an assay based on the inhibition of cleavage result was obtained when the experiment was repeated in of constitutively active g-secretase-dependent Notch1 MNS70 cells (Supplementary Figure S1). RNA was (Weng et al., 2003; pcDNA3-DE). Notch pathway extracted from lysates and subjected to real-time PCR activity was monitored using a reporter construct with analysis, confirming that upregulation of Hes1 had

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1494 Cycloheximide indeed occurred in the Shh treated cells used for luciferase –– ++ analysis (Figure 5b). Hey2 acted as an endogenous control for Notch pathway activation (Figure 5c). Mutant Shh-N Mutant Shh-N Mutant Shh-N To further investigate the apparent RBP-Jk indepen- 4.4 4.4 dence of the Hes1 response to Shh, the action of RBP-Jk kb kb was directly antagonized during Shh stimulation. This was 2.4 2.4 achieved by transfection of a dominant-negative form of RBP-Jk (dnRBP-Jk), which has a mutation in the Hes1 1.4 1.4 Hes1 DNA binding domain and inhibits signalling (Blokzijl 19.3 1.8 2.0 2.6 et al., 2003). Shh still induced Hes1 inthepresenceof

GAPDH GAPDH 100 90 80 Control Shh-full Ihh-full Dhh-full 70 4.4 kb 60 50 2.4 40 Hes1 30 1.4 20 % of activation without drug % of activation 8.5 1.7 11.1 10 0 0 5 10 15 20 25 30 35 40 GAPDH DAPT concentration (µM) Figure 2 Hes1 is a transcriptional target of Hedgehog signalling. Northern blots probed with Hes1. Numbers represent fold change compared with control lane (left)by GAPDH normalized 0.06 densitometry. (a) Hes1 mRNA is upregulated in 10T1/2 mesodermal cells following treatment with recombinant Sonic Hedgehog 0.05 (Shh)-N conditioned media; however, this response is inhibited in the presence of cycloheximide (3 day time point). ‘Mutant’ refers to Shh null-mutant control. (b)Regulation of Hes1 by Shh-N is also 0.04 observed after stimulation of MNS70 neural cells (2 days post- transfection). (c)In addition to being controlled by full-length Shh 0.03 (Shh-full), Hes1 is also strongly upregulated 4 days after transfection with full-length Desert Hedgehog (Dhh-full)and marginally by full-length Indian Hedgehog (Ihh-full). 0.02

0.01 Figure 3 Induction of Hes1 by Sonic Hedgehog (Shh)does not require g-secretase-mediated cleavage of Notch ligands. (a) N-[N- 0.00 (3,5-Difluorophenacetyl-L-alanyl)] S-phenylglycinc t-butyl ester Shh – + – + (DAPT)is a potent inhibitor, with 1 mM strongly repressing Notch DAPT– – + + signalling. 10T1/2 Cells were transfected with the constitutively active g-secretase-dependent Notch construct pcDNA3-DE, and 0.05 RBP-Jk mediated luciferase activity measured after 2 days. Each point is the geometric mean of 3 replicates with firefly luciferase activity normalized to renilla activity, scaled so that non-inhibited 0.04 Notch pathway activity is 100%. Error bars are 71 geometric standard deviation. (b)In 10T1/2 cells stimulation with Shh caused a 5.8-fold increase in Hes1 mRNA level 3 days post-treatment by real-time PCR. In parallel wells, where 10 mM DAPT was included 0.03 in the media a 7.3-fold change due to Shh was observed. Media (7Shh, 7DAPT)was changed halfway through the culture period. (c)When the experiment described in ( b)was repeated in MNS70 0.02 cells, Shh stimulation lead to a 2.5-fold increase in Hes1 without drug, and a 22.4-fold increase in the presence of DAPT. Data in (b and c)are geometric means of Hes1 level relative to GAPDH Hes1/GAPDH mRNA level0.01 Hes1/GAPDH mRNA level from four independent biological replicates, with TaqMan reactions for each performed in quadruplicate. Error bars are equivalent to 7 1 standard deviation, and conservatively take into account 0.00 variation form real-time PCR technical replicates as well as biological replicate variation. Variation statistics were combined Shh – + – + using the ‘square root of sum-of-squares’ technique. DAPT– – + +

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1495 Phase Contrast DAPI 1000

800 Shh DAPT – – 600

400

200 to pcDNA3 vector control to pcDNA3 vector Shh DAPT relative FF/Ren Luciferase, + – 0

0.18 0.16 0.14 Shh DAPT 0.12 – + 0.10 0.08 0.06 0.04 Shh DAPT Hes1/GAPDH mRNA level 0.02 + + 0.00

0.0004 Figure 4 Sonic Hedgehog (Shh)prevents formation of N-[N- (3,5-Difluorophenacetyl-L-alanyl)] S-phenylglycinc t-butyl ester (DAPT)induced morphology in MNS70 cells. MNS70 cells treated 0.0003 as described for Figure 3c. (a–d)Live cell images by phase contrast microscopy. (e–h)Fixed cells showing nuclei stained with DAPI (images inverted). An increase in cell density is evident after 0.0002 treatment with Shh, particularly with regard to the small cells with dark-staining nuclei (f). Blocking canonical Notch signalling with DAPI leads to a predominance of large cells with diffuse nuclei, and small cells in clusters on top of the monolayer (c)that are 0.0001 poorly adhered and wash away during fixing (g). Treatment with Shh in the presence of DAPT maintains the morphology of mixed mRNA level Hey2/GAPDH cell sizes, including many small cells with dense nuclei that remain 0.0000 after washing (d and h). dnRBP-Jk,withnodetectabledecreaseintheresponse (Figure 6a). Luciferase-based control assays confirmed (Shh-N) (mutant) pcDNA3

that dnRBP-Jk is a potent inhibitor of canonical Notch (Notch IC) signalling under our experimental conditions (Figure 6b). rShh media Control media pcDNA3-ICTX

This provides further evidence that the induction of Hes1 pShh-N-PMT21 64-Shh-N-PMT21 by Shh is via a non-canonical mechanism. ∆ p Figure 5 The Sonic Hedgehog (Shh)-mediated Hes1 response Induction of Hes1 by Shh requires signalling through appears not to involve the transcription factor RBP-Jk.10T1/2cells Smoothened were transiently transfected with the RBP-Jk reporter pGA981-6 Since it appears that the increase in Hes1 mRNA in and stimulated with Shh by transfection (Shh-N)or by addition of response to Shh does not use the machinery of the recombinant Shh (rShh)conditioned media. ( a)Shh treatment does not increase luciferase, while the Notch-IC positive control construct canonical Notch pathway, we determined whether the causes a dramatic elevation. Each point represents the geometric response required the canonical Hedgehog signalling mean normalized luciferase activity of four biological replicates, pathway. 10T1/2 cells were stimulated with Shh or the scaled relative to pcDNA3 vector having a value of 1. Error bars are null-mutant control in the presence or absence of cyclo- 71 geometric standard deviation. (b)Shh-treated cells show Hes1 pamine. Cyclopamine is a potent inhibitor of Smo, the upregulation by real-time PCR. RNA for Hes1 analysis was extracted from the luciferase assay lysates, so data is for the transducer of signalling after reception of Hedgehog mole- identical cells investigated in panel a. Each point represents four cules by Patched (Chen et al., 2002; Frank-Kamenetsky biological replicates. (c)Notch-IC treated cells show upregulation of et al., 2002). Cyclopamine signiﬁcantly inhibited the Hes1 endogenous Hey2 by real-time PCR, conﬁrming that even though response, as measured by real-time PCR (Figure 7), Hes1 is not increased with this treatment, the cells do indeed respond to Notch signalling (samples as for b). Geometric means and error indicating that Smo function is required for the induction bars for b and c calculated as in Figures 3b and c. of Hes1 by Shh.

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1496 0.035 0.18 0.16 0.030 0.14 0.025 0.12 0.020 0.10

0.015 0.08 0.06 0.010 0.04 Hes1/GAPDH mRNA level Hes1/GAPDH mRNA level 0.005 0.02

0.000 0.00 Shh – + – + Shh – + – + dnRBP-Jκ – – ++ Cyclopamine– – + + Figure 7 Cyclopamine inhibits the induction of Hes1 by Sonic 160 Hedgehog (Shh). Real-time PCR analysis of Hes1 mRNA levels 3 days post-treatment. Each point represents four biological repli- 140 cates, each of which had TaqMan analysis performed in quadruplicate. Geometric means and error bars calculated as in 120 Figures 3b and c. 100 80 regulate cell cycle. Other identiﬁed genes, such as those 60 involved in adhesion and migration, may contribute to later features such as invasion and metastasis. A subset

to empty vector control to empty vector 40 of the identified target genes, which are currently FF/Ren Luciferase, relative FF/Ren Luciferase, 20 unnamed and for which little biology is known, await further investigation. 0 A number of the identified Shh target genes display Notch-IC – – + + abnormal expression in tumours in human patients, dnRBP-Jκ – ++– indicating that they may be important in cancer Figure 6 Directly inhibiting RBP-Jk action does not prevent the aetiology and/or ongoing tumour survival. A key Sonic Hedgehog (Shh)-mediated Hes1 response. (a)10T1/2 cells example is the metal transport protein Ceruloplasmin, were transfected with a dominant-negative mouse RBP-Jk which is associated with a number of cancers (Chakra- (dnRBP-Jk)expression construct (pMIKneo-RBP(R218H)or empty vector control), before addition of Shh-conditioned media borty et al., 1984; Kunapuli et al., 1987; Lowndes and (or control media)the following day. At 3 days post-transfection Harris, 2004)and is considered a diagnostic serum RNA was harvested and Hes1 levels assessed by real-time PCR. marker in advanced stage solid malignant tumours Data displayed as a scatterplot to show that though there is (Senra Varela et al., 1997). Some Shh target genes seen considerable variation in the magnitude of the Hes1 response to in the current work are characteristic of desmoplastic Shh, it is always dramatic (nine biological replicates per treatment). Horizontal bars show geometric mean for each treatment. compared with classic medulloblastoma in human Comparison of columns 4 and 2 shows that there is no detectable patients (Pomeroy et al., 2002). Genes that fit this inhibition of the Hes1 response in the presence of dnRBP-Jk.(b) category include Decorin, Nidogen, Selenoprotein P and dnRBP-Jk is functional under our experimental conditions, as it Matrix gla protein (MGP). Desmoplastic medulloblas- acts as a potent inhibitor of the Notch-IC-mediated stimulation of reporter pGA981-6. Each point represents the geometric mean of toma is associated with Hedgehog pathway activation, three biological replicates with firefly luciferase activity normalized both in NBCCS patients and sporadic cases (Pietsch to renilla activity, and scaled relative to basal (empty vector et al., 1997). Selenoprotein P and Decorin have pre- treatment)having value of one. viously been noted as differentially expressed during tumour progression in a mouse glioma model (Johans- Discussion son et al., 2005). MGP and another prominent gene in this work, Igfb4, distinguish high-grade recurrent glioma Abnormal activation of Hedgehog signalling is involved from primary glioma in patients (van den Boom et al., in a number of solid tumour types, yet little is known 2003). Other similarities to neural tumour models are about the specific downstream effects that mediate also apparent, including changes in expression of Hedgehog influenced tumorigenesis. Here, we have Pleiotrophin and Klf4, which have recently been shown identified and validated targets of Hedgehog signalling as differentially expressed in granule cell precursors of in two established cell models with stem cell-like the Patched þ /Àmouse model as they progress to characteristics. Analysis of these genes shows many are medulloblastoma (Oliver et al., 2005). involved in processes that may influence tumour Comparison with other animal models of Hedgehog formation. Examples are genes involved in decisions of pathway disruption has also uncovered similarities cell fate and proliferation, particularly those that with the Shh target genes in our mesodermal model.

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1497 Growth arrest and DNA-damage-inducible 45 a (Gad- preliminary studies of signalling pathway inhibition in d45a)is elevated in rhabdomyosarcoma and medullo- both murine and human medulloblastoma suggest that blastoma arising in Patched þ /Àmice (Kappler et al., simultaneously inhibiting Notch and Hedgehog signal- 2004a, b). However, it remains unclear whether this gene ling is more effective than inhibiting either pathway in is transcriptionally regulated by the Hedgehog pathway, isolation (Hallahan et al., 2004). or if the upregulation simply reflects observed changes The dramatic change in morphology of MNS70 cells in Trp53 expression in the tumours, as Gadd45a is after treatment with DAPT reinforces the hypothesis associated with p53-mediated cell cycle arrest (Amund- that Notch signalling is important in the maintenance of son et al., 1998). The fact that Gadd45a mRNA levels neural stem characteristics (Kageyama et al., 2005). increased in response to Shh in 10T1/2 cells provides That the effect of DAPT could be abrogated by Shh evidence for the former. supports our finding that Shh acts below the level of The interplay between key developmental signalling Notch ligand cleavage to complement at least a subset of pathways, evident in this study from transcriptional outcomes normally mediated via Notch ligands. events mediated by Hedgehog pathway activation, In the experiment shown in Figure 5, Notch-IC indicates that some targets of Hedgehog signalling may dramatically enhanced activity of the RBP-Jk motif represent common end points whereby two or more reporter in 10T1/2 cells, though it did not increase oncogenic signalling networks can ultimately control the endogenous Hes1 mRNA levels. A similar result was same molecules. Further analysis of one such example, seen in the neural precursor line (Supplementary Figure involving the Notch effector Hes1, has uncovered a novel S1). This insensitivity of endogenous Hes1 to activated mechanism for Hes1 regulation. In particular, the finding Notch1 has previously been observed in several cell that Hes1 represents a common meeting point between types, including 10T1/2, and suggests that Hes1 is the Hedgehog and Notch pathways in both mesodermal commonly not the primary target of canonical Notch and neural cells has implications for both tumour biology signalling via this receptor (Shawber et al., 1996; Iso and normal embryonic development. et al., 2001). Previous work eluding to the regulation of Hes1 by Experiments involving blockage of protein synthesis Shh includes the discovery that hairy, a Hes gene were informative for Hes1, in that drug treatment itself homologue, is regulated by Cubitus interruptus (the had only a small effect on Hes1 mRNA levels, and equivalent of the vertebrate Gli proteins)in Drosophila prevented the induction of Hes1 by Shh. This indicates (Hays et al., 1999; Fu and Baker, 2003; Kwon et al., that the response is either indirect, or Hes1 requires a 2004). Other studies in the fly have indicated links labile cofactor at some level of its regulation or between Hedgehog and Notch signalling (Alexandre production. Hes1 protein is known to have a very short et al., 1999; Baonza and Freeman, 2001). Solecki et al. half-life, and is a key repressor of its own promoter (2001)previously noted in studies of cultured cerebellar (Hirata et al., 2002), so the fact that blocking protein granule cell precursors that Hes1 was upregulated in synthesis alone did not increase Hes1 mRNA levels to response to Jagged (Notch ligand)or Shh stimulation, those seen with Shh stimulation suggests that the but had not considered the latter was a Notch- mechanism of induction by Shh does not simply involve independent effect. Our evidence indicates a novel relief of basal Hes1 self-repression. interaction between the Hedgehog and Notch signalling The regulation of Hes1 via Notch signalling has been pathways. We demonstrate that the regulation of Hes1 by extensively studied, particularly with regard to the role Shh does not involve g-secretase-mediated Notch clea- of RBP-Jk. It has been known for some time that other vage, and likely involves transcription factors other than mechanisms must also be involved. For example, Hes1 is RBP-Jk. This appears to be a separate effect to the recent expressed prior to known activity of the Notch pathway discovery of Jagged upregulation via Shh/Gli3 (McGlinn in neural tissue (Kageyama et al., 2005), and previous et al., 2005). Therefore, in mammals the Hedgehog studies have pointed to Notch pathway signalling pathway influences the Notch pathway at multiple points. events mediated independently of RBP-Jk (reviewed by The exact molecular basis of Hes1 regulation by Shh Martinez Arias et al., 2002). It will be interesting to remains to be elucidated. We have investigated several determine the extent to which Shh effectors influence Hes1-promoter reporter constructs (mouse and human), such phenomena. Our observed inhibition of the Hes1 but have been unable to demonstrate activation of response to Shh by cyclopamine shows that the newly these in response to Shh in 10T1/2 cells. Data for a identified mechanism controlling Hes1 expression construct containing 856 bp of Hes1 upstream sequence does involve canonical Hedgehog signalling, as it is (Solecki et al., 2001), which includes the RBP-Jk dependent on Smo function. responsive region, are shown in Supplementary Figure Given that Notch signalling is crucial to the main- S2. Thus, it is likely that the Hedgehog response tenance of several self-renewing stem cell types in vivo element(s)reside, at least in part, outside of currently (Duncan et al., 2005; Kageyama et al., 2005; Woodward recognized Hes1 promoter regions. et al., 2005), the finding that Hes1 is also regulated by Interactions between cellular signalling pathways Shh may indicate a functional convergence point in have implications for future anticancer therapies. If tumour biology when either of these pathways is tumour cells feature activation of several signalling abnormally activated. Combined with the recent finding pathways, then targeting one pathway alone may not be that Hes1 directly represses the promoter of p27kip1 sufficient to stop tumour growth. Supporting this idea, (Murata et al., 2005), a key regulator of the G1-S cell

Oncogene Sonic Hedgehog regulates Hes1 via a novel mechanism WJ Ingram et al 1498 cycle checkpoint, Hes1 warrants further investigation as coding sequence), using GenBank (NCBI) information. Probe to whether it is a potential target for therapeutic sequences were isolated by RT–PCR, and after cloning and intervention. sequencing PCR products were labelled with 32P using Megaprime (GE Healthcare, Chalfont St Giles, UK). Quantitative real-time PCR was performed using the Prism 7000 Sequence Detection System/SDS software (ABI, Foster Materials and methods City, CA, USA). Synthesis of cDNA used MMLV-RT (Invitrogen). Samples were run in triplicate or quadruplicate, Cell culture, transfection and stimulation with recombinant Shh using GAPDH control reagents (ABI)for normalization. 10T1/2 cell culture, mouse Shh-N and null-mutant constructs, Hes1 was assayed using Mm00468601_m1 and Hey2 using transient transfection, conditioned media production and use, Mm00469280_m1 (ABI). and alkaline phosphatase assays were performed as previously described (Ingram et al., 2002). Notch pathway constructs are as described in the Results section. MNS70 cells were grown Luciferase reporter assays Firefly Luciferase assays were performed using the Dual- on poly-D-lysine (Sigma, St Louis, MO, USA), in 1:1 DMEM/ F12 containing 8.7% fetal bovine serum and 4.3% horse serum Luciferase Reporter Assay System (Promega, Madison, WI, (Invitrogen, Carlsbad, CA, USA). For inhibition experiments USA), and included a Renilla (Ren) luciferase construct Shh stimulation of 10T1/2 cells was achieved using conditioned (pRL-SV40; Promega)as an internal control. media, while stimulation of MNS70 cells was with 2 mgmlÀ1 recombinant Shh-N (R&D systems, Minneapolis, MN, USA). Protein synthesis inhibition assay Transfection of full-length murine Shh, Ihh and Dhh was In order to determine the optimal treatment regime for achieved using cDNAs cloned into pcDNA3 (Invitrogen). cycloheximide inhibition of protein synthesis in 10T1/2 cells, DAPI staining was performed after fixing in 4% PFA. an assay was developed using translation of Ren luciferase as a quantifiable marker of drug function. Cells were pre-treated RNA isolation with cycloheximide (Sigma)for various time periods/concen- À2 RNA was isolated using the RNeasy kit with DNase digestion trations before transfection of 15 ng cm pRL-SV40, then (Qiagen, Hilden, Germany). For RNA extraction from cells in maintained in media with the drug and harvested 3 days post- Promega luciferase lysis buffer, samples were diluted 14-fold in transfection. Cells in which cycloheximide was removed Qiagen RLT buffer before continuing the RNeasy procedure. post-transfection acted as a positive control for luciferase expression, while comparison with non-cycloheximide treated cells showed that the presence of cycloheximide did not affect Large-scale gene-expression profiling transfection efficiency. Inhibition of luciferase activity was Microarrays printed with the mouse Compugen oligonucleo- achieved at 1 mgmlÀ1. Pre-treatment was found to be essential tide library were manufactured by the IMB SRC Microarray for full inhibition, with 2 h being sufficient (data not shown). Facility, and contained 21 013 elements. RNA harvesting, Ethanol alone acted as a solvent control. preparation and labelling steps were performed in parallel for Shh stimulated and pair-matched controls. Hybridizations were performed as described previously, using balanced dye- Use of pathway antagonists swap designs (Ingram et al., 2002). Six chips were hybridized DAPT, in dimethyl sulfoxide, was obtained from Calbiochem with samples derived from transfections with three indepen- (San Diego, CA, USA). Cyclopamine (Merck, Whitehouse dent sets of plasmid preparations. RNA was harvested 4 days Station, NJ, USA)was dissolved in methanol. Both drugs were post-transfection. Spot intensities were quantified using used at 10 mM for inhibition studies. Solvent alone was used for ImaGene (BioDiscovery, Marina del Rey, CA, USA). Analysis negative controls. All transfections involving dnRBP-Jk was performed using Excel (Microsoft, Redmond, WA, USA), contained 75% pMIKneo-RBP(R218H)/empty pMIKneo, after Lowess normalization in GeneSpring (Silicon Genetics, with the remaining 25% of the DNA amount made up of Redwood City, CA, USA). Genes with significant signal on pGA981-6, pcDNA3-ICTX or corresponding empty vectors as one or both channels were distinguished from non-informative appropriate. genes using a threshold two standard deviations above the average local background signal, in accordance with Hegde Acknowledgements et al. (2000)and Li et al. (2002). Signal strength on a channel had to be above this threshold on at least four replicate chips The following people kindly provided plasmids: Dr J Aster to be considered informative. Genes were considered differen- (pcDNA3-DE and pcDNA3-ICTX), Dr J Kitajewski tially regulated if the geometric mean of normalized ratios for (pGA981-6), Dr U Lendahl (pMIKneo-RBP(R218H)) and induction or repression was above 1.5-fold. Dr D Solecki (pGL2-hHes1-prom-850). The MNS70 line was a gift of Dr M Nakafuku and Dr H Sasaki. D Ingram wrote Northern blotting and quantitative real-time RT–PCR software for the automated batch handling of microarray data Northern blotting was performed as described previously files and provided helpful advice. Supported by the National (Ingram et al., 2002). Probes to genes of interest were designed Health and Medical Research Council of Australia, and the to the 30-UTR (or where not possible to a unique region of the ARC Centre for Functional and Applied Genomics.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene