Volume 10 Number 1 January 2008 pp. 89–98 89 www.neoplasia.com\

RESEARCH ARTICLE

An Integrated Approach Identifies Enrico De Smaele*, Caterina Fragomeli*,3, Elisabetta Ferretti*,3, Marianna Pelloni*,3, Nhlh1 and Insm1 as Sonic Agnese Po*, Gianluca Canettieri*, Sonia Coni*, Lucia Di Marcotullio*, Azzura Greco*, – † Hedgehog regulated Marta Moretti*, Concezio Di Rocco , ‡ in Developing Simona Pazzaglia , Marella Maroder*, 1,2 Isabella Screpanti*, Giuseppe Giannini* and and Alberto Gulino*,§

*Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy; †Neurosurgery Institute, Catholic University, Rome, Italy; ‡Biotechnology Unit, Ente Per Le Nuove Technologie, l'Energia e l'Ambiente (ENEA), Centro Ricerche Casaccia, Casaccia, Rome, Italy; §Neuromed Institute, Pozzilli (Isernia), Italy

Abstract Medulloblastoma (MB) is the most common malignant brain tumor of childhood arising from deregulated cerebel- lar development. (Shh) pathway plays a critical role in cerebellar development and its aberrant expression has been identified in MB. expression profiling of cerebella from 1- to 14-day-old mice unveiled a cluster of genes whose expression correlates with the levels of Hedgehog (HH) activity. From this cluster, we identified Insm1 and Nhlh1/NSCL1 as novel HH targets induced by Shh treatment in cultured progenitors. Nhlh1 promoter was found to be bound and activated by Gli1 factor. Remarkably, the expression of these genes is also upregulated in mouse and human HH–dependent MBs, suggesting that they may be either a part of the HH-induced tumorigenic process or a specific trait of HH-dependent tumor cells.

Neoplasia (2008) 10, 89–98

Introduction A major regulator of this process is the Hedgehog (HH) pathway Medulloblastoma (MB) is the most common malignant pediatric brain [7]. Sonic Hedgehog (Shh) is a glycoprotein secreted by Purkinje tumor, whose prognosis has not improved significantly in the last two cells, which binds to the transmembrane receptor Patched (Ptc) on decades, despite multimodal therapy (surgery, radiation, and chemother- apy), thus justifying the continuous effort in better characterizing the mo- Abbreviations: EGL, external granule layer; EMSA, electrophoretic mobility shift assay; lecular mechanisms involved in tumor initiation and progression [1,2]. GCP, granule cell precursor; GST, glutathione-S-transferase; HH, Hedgehog; MB, MB is commonly recognized to originate from cerebellar granule medulloblastoma; Ptc, Patched; QPCR, quantitative polymerase chain reaction; or other precursors that fail to differentiate and keep proliferating [3]. Shh, Sonic Hedgehog Address all correspondence to: Alberto Gulino, Department of Experimental Medi- Indeed, the transcriptional pattern of MB is similar to that of the cine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy. developing mouse cerebellum (5–10 days postpartum), thus support- E-mail: [email protected] ing the concept that MB is formed from immature cerebellar precur- 1This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Tele- sor cells that retain most of the undifferentiated characteristics [4]. thon grant GGP07118, the Ministry of University & Research and National Research Council, the Ministry of Health, the Center of Excellence for Biology and Molecular Cerebellar development in mammals largely occurs in the early Medicine, Pasteur Institute, Cenci Bolognetti Foundation, University La Sapienza, postnatal period. In mice, proliferation and expansion of the most Rome, and the Rome Oncogenomic Center. G. C. was supported by the Ministry external layer of the cerebellum, i.e., the external granule layer of University & Research and National Research Council, Project Rientro dei cervelli. 2This article refers to supplementary material, which is designated by Table W1 and is (EGL), start at birth and peak by 8 to 10 days postpartum (P) [5]. available online at www.neoplasia.com. Subsequently, EGL cell proliferation starts exhausting, most of the 3These authors contributed equally. cells migrate through the molecular and Purkinje cell layers, reach Received 27 September 2007; Revised 13 November 2007; Accepted 15 November 2007 their final resting state, and extend dendrites in the internal granule Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 layer until EGL disappears and differentiation is completed [6]. DOI 10.1593/neo.07891 90 Identification of HH-Dependent Genes Involved in MB De Smaele et al. Neoplasia Vol. 10, No. 1, 2008 granule cell precursors (GCPs) and, through the activation of down- periments. Data analysis was performed using Time-Course Experi- stream transcription factors of the Gli family, promotes proliferation of ment AMDA version 2.1.0 (Milan, Italy). the EGL progenitors [8]. Interestingly, at the stage of maximal GCP pro- Real-time quantitative polymerase chain reaction (QPCR) was per- liferation, HH is maximally activated, as documented by the increase of formed as previously described [13] using the ABI Prism 7900HT its target genes such as N-myc,CyclinD2(Ccnd2), and PDGFRα [4,9]. System (Applied Biosystems, Monza, Italy), with Assay-on-Demand Aberrant activation of the Shh pathway has been linked to MB reagents (Applied Biosystems). mRNA quantification was expressed formation [10,11]. Germline of Ptch1 (the Shh receptor in arbitrary units as sample/calibrator ratio or sample/mean of con- which acts as a repressor in the absence of the ligand) is responsible trols ratio. All values were normalized to endogenous controls: glycer- for the Gorlin syndrome that exhibits high MB incidence [12]. Mu- aldehyde-3-phosphate dehydrogenase, β-actin, and hypoxanthine– tations of the HH pathway negative regulators (e.g., Ptch1, SuFu, guanine phosphoribosyltransferase. and Ren/KCTD11) were also reported in sporadic MB (reviewed in the works of Wetmore _Hlt183838176[[10], Ferretti et al. [11], and Di Marcotullio et al. [13]), as well as in the upregulation of Shh Constructs and Luciferase Assays target genes, such as Gli1 [10]. Similarly, different mouse models that pGL4 and pRLTK reporters were from Promega (Madison, IL). exhibit increased rate of MB underscore the direct relationship be- pGL4-5xmutGliBS, pGL4-5xmGliBS, pGL4-3xhGliBS, and pGL4- tween Shh/Ptc1 signaling and MB [10]. Shh transcriptional targets 5xhGliBS were generated by ligation of synthetic oligos (sequences involved in the mitogenic response of mice GCPs (e.g., Ccnd2 and are shown in Figure 4A) into pGL4. pCMVGliHA was previously de- N-myc) were confirmed to be implicated in MB development scribed [13]. Flag-tagged Gli1 was cloned into pCDNA (Invitrogen, – [14,15], further highlighting the role of altered Shh signaling in MB. Carlsbad, CA) by PCR using the Not I site. pGEX GliZF was cloned To further characterize the contribution of the HH pathway in by PCR and fused in-frame in pGEX4t3 (Amersham, Milan, Italy). All cerebellar development and MB and to search for novel Shh targets constructs were verified by sequencing. potentially useful for patient stratification and/or molecular therapies For luciferase experiments, HEK293T cells were transfected with we: 1) used profiling of mouse cerebella at different Lipofectamine 2000 (Invitrogen, Milan, Italy) according to the man- ’ time points during postnatal development (P1 through P14) to iden- ufacturer s instructions. About 24 to 48 hours later, luciferase assay tify subsets of genes specifically upregulated in the time window in was performed using a dual-luciferase kit (Promega, Milan, Italy). which HH pathway is strongly activated; 2) selected several of these genes and verified if they are induced in cultured cerebellar GCPs Electrophoretic Mobility Shift Assay treated with Shh; and 3) analyzed mouse and human MB samples The double-stranded DNA fragments corresponding to the mouse for the expression levels of these genes. and human promoter regions (Figure 3A) were PCR-amplified and Through this approach, we have identified Insm1 and Nhlh1/ purified by electrophoresis and gel extraction kit (Qiagen, Milan, NSCL1 as new HH target genes that are overexpressed in MB. Italy). The canonical Gli binding sequence (Figure 3A) was produced Through the identification of functional Gli binding sites on the pro- by annealing complementary oligos. Fragments were 32P-labeled, moters of mouse and human Nhlh1, we also highlighted a direct reg- purified through G30 columns (Amersham Pharmacia, Milan, Italy), ulation of this gene by Gli transcription factors. and used for electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared from 293T cells transfected with pCDNAFlagGli1 or an empty vector. Glutathione S-transferase (GST) Materials and Methods fusion were produced in BL21 cells following isopropyl- β-D-1-thiogalactopyranoside (IPTG) induction, cell lysis, and bind- Tissue Samples, Cell Cultures, and Treatments ing to glutathione–agarose beads (Amersham Pharmacia) according to Surgical specimens of human MBs were collected with the approval manufacturer’s instructions. of the Institutional Review Board. Control human cerebella RNA were EMSA was performed according to standard procedures. Binding from Biocat (Heidelberg, Germany) and Ambion (Foster City, CA). reactions contained 20,000 counts per minute 32P-labeled probe and Mouse cerebella and MB specimens were obtained from wild-type 15 μg nuclear extract (or the indicated amount of GST fusion pro- +/− and Ptc1 CD1 mice [16]. Animal handling was according to the tein) in binding buffer. In some experiments, 2 μgofrabbitanti- guidelines of Sapienza University of Rome. Mouse GCP cultures were GLI1 (H-300; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse prepared cultured and treated as previously described [17]. Recombinant anti–FLAG-M2 (Sigma) antibodies were added to the sample. Com- Shh N-terminal peptide was from R&D Systems (Minneapolis, MN). petition experiments were performed by adding 50× or 100× molar HEK293T cells were maintained in DMEM containing 10% excess of cold oligonucleotides. Complexes were resolved on a non- FBS, glutamine, and antibiotics. 5% PAGE, dried, and exposed for autoradiography. Microarrays and Real-Time Quantitative Polymerase Chain Reaction Analysis Western Blot Analysis Total RNA from P1 to P14 mice cerebella (six mice at each time Tissue samples were lysed in standard radioimmunoprecipitation point) was used. RNA extraction, cDNA synthesis, cRNA label- assay buffer plus protease inhibitors (Roche, Mannheim, Germany). ing, hybridization, and scanning were performed according to the Lysates were separated on SDS-PAGE and immunoblotted using stan- manufacturer’s instructions (Affymetrix, Santa Clara, CA). For each dard procedures. Anti-NSCL1 (AB5698; Chemicon, Milan, Italy), of the duplicate microarray hybridization, RNA from three mice anti–β-actin, and HRP-conjugated secondary antisera (Santa Cruz of the same time point was pooled. GeneChip Murine Genome Biotechnology, Heidelberg, Germany) were used, followed by en- U74Av2 Set microarrays (Affymetrix) were used in all microarray ex- hanced chemiluminescence (ECL; Amersham). Neoplasia Vol. 10, No. 1, 2008 Identification of HH-Dependent Genes Involved in MB De Smaele et al. 91

Results Chip Murine microarray (Affymetrix). RNA samples obtained from six mice cerebella collected at each time point (P1, P2, P7, and P14) were Gene Expression Profiling of Developing Mouse Cerebellum used. Analysis of about 12,000 genes present in the microarrays allowed Quantitative analysis of Gli1 mRNA expression levels throughout us to identify several clusters of genes with diverse trends of expression mouse postnatal development shows a peak between P7 and P10 (Fig- during cerebellar development (Figure 1B and not shown). We focus ure 1A), indicating the highest HH pathway activation in this timeframe. here on the analysis of a subset of genes specifically upregulated be- Because putative HH target genes are expected to be regulated in an tween days 2 and 14, with a peak around day 7, and reduced expression overlapping period, we have analyzed gene expression in mouse cere- afterwards (Figure 1B). As expected, some of the genes that belong to bella during the first 2 weeks of development, by means of the Gene- this cluster are known targets of HH pathway (e.g., Ccnd2, PDGFRα,

Figure 1. Identification of genes whose expression mirrors Hedgehog activity during mouse cerebellar development. (A) Modulation of Gli1 expression in mouse cerebella during development. (B) Cluster of genes with peak of expression at P7, from microarrays data. (C) Expression levels of Shh target genes during cerebellar development (data from microarrays). (D) QPCR analysis on the same mRNA samples used for microarray hybridization (triplicate experiments). 92 Identification of HH-Dependent Genes Involved in MB De Smaele et al. Neoplasia Vol. 10, No. 1, 2008

N-myc,andGli1 itself [18,19]) (Figure 1C ). Table W1 shows a selec- tion in Shh-treated GCPs (Figure 2, A and D). Nhlh1 levels in con- tion of the most strongly modulated genes belonging to this cluster. trol GCPs decrease significantly throughout the time of culture (from The developmentally regulated expression of several genes (including 24 to 96 hours) (Figure 2B), suggesting that Nhlh1 expression is sus- HH targets such as Ccnd2) was further confirmed by QPCR analysis on tained by in vivo signals that are progressively lost during the in vitro the same mRNA samples used in the microarrays (representative ex- culture. One of these in vivo signals is represented by Shh: in fact, the periments in Figure 1D and not shown). Between the most strongly addition of Shh antagonizes silencing of the Nhlh1 gene in cultured upregulated genes were the transcription factors neural basic helix– GCPs, suggesting that Shh activates Nhlh1 transcription (Figure 2B). loop–helix 1 (Nhlh1) [20] and insulinoma-associated 1/IA-1 (Insm1) Moreover, Insm1 expression levels significantly increase upon Shh [21] (Figure 1D). treatment (Figure 2C ), whereas the levels of several additional genes examined, including Pax6 (Figure 2E ), Nfib,andHK2 (data not Gene Expression Analysis in Shh-Treated Cultured shown), were not significantly modulated. Cerebellar GCPs These data suggest that Nhlh1 and Insm1 are targets of the HH The main cerebellar HH target cell is the GCP, which also repre- signaling pathway. sents the most abundant cerebellar cell population. Therefore, fol- lowing the hypothesis that several genes upregulated at P7 during Nhlh1 Is a Direct Target of Gli1 Transcription Factor cerebellar development should also be HH targets, we measured ex- We next analyzed Insm1 and Nhlh1 promoter regions for the pres- pression levels in primary GCPs (from P4 mice) cultured in the pres- ence of putative Gli-responsive sites. Whereas analysis of the Insm1 ence or in the absence of recombinant Shh-N . A significant promoter did not reveal potential Gli binding sites (not shown), anal- increase in Gli1 and Ccnd2 mRNA levels confirmed the HH activa- ysis of the promoter region of mouse Nhlh1 allowed the identification

Figure 2. Shh induces transcription of Nhlh1 and Insm1.(A–D) Primary GCPs from P4 mice cerebella were cultured with or without Shh for 4 days (1d–4d). Relative levels of (A) Gli1, (B) Nhlh, (C) Insm, (D) Ccnd2, and (E) Pax6 at the different time points are represented (averages from triplicate experiments; a.u., arbitrary units). Neoplasia Vol. 10, No. 1, 2008 Identification of HH-Dependent Genes Involved in MB De Smaele et al. 93 of a potential Gli binding site, located approximately 700-bp (−702/ transfected 293T cells and a DNA probe containing the mouse pro- −690) upstream of the transcriptional start site, which was partially moter fragment spanning between −736 and −615 bp (shown in conserved also in the upstream region of the human sequence (−695/ Figure 3A). Figure 3B shows that Gli1 binds the promoter frag- −685; see Figures 3A and 4A). ment (mNhlh) and this binding is competed by either a 50× cold To verify whether Nhlh1 promoter was actually bound by Gli1, probe excess or addition of anti-Gli1 or anti-Flag antibodies. The we performed EMSA using nuclear extracts from Flag-tagged Gli1- presence of a Gli-promoter complex was confirmed by the efficient

Figure 3. Gli transcription factor binds in vitro to the putative Gli-binding sequences on the Nhlh1 promoters. (A) Mouse (1) and human (2) Nhlh1 promoter sequence containing the Gli-responsive regions (underlined). Sequence (3) is a canonical GliBS–containing fragment [22]. Double-stranded oligonucleotides containing these sequences were used in the following EMSA. (B) EMSA of the mouse Nhlh probe (P) was performed using lysates from cells transfected with Gli1–Flag–expressing vector. The shifted complex (S) is competed with a 50× excess of cold probe or by incubation with antibodies α-Gli1 or α-flag. (C) EMSA using recombinant GST–Gli (Gli1 zinc-finger fragment: aa 242–424). Lanes 1 to 8: GST–Gli binds with good affinity and in linear scale to the labeled mNhlh probe (lanes 2–5: used 2–0.2 μgofGST– Gli), whereas GST does not bind (lane 6: used 2 μg). Binding can be competed by 100× unlabeled probe (lanes 7 and 8). Lanes 9 to 13: binding of GST–Gli to a canonical GliBS (lane 9: free probe; lanes 10 and 11: binding to GST–Gli (2 and 1 μg); lane 12: 100× cold com- petition; lane 13: binding to GST alone). (D) Same experiment as in (C) lanes 1 to 8, but using the hNhlh probe. 94 Identification of HH-Dependent Genes Involved in MB De Smaele et al. Neoplasia Vol. 10, No. 1, 2008 and quantitative binding of recombinant GST-fused Gli1 zinc-finger tor, whereas activity of mutant GliBS (mutGliBS, unable to bind to the mNhlh probe (which was competed by a 100× excess cold Gli1) or the empty reporter was not modulated (Figure 4B). Simi- probe), whereas the corresponding GSTalone did not bind at all (Fig- larly, human GliBS reporters significantly enhanced luciferase tran- ure 3C ). As a positive control, binding of a canonical GliBS-containing scription in response to increasing amounts of transfected Gli1 fragment [22] to the same GST–Gli1 protein indicates a comparable (Figure 4C ). affinity of the two using the human Nhlh1 promoter probe (hNhlh: These results indicate that Nhlh is a direct target of HH signaling position −730/−607), whose complex with GST–Gli1 was competed pathway and is transcriptionally activated by Gli1. by an excess of cold probe (Figure 3D), whereas GST alone failed to bind DNA. Expression Analysis of Insm1 and Nhlh1 in MB Samples To verify whether the interaction between Gli1 and these Gli- To test whether Insm1 and Nhlh1, modulated during cerebellar binding sites was able to trigger transcription, luciferase reporter con- development, could also be relevant in MB, we evaluated their ex- − − structs containing the Gli-binding sites from either mouse (mNhlh- pression in tumors obtained from Patched1+/ mice (Ptc+/ ) in which GliBS) or human (hNhlh-GliBS) Nhlh1 promoters (Figure 4A) were GCPs develop HH-dependent MB due to the loss of the receptor used in luciferase assays. mNhlh-GliBS reporter increased luciferase Ptc, which leads to the constitutive activity of the pathway [16,23]. levels following cotransfection with increasing amounts of Gli1 vec- Confirming HH pathway activation in these tumors, all MB from − Ptc+/ animals exhibited high levels of HH activity compared to adult and P7 cerebella as documented by Gli1 (Figure 5D)andCcnd2 mRNA levels (Figure 5C ). Remarkably, we observed a dramatic in- crease of both Insm1 and Nhlh1 mRNA levels in all MBs (Figure 5, A– C), suggesting that HH-triggered expression of these genes is conserved during cerebellar tumorigenesis in vivo. The high Nhlh1 levels expressed in mouse MB samples were also mirrored by increased protein levels when compared either to normal adult control (Figure 5E )ortoprimary granule cells obtained from P4 mice cerebella (Figure 5F). Interestingly, relative levels of Nhlh1 protein samples from P2 and P7 cerebella con- trolswerehigherthaninadult,inkeepingwithmRNAexpressionlevels showninFigure1D. Next, we investigated whether the deregulated expression of Insm1 and Nhlh1 observed in mouse HH–dependent MB was also relevant in the multifactorial carcinogenesis of sporadic human MBs. To this purpose, we analyzed a set of 16 human sporadic MBs (Figure 6). Insm1 was expressed at higher levels in tumors compared to either adult (P < .05) or fetal cerebella (Figure 6A). Likewise, Nhlh1 was overexpressed in all tumor samples compared to adult (P <.005) and fetal cerebella (Figure 6B). Most MBs respond to the HH inhibitor cyclopamine with a block of proliferation, implying a certain degree of basal HH activity [19]. However, we recently reported that human sporadic MBs might be subclassified as low- or high-Gli1–expressing, indicating a different level of HH activation [17]. We have therefore subdivided our series of MBs in two subsets: MBs with Gli1 levels at least two standard deviations above the mean value of the adult cerebella (Glihigh subset) and MBs with Gli1 levels within or below the range of normal cerebella (Glilow) (Figure 6C ). We then analyzed Insm1 and Nhlh1 levels in these two subsets. Al- though Insm1 levels in the Glihigh subset were more significantly in- creased (P < .02) compared to adult cerebella, we could not correlate Gli1 and Insm1 expression levels on the whole MB population (Fig- low Figure 4. Gli-Binding sites on Nhlh are transcriptionally activated ure 6D), because of the high Insm1 levels found frequently in Gli by Gli1. (A) Comparison of a canonical Gli–binding site [22] and tumors (Figure 6, A and C ), suggesting a more complex regulation of Gli-binding sites on mouse and human Nhlh promoters (mNhlh- Insm1 in MB, in addition to HH control. Instead we found a signif- GliBS and hNhlh-GliBS, respectively). At the bottom, a mutated icant correlation between Gli1 and Nhlh1 levels (P < .02) in the two binding site (mutGliBS) was used as a negative control. (B) Activa- MB subset (Figure 6E ), consistent with a direct role of HH in the mNhlh tion of a -GliBS luciferase reporter (five copies of the se- regulation of Nhlh1 expression. quence), following cotransfection with increasing amounts (5, 20, and 50 ng) of Gli1-expressing vector. Empty reporter and mut- GliBS (five copies) are cotransfected as a control with 50 ng of Gli1 plasmid. (C) Same assay as in (B), but using hNhlh-GliBS (three or Discussion five copies) and mutGliBS as reporters (average ± SD from at least Our work identifies the developmentally regulated genes Insm1 and three experiments). Nhlh1 as novel targets of HH activity. Most relevantly, the expression Neoplasia Vol. 10, No. 1, 2008 Identification of HH-Dependent Genes Involved in MB De Smaele et al. 95

Figure 5. Increased Insm1 and Nhlh1 expression in Hedgehog-dependent mouse MB. (A–D) Insm1, Nhlh1, Ccnd2, and Gli1 mRNA ex- pression in different MB samples compared to P7 and adult cerebellum controls (average value from six cerebella). Quantitations have been performed in triplicate experiments (a.u., arbitrary units). (E–F) Nhlh1 protein levels in MBs and mouse cerebella correlate with mRNA expression. Western blot showing Nhlh1 protein levels in: (E) four mouse MB and normal adult, P2 and P7 cerebella; (F) mouse MB, GCPs and adult. β-Actin is shown as a loading control. of these genes is upregulated in both mouse and human MBs, in fied intermediate transcription factors. To date, the transcriptional which HH pathway is activated, suggesting that they may be either activation of Insm1 in neuroendocrine tissues and tumors has been a part of the HH-induced tumorigenic process or a specific trait of attributed to the formation of basic helix–loop–helix (bHLH) pro- HH-dependent tumor cells. tein heterodimers, NeuroD/E47 and neurogenin3/E47 [27], on Insm1 encodes a zinc-finger DNA-binding protein and exhibits a the E-box of Insm1 promoter [24,28]. Whether these factors are in- restricted expression pattern, including fetal brain [21,24], pancreas volved in the regulation of Insm1 by HH remains to be elucidated. [25], and neuroendocrine tumors [21,26]. Interestingly, the link between HH pathway and Insm1 we have shown We report here on the activation of Insm1 expression by HH sig- in brain tumors is consistent with the Insm1 expression in the majority of naling. The Insm1 promoter does not present Gli-binding sites, sug- small cell lung cancers [26], which also exhibit a high frequency of HH gesting that the mechanisms of activation by Shh may be due either pathway activation [29]. Furthermore, increased Insm1 had been previ- 1) to the presence of Gli-binding sites in other cis-regulatory regions ously detected in desmoplastic MB, together with N-myc, Ptc,andGli1, of the gene or 2) to indirect regulation, through some yet unidenti- which well fits with increased HH activation in this subclass of MB [30]. 96 Identification of HH-Dependent Genes Involved in MB De Smaele et al. Neoplasia Vol. 10, No. 1, 2008

The biologic functions of Insm1 are not well defined: it has been (e.g., neurogenin, NeuroD, and Math1) are expressed in neuronal cells shown that Insm1 acts as a transcriptional repressor on NeuroD and and are involved in the specification of neuronal lineages. Nhlh1 acts on Insm1 itself, generating an autoregolatory loop [31]. It was also as a transcriptional repressor through dimerization with E47 (a class I shown that Insm1 is required for the differentiation of endocrine bHLH) and interacts with the Lim-only family members LMO4 and pancreas [25,28], whereas its role in cerebellar or neuronal develop- LMO2 that modulate its activity [33]. ment has not been identified yet [25]. Likewise, the role of Insm1 in So far, analysis of Nhlh1 transcripts [20,34] or use of lacZ reporter MB needs to be clarified. The regulatory loop between Insm1 and [35] indicate that Nhlh1 expression is confined to the nervous sys- NeuroD, a factor important in the differentiation and survival of tem. Nhlh1 expression has been observed in both the embryonic and postmitotic cerebellar granule cells, may explain, in part, its role in postnatal developing cerebellum. Between E14.5 and E18.5, Nhlh1 neuronal development and tumorigenesis [32]. expression is restricted to migrating GCP of the cerebellar epitheli- As for the second HH target Nhlh1, this gene was originally cloned um, which originates from the rhombic lip to form the EGL [35,36]. because of its homology within the bHLH motif to the oncogenic he- Postnatally, Nhlh1 is expressed in the premigratory zone of the matopoietic transcription factor SCL/Tal1 [20]. Nhlh1 belongs to EGL with maximal expression between P7 and P10 [37], in agree- class II of the bHLH protein family of transcriptional regulators [27]. ment with our findings (Figure 1D). At P10, Nhlh1 transient expres- Class II bHLHs exhibit tissue-restricted expression and some of them sion is also detected in the newly formed internal granule layer [35],

Figure 6. (A–C) Insm1, Nhlh, and Gli1 expression levels in highGli and lowGli human MBs compared to fetal and adult control cerebellar tissue. (D–E) Regression curves for Insm1 (no correlation) and Nhlh1 (P < .05) relative to Gli1. Neoplasia Vol. 10, No. 1, 2008 Identification of HH-Dependent Genes Involved in MB De Smaele et al. 97 suggesting that it may be involved in GCP proliferation and in the [7] Fuccillo M, Joyner AL, and Fishell G (2006). Morphogen to mitogen: the mul- onset of differentiation, but not in the maintenance of the differen- tiple roles of hedgehog signalling in vertebrate neural development. Nat Rev Neurosci 7 (10), 772–783. tiated state [36]. [8] Ruiz i Altaba A, Palma V, and Dahmane N (2002). Hedgehog–Gli signalling Such a role at the beginning of the differentiation process is also and the growth of the brain. Nat Rev Neurosci 3 (1), 24–33. supported by the expression pattern of Nhlh1 in early differentiating [9] Kenney AM, Cole MD, and Rowitch DH (2003). Nmyc upregulation by sonic embryonic , where Nhlh1 is expressed in cells that have just hedgehog signaling promotes proliferation in developing cerebellar granule neu- – become postmitotic [34]. Interestingly, expression of Nhlh1 in postmi- ron precursors. Development 130 (1), 15 28. [10] Wetmore C (2003). Sonic hedgehog in normal and neoplastic proliferation: in- totic neurons has to be downregulated when the cells have migrated to sight gained from human tumors and animal models. Curr Opin Genet Dev 13 their final position to allow them to become differentiated [34]. (1), 34–42. The identification of Nhlh1 as a novel target of HH pathway, un- [11] Ferretti E, De Smaele E, Di Marcotullio L, Screpanti I, and Gulino A (2005). derscores its potential role in the activity of Shh. HH signaling plays Hedgehog checkpoints in medulloblastoma: the 17p par- – a complex role in driving the development of lineage-committed cer- adigm. Trends Mol Med 11 (12), 537 545. [12] Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram ebellar GCPs, being mostly responsible for the expansion of an early A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, et al. (1996). of pool of progenitor cells, which, subsequently, terminally differentiate the human homolog of Drosophila patched in the nevoid basal cell carcinoma syn- into postmitotic mature granule cells. HH window of activity is over- drome. Cell 85 (6), 841–851. lapping with the expression of Nhlh1, suggesting that this regulatory [13] Di Marcotullio L, Ferretti E, De Smaele E, Argenti B, Mincione C, Zazzeroni F, loop might be involved in the transition from proliferation to early Gallo R, Masuelli L, Napolitano M, Maroder M, et al. (2004). REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma. differentiation of progenitor cells. Proc Natl Acad Sci USA 101 (29), 10833–10838. Whether this process may be relevant for cerebellar tumorigenesis, [14] Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, Lin SM, which stems from disequilibrium between proliferation and differen- Wickramasinghe R, Scott MP, and Wechsler-Reya RJ (2003). Transcriptional tiation of GCP, is an outstanding question that needs to be appropri- profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation – ately addressed. We have described a deregulated expression of Nhlh1 of neuronal precursors. Proc Natl Acad Sci USA 100 (12), 7331 7336. [15] Park PC, Taylor MD, Mainprize TG, Becker LE, Ho M, Dura WT, Squire J, in both human and murine MB, with increased levels of both and Rutka JT (2003). Transcriptional profiling of medulloblastoma in children. mRNA and protein. Furthermore, we show that Nhlh1 expression J Neurosurg 99 (3), 534–541. correlates with the strength of HH pathway activity. Nhlh1 expres- [16] Pazzaglia S, Mancuso M, Atkinson MJ, Tanori M, Rebessi S, Majo VD, Covelli V, sion may be a feature of HH-dependent tumors in which a deregu- Hahn H, and Saran A (2002). High incidence of medulloblastoma following X-ray– – lation of the signals sustaining the transition from proliferation irradiation of newborn Ptc1 heterozygous mice. Oncogene 21 (49), 7580 7584. [17] Ferretti E, Di Marcotullio L, Gessi M, Mattei T, Greco A, Po A, De Smaele E, toward differentiation of GCP would have occurred during the tu- Giangaspero F, Riccardi R, Di Rocco C, et al. (2006). Alternative splicing of the morigenic process. These may speculatively include uncoordinated ErbB-4 cytoplasmic domain and its regulation by hedgehog signaling identify activation versus silencing of Nhlh1 at the proliferation/differentiation distinct medulloblastoma subsets. 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Symbol Description Ratio P7/P2 Ratio P14/P7

Transcription Factors Nhlh1 Nescient helix–loop–helix 1 19.39 0.13 Insm1 Insulinoma-associated 1 10.96 0.19 Myb Myeloblastosis oncogene 5.64 0.13 Nfib Nuclear factor I/B 3.90 0.44 Mycn v-myc myelocytomatosis viral–related oncogene, neuroblastoma-derived (avian) 3.78 0.14 Lhx1 LIM homeobox protein 1 3.41 0.17 Pax6 Paired box gene 6 3.36 0.43 Tead2 TEA domain family member 2 3.20 0.25 Gli2 GLI-Kruppel family member GLI2 2.82 0.32 Sox18 SRY-box containing gene 18 2.82 0.36 E2f8 E2F transcription factor 8 2.68 0.35 Trp53 Transformation-related protein 53 2.64 0.27 Cell cycle, cell division, repair, DNA replication, and proliferation Ccna2 Cyclin A2 8.91 0.10 Ccnb1 Cyclin B1 8.70 0.15 Mki67 Antigen identified by monoclonal antibody Ki 67 8.26 0.18 Cks2 CDC28 protein kinase regulatory subunit 2 8.12 0.09 Aspm Asp (abnormal spindle)-like, –associated (Drosophila) 7.48 0.13 Nusap1 Nucleolar and spindle-associated protein 1 7.17 0.11 Cdc20 Cell division cycle 20 homolog (Saccharomyces cerevisiae) 6.89 0.11 Ect2 ect2 oncogene 6.82 0.14 Cdca7 Cell division cycle associated 7 6.44 0.16 Bub1 Budding uninhibited by benzimidazoles 1 homolog (S. cerevisiae) 6.36 0.14 Cdc6 Cell division cycle 6 homolog (S. cerevisiae) 6.36 0.14 Cdca8 Cell division cycle–associated 8 6.05 0.17 Pold1 Polymerase (DNA-directed), delta 1, catalytic subunit 5.55 0.18 Ccnb2 Cyclin B2 5.17 0.15 Cdc2a Cell division cycle 2 homolog A (Shizosaccharomyces pombe) 5.03 0.14 Uhrf1 Ubiquitin-like, containing PHD and RING finger domains, 1 4.89 0.17 Nek2 NIMA (never in mitosis gene a)–related expressed kinase 2 4.87 0.27 Cdk2ap1 CDK2 (cyclin-dependent kinase 2)–associated protein 1 4.70 0.49 Cdca7l Cell division cycle–associated 7 like 4.12 0.22 Fen1 Flap structure–specific endonuclease 1 4.07 0.25 Mad2l1 MAD2 (mitotic arrest-deficient, homolog)–like 1 (yeast) 3.49 0.25 Ccnf Cyclin F 3.45 0.29 Cdc25c Cell division cycle 25 homolog C (S. cerevisiae) 3.38 0.28 Rfc4 Replication factor C (activator 1) 4 3.14 0.23 Rad51ap1 RAD51-associated protein 1 3.09 0.28 Brca1 Breast cancer 1 2.64 0.38 Xrcc5 X-ray repair- complementing defective repair in Chinese hamster cells 5 2.61 0.58 Cks1b CDC28 protein kinase 1b 2.56 0.16 Pcna Proliferating cell nuclear antigen 2.54 0.36 Cell adhesion, extracellular matrix, junctions, cell migration, and axon guidance Mfap4 Microfibrillar-associated protein 4 9.57 0.11 Anln Anillin, actin binding protein (scraps homolog, Drosophila) 7.42 0.37 Efs Embryonal Fyn–associated substrate 5.34 0.34 Plxnb2 Plexin B2 3.74 0.18 Cytoskeleton and cytoskeleton binding Kif2c Kinesin family member 2C 6.79 0.14 Kif11 Kinesin family member 11 5.84 0.16 Kif20a Kinesin family member 20A 5.41 0.20 Tnnt1 Troponin T1, skeletal, slow 4.95 0.51 Birc5 Baculoviral IAP repeat–containing 5 4.90 0.18 Kif4 Kinesin family member 4 4.35 0.22 Kif23 Kinesin family member 23 4.07 0.22 Vil2 Villin 2 3.74 0.40 Myl1 Myosin, light polypeptide 1 3.52 0.27 Kif22 Kinesin family member 22 2.86 0.41 Cell metabolism Hk2 Hexokinase 2 8.16 0.29 Tyms Thymidylate synthase 5.93 0.16 Klk1b1 Kallikrein 1–related peptidase b1 4.38 0.27 Tk1 Thymidine kinase 1 3.72 0.22 Usp1 Ubiquitin-specific peptdiase 1 3.60 0.34 Rrm2 Ribonucleotide reductase M2 3.52 0.15 Ttk Ttk protein kinase 3.39 0.29 Ddah2 Dimethylarginine dimethylaminohydrolase 2 3.07 0.50 Klk1 Kallikrein 1 2.90 0.36 Table W1. (continued ) Symbol Description Ratio P7/P2 Ratio P14/P7

RNA metabolism, transcription machinery, and polymerase Mcm3 Minichromosome maintenance–deficient 3 (S. cerevisiae) 6.28 0.12 Tgif TG interacting factor 4.86 0.17 Hmgb2 High mobility group box 2 4.61 0.16 Fignl1 Fidgetin-like 1 4.36 0.11 Ezh2 Enhancer of zeste homolog 2 (Drosophila) 4.35 0.25 Mcm5 Minichromosome maintenance–deficient 5, cell division cycle 46 (S. cerevisiae) 4.01 0.17 Mcm7 Minichromosome maintenance–deficient 7 (S. cerevisiae) 3.97 0.22 Mcm4 Minichromosome maintenance–deficient 4 homolog (S. cerevisiae) 3.55 0.25 Mcm2 Minichromosome maintenance–deficient 2 mitotin (S. cerevisiae) 3.37 0.25 Pola1 Polymerase (DNA-directed), alpha 1 3.07 0.26 Nt5dc2 5′-Nucleotidase domain containing 2 3.01 0.30 Mcm10 Minichromosome maintenance–deficient 10 (S. cerevisiae) 2.94 0.35 Nsbp1 Nucleosome binding protein 1 2.71 0.26 Recc1 Replication factor C 1 2.62 0.59 Sfrs1 Splicing factor, arginine/serine–rich 1 (ASF/SF2) 2.60 0.20 Solt SoxLZ/Sox6 leucine zipper binding protein in testis 2.52 0.36 Signal transduction and kinases Csnk2a1 Casein kinase II, alpha 1 polypeptide 4.42 0.29 Map3k1 Mitogen-activated protein kinase kinase kinase 1 4.39 0.36 Aurka Aurora kinase A 4.27 0.22 Prkcn Protein kinase C, nu 3.96 0.35 Rnd2 Rho family GTPase 2 3.28 0.49 Rab3d RAB3D, member RAS oncogene family 3.27 0.49 Sstr2 Somatostatin receptor 2 3.23 0.31 Plk1 Polo-like kinase 1 (Drosophila) 3.08 0.35 Plk4 Polo-like kinase 4 (Drosophila) 2.89 0.22 Membrane structure and functions, receptors and ligands, secretion, and ion channels Igfbpl1 Insulin-like growth factor binding protein–like 1 9.07 0.20 Sema7a Sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A 8.81 0.45 Sstr2 Somatostatin receptor 2 6.98 0.15 Slc29a1 Solute carrier family 29 (nucleoside transporters), member 1 6.27 0.36 Tmem123 Transmembrane protein 123 4.78 0.58 Vps37b Vacuolar protein sorting 37B (yeast) 4.61 0.49 Plp2 Proteolipid protein 2 4.19 0.17 Tmpo Thymopoietin 3.95 0.06 Kcne1l Potassium voltage-gated channel, Isk-related family, member 1–like 3.45 0.35 Lbr Lamin B receptor 3.40 0.50 Clic1 Chloride intracellular channel 1 3.17 0.34 Cxcr4 Chemokine (C-X-C motif) receptor 4 2.74 0.28 Cd63 Cd63 antigen 2.63 0.42 WNT–Shh pathways Sfrp1 Secreted frizzled–related sequence protein 1 5.39 0.21 Fzd2 Frizzled homolog 2 (Drosophila) 3.12 0.21 Nuclear protein Smc2l1 SMC2 structural maintenance of 2–like 1 (yeast) 5.99 0.17 Nde1 Nuclear distribution gene E homolog 1 (Aspergillus nidulans) 5.93 0.40 Smc4l1 SMC4 structural maintenance of chromosomes 4–like 1 (yeast) 5.81 0.21 Top2a Topoisomerase (DNA) II alpha 5.74 0.13 Lmnb1 Lamin B1 5.38 0.06 Smarca5 SWI/SNF–related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 2.95 0.36 H2afv H2A histone family, member V 2.94 0.48 C79407 Expressed sequence C79407 2.83 0.41 Rad51 RAD51 homolog (S. cerevisiae) 2.75 0.35 Others AI506816 Expressed sequence AI506816 5.99 0.15 Prc1 Protein regulator of cytokinesis 1 5.15 0.15 Spata13 Spermatogenesis-associated 13 5.01 0.55 Trim59 Tripartite motif–containing 59 4.51 0.23 Dtl Denticleless homolog (Drosophila) 4.35 0.25 Tera Teratocarcinoma-expressed, serine-rich 3.38 0.26 Anp32e Acidic (leucine-rich) nuclear phosphoprotein 32 family, member E 3.31 0.28 Racgap1 Rac GTPase–activating protein 1 3.01 0.20 Nedd1 Neural precursor cell expressed, developmentally downregulated gene 1 2.96 0.33

Ratios between signal at P7 and signal at P2 and P14/P7 are indicated. Numbers are averages from duplicate experiments. Only genes modulated more than 2.5-fold are shown.