Abstract We have recently generated a novel medulloblastoma (MB) mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1^+/−/Tis21^KO). Its main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs). By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1^+/−/Tis21 wild-type vs. Ptch1^+/−/Tis21 knockout), among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets. The data analysis using bioinformatic tools revealed: (i) a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype, i.e., the neural cell type involved in group 3 MB; (iii) the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs. Keywords: medulloblastoma model, cerebellar precursor cell, drug target, Sonic Hedgehog, primary cilium, neural migration, retina, chemokines Introduction About 30% of medulloblastomas (MBs), the pediatric tumor of the cerebellum, originates from the granule neuron precursor cells (GCPs) located in the external granular layer (EGL), at the surface of the developing cerebellum, in consequence of hyperactivation of the Sonic Hedgehog (Shh) pathway (Kadin et al., [31]1970; Schüller et al., [32]2008; Yang et al., [33]2008; Gibson et al., [34]2010; Northcott et al., [35]2012). Other MB subtypes may originate from neural precursors of the cerebellar embryonic anlage, different from GCPs and dependent on Wnt signaling, or from GCPs with activation of different pathways (group 3), or also from neural precursors of unknown origin (group 4; Northcott et al., [36]2012). GCPs intensely proliferate postnatally in the EGL, before exiting the cell cycle and migrating inward to form the mature internal granular layer (IGL; Hatten, [37]1999). GCPs in the EGL are forced to divide by the proliferative molecule Shh, secreted by Purkinje neurons (Dahmane and Ruiz i Altaba, [38]1999; Wallace, [39]1999; Wechsler-Reya and Scott, [40]1999). It is believed that the prolonged mitotic activity of the GCPs, consequent to hyperactivation of the Shh pathway, makes them potential targets of transforming insults (Wang and Zoghbi, [41]2001). We have previously shown that mice lacking one allele of Ptch1, which develop MB with low frequency as result of the activation of the Shh pathway (Hahn et al., [42]1998), when crossed with mice knockout for the MB suppressor Tis21 develop MB with very high frequency (Farioli-Vecchioli et al., [43]2012a,[44]b). We identified as responsible for this effect a defect of migration of the GCPs that, remaining for a longer period in the EGL under the proliferative influence of Shh, developed tumor more frequently. Whole-genome analyses of expression and function indicated that the key molecule responsible for the lack of migration of GCPs is the chemokine Cxcl3 (Farioli-Vecchioli et al., [45]2012a). Together with Cxcl3, we identified other 187 gene sequences, 163 of which have a functional product, whose expression in double mutant Ptch1 heterozygous/Tis21 knockout mice was modified, relative to Ptch1 heterozygous mice in Tis21 wild-type background (single mutants; Farioli-Vecchioli et al., [46]2012a). The set of genes whose expression significantly differs in the comparison Ptch1^+/−/Tis21 wild-type vs. Ptch1^+/−/Tis21^KO will be hereafter defined as Set A (Figure [47]1). Figure 1. [48]Figure 1 [49]Open in a new tab A Venn diagram showing four genotype pairwise comparisons and the intersection of their differentially expressed gene/sequences set A–D. Set A corresponds to the pairwise comparison Ptch1^+/−/Tis21^KO vs. Ptch1^+/−/Tis21^+/+; Set B refers to Ptch1^+/−/Tis21^+/+ vs. wild type; Set C concerns Ptch1^+/−/Tis21^KO vs. Ptch1^+/+/Tis21^KO; Set D represents the double-knockout contribution in background wild type. Here, we aimed to expand the functional investigation of the previous whole-genome analysis of gene expression alterations occurring at the onset of tumorigenesis in the GCPs, in order to further examine the set of genes whose expression is modified in Ptch1 heterozygous/Tis21 knockout double mutant mice relative to Ptch1 heterozygous/Tis21 wild-type mice (Set A). Given that Tis21 mutation has a strong tumorigenic effect in Ptch1 heterozygous background, with a high increase of MB frequency, we assumed that the transcriptional changes occurring in the Set A of 163 genes after Tis21 ablation in Ptch1 background were at the origin of the increased tumorigenicity observed. These genes, referred to as Tis21-dependent—given that their expression is by definition modified by the ablation of Tis21 in Ptch1 heterozygous background—will be divided in up-regulated and down-regulated, relative to Ptch1 heterozygous/Tis21 wild-type mice. It is worth noting that among the genes in Set A whose expression is down-regulated abound those with tumor-inhibitory activity (e.g., Pag1, PadI4, Lats2, and Cxcl3), while among the up-regulated genes are present tumor facilitators (e.g., Rab18, Dek). Phenotypically, our genomic data refer to GCPs at a very early pre-neoplastic stage, having been isolated from 7 day-old mice, i.e., when the neoplastic lesions have not yet emerged. The data analyzed interestingly lead to: (i) a link between the Shh signaling and the impairment of the GCPs migration, through a Shh-dependent deregulation of the receptor-mediated endocytosis pathway; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype/toward the neural cell type involved in group 3 MB; (iii) the identification of a subset of putative drug targets for MB involved in the regulation of cell cycle, Pdgf, Rapamycin target protein 1 and Hippo signaling pathways. Finally, our findings indicate a role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms. Materials and methods Gene expression array Genome-wide expression study design and experimental procedures, of GCPs isolated from the EGL of P7, mice were previously performed with Whole Mouse Genome Microarrays (Agilent Technologies), as described in Farioli-Vecchioli et al. ([50]2012a). GCPs were isolated from Ptch1 heterozygous/Tis21 knockout double mutant and Ptch1 heterozygous/Tis21 wild-type mice of either sex (Farioli-Vecchioli et al., [51]2012a). In order to extract the mRNA from GCPs for microarray analysis, for each of the four genotypes were used 4 replicates of GCPs isolated from 3-4 mice each, for a total of about 64 mice (Farioli-Vecchioli et al., [52]2012a). The experiments and all animal procedures were completed in accordance with the current European (directive 2010/63/EU) Ethical Committee guidelines and approved by the Ethical Committee of the Italian Ministry of Health (authorized protocol number 14/2009 dated 14/12/2009, expiry date 14/12/2012, according to Law Decree 116/92). Experiments performed after 14/12/2012 are authorized by the Ethical Committee of the Italian Ministry of Health by protocols 307/2013-B and 193/2015-PR expiring 30/03/2020. Functional data analysis Raw data from microarrays experiments were processed and analyzed using GeneSpringGX 12.5 (Agilent Technologies), as already described (Farioli-Vecchioli et al., [53]2012a). Pathway enrichment analysis of Set B and Set D genes was performed with MetaCore™ by Thomson Reuters (Ekins et al., [54]2007). Pathways with corrected enrichment p-value p < 0.05 were considered significant. MetaCore™ integrated software for functional analysis and its manually curated database have also been used for functional annotation of Tis21-dependent Set A genes, together with DAVID Bioinformatics Resources version 6.7 public database by the National Center for Biotechnology Information (NIH) (Huang da et al., [55]2009a,[56]b), Mouse Genome Database (MGD) by The Jackson Laboratory, Bar Harbor, Maine (Blake et al., [57]2014), Cerebellar Development Transcriptome Data Base (CDT-DB) by NIJC, RIKEN-BSI, Japan (Sato et al., [58]2008) and Universal Protein Resource (UniProt) by the UniProt Consortium (Consortium, [59]2014). Drug target analysis To identify potential drug targets among the Set A differentially expressed genes, we have used the drug target selection tool via gene list analysis by MetaCore™ (Thomson Reuters) (Ekins et al., [60]2007) and Thomson Reuters Cortellis Drug Viewer tool (also available on MetaCore™ platform) via pathway analysis. The search has been performed among human primary/direct (Table [61]3) and secondary/indirect (Table [62]4) drug targets (see OrthoDB Kriventseva et al., [63]2015, for the comparison between human and mouse orthologs). The drugs have been taken into account for their targets and not for their use, so not only anti-neoplastic agents are listed in Tables [64]3–[65]6. Cortellis™ drugs results were compared with records contained in public databases such as DrugBank version 4.2 (Knox et al., [66]2011; Law et al., [67]2014), PubChem Compound by NIH (Bolton et al., [68]2010) and Naturally Occurring Plant based Anticancerous Compound-Activity-Target (Mangal et al., [69]2013). Finally, to further annotate Set A list genes with respect to known drug-gene interactions and potential druggability, in both mouse and human, we have used the search tools on The Drug Gene Interaction Database (DGIdb) (Griffith et al., [70]2013) via gene list (Figure [71]3, Tables [72]4, [73]5). Table 3. A list containing the direct targets and their drugs obtained using MetaCore™ (i.e., ATP1A1) and Cortellis™ db. MetaCore™/Cortellis™ Therapeutic Drug-target direct Interactions Gene Symbol Target Drug Effect Pubmed ATP1A1 ATP1A1 Trichlormethiazide intracellular Inhibition Istaroxime Inhibition 12904068, 12388640, 17239702 HSD11B2 11 beta-Hydroxysteroid dehydrogenase Enoxolone Inhibition 8504732, 16012754, 16309197, 16391818, 17566745, 18721818, 18600055, 19949310, 24613819 H19 H19 BC-822 Inhibition PDGF PDGF CR002 Inhibition 17308324, 16510766, 12937299 AGTR2 AGTR2 XR-510 Inhibition PD-123319 Inhibition MP-157 Inhibition EMA401 Inhibition 23489258 ACACA Acetyl-CoA carboxylase 1 isoform 1 CP-640186 Inhibition 12842871, 15341732, 23358327, 15934915 CP-610431 Inhibition 12842871 SRPK2 SRPK2 SRPIN340 Inhibition 16840555, PMC2916360 GBF1 GBF1 Brefeldin A Inhibition 3192548, PMC160661, 2004424 [74]Open in a new tab The Cortellis™ drugs list has been compared and expanded using others public databases as described in Materials and Methods section. Table 4. A list containing the indirect drug-target interactions obtained using MetaCore™. MetaCore™Secondary Drug-target Interactions Drug Gene Symbol Target Effect Pubmed Lestaurtinib extracellular region CDC42BPB CDC42BPB Inhibition NLK NLK Inhibition LATS2 LATS2 Inhibition FRK FRK Inhibition TAOK2 TAO2 Inhibition SRPK2 SRPK2 Inhibition SIK2 QIK Inhibition Midostaurin intracellular LATS2 LATS2 Unspecified 19654408 FRK FRK Unspecified 19654408 SRPK2 SRPK2 Unspecified 19654408 SIK2 QIK Unspecified 19654408 Ca('2+) cytosol DGKB Diacylglycerol kinase, beta Activation 9003401, 16288460, 10206945, 12832407 PADI4 PAD4 Activation 11798170, 15247907 DGKA DGK-alpha Activation 9003401, 16288460, 10206945, 12832407 DGKG Diacylglycerol kinase, gamma Activation 9003401, 16288460, 10206945, 12832407 VDAC1 VDAC 1 Activation 12022949, 16597621 Estradiol extracellular region BTG2 BTG2 Inhibition 12959972 RGS5 RGS5 Inhibition 17374850 CXCL12 SDF-1 Activation 17362937, 17555868 HSD11B2 HSD11B2 Inhibition 9623596, 10503719 Dabrafenib intracellular FRK FRK Inhibition TAOK2 TAO2 Inhibition SIK2 QIK Inhibition Vandetanib intracellular CDC42BPB CDC42BPB Unspecified 22037378 FRK FRK Unspecified 22037378 SIK2 QIK Inhibition 22037377, 22037378 Tozasertib intracellular TAOK2 TAO2 Unspecified 22037378 SRPK2 SRPK2 Unspecified 18183025,22037378 SIK2 QIK Unspecified 18183025,22037378 Retinoic acid extracellular region PADI4 PAD4 Activation 10488123 ADAMTS5 Aggrecanase-2 Activation 17255106 Uprosertib intracellular NLK NLK Inhibition FRK FRK Inhibition Dexamethasone extracellular region SLC25A15 SLC25A15 (ORC1) Activation 12055339, 11997094 HSD11B2 HSD11B2 Activation 16872738 Zn('2+) nucleus ZNF286A ZNF286A Unspecified 11347906 BSN Bassoon Unspecified 9806829, 9679147, 9679147 Pazopanib extracellular region FRK FRK Unspecified 22037378 TAOK2 TAO2 Unspecified 22037378 Masitinib intracellular FRK FRK Inhibition 22037378 Pyridoxal Phosphate intracellular SLC25A15 SLC25A15 (ORC1) Inhibition 12807890 ACACA ACACA Inhibition 16249179 Fedratinib intracellular TAOK2 TAO2 Inhibition SIK2 QIK Inhibition Nilotinib intracellular CDC42BPB CDC42BPB Unspecified 22037378 FRK FRK Inhibition 22037378 Epigallocatechin-3-gallate extracellular region CXCL1 GRO-1 Inhibition TAK-901 intracellular FRK FRK Inhibition SIK2 QIK Inhibition INK128 intracellular FRK FRK Inhibition SIK2 QIK Inhibition Ibrutinib intracellular FRK FRK Inhibition Zn('2+) extracellular region ADAMTS10 ADAM-TS10 Activation 11867212 ADAMTS5 Aggrecanase-2 Activation 11867212 TG100115 intracellular LATS2 LATS2 Unspecified Perphenazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Pamapimod intracellular NLK NLK Unspecified Eprosartan extracellular region AGTR2 AGTR2 Inhibition 2016730, 8515425 Aclarubicin intracellular TIMP1 TIMP1 Activation 12118337 Promazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Phenoxybenzamine extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Carbenoxolone intracellular HSD11B2 HSD11B2 Inhibition 10566667, 10664531, 12190302 Anisindione intracellular ACACA ACACA Unspecified 4067989 Maprotiline extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Irbesartan extracellular region AGTR2 AGTR2 Inhibition 2329553 Prazepine extracellular region ATP1A1 ATP1A1 Inhibition 2576298 lorcainide extracellular region ATP1A1 ATP1A1 Inhibition 10593659 Pirfenidone intracellular TIMP1 TIMP1 Inhibition 17086734 NO extracellular region SLC6A6 SLC6A6 Activation 11698241, 14992266, 15166008 Taurine extracellular region SLC6A6 SLC6A6 Inhibition 9130593, 10936171, 12437590 Fenoprofen intracellular ACACA ACACA Inhibition 1347398 Meloxicam extracellular region TIMP1 TIMP1 Inhibition 16536903 Promethazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Biotin intracellular ACACA ACACA Activation 12421859, 14749229, 15539280 Furosemide intracellular HSD11B2 HSD11B2 Inhibition 9724039 Dimethyl sulfoxide intracellular PADI4 PAD4 Activation 10488123 Thiram intracellular HSD11B2 HSD11B2 Inhibition 12901862 Disopyramide extracellular region ATP1A1 ATP1A1 Inhibition 10593659 Fluoxymesterone intracellular HSD11B2 HSD11B2 Inhibition Digitoxin extracellular region ATP1A1 ATP1A1 Inhibition 11525233 Methylene blue intracellular GUCY1A3 GUCY1A3 Inhibition 12031394, 12729841 Heparin extracellular region CXCL12 SDF-1 Inhibition 10570220, 15741341, 17264079, 17726466 Atenolol extracellular region ATP1A1 ATP1A1 Inhibition 11201505 Hg('2+) extracellular region SLC25A15 SLC25A15 (ORC1) Inhibition 12807890 Procainamide extracellular region ATP1A1 ATP1A1 Inhibition 10593659 Trazodone extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Nicotinic acid intracellular ACACA ACACA Inhibition 8857918, 10195935, 11171611, 12873710, 15030302 Imatinib intracellular FRK FRK Unspecified 15711537, 18183025, 22037378 Sotalol extracellular region ATP1A1 ATP1A1 Unspecified 11201505 Tocainide extracellular region ATP1A1 ATP1A1 Inhibition TRV-120027 extracellular region AGTR2 AGTR2 Unspecified Ruxolitinib intracellular TAOK2 TAO2 Unspecified 22037378 Ruboxistaurin intracellular LATS2 LATS2 Unspecified 22037378 Cisplatin extracellular region ATP1A1 ATP1A1 Inhibition Trifluoperazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Doxycycline intracellular TIMP1 TIMP1 Activation 20874665, 17122880, 17145119, 19442784 Losartan extracellular region AGTR2 AGTR2 Inhibition 1469703, 1875348, 1920360, 7473594, 7562905, 7606390, 7990105, 8064808, 8246245, 8277505, 8277506, 8340920, 8421274, 11310611, 11749394, 12213451, 16039125, 7990105, 14761190 Silmitasertib intracellular SRPK2 SRPK2 Inhibition Chlorpromazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 R547 intracellular TAOK2 TAO2 Unspecified 22037378 17alpha-Hydroxyprogesterone extracellular region HSD11B2 HSD11B2 Inhibition 10566667 Alprazolam extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Digoxin extracellular region ATP1A1 ATP1A1 Inhibition 11072868 CFI-400945 intracellular NLK NLK Unspecified Clotiapine extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Indenolol extracellular region ATP1A1 ATP1A1 Inhibition 1685314, 11201505 Testosterone extracellular region ACACA ACACA Activation 16579987 Aldosterone extracellular region ATP1A1 ATP1A1 Activation 16767692, 10718622 Encainide extracellular region ATP1A1 ATP1A1 Inhibition 2168576, 10593659 Regorafenib intracellular FRK FRK Inhibition Cholic acid intracellular HSD11B2 HSD11B2 Inhibition 12015312 BMS-387032 intracellular TAOK2 TAO2 Unspecified Angiotensin II extracellular region AGTR2 AGTR2 Activation 16570916, 2362273 Tivozanib intracellular FRK FRK Inhibition Clofilium extracellular region ATP1A1 ATP1A1 Inhibition 15637453 Amoxapine extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Tranilast intracellular TIMP1 TIMP1 Inhibition 16393468 Zn('2+) cytosol RBM5 RBM5 Unspecified 10352938 Androstanolone extracellular region RPS12 RPS12 Inhibition 15525599 Ketoconazole intracellular HSD11B2 HSD11B2 Inhibition 10664531 Thioridazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Retinoic acid intracellular GCNT3 GCNT3 Activation 15591039 Prochlorperazine extracellular region ATP1A1 ATP1A1 Unspecified 6167716 Latanoprost extracellular region TIMP1 TIMP1 Activation 12454040 Piperacillin intracellular LACTB LACTB Inhibition Afatinib intracellular FRK FRK Inhibition Afuresertib intracellular NLK NLK Inhibition Topiramate intracellular ACACA ACACA Activation 16415917 Nebentan extracellular region AGTR2 AGTR2 Unspecified Celiprolol intracellular ATP1A1 ATP1A1 Inhibition 1685314, 11201505 Disulfiram intracellular HSD11B2 HSD11B2 Inhibition 12901862 Cimetidine intracellular PADI4 PAD4 Unspecified Candesartan extracellular region AGTR2 AGTR2 Unspecified 16039125 Progesterone intracellular HSD11B2 HSD11B2 Inhibition 9623596, 10566667, 10664531, 12915667, 9055382 Loxapine extracellular region ATP1A1 ATP1A1 Inhibition 2576298 Nadolol extracellular region ATP1A1 ATP1A1 Inhibition 11201505 Hydrochlorothiazide intracellular HSD11B2 HSD11B2 Inhibition 10334975 Deoxycholic acid intracellular HSD11B2 HSD11B2 Inhibition 12015312 Metoprolol extracellular region ATP1A1 ATP1A1 Inhibition 11201505 D-Glucose extracellular region SLC6A6 SLC6A6 Inhibition 10516137, 12870156 Paricalcitol PTH PTH Inhibition 9697664, 16797393 [75]Open in a new tab The only exception is the record related to the PTH gene that has been taken from Cortellis Drug Viewer list and compared with others public databases, as described in Materials and Methods section. Table 6. A sub-list of Set A genes with the correspondent selected druggable gene category and source of information obtained using the Search Druggable Gene Categories tool on DGIdb, where a druggable gene category is a group of genes that are thought to be potentially druggable by various methods of prediction. DGIdB Druggable Targets Search term Gene Druggable Gene Category Sources ACACA ACACA-acetyl-CoA carboxylase alpha DRUGGABLE GENOME HopkinsGroom RussLampel DRUG RESISTANCE GO ADAMTS10 ADAMTS10-ADAM metallopeptidase with thrombospondin type 1 motif, 10 PROTEASE GO dGene NEUTRAL ZINC METALLOPEPTIDASE GO DRUGGABLE GENOME RussLampel dGene ADAMTS5 ADAMTS5-ADAM metallopeptidase with thrombospondin type 1 motif, 5 DRUGGABLE GENOME RussLampel dGene PROTEASE GO dGene NEUTRAL ZINC METALLOPEPTIDASE GO AGTR2 AGTR2-angiotensin II receptor, type 2 G PROTEIN COUPLED RECEPTOR dGene GO HopkinsGroom DRUGGABLE GENOME dGene RussLampel HopkinsGroom AKAP2 AKAP2-A kinase (PRKA) anchor protein 2 KINASE GO ATP1A1 ATP1A1-ATPase, Na/K transporting, alpha 1 polypeptide DRUGGABLE GENOME RussLampel HopkinsGroom TRANSPORTER HopkinsGroom GO ION CHANNEL HopkinsGroom ABC TRANSPORTER GO DRUG RESISTANCE GO BTG2 BTG2-BTG family, member 2 DNA REPAIR GO CDC27 CDC27-cell division cycle 27 homolog (S. cerevisiae) TUMOR SUPPRESSOR GO CDC42BPB CDC42BPB-CDC42 binding protein kinase beta (DMPK-like) DRUGGABLE GENOME RussLampel dGene HopkinsGroom SERINE THREONINE KINASE dGene GO KINASE GO HopkinsGroom CXCL1 CXCL1-chemokine (C-X-C motif) ligand 1 GROWTH FACTOR GO CXCL12 CXCL12-chemokine (C-X-C motif) ligand 12 CELL SURFACE GO EXTERNAL SIDE OF PLASMA MEMBRANE GO GROWTH FACTOR GO DAZL DAZL-deleted in azoospermia-like TUMOR SUPPRESSOR GO DEK DEK - DEK oncogene DNA REPAIR GO DGKQ DGKQ-diacylglycerol kinase, theta 110 kDa KINASE GO TRANSCRIPTION FACTOR BINDING GO DPP10 DPP10-dipeptidyl-peptidase 10 (non-functional) DRUGGABLE GENOME RussLampel HopkinsGroom dGene PROTEASE HopkinsGroom GO dGene EFNA4 EFNA4-ephrin-A4 KINASE GO TYROSINE KINASE GO ERG ERG-v-ets erythroblastosis virus E26 oncogene homolog (avian) CLINICALLY ACTIONABLE FoundationOneGenes FOXF2 FOXF2-forkhead box F2 TRANSCRIPTION FACTOR COMPLEX GO TRANSCRIPTION FACTOR BINDING GO FRK FRK-fyn-related kinase KINASE GO HopkinsGroom TYROSINE KINASE GO dGene DRUGGABLE GENOME HopkinsGroom dGene RussLampel GPR82 GPR82-G protein-coupled receptor 82 G PROTEIN COUPLED RECEPTOR dGene GO HopkinsGroom DRUGGABLE GENOME dGene RussLampel HopkinsGroom GTPBP4 GTPBP4-GTP-binding protein 4 TUMOR SUPPRESSOR GO GUCY1A3 GUCY1A3-guanylate cyclase 1, soluble, alpha 3 DRUGGABLE GENOME HopkinsGroom RussLampel HSD11B2 HSD11B2-hydroxysteroid (11-beta) dehydrogenase 2 DRUGGABLE GENOME RussLampel HopkinsGroom DRUG RESISTANCE GO SHORT CHAIN DEHYDROGENASE REDUCTASE HopkinsGroom IPO7 IPO7-importin 7 TRANSPORTER GO JMY JMY-junction mediating and regulatory protein, p53 cofactor TUMOR SUPPRESSOR GO DNA REPAIR GO LARS LARS-leucyl-tRNA synthetase DRUGGABLE GENOME HopkinsGroom RussLampel LATS2 LATS2-LATS, large tumor suppressor, homolog 2 (Drosophila) SERINE THREONINE KINASE GO dGene KINASE GO HopkinsGroom TUMOR SUPPRESSOR GO DRUGGABLE GENOME HopkinsGroom dGene RussLampel NAPEPLD NAPEPLD - N-acyl phosphatidylethanolamine phospholipase D PHOSPHOLIPASE GO NLK NLK-nemo-like kinase KINASE HopkinsGroom GO DRUGGABLE GENOME HopkinsGroom dGene RussLampel SERINE THREONINE KINASE dGene GO TRANSCRIPTION FACTOR BINDING GO PA2G4 PA2G4-proliferation-associated 2G4, 38 kDa DRUGGABLE GENOME RussLampel HopkinsGroom TUMOR SUPPRESSOR GO PROTEASE HopkinsGroom PAFAH1B1 PAFAH1B1-platelet-activating factor acetylhydrolase 1b, regulatory subunit 1 (45 kDa) TUMOR SUPPRESSOR GO PDGFD PDGFD-platelet-derived growth factor D GROWTH FACTOR GO PPP1R13L PPP1R13L-protein phosphatase 1, regulatory subunit 13 like TRANSCRIPTION FACTOR BINDING GO PTH PTH-parathyroid hormone HORMONE ACTIVITY GO RBM5 RBM5-RNA binding motif protein 5 DRUGGABLE GENOME HopkinsGroom RussLampel RIPK3 RIPK3-receptor-interacting serine-threonine kinase 3 DRUGGABLE GENOME RussLampel dGene SERINE THREONINE KINASE GO dGene KINASE GO SENP7 SENP7-SUMO1/sentrin specific peptidase 7 PROTEASE dGene GO DRUGGABLE GENOME dGene SIK2 SIK2-salt-inducible kinase 2 SERINE THREONINE KINASE GO dGene KINASE GO HopkinsGroom DRUGGABLE GENOME RussLampel HopkinsGroom dGene SLC25A15 SLC25A15-solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15 TRANSPORTER GO DRUGGABLE GENOME RussLampel SLC6A6 SLC6A6-solute carrier family 6 (neurotransmitter transporter, taurine), member 6 DRUGGABLE GENOME RussLampel HopkinsGroom TRANSPORTER GO HopkinsGroom SMG1 SMG1-smg-1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans) PHOSPHATIDYLINOSITOL 3 KINASE HopkinsGroom DRUGGABLE GENOME HopkinsGroom RussLampel dGene SERINE THREONINE KINASE dGene GO KINASE GO DNA REPAIR GO SRPK2 SRPK2-SRSF protein kinase 2 SERINE THREONINE KINASE GO dGene KINASE GO TUMOR SUPPRESSOR GO DRUGGABLE GENOME RussLampel dGene SYT11 SYT11-synaptotagmin XI TRANSPORTER GO TAF7 TAF7-TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 55 kDa TRANSCRIPTION FACTOR COMPLEX GO HISTONE MODIFICATION GO TRANSCRIPTION FACTOR BINDING GO TAOK2 TAOK2-TAO kinase 2 KINASE HopkinsGroom GO DRUGGABLE GENOME HopkinsGroom RussLampel dGene SERINE THREONINE KINASE dGene GO TUMOR SUPPRESSOR GO TIMP1 TIMP1-TIMP metallopeptidase inhibitor 1 PROTEASE INHIBITOR GO dGene DRUGGABLE GENOME dGene TOMM22 TOMM22-translocase of outer mitochondrial membrane 22 homolog (yeast) TRANSPORTER GO UPF3B UPF3B-UPF3 regulator of nonsense transcripts homolog B (yeast) TRANSPORTER GO USP36 USP36-ubiquitin specific peptidase 36 PROTEASE dGene GO DRUGGABLE GENOME dGene VDAC1 VDAC1-voltage-dependent anion channel 1 TRANSPORTER GO ION CHANNEL GO ZC3H12D ZC3H12D-zinc finger CCCH-type containing 12D TUMOR SUPPRESSOR GO [76]Open in a new tab Figure 3. [77]Figure 3 [78]Open in a new tab Drug targets belonging to the Set A discussed in the main text. Each gene product is labeled with a thermometer indicating the gene expression changes: downward thermometers have a blue color showing down-regulated expression, whereas upward thermometers have a red color showing up-regulated expression. The most part of the figure objects are deregulated also in other two pair comparisons. For this reason, the thermometer number 1 is related to the pairwise comparison Ptch1^+/−/Tis21^+/+ vs. wild type or Set B, the thermometer number 2 is related to the pairwise comparison Ptch1^+/−/Tis21^−/− vs. wild type or Set D, while the thermometer number 3 is related to the pairwise comparison Ptch1^+/−/Tis21^KO vs. Ptch1^+/−/Tis21^+/+ or Set A. See Figure [79]4 for the set of symbols, objects and interactions between objects indicated in this figure. Table 5. A drug-target interactions list and the correspondent source of information obtained using the Search Drug-Target Interactions tool on DGIdb, where a drug-gene interaction is a known interaction between a known drug compound and a target gene. DGIdB Drug-Gene Interactions Search Term Gene Drug Interaction Type Source Source Version ACACA ACACA-acetyl-CoA carboxylase alpha TOFA n/a GuideToPharmacologyInteractions 04-mar-15 BIOTIN n/a DrugBank 3 ADAMTS5 ADAMTS5-ADAM metallopeptidase with thrombospondin type 1 motif, 5 n/a DrugBank 3 n/a DrugBank 3 BATIMASTAT n/a DrugBank 3 AGTR2 AGTR2-angiotensin II receptor, type 2 [80]PD123319 n/a GuideToPharmacologyInteractions 04-mar-15 [125I][81]CGP42112 n/a GuideToPharmacologyInteractions 04-mar-15 COMPOUND 21 [PMID: 22802221] n/a GuideToPharmacologyInteractions 04-mar-15 [82]CGP42112 n/a GuideToPharmacologyInteractions 04-mar-15 ANGIOTENSIN III n/a GuideToPharmacologyInteractions 04-mar-15 ANGIOTENSIN II n/a GuideToPharmacologyInteractions 04-mar-15 NOVOKININ n/a GuideToPharmacologyInteractions 04-mar-15 ANGIOTENSIN A n/a GuideToPharmacologyInteractions 04-mar-15 CANDESARTAN CILEXETIL Antagonist TTD 4.3.02 (2011.08.25) TAK-491 Antagonist TTD 4.3.02 (2011.08.25) PS433540 Antagonist TTD 4.3.02 (2011.08.25) AZILSARTAN Antagonist TTD 4.3.02 (2011.08.25) AZILSARTAN MEDOXOMIL Antagonist TTD 4.3.02 (2011.08.25) TAK-591 Antagonist TTD 4.3.02 (2011.08.25) TASOSARTAN Antagonist DrugBank 3 ATP1A1 ATP1A1-ATPase, Na/K transporting, alpha 1 polypeptide ALMITRINE n/a TEND 01-Aug-2011 BEPRIDIL n/a TEND 01-Aug-2011 ACETYLDIGITOXIN n/a TEND 01-Aug-2011 OUABAIN n/a TEND 01-Aug-2011 DIGOXIN n/a TEND 01-Aug-2011 DIGITOXIN n/a TEND 01-Aug-2011 DESLANOSIDE n/a TEND 01-Aug-2011 ACETYLDIGITOXIN n/a GuideToPharmacologyInteractions 04-mar-15 DESLANOSIDE n/a GuideToPharmacologyInteractions 04-mar-15 DIGITOXIN n/a GuideToPharmacologyInteractions 04-mar-15 DIGOXIN n/a GuideToPharmacologyInteractions 04-mar-15 OUABAIN Binder TTD 4.3.02 (2011.08.25) LUMEFANTRINE Binder TTD 4.3.02 (2011.08.25) CICLOPIROX Binder TTD 4.3.02 (2011.08.25) ALUMINIUM Binder TTD 4.3.02 (2011.08.25) CHLOROPROCAINE Blocker TTD 4.3.02 (2011.08.25) ARTEMETHER Binder TTD 4.3.02 (2011.08.25) ALMITRINE Binder TTD 4.3.02 (2011.08.25) DESLANOSIDE Binder TTD 4.3.02 (2011.08.25) DIGITOXIN Inhibitor DrugBank 3 HYDROFLUMETHIAZIDE Other/Unknown DrugBank 3 BEPRIDIL Inhibitor DrugBank 3 DIGOXIN n/a DrugBank 3 ACETYLDIGITOXIN Inhibitor DrugBank 3 ALUMINIUM Binder DrugBank 3 MAGNESIUM n/a DrugBank 3 ETHACRYNIC ACID Inhibitor DrugBank 3 DIAZOXIDE Other/Unknown DrugBank 3 TRICHLORMETHIAZIDE Inhibitor DrugBank 3 POTASSIUM n/a DrugBank 3 DESLANOSIDE Inhibitor DrugBank 3 ALMITRINE Binder DrugBank 3 OUABAIN Inhibitor DrugBank 3 BRETYLIUM Inhibitor DrugBank 3 CEACAM3 CEACAM3-carcinoembryonic antigen-related cell adhesion molecule 3 ARCITUMOMAB Antibody TTD 4.3.02 (2011.08.25) CXCL12 CXCL12-chemokine (C-X-C motif) ligand 12 TINZAPARIN Binder DrugBank 3 DEPTOR DEPTOR - DEP domain containing MTOR-interacting protein AZD8055 Inhibitor MyCancerGenome 13-mar-13 MLN0128 Inhibitor MyCancerGenome 13-mar-13 OSI-027 Inhibitor MyCancerGenome 13-mar-13 ERLOTINIB Antagonist DrugBank 3 FRK FRK-fyn-related kinase REGORAFENIB Inhibitor MyCancerGenomeClinicalTrial 30-Feburary-2014 HSD11B2 HSD11B2-hydroxysteroid (11-beta) dehydrogenase 2 NADH n/a DrugBank 3 LARS LARS-leucyl-tRNA synthetase L-LEUCINE n/a DrugBank 3 PADI4 PADI4-peptidyl arginine deiminase, type IV N-[(1S)-1-(AMINOCARBONYL)-4-(ETHANIMIDOYLAMINO)BUTYL]BENZAMIDE n/a DrugBank 3 L-CITRULLINE n/a DrugBank 3 PDGFD PDGFD-platelet derived growth factor D SUNITINIB Inhibitor MyCancerGenomeClinicalTrial 30-Feburary-2014 PPP1R13L PPP1R13L-protein phosphatase 1, regulatory subunit 13 like CISPLATIN n/a PharmGKB 12-Jul-2012 CYCLOPHOSPHAMIDE n/a PharmGKB 12-Jul-2012 THALIDOMIDE n/a PharmGKB 12-Jul-2012 PLATINUM COMPOUNDS n/a PharmGKB 12-Jul-2012 PLATINUM n/a PharmGKB 12-Jul-2012 PTH PTH-parathyroid hormone NORLEUCINE n/a DrugBank 3 SLC25A15 SLC25A15-solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15 L-ORNITHINE n/a DrugBank 3 SRPK2 SRPK2-SRSF protein kinase 2 PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER n/a DrugBank 3 PURVALANOL n/a DrugBank 3 ADENINE n/a DrugBank 3 ADENOSINE-5′-DIPHOSPHATE n/a DrugBank 3 TAOK2 TAOK2-TAO kinase 2 PHOSPHONOSERINE n/a DrugBank 3 VDAC1 VDAC1-voltage-dependent anion channel 1 DIHYDROXYALUMINIUM Inhibitor DrugBank 3 [83]Open in a new tab Results and discussion Whole-genome expression changes underlying Tis21-dependent activity in GCPs during cerebellum development By using oligonucleotide microarrays, we monitored the transcriptomic profiles belonging to GCPs isolated at postnatal day 7 (P7), i.e., cells under the proliferative and tumorigenic influence of Shh deregulated signaling in EGL. When expression profiles of genes from either Ptch1 heterozygous GCPs in Tis21 wild-type background (Ptch1^+/−/Tis21^+/+) or double mutant (Ptch1^+/−/Tis21^KO) GCPs were compared with the control wild-type (Ptch1^+/+/Tis21^+/+), a consistent subset of genes showed a significant change in expression level, i.e., 2599 in Ptch1^+/−/Tis21^+/+ (Figure [84]1 Set B) and 3525 in Ptch1^+/−/Tis21^KO (Figure [85]1 Set D). Instead, the contribution of Ptch1^+/− in Tis21 Knockout background was exemplified by 5381 differentially expressed genes (Figure [86]1 Set C; Ptch1^+/−/Tis21^KO vs. Ptch1^+/+/Tis21^KO). Here we analyze and discuss mainly those genes that were differentially expressed in the pairwise comparison Ptch1^+/−/Tis21^KO vs. Ptch1^+/−/Tis21^+/+ (Set A; Figure [87]1; Supplementary Table [88]1), to identify the contribution by Tis21 in Ptch1 heterozygous background. These genes are critical as they underlie the great increase of MB frequency observed in Ptch1 heterozygous mice ablated of Tis21 (Ptch1^+/−/Tis21^KO), relative to Ptch1 heterozygous mice in a wild-type background (Ptch1^+/−/Tis21^+/+). Tis21-dependent mechanisms underlying the onset of Shh-type MB in GCPs during pre-neoplastic development involve a set of 188 sequences (Figure [89]1 Set A). Among them, about 170 encode for proteins with a known function. In particular, 13 genes belonging to a subset of set A (Figure [90]1) showed a change of expression that was influenced exclusively by the ablation of Tis21: Tigar, Dsc2, Padi4, Serbp1, Lnx1, Pag1, Olfr670, Mcemp1, Cldn22, Slc25a15, Pth, Pdgfd and Cxcl3. The validation of some of these genes has already been performed by quantitative real-time PCR (Farioli-Vecchioli et al., [91]2012a). Functional analysis of Ptch1 heterozygous/Tis21-null mouse model deregulated genes Deregulated genes in our pre-neoplastic model mainly belong to developmental pathways that affect different cellular processes such as cell cycle regulation, proliferation, cell adhesion, cytoskeleton remodeling, apoptosis, survival and differentiation (Table [92]1). In particular, genes belonging to developmental signaling cascades, differentially expressed in our Shh-deregulated model, depend on the Ptch1 mutation contribution as inferred by set B vs. set D data analysis (Figure [93]2). As well known in the literature, in fact, developmental cascades, when deregulated, acquire oncogenic effect. Neuronal development and tumorigenesis rely on cell communication via identical signaling pathways, resulting in a complex signaling network that creates a breeding ground for tumor-initiating events (Peifer and Polakis, [94]2000; Schwartz and Ginsberg, [95]2002; Clark et al., [96]2007; Katoh, [97]2007; Neth et al., [98]2007; Guo and Wang, [99]2009; Rodini et al., [100]2010; Mimeault and Batra, [101]2011; Roussel and Hatten, [102]2011; Akhurst and Hata, [103]2012; Manoranjan et al., [104]2012). Table 1. The most informative deregulated genes belonging to the Set A and associated with the influence of Tis21 gene in background Ptch1 heterozygous (GCPs at P7). Process Text type Down-regulated in Set A Up-regulated in Set A Enrichment probability Cell Cycle NS Wtap, Sik2, Rab11fip4, Lats2, Zc3h12d, Sema4b, Tigar Pa2g4, Srpk2, Eif3a, Eif3c, Eif2c1, Taok2, Mphosph10, Rrp1, Ipo7, Taf7, Cdc27, Ckap5 0.000004 Cytoskeleton NS Cdc42bpb, Sik2 Ehbp1, Akap2, Rab18, Ckap5, Emd 1 Protein Ubiquitination NS Lnx1, Nfx1 Ube2o, Cdc27, Smurf2, Usp36 0.03787 Cell Proliferation NS Pag1, Gcnt3, Sema4b Agtr2, Eif2c1, Gtpbp4, Rps12, Slc6a6 0.69694 Apoptotic Process NS Tigar, Ppp1r13l, Serpina3g Vdac1, Ripk3, Rbm5, Isoc2b, Sltm, Cxcl12 0.32304 Cell Adhesion NS Col4a6, Col23a1, Dsc2, Cldn22, Egflam – 0.39940 Cell differentiation NS Zfhx2os, Dazl Deptor, Foxf2, Lhx5 0.45675 Primary Cilium MT Ccdc157, Ccdc171, Rab11fip2, Rab11fip4, Cxcl3 Syne2, Rgs5 0.00001 Vescicle-mediated transport MT Rab11fip4, Rab11fip2, Cxcl3 Ehbp1, Zfyve20, Cxcl12, Sgsm2, Ckap5, Vps35, Rab18, Smurf2 0.00574 Retinal Development MT H19, Col4a6, Rab11fip4, Bsn, Efna4, Egflam Vdac1, Taf7, Emd, 9130401M01Rik, Taok2, Hist3h2ba, Tomm22, Vps35, Slc6a6, Pafah1b1, Akap2, Raly, Rps12, Nlk, Pa2g4, Srpk2, Dgkq, Cdc27, Syne2, Ripk3 0.000000 Developmental Process SD Gpr82, Smg1, H19, Mettl14, Fat4, Sema4b, Lats2 Rgs5, Sgsm2, Emd, Rab18, Vps35, Nlk, Gigyf2, Kctd5, Ankrd11, Cxcl12, Pdgfd 0.00007 Cell Migration MT Cxcl3, Jmy, Efna4, Timp1, Frk, Prrx1, Rab11Fip2 Cxcl12, Pdgfd, Pafah1b1 0.00008 Epigenetic Modulation SD Hist2h2bb, Cbx3, Padi4 Hist3h2ba, Ankrd11, Ankrd24, Ankrd26, Brwd1, Dek, Anp32a, Taf7, Pa2g4, Emd, Ipo7 0.000000 RNA Processing SD Wtap, Mettl14 Rbm5, Raly, Srpk2, Ddx23, Dek, Htatsf1 0.00312 Nonsense-mediated mRNA decay SD Smg1, Upf3b – 0.00755 Ribonucleoprotein complex biogenesis and assembly SD – Eif3a, Eif3c, Eif2c1, Rmnd1, Rrp1, Gtpbp4, Rps12, Mphosph10, Ipo7 0.00003 Shh-type MB genes MT Gli1[105]^* Pdgfd 0.0164 Group 3 MB genes MT Nlk, Ppp2r2b[106]^*, Raf1[107]^* 0.0161 [108]Open in a new tab The genes are clustered in pathways of involvement, according to the functional analysis methods following described. Many genes overlap different clusters. We divide the genes into two categories: down-regulated and up-regulated, depending on whether the mutation of Tis21 causes in double mutant mice an increase or decrease of expression, respectively, relative to Ptch1 heterozygous/Tis21 wild-type mice. ^* Genes whose levels are changed in Set A although not significantly accordingly to the statistical analysis performed. The functional classes involved in our study have been discussed in the main text (MT), in the [109]Supplementary Data (SD) or are not shown (NS). The enrichment probability of the deregulated genes in each functional class has been calculated using the Fisher Exact Text. Figure 2. [110]Figure 2 [111]Open in a new tab A molecular overview of our Shh-deregulated mouse model, characterized by the genetic disruption of hedgehog signaling resulting from a heterozygous mutation in the negative regulators Ptch1, whose effects are enhanced by the homozygous deletion of the Tis21 tumor suppressor gene. The figure shows the proteins mainly involved in neural developmental pathways, encoded by the differentially expressed genes of set B and set D and shown in their subcellular compartments. Each differentially expressed pathway object is labeled with a thermometer that indicates the gene expression changes: downward thermometers have a blue color and indicate down-regulated expression, whereas upward thermometers have a red color and indicate up-regulated expression. The thermometer number 1 is related to the pairwise comparison Ptch1^+/−/Tis21^+/+ vs. wild type Set B, while the thermometer number 2 is related to the pairwise comparison Ptch1^+/−/Tis21^−/− vs. wild type Set D. Signaling cross-talks between developmental pathways involved in pre-neoplastic GCPs development are initiated by different growth factors and cytokines, the most part of which interacts with G-protein coupled receptors. This is supported by a strong up-regulation of many members of heterotrimeric G-proteins, their target molecules and regulators (data not shown in figure). Among those reported in literature as involved in MB (Roussel and Hatten, [112]2011), developmental signaling pathways appear to be up-reregulated in our experimental data, according to the far fewer percentages of negative signature genes (Chen et al., [113]2013) and the over-representation of WNT and axonal guidance genes present in human MB Shh-type (Northcott et al., [114]2011). Interestingly, a β-Catenin-Gli1 balanced interaction has been recently reported to regulate Shh-driven MB tumor growth in Ptch1 heterozygous mice in vitro (Zinke et al., [115]2015), while Sox7, a transcription factor that is known to reduce Wnt/β-Catenin stimulated transcription in a dose-dependent manner (Takash et al., [116]2001), is up-regulated in Set B and Tis21 ablation enhances its up-regulation in Set D (data not shown in figure). Furthermore, Smo-dependent non-canonical Shh pathways (Jenkins, [117]2009), which have been reported to modulate cytoskeleton-dependent processes and fluctuation of Ca^2+ through the plasma membrane in mammalian neurons (Brennan et al., [118]2012) and suggested in possible association with MB (Briscoe and Therond, [119]2013), are put in light here for the first time as related to the MB Shh-type mouse model. Evidences of a deregulated Slit-Robo pathway, which is implicated in neuronal migration (Wong et al., [120]2001; Marillat et al., [121]2004), are present in our data with the up-regulation of the axon guidance receptor Robo4. The ligand of Robo4, Slit2, has been linked to the inhibition of MB cell invasion (Werbowetski-Ogilvie et al., [122]2006). Proteins belonging to the ubiquitin-dependent degradation of GCPs cell cycle regulators [24] have their genes up-regulated in our model, in particular a number of ubiquitin-conjugating enzymes and some constituents of the SCF (Skip1, Cullin1, F-box)-E3 ubiquitin ligase complex. Among them, a substrate recognition component of the SCF-type E3 ubiquitin ligase, the F-box protein Fbw7, which has been linked to a premature migration of GCPs in conditional Fbw7-knockout mice [30]. An up-regulation genes coding for proteins involved in palmitoylation (i.e., HHAT) and transport of Shh (i.e., DISP1) is noticed in in Set D, where Ptch1 sterol-sensing domain seems to control Smoothened activity through Ptch1 vesicular trafficking [34]. Retinoblastoma-associated protein (Rb), as well as its downstream effectors E2F3 and E2F5, has its correspondent gene up-regulated in set D, where the deregulation of the Rb/E2F tumor suppressor complex in MB Shh-driven has been already associated to the E2F1-dependent regulation of lipogenic enzymes in primary cerebellar granule neuron precursors (Bhatia et al., [123]2011). Figure [124]4 below shows the set of symbols whereby network objects and interactions between objects are indicated in this figure. At the same time, Tis21 ablation is responsible for the delayed migration of pre-neoplastic precursors outside the EGL, which corresponds to a delayed cell differentiation and represents the key step for MB Shh-type formation. In fact, where GCPs proliferate for a prolonged period in EGL, they became the target of neoplastic transforming insults (Farioli-Vecchioli et al., [125]2012a,[126]b). Moreover, we have noticed evidence for the involvement of the primary cilium in our GCPs pre-neoplastic model, mainly in Set B but also in Set A data (Figure [127]3), and also evidence of Smo-dependent non-canonical Shh pathways. A link between Shh signaling at primary cilium and clathrin-mediated endocytotic trafficking/cytoskeletal remodeling will also be discussed. Figure 4. [128]Figure 4 [129]Open in a new tab The figure shows symbols, objects and interactions between objects indicated in Figures [130]2, [131]3. Another observation is related to the mitogen role of Shh signaling, not only in the developing cerebellum but also in the neuronal tube and overall in the retinal cell specification. In fact, a large number of deregulated genes in our Set A are also involved in the delayed differentiation of retinal cell types. Notably, it has been previously shown a parallelism between MB and retinal development; in fact, the analysis of cell populations in MB-derived from GCPs (particularly the group 3) suggests the occurrence of a potential aberrant trans-lineage differentiation into retinal neuronal precursors (Kool et al., [132]2008; Hooper et al., [133]2014). Here, given the large number of genes comprised in set A that is deregulated during the retinal cell development, we want to focus our attention on the timing of exit from the cell cycle, a crucial step in retinal cell development and differentiation, which is under the influence of Shh signaling, as it occurs in the development of MB Shh-type (Dyer, [134]2004). These considerations will be taken into account for a parallel comparison with our model data. Finally, a fine regulation at RNA processing, ribosomes and vesicle trafficking level but also an epigenetic modulation were noticed in set A deregulated genes (Table [135]1). In the following paragraphs, we will discuss the most informative deregulated coding genes belonging to the set A according to their functional clusters (Figure [136]3 and Table [137]1), the most of which overlap different clusters. Among them, there is a large number of already known genes as oncogenes and tumor suppressors. In doing this, we have also taken into account the genes which are deregulated in consequence of the Ptch1 heterozygous. Primary cilium roles in GPCs proliferation and differentiation Primary cilia are sensory non-motile microtubule-based organelles (Lee and Gleeson, [138]2010) protruding from the surface of GCPs in the EGL at early post-natal stages (Del Cerro and Snider, [139]1972), whose requirement for Shh-induced expansion and cerebellar development has been proved using mutants of genes involved in the ciliary formation and maintenance (Chizhikov et al., [140]2007; Spassky et al., [141]2008). Among them, the genetic ablation of primary cilia by removing Kif3a (which encodes the microtubule plus end-directed kinesin motor 3A protein), blocked MB formation driven by a constitutively active Smoothened protein (Han et al., [142]2009). Therefore, Kif3a down-regulation blocks MB Shh-type formation in a primary cilia-dependent manner; moreover, its activity is not required for GCPs differentiation (Chizhikov et al., [143]2007). In our model, we observe that Kif3a is up-regulated in Ptch1 heterozygous mice, irrespective of the presence or absence of Tis21, which is therefore not involved in the Kif3a-dependent phenotype (Figure [144]2). This is consistent with the finding that Kif3a is required for the proliferation of the GCPs (Chizhikov et al., [145]2007) and with our observation that Tis21 in cerebellum regulates the migration of the GCPs but not their proliferation, while the opposite occurs for Ptch1. Nevertheless, in our model, several genes encoding for the coiled-coil domain containing proteins are deregulated in Set A, and thus are dependent on Tis21, i.e., Ccdc157 and Ccdc171 (Table [146]1). One-fourth of the deregulated genes in Set A corresponds to coiled-coils proteins (data not shown), whose highly versatile protein folding motif is related to different biological processes, from subcellular infrastructure maintenance to trafficking control (Burkhard et al., [147]2001; Rose et al., [148]2005) and cilia-related (McClintock et al., [149]2008; Munro, [150]2011). A coiled-coil containing protein is also Rab11 family-interacting protein 4 encoded by Rab11fip4 (Muto et al., [151]2006, [152]2007), whose role in our model will be discussed more in detail in other paragraphs together with the functional product of Rab11fip2, and their wide implication in Shh signaling at primary cilium as a protein involved in microtubule-based vesicle trafficking. Another protein, Nesprin-2 encoded by Syne2, is known to mediate centrosome migration and is essential for early ciliogenesis and formation of the primary cilia by the interaction with the coiled-coil domain of Meckelin protein (Dawe et al., [153]2009). Notably, Ccdc157, Ccdc171, Rab11fip2, and Rab11fip4 are significantly down-regulated in Set A, while Syne2 is up-regulated. Also a novel repressor of hedgehog signaling, whose gene Rgs5 is up-regulated in set A, has been proven to be present with Smo in primary cilia (Mahoney et al., [154]2013). This would suggest that Tis21-dependent tumorigenesis in a (proliferation-independent) way involves ciliogenesis. This latter may be also enhanced by Syne2 after Tis21 ablation. Evidences of direct involvement of Shh signaling on the increase of Ca^2+ levels (Ca^2+ spikes) have been shown at the primary cilium of chicken embryonic spinal neurons. In this system has been observed that Shh (a recombinant N-terminal molecule) may recruit second messengers (i.e., calcium—Ca^2+—and inositol triphosphate) by a non-canonical pathway, through the activation of the Smoothened protein, which translocates to the cilium and becomes activated by phosphorylation at its C-terminal from a G-protein-coupled receptor kinase 2 (Riobo et al., [155]2006; Belgacem and Borodinsky, [156]2011; Brennan et al., [157]2012). Belgacem and Borodinsky ([158]2011) proposed a model in which the primary cilium acts as a subcellular compartment for Shh signaling allowing the spatiotemporal integration of the second messengers through a Smoothened-dependent recruitment of G[i] proteins and Phospholipase C that in turn increases inositol triphosphate levels. The opening of Inositol triphosphate receptors-operated stores and the following activation of Transient receptor potential cation channel 1 (Trpc1) results in an increased Ca^2+ spike activity. This model could fit with our data (Figure [159]3) in which Plc-gamma2, Ip3r3, Trpc1, Trpc2, and Trpc3 are up-regulated in Ptch1 heterozygous mice (but independently from Tis21). Notably, this is the first time that such regulation is observed directly in a mouse model of Shh-type MB, as it had been previously only suggested (Briscoe and Therond, [160]2013). Furthermore, the authors suggested that the Smoothened-dependent Ca^2+ spike activity is necessary for Shh-induced differentiation of spinal postmitotic neuron. Moreover, the role of second messenger signaling in the regulation of cerebellar granule cell migration has been studied in different mouse models (Komuro et al., [161]2015), which highlighted the direct evidence of the role of Ca^2+ signaling in granule cell turning and modulation of their migration rate. The revision of these studies, performed by Komuro et al. ([162]2015), suggested the role of Ca^2+ as potential therapeutic target for some deficits in granule cell migration since its downstream effectors control the assembly and disassembly of cytoskeletal elements. In the last years, the discovery of the role of the primary cilium in Shh signaling captured the attention of the scientific community, leading to test a large number of molecules that modulate SMO cilial translocation acting on different therapeutic potential targets in different types of cancer among which MB (Amakye et al., [163]2013). Loss of cilia in cancer has been suggested to be responsible for an insensitivity of cancer cells to environmental repressive signals, based in part on derangement of cell cycle checkpoints governed by cilia and centrosomes (Plotnikova et al., [164]2008). The importance of the role of cilia in Shh-driven medulloblastoma allografts derived from Ptch1^+/−P53^−/− mice has been shown using a Shh antagonist, i.e., arsenic trioxide (a therapeutic agent for acute promyelocytic leukemia), which inhibits the growth of tumor through the prevention of Shh ciliary accumulation and the reduction of the stability of the Gli2 transcriptional effector (Kim et al., [165]2010). Receptor-mediated endocytosis mechanisms, microtubule-based vesicle recycling and intracellular membrane trafficking Other genes deregulated in Set A are involved in endocytic trafficking clathrin-dependent (Figure [166]5), and a certain number is related to cytoskeletal remodeling and primary cilium that could be very interesting for their implications for target therapy. Figure 5. [167]Figure 5 [168]Open in a new tab Drug targets percentage per functional class of the Set A deregulated genes. The drug targets identified in Set A are showed as percentage with respect to the functional classes to which they belong. The clathrin-dependent endocytic mechanism is a receptor-mediated endocytosis type, which involves clathrin-coated vesicles, early endosomes, microtubule-based vesicle trafficking, lysosomes and recycling transport vesicles in its pathway (Le Roy and Wrana, [169]2005). Evidences of a deregulated clathrin-mediated endocytosis pattern have been reported for MB group 4 during early human neurogenesis (Hooper et al., [170]2014), and detected in our Set B (data not shown) as well as in Set A, where a large number of deregulated genes belongs to this signaling: Rab11fip4 (Horgan and McCaffrey, [171]2009), Ehbp1 (Guilherme et al., [172]2004), Rab11fip2 (Fan et al., [173]2004), Zfyve20 (de Renzis et al., [174]2002; Naslavsky et al., [175]2004), Cxcl12 (Fan et al., [176]2004; Teicher and Fricker, [177]2010; Zhu et al., [178]2012), Sgsm2 (Yang et al., [179]2007), Ckap5 (Foraker et al., [180]2012), Vps35 (Foraker et al., [181]2012), and Rab18 (Foraker et al., [182]2012). Rab11fip4 is known to interact with Rab11 (a Rab GTPase involved in the regulation of intracellular membrane trafficking), which in turn interacts with Rab-coupling protein, a protein involved in the formation of lamellipodia, which facilitates invasive cancer cell migration (Kelly et al., [183]2012). Ehbp1 gene product is involved in actin reorganization coupled to endocytosis clathrin-mediated (Guilherme et al., [184]2004). Zfyve20 is known to regulate structure, sorting, endocytic/recycling pathway of the early endosomal compartment by the interaction with Rab4 and Rab5 (de Renzis et al., [185]2002; Naslavsky et al., [186]2004). Vps35, among other genes involved in intracellular membrane trafficking, is deregulated in Set A. It encodes for the vacuolar protein sorting-associated protein 35, a component of the trimeric cargo-selective retromer complex, required for the efficient endosome-to-Golgi recycling of many membrane proteins among which Wntless. It is also known to regulate endosomal tubule dynamics (Harbour et al., [187]2010; Berwick and Harvey, [188]2014). The oncogene Rab18 is required for normal ER structure and the regulation of endocytic traffic (Lütcke et al., [189]1994; Gerondopoulos et al., [190]2014). This Rab GTPasi is also known as a MB antigen (Behrends et al., [191]2003). Interestingly, Rab18 has been localized at lipid droplets (LDs) level (Martin et al., [192]2005), and its overexpression causes close apposition of LDs to membrane cisternae connected to the rough ER (LD-associated membrane or LAM) (Ozeki et al., [193]2005). Indeed, de novo lipid synthesis has been found in certain highly proliferative and aggressive tumors such as MB Shh-type, suggesting that the Shh-E2F1-FASN axis regulating de novo lipid synthesis in cancers could be used as therapeutic target in hedgehog-dependent tumors (Bhatia et al., [194]2011). Remarkably, Rab11fip2 encodes for a Rab11 family-interacting protein 2, which directly interacts with the actin-based myosin Vb motor protein regulating Cxcr2 recycling and receptor-mediated chemotaxis; hence, Rab11fip2 links Rab11 to molecular motor proteins and cell migration (Jones et al., [195]2006; Horgan and McCaffrey, [196]2009). Moreover, the passage of the internalized Cxcr2 through the Rab11-recycling system appears to have a key role in the physiological response to a chemokine that follows to the formation of clathrin-coated vesicles; in fact, Rab11 family-interacting protein 2 can form a complex with AP-2 that is a major clathrin adaptor complex in cells (Cullis et al., [197]2002; Fan et al., [198]2004; Le Roy and Wrana, [199]2005). Furthermore, the Rab11 involvement into the regulation of vesicular trafficking during primary ciliogenesis has been already put in light (Knödler et al., [200]2010; Hsiao et al., [201]2012). A consequence of the above findings, indicating an interaction between Cxcr2 and the clathrin pathway, is that, since Cxcl3 binds the Cxcr2 receptor (Zlotnik et al., [202]2006), we can infer that this chemokine receptor-mediated chemotaxis mechanism is clathrin-dependent and linked through Rab11fip2 to the primary cilium, in which the Shh signaling takes part. Other evidence of an involvement of Rab11fips and Shh signaling derives from Rab11fip4 (see the retina development section). This protein seems to be involved in the regulation of the membrane trafficking system through interaction with other small GTPases, among which could be Ras-related protein Rab-23, and in the negative regulation of Shh signaling at the primary cilium (Muto et al., [203]2007; Hsiao et al., [204]2012). All this points to an important link between Shh signaling, operating through the primary cilium, and GCPs impaired cell migration, through a clathrin-Cxcl3-Cxcr2-mediated chemotaxis and microtubule-based endocytic vesicle recycling trafficking. Furthermore, the findings of a study about the role of endosomes around mother centriole appendages, and their Rab11-dependent recycling activity that requires centrosome-associated endosome proteins (Hehnly et al., [205]2012), seem to be in line with our data (see drug target section). Interestingly, they showed that (i) the appendages of the mother centriole and recycling endosomes are in intimate contact, as first evidence for a novel centrosome-anchored molecular pathway and regulation of endosome recycling; (ii) there is a structural association between the endosome and the centrosome with new and unexpected implications for recycling endosome functions, such us that one related to cilia formation; (iii) it is also possible that Rab11, and other endosome-associated molecules bound to the centrosome, may play dual roles in endosome and centrosome function (Hehnly et al., [206]2012). Retinal development In mice, retinal development occurs between E11.5 and P8, as uncommitted neuroblasts leave the cell cycle and commit to retinal cell fates (Mu et al., [207]2001). Thanks to mice models, it is known that aberrant proliferation during the development of the neural tube, of cerebellum and retina, leads to embryonal and early postnatal tumors (Dyer, [208]2004). The potent mitogen Shh positively controls the proliferation of their neuronal precursor cells (Martí and Bovolenta, [209]2002). In particular, Shh signaling plays a pivotal role in regulating the proliferation of retinal progenitor cells (RPCs) and the differentiation of retinal ganglion cells (RGCs) during vertebrate retinal development, acting in a cell-specific manner; namely, in mouse Shh is required as positive regulator of RPCs proliferation and as negative regulator of RGCs production, by inhibiting cell-cycle exit (Wang et al., [210]2005; Wallace, [211]2008). A further molecular target which may be responsible for the regulation of retinal cell proliferation and hence for cancer cell proliferation was suggested to be Rb; in fact, the levels of Rb protein appear critical for the development of retinal tumors (Sicinski et al., [212]1995). The rationale for this is that Rb, when active, inhibits the cycle at the G1 checkpoint, prior to cell differentiation, whereas its inactivation, exerted by phosphorylation from cyclinD1/CDK4, is known to start the cell cycle progression. Thus, high levels of Rb may be more difficult to inactivate and vice-versa, thus critically linking the Rb-dependent developmental regulation of proliferation during neurogenesis to cancer cell proliferation for certain types of tissues (Sicinski et al., [213]1995). Indeed, in our model we observe that in GCPs heterozygous for Ptch1 (set B: Ptch1^+/−/Tis21^+/+ vs. Ptch1^+/+/Tis21^+/+) cyclin D1 expression does not change while Rb does increase. This suggests that in Shh-driven neoplastic GCPs the increase of Rb protein is compensatory. In the case of MB, a parallel between developmental neurobiology and oncology was suggested for the proliferating progenitor cells of the retina and cerebellar granule neurons, where the failure to exit the cell cycle leads to aberrant cell proliferation during development in mice (Romer and Curran, [214]2004). Notably, MB-types Cluster D and E (also referred to as groups 4 and 3, respectively) have been found to be marked by a deregulated expression of retinal photoreceptor genes suggesting a distinct origin (i.e., non-cerebellar) from stem cells during the embryonic development, with respect to MB Shh-type in human (Kool et al., [215]2008). In this context, concerning the contribution of Tis21 deletion to the MB development, in Set A we noticed a great number of deregulated genes that have been previously described as involved in retinal development. This comparison could be useful to suggest some common mechanisms related to the progenitor cells cell-cycle exit failure. A consistent subset of Set A genes has been previously described as being involved in cellular expression patterns of mouse early retinal development; these gene were previously recognized by analyzing the outer retinal neuroblastic layer, which in early developmental stages consists almost entirely of mitotic progenitor cells: Vdac1, Taf7, Emd, 9130401M01Rik, Taok2, Hist3h2ba, Tomm22, Vps35, H19, Slc6a6, Pafah1b1, Akap2, Raly, Rps12, Nlk, Pa2g4 and Srpk2 (Blackshaw et al., [216]2004). These genes are all up-regulated in Set A except for H19. Furthermore, a number of other genes that were identified in other studies as being involved in retinal development, are down-regulated in set A: Col4a6 (Bai et al., [217]2009), Rab11fip4 (Muto et al., [218]2007), Bsn (Dick et al., [219]2003), Efna4 (Marcus et al., [220]1996; Poliakov et al., [221]2004; Triplett and Feldheim, [222]2012), Egflam (whose product is also known as Pikachurin) (Omori et al., [223]2012); conversely, other retinal genes up-regulated in set A are: Dgkq (Pilz et al., [224]1995), Cdc27 (Leung et al., [225]2008), Syne2 (Yu et al., [226]2011), Slc6a6 (Vinnakota et al., [227]1997; Warskulat et al., [228]2007), Ripk3 (Trichonas et al., [229]2010). The genes listed above belong to different functional clusters, and some of them will be discussed more in detail in their paragraph of pertinence. Interestingly, the mouse Rab11fip4, whose product regulates the Rab GTPases and is predominantly expressed in the developing neural tissues, among which retina, acts as regulator of RPCs cell-cycle exit and their subsequent differentiation (Muto et al., [230]2007). Rab11fip4 seems to be involved in the regulation of membrane trafficking system through interaction with other small GTPases and in the negative regulation of Shh signaling (Muto et al., [231]2007). Moreover, the Syne2 gene product is known to mediate nuclear migration during mammalian retinal development connecting the nucleus with dynein/dynactin and kinesin proteins (Yu et al., [232]2011). This comparison is in line with the evidence that progenitors from the developing cerebral cortex, cerebellum and retina share a common expression program, suggesting a common evolutionary origin of the different progenitors cells (Livesey et al., [233]2004), and implying a possible common differentiation program. Our data suggest that this developmental process, possibly at the steps of migration and differentiation, is regulated by Tis21. Migration The migration of the GCPs is known to be induced by responses to local environmental cues. However, the alterations of migratory behavior may also depend on intrinsic programs (Komuro et al., [234]2013). In our study, we observed in Set A the differential expression of a great number of genes belonging to migration, cell adhesion and differentiation mechanisms. Among the genes implicated in migration seven are down-regulated after ablation of Tis21 in Shh-activated background, namely, Cxcl3, Jmy, Efna4, Timp1, Frk, Prrx1, and Rab11fip2, and three are up-regulated: Cxcl12, Pdgfd, and Pafah1b. Cxcl3 is a chemokine known to have chemotactic activity for neutrophils (Wuyts et al., [235]1999). Chemotaxis but also angiogenesis seems to be activated by the interaction with its receptor (Addison et al., [236]2000; Zlotnik et al., [237]2006). In our previously published data, Cxcl3 has been identified as a gene whose transcription is regulated positively by Tis21 (which directly associates to the Cxcl3 promoter), suggesting that the evident role played in vivo by Tis21 as inducer of the migration of the GCPs out of the EGL might occur through its functional product; this implies Cxcl3 as a new pharmacological target for medulloblastoma therapy, also considering that MB lesions were reduced using this chemokine as treatment (Farioli-Vecchioli et al., [238]2012a,[239]b). Recently, the anti-cancer role of this chemokine has also been recognized for non-small lung cancer, mediated by the activity of interleukin-27 (Airoldi et al., [240]2015). Jmy, a junction mediating and regulatory protein, is known to induce cell motility by promoting actin assembly and regulating cadherins in the cytoplasm; in DNA damage conditions it undergoes nuclear accumulation, where acts as p53-cofactor promoting apoptosis (Shikama et al., [241]1999; Coutts et al., [242]2007; Zuchero et al., [243]2009). Together, these findings suggest that the ability of Jmy to regulate actin and cadherin, by coordinating cell motility with the p53 response, could underlie a common pathway (Coutts et al., [244]2009). Furthermore, actin assembly has been found to regulate the nuclear import of Jmy in response to DNA damage (Zuchero et al., [245]2012). Thus, an abnormal activity and/or localization of Jmy could contribute to tumor invasion, thus making this gene as a potential tumor therapeutic target (Wang, [246]2010). In theory, the down-regulation of Jmy may contribute to enhance the rate of MB in our mouse model, possibly by controlling the migration of the GCPs as shown for Cxcl3. Regarding Efna4, this gene encodes for the EphrinA4 protein that has been linked with the migration of neuronal cells during development (Poliakov et al., [247]2004). A recent study showed that EphrinA4 and the Ephrin type-B receptor 4 are almost exclusively expressed in Shh MBs, while other Ephrins are expressed in non–Shh MBs; furthermore, a strategy of overexpression and silencing, applied specifically to EphrinB1, showed its ability to control the migration of MB cell lines (McKinney et al., [248]2015). Further evidence of the migration-promoting activity of EphrinA4 has been observed in human glioma cells, where it acts by accelerating a canonical FGFR signaling pathway (Fukai et al., [249]2008). Timp1 protein product is a metalloproteinase inhibitor known to be involved in cell adhesion and migration of human neural stem cells (hNSCs). In fact, through its cell surface receptor CD63, TIMP-1 activates β1 integrin, FAK (focal adhesion kinase) and the PI3K (phosphoinositide 3-kinase) signal transduction pathway, resulting in the migration of hNSCs (Lee et al., [250]2014). Due to its chemoattractant properties, TIMP-1 has been identified in the same study as a therapeutic target for human glioma. The Frk gene product is a Src kinase known as Tyrosine-protein kinase FRK, which controls the migration and invasion of human glioma cells by regulating JNK/c-Jun signaling (Zhou et al., [251]2012). Moreover, the Tyrosine-protein kinase FRK acts as a tumor suppressor in breast cancer by regulating the stability of PTEN, as the loss of Rak (i.e., Frk) induced tumorigenicity in immortalized normal mammary epithelial cells (Yim et al., [252]2009). In mouse brain, Pten is known to be expressed starting at approximately postnatal day 0 (Lachyankar et al., [253]2000) and has also been correlated with the regulation of neuronal precursor cell migration (Li et al., [254]2002). In Set B Pten is up-regulated. The paired-related homeobox transcription factor 1, which is encoded by Prrx1, is an epithelial-mesenchymal transition (EMT) inducer in embryos, where this process is required for the formation of tissues for which cells originate far from their final destination (Ocaña et al., [255]2012). EMT is modified and exploited by cancer cells for metastatic dissemination and also in cancer cells. In particular, the loss of Prrx1 has been related to the ability of cancer cells to acquire tumor-initiating abilities concomitantly with stem cells properties (Ocaña et al., [256]2012). Furthermore, paired-related homeobox transcription factor 1 has been found to promote tenascin-C–dependent fibroblast migration when its expression was induced by Focal adhesion kinase 1 (McKean et al., [257]2003). Fak is up-regulated in both Set B and Set D. Very interesting is the down-regulation of Rab11fip2, whose role in the endocytic recycling pathway has been linked to cell migration (Jones et al., [258]2006) as previously discussed (Section Receptor-Mediated Endocytosis Mechanisms, Microtubule-Based Vesicle Recycling and Intracellular Membrane Trafficking). Among the genes up-regulated in Set A related to migration there is Cxcl12, which encodes for a deeply studied chemokine involved in different mechanisms in cancer development and metastatic invasion (Duda et al., [259]2011; Hattermann and Mentlein, [260]2013), but also described as involved in the migration of neuronal cells through both its receptor, C-X-C chemokine receptor type 4 and Atypical chemokine receptor 3 (Tiveron and Cremer, [261]2008; Memi et al., [262]2013; Yang et al., [263]2013). Cxcl12 appears to exert an action opposite to Cxcl3, as it promotes the localization of the GCPs to the EGL by chemoattraction, being released from meninges (Klein et al., [264]2001; Zhu et al., [265]2002). Thus, the upregulation of Cxcl12, consequent to the ablation of Tis21, synergizes with the downregulation of Cxcl3 in preventing the migration of the GCPs from the EGL. Notably, the chemoattraction of cerebellar granule cells by Cxcl12, whose receptor C-X-C chemokine receptor type 4 is coupled to a G protein, is selectively inhibited by the soluble EphB receptor; this inhibition is blocked by a truncated PDZ-RGS3 lacking the RGS domain, which activates the G-proteins. Therefore, this points to the existence of a pathway connecting B ephrins and Cxcl12 to the regulation of G protein–coupled chemoattraction, and leads to a model for regulation of migration in cerebellar development (Lu et al., [266]2001). In this regard, in our model (Set A) we have detected not only a down-regulation of Efna4, which is a cell surface GPI-bound ligand for Eph receptors, but also the up-regulation of a regulator of heterotrimeric G protein signaling, i.e., Rgs5. Therefore, in our model the decrease of Cxcl3 and Efna4 and the increase of Cxcl12 after ablation of Tis21 in Ptch1 heterozygous mice would synergize in impairing the migration of GCPs from the EGL. As mentioned above, the increase of Cxcl12 in the Tis21-null EGL GCPs would prevent their migration by chemoattraction (Zhu et al., [267]2002). Moreover, the PDGFR pathway members have been recently studied in correlations with MB Shh-driven and MB cell migration, and an upregulation of PDGFRA (receptor for PDGF-A), PDGF-D (ligand for PDGFRB) and Cxcl12 has been observed in Shh-type MB (vs. non Shh MBs). In particular, the activation of the PDGFR pathway has been shown to activate the Cxcl12 receptor (i.e., CXCR4, via inhibition of GRK6) and thus cell migration (Yuan et al., [268]2013). A relevant caveat of that study is that the direction of cell migration, relative to the EGL, was not assessed, having the analysis been conducted only in vitro (Yuan et al., [269]2013). In our model, we noticed an up-regulation of Pdgfd and Cxcl12 genes in Set A, while both Pdgfra and Pdgfrb and also Cxcl12 are up-regulated in Set B and D (see Figure [270]3). This indicates that the heterozygosity of Ptch1 is in itself a condition inducing the expression of both the Cxcr4 and Pdgfr pathways, and that the additional ablation of Tis21 further enhances their activation. Finally, Pafah1b1, up-regulated in Set A, encodes for Lis1, a microtubule regulator that is required for correct neuronal migration during development (Hippenmeyer et al., [271]2010; Escamez et al., [272]2012). Epigenetic modulation The transcriptional reprogramming of the epigenetic patterns is among the causes of tumorigenesis. Nowadays, the epigenetic regulation of transcription and genome organization in MB Shh-type pathogenesis is extensively studied (Batora et al., [273]2014; Hovestadt et al., [274]2014; Shi et al., [275]2014). We have previously provided a first functional genomic analysis of epigenetically regulated genes or interacting proteins for our mouse model Tis21 KO, identified in background either Ptch1 heterozygous or wild-type (Farioli-Vecchioli et al., [276]2012b). We also have recently shown that the Tis21 protein binds to histone deacetylases (HDAC1/4/9) in GCPs, where they are required for the Tis21-dependent inhibition of cyclin D1 expression (Micheli et al., [277]2016). In the present analysis, a great number of genes encoding proteins involved in epigenetic regulation appear to be deregulated in Set A (three down-regulated and 12 up-regulated). They mostly belong to the category of the Histone modification rather than to the Chromatin remodeling one (Table [278]2), which categories are described in (Arrowsmith et al., [279]2012; Plass et al., [280]2013). Our data are in line with those previously published highlighting a great number of deregulated or mutated histone modifiers involved in medulloblastoma (Northcott et al., [281]2012) and their importance as oncogenes or as tumor suppressors (Cohen et al., [282]2011). Furthermore, we highlight also the deregulation of five Histone modifier Regulators, up-regulated in Set A, namely: Emd (Berk et al., [283]2013), Anp32a (Seo et al., [284]2001; Fan et al., [285]2006; Kular et al., [286]2009), Taf7 (Gegonne et al., [287]2001, [288]2013; Kloet et al., [289]2012), Pag2g4 (Zhang et al., [290]2003), Ipo7 (Jäkel et al., [291]1999; Mühlhäusser et al., [292]2001). A detailed description of Set A genes involved in epigenetic modulation is presented in [293]Supplementary Data, at section “Epigenetic modulation.” Table 2. Regulators of the epigenome identified in Set A. Functional Groups Subgroups Down-regulated in Set A Up-regulated in Set A Histone modification Histones Hist2h2bb (Marzluff et al., [294]2002; González-Romero et al., [295]2010) Hist3h2ba (Marzluff et al., [296]2002; González-Romero et al., [297]2010) Writers Cbx3 (Takanashi et al., [298]2009) - Editors Padi4 (Wang et al., [299]2004, [300]2012; Chang et al., [301]2009; Tanikawa et al., [302]2012; Christophorou et al., [303]2014; Deplus et al., [304]2014) Ankrd11 (Behrends et al., [305]2003; Zhang et al., [306]2004; Plass et al., [307]2013; Gallagher et al., [308]2015), Ankrd24 (Plass et al., [309]2013), Ankrd26 (Plass et al., [310]2013) Readers Cbx3 (Arney and Fisher, [311]2004) Brwd1 (Huang et al., [312]2003; Bai et al., [313]2011; Arrowsmith et al., [314]2012; Filippakopoulos and Knapp, [315]2012; Plass et al., [316]2013) Chromatin remodeling Nucleosome remodeling factor Padi4 (Christophorou et al., [317]2014) - Chromatin remodeling factor - Dek (Cavellán et al., [318]2006; Hu et al., [319]2007; Privette Vinnedge et al., [320]2013; Saha et al., [321]2013; Hooper et al., [322]2014) [323]Open in a new tab Histones and enzymes involved in histone modification and chromatin remodeling are showed in the table. They have been classified in subgroups according to their function and divided in enzymes that transfer the modification or writers, enzymes that modify or revert a modification also called editors or erasers and enzymes that mediate the interactions of proteins or protein complexes with the modification known as readers (Arrowsmith et al., [324]2012; Plass et al., [325]2013). Drug targets The increasing recognition of the heterogeneity of molecular basis underlying cancer allows the identification and development of molecularly targeted agents and their transfer to the patients (Rask-Andersen et al., [326]2011; Saletta et al., [327]2014). Here we provide a drug target identification through the genomic analysis of deregulated MB Shh-type Tis21 knockout-dependent genes in Set A and, where possible, the identification of potentially druggable targets (Figure [328]3, Tables [329]3–[330]5), performed using the methods described in the appropriate section. This analysis has given as a result 20 primary, 31 secondary, 53 druggable targets and 18 gene targets among Set A elements (Tables [331]3–[332]6). Their distribution within the functional classes is showed in Figure [333]5. and the putative drug targets identified in SetA, here discussed, are shown in Figure [334]3. Among the Set A genes showing a change of expression influenced exclusively by the ablation of Tis21, there is Pdgfd that has been discussed in developmental (see [335]Supplemtary Data) and migration processes as up-regulated gene. Since the overactivity of PDGF signaling can drive tumorigenesis (Pietras et al., [336]2003), and since PDGF-D in particular has been found to be a potent transforming and angiogenic growth factor (Li et al., [337]2003) highly expressed in Shh-type medulloblastoma (Yuan et al., [338]2013), we propose targeting PDGF-D as therapeutic strategy for medulloblastoma Shh-type, as already studied in other tumors (Heldin, [339]2013). Another very interesting drug target could be the phosphatidylinositol 3-kinase-related kinase Smg1, which here has been discussed in developmental and nonsense-mediated decay processes (Supplementary Material) as down-regulated in Set A (up-regulated in Set D). This protein seems to act antagonistically with mTOR signaling (González-Estévez et al., [340]2012; Du et al., [341]2014), and this is in functional synergy with the up-regulation in Set A of Deptor, which negatively regulates mTOR signaling (Beauchamp and Platanias, [342]2013). Also Deptor is one of the druggable target identified in our study. Taken together, these findings seem to support the importance of mTOR pathway and its upstream PDGF signaling in the pathogenesis of medulloblastoma (Mohan et al., [343]2012). Other two druggable targets are regulators of cell cycle and developmental processes: Sik2 and Lats2. The functional product of Sik2 is localized at the centrosome where its absence leads to a delay of G1/S transition (Ahmed et al., [344]2010), while Lats2 encodes for a centrosomal protein (a serin threonin kinase) whose loss leads to centrosome fragmentation (Yabuta et al., [345]2007), also involved in the regulation of cell cycle G1/S checkpoint, for its relevance to MB tumorigenesis due to its influence in the reduction of expression of cyclin-D1 and N-CoR (Park et al., [346]2012; Lit et al., [347]2013). The deregulation of centrosome and cilia biogenesis have been already described in different human diseases, in particular, in cancer where a derangement of cell cycle checkpoints is governed by cilia and centrosomes (Plotnikova et al., [348]2008; Nigg and Raff, [349]2009; Bettencourt-Dias et al., [350]2011). In addition to that, Sik2 has been characterized as negative regulator of Hippo signaling in Drosophila (Wehr et al., [351]2013). In our data, other two regulators of Hippo signaling appear to be down-regulated after ablation of Tis21, Lats2, and Fat4 that we discuss for their role in the developmental process ([352]Supplementary Data); both act also as tumor suppressors. This evidence supports the involvement of Hippo signaling (Roussel and Hatten, [353]2011) and centrosome assembly in the pathogenesis of MB. Another putative drug target belonging to developmental processes, Rgs5, encodes for an endogenous repressor of Shh signaling and has been proposed in a recent study as potential therapeutic target in Hh-mediated diseases. In fact, it was shown that (i) Rgs5 inhibits the Shh-mediated signaling by activating the GTP-bound Gαi downstream of Smo and (ii) a physical complex between Rgs5 with Smo is present in primary cilia (Mahoney et al., [354]2013). The apoptosis is not the only form of cellular death in which the deregulated genes in Set A are involved. In fact, the Ripk3 functional product, a receptor-interacting protein kinase 3, has been reported to contribute to both apoptotic and necroptotic cell death, depending on target availability (Cook et al., [355]2014; Vanden Berghe et al., [356]2014). Since many anti-cancer drugs are inducers of apoptosis, the induction of RIP3-dependent necrosis is an attractive strategy to circumvent apoptosis resistance of cancer cells (Moriwaki and Chan, [357]2013) that is currently under investigation (Moriwaki et al., [358]2015). As Ripk3 expression is induced after knockout of Tis21 in Shh-activated background, we may hypothesize that Ripk3 plays in our model a tumor suppressor role. The therapeutic advantage of targeting the ubiquitin-proteasome system has already being successfully investigated with proteasomal inhibitors in Shh-type MB with in vivo preclinical studies (Ohshima-Hosoyama et al., [359]2011) and in a preliminary study with personalized targeted therapy for pediatric brain tumors among which MB (Wolff et al., [360]2012). However, the targeting of specific enzymes regulating the ubiquitylation process, e.g., SKP2, a SCF ubiquitin ligase, up-regulated in Set D (Figure [361]3), has been recently proposed as a more specific approach than the previous one (Hede et al., [362]2014). Two genes belonging to the ubiquitin-dependent degradation processes are up-regulated in Set A and have been identified in this study as putative drug target: Ups36 and Cdc27. Usp36, encoding for a deubiquitinating enzyme (Quesada et al., [363]2004), has been detected as overexpresed in human ovarian cancer compared to normal ovaries (Li et al., [364]2008). An Usp36 homolog, recently detected in Drosophila stem cells, has been linked to the deubiquitylation of histone H2B and to the silencing of key differentiation genes, including genes target of Notch (Buszczak et al., [365]2009). Whereas the anaphase-promoting complex component Cdc27 is an E3 ubiquitin ligase able to control cell cycle progression at the G1 to S transition, and also to induce the non-proteolytic disassembly of the spindle checkpoint during mitosis (Jin et al., [366]2008; Manchado et al., [367]2010; Pawar et al., [368]2010). For these reasons, Cdc27 functional product has been identified as a molecular target of the curcumin-induced cell cycle arrest and apoptosis in Shh-type MB (Lee and Langhans, [369]2012). The therapeutic properties of this natural substance were already shown in Shh-driven MB models, highlighting its ability to inhibit the Shh signaling, to reduce the level of β-catenin and to inhibit HDAC4 (Elamin et al., [370]2010; Lee et al., [371]2011). Thus, the up-regulation of Cdc27 after ablation of Tis21 may be a tumorigenic factor. Of interest as drug target among the genes belonging to the migration processes is Timp1. Its signal trasduction pathway results in the migration of hNSCs, showing chemoattractant properties and being identified in the same study as a therapeutic target for human glioma (Lee et al., [372]2014). Here we suggest the same prospective use of Timp1 in MB Shh-type. The Cdc42bpb produces a Rho GTPase activated serine/threonine kinase, which, by regulating the (non-muscle) cytoskeletal actomyosin, influences cell shape and promotes motility and migration (Tan et al., [373]2008, [374]2011), thus acting also as a key regulator of tumor cell invasion. Cdc42bpb is therefore an interesting drug target (Heikkila et al., [375]2011; Unbekandt et al., [376]2014). It's down-regulation in our Set A data is in line with the GCPs migration failure, consistently with the deregulation of others migration regulators discussed above. Genes involved in the RNA processing are Smg1 and Upf3b, respectively down- and up-regulated in Set A. Interestingly, both genes encode for proteins involved in alternative splicing (AS) coupled with Nonsense-Mediated Decay (NMD) mechanism by which NMD controls transcript abundance by regulating AS; in fact, the SURF complex, which includes the SMG1–UPF1–eRF1–eRF3 proteins, forms a bridge between the ribosome and the downstream exon-junction complex (EJC) associated with UPF3b and UPF2 (Chamieh et al., [377]2008; McIlwain et al., [378]2010). Moreover, Smg1 has been showed to be involved in the predisposition to tumor formation and inflammation in Smg1 heterozygous mice; this mouse model presents elevated basal tissue and serum cytokine levels—indicating low-level inflammation—and can progress to chronic inflammation or enhanced cancer development. Therefore, this is a model of inflammation-enhanced cancer development (Roberts et al., [379]2013), also suggesting that Smg1 is a tumor suppressor (Du et al., [380]2014). To our knowledge, the possibility to target inflammation through Smg1 has never been applied to medulloblastoma until now. Furthermore, in our data Il1b, for example, is up-regulated in Set D and B (See Figure [381]2); also Cxcl12 is up-regulated in Set D, B, and also in Set A, as mentioned previously, while Cxcl3 is down-regulated in Set A. It is also worth noting the existence of an anticoagulant inhibitor of CXCL12/CXCR4 axis, the Tinzaparin, as well as other heparinoids that have been studied in brest cancer models for their heparinoid-mediated inhibition of chemotaxis activity (Harvey et al., [382]2007; Mellor et al., [383]2007). As Cxcl12 favors the localization of the GCPs to the EGL by chemoattraction, an inhibitor of Cxcl12 might achieve an opposite effect, similar to the promigratory effect exerted by Cxcl3. Summary and conclusions During cerebellar development, Shh is expressed in Purkinje cells and regulates the proliferation of GCPs acting as mitogen (Dahmane and Ruiz i Altaba, [384]1999; Wallace, [385]1999; Wechsler-Reya and Scott, [386]1999). GPCs undergo a prolonged mitotic activity in the EGL during the early postnatal period in the mouse. After this intense proliferative phase, around the second postnatal week in mouse, GPCs exit the cell cycle and migrate radially to the IGL, along the Bergmann glia. During this inward migration, differentiation of post-mitotic GPCs takes place, allowing the mature granule neurons to expand the IGL, where they extend dendrites (Dyer, [387]2004; Rodini et al., [388]2010; Wang and Wechsler-Reya, [389]2014). As a consequence of a hyperactivation of the Shh pathway occurring in the Ptch1^+/− MB mouse model, the prolonged mitotic activity of GCPs makes them potential targets for transforming insults, leading to MB (Wang and Zoghbi, [390]2001). Mutations in Ptch1 occur in sporadic human MB and promote the tumor in Ptch1 heterozygous mouse models at a rate of approximately 8% within 12 weeks of age and up to 30% between 12 and 25 weeks of age (Goodrich et al., [391]1997). As previously described (Farioli-Vecchioli et al., [392]2012a), the ablation of Tis21 in our mouse model strikingly enhances, from 25 to 80%, the incidence of MB spontaneously occurring in Ptch1 heterozygous mice. We observe that the whole balance between division, differentiation and death is disrupted in Ptch1 heterozygous GCPs lacking Tis21, leading to an increase in tumor incidence (Farioli-Vecchioli et al., [393]2012a). In particular, Tis21 seems to act on the timing of migration of GPCs from EGL to IGL, causing an extended period of localization in the proliferative region, decrease of differentiation and increase of apoptosis (Farioli-Vecchioli et al., [394]2012a,[395]b). These cellular changes are mirrored in gene expression changes occurring in Set A, shown in Table [396]1. We observe that the most significant enrichments of genes whose expression is altered in Set A, fall in the functional pathways of developmental signaling, retinal development, cell migration, epigenetic modulation, and primary cilium-related activities. We will summarize some consideration on these functions and also about the ubiquitin-dependent degradation (Table [397]1), in relation to the possible drug targets available. Cell cycle regulation and cell proliferation of the GCPs (Set A) We have shown that no change in the proliferation of GCPs occurs after ablation of Tis21 (Farioli-Vecchioli et al., [398]2012a). Moreover, in a recent study, we have demonstrated that the proliferation of the GCPs is not ruled by Tis21 but by the family-related gene Btg1 (Ceccarelli et al., [399]2015). Indeed, if we analyze the type of expression changes occurring in the whole array of genes of Set A that either directly or indirectly regulate the proliferation and/or the cell cycle of the GCPs (Table [400]7) we find that there is upregulation as well downregulation of genes that affect either positively or negatively this process, evidently resulting in no net change of proliferation of the GCPs. Nonetheless, the defect of migration of the Tis21-null GCPs forces them to stay a longer period in the EGL under the control of Shh influence, possibly leading to different types of alterations in cell division, including the control of centrosome assembly (see below). Table 7. The table shows a sub-set of Set A deregulated genes which can influence proliferation in a direct or indirect manner. Genes Functional classes Expression level Effect on proliferation Oncosuppressor gene Gtpbp4 Proliferation Up + Ipo7 Cell Cycle Up + Eif3a Cell Cycle Up + Eif3c Cell Cycle Up + Cdc27 Cell Cycle Up + Ckap5 Cell Cycle Up + Ankrd11 Epigenetic Up + Mphosph10 Cell Cycle Up + Rps12 Proliferation Up + Rrp1 Cell Cycle Up + Srpk2 Cell Cycle Up + Taf7 Cell Cycle Up + Taok2 Cell Cycle Up + Slc6a6 Proliferation Up + Agtr2 Proliferation Up − Pa2g4 Cell Cycle Up − Eif2c1 Cell Cycle; Proliferation Up − X Pag1 Proliferation Down − X Rab11fip4 Cell Cycle Down − Lats2 Cell Cycle Down − X Tigar (9630033F20Rik) Cell Cycle Down − Sema4b Cell Cycle; Proliferation Down − Zc3h12d Cell Cycle Down − X Gcnt3 Proliferation Down + Sik2 Cell Cycle Down + Wtap Cell Cycle Down + [401]Open in a new tab Cilium, GCPs migration, clathrin motility, and centrosome assembly (Set A) We have shown a link between the Shh signaling, operating through the primary cilium, and the impairment of cell migration, i.e., the main phenotype observed in Ptch^+/−/Tis21^KO mice [17]. In fact, the primary cilium, as mentioned above, is a sensory non-motile microtubule-based organelle which acts as a subcellular compartment for Shh signaling through a Smoothened-dependent recruitment of G[i] proteins (Belgacem and Borodinsky, [402]2011). These include the Rab11 family, which impacts on cell motility, and whose components Fip4 and Fip2 are down-regulated in Set A. Remarkably, Rab11Fip2 interacts with the myosin Vb motor protein (Horgan and McCaffrey, [403]2009) that regulates the recycling of C-X-C chemokine receptor type 2, the receptor of Cxcl3, and the receptor-mediated chemotaxis, as confirmed by Raman et al. ([404]2014). As we have pointed out previously, Cxcl3 induces the migration of GCPs out of the EGL and its decrease in Set A is at the origin of the increase of tumorigenesis in Tis21 KO model (Farioli-Vecchioli et al., [405]2012a). All this points to a link between the decrease of the Cxcl3-Cxcr2 function in Set A and the clathrin-mediated chemotaxis and microtubule-based migration. Such type of reasoning could be extended to the whole set of coiled coil molecules present in the cilium whose expression is altered in Set A, further suggesting that the ablation of Tis21 in Set A could trigger an impairment of GCPs migration acting at more than one level. Another interesting connection is with the cilium-based GTPase Rab11Fip4; in fact, since Rab11Fip4 induces Gli3 (Muto et al., [406]2007) which is a negative regulator of Shh signaling, the ablation of Tis21, by down-regulating Rab11fip4, may enhance the Shh pathway activity, thus conferring more penetrance to the Shh stimulus. Moreover, the presence of cilia is in itself necessary for the development of Shh-type MB, and the formation of cilia might be enhanced by the upregulation in Set A of Syne2 (Chizhikov et al., [407]2007). We also noticed several deregulated genes in Set A related to an evident deregulation of centrosome assembly (Akap2, Syne2, Ckap5, Sik2, Emd, and Lats2). Since the basal bodies, microtubule-based structures, are required for the formation of cilia (also non-motile ones) but also for the pericentriolar material at the core of the centrosome (Nigg and Raff, [408]2009), our results could confirm the previously reported evidences of a deregulation of centrosome and cilia biogenesis that have been described in different types of cancer, where a derangement of cell cycle checkpoints is governed by cilia and centrosomes (Plotnikova et al., [409]2008; Nigg and Raff, [410]2009; Bettencourt-Dias et al., [411]2011). Primary cilium (Set B) In reference to set B (comparison of Ptch1 heterozygous mice vs. wild-type, thus without involvement of Tis21) our attention was captured by mechanisms that could regulate cell cycle machinery in a primary cilia-dependent fashion. These are suggestive of a possible involvement of Smo-dependent non-canonical Shh-pathways, namely concerning our data showing for the first time that Plc-gamma2, Ip3r3, Trpc1, Trpc2, and Trpc3 are up-regulated in Ptch1 heterozygous mice. These genes belong to the described Smo-dependent non-canonical Shh pathways (Figure [412]3) that have been reported to modulate cytoskeleton-dependent processes (Jenkins, [413]2009) and Ca^2+ spikes (Brennan et al., [414]2012). In particular, a model in which the subcellular compartment (i.e., primary cilium) for Shh signaling allows the spatiotemporal integration of second messengers has been proposed (Belgacem and Borodinsky, [415]2011), and the role of Ca^2+ signaling in granule cell turning and in modulation of their migration rate has been suggested as potential therapeutic target for some deficits in granule cell migration, since its downstream effectors control the assembly and disassembly of cytoskeletal elements (Komuro et al., [416]2015). The presence of the key components of the Shh pathway in cilia has been assessed, as well as the anterograde and retrograde traffic regulating its signaling (Goetz and Anderson, [417]2010). We have taken in consideration the role of primary cilia in GCPs, where their presence has been assessed in the EGL at early post-natal stages (Del Cerro and Snider, [418]1972), as well as their requirement for Shh-induced expansion and cerebellar development (Chizhikov et al., [419]2007; Spassky et al., [420]2008). Exploring this scenario, in our MB mouse model we have highlighted some other cilia-related protein targets modified in Set B—but not in Set A—such as Tctex-1, identified as a novel “checkpoint” for G1-S transition controlling ciliary resorption, cell cycle S-phase entry and fate of neural progenitors of developing neocortex (Li et al., [421]2011; Sung and Li, [422]2011). Possible lineage switch of pGCPs in Set A and retinal development An intriguing observation concerns the fact that three genes in Set A whose expression is significantly modified - namely, Nlk, Raf1, and Ppp2r2b—are markers for group 3 medulloblastoma (Kool et al., [423]2008; Gibson et al., [424]2010; Northcott et al., [425]2011, [426]2012c; Taylor et al., [427]2012; Hooper et al., [428]2014). Moreover, Nlk is among the genes of Set A modified in retinal development, and it has been suggested that cerebellar and retinal progenitor cells have common evolutionary origin [76]. It is also worth noting that, according to references (Kool et al., [429]2008; Hooper et