Functional Genomics Identifies Tis21-Dependent Mechanisms and Putative Cancer Drug Targets Underlying Medulloblastoma Shh-Type Development

We have recently generated a novel medulloblastoma (MB) mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+/−/Tis21KO). 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.

The protein encoded by the Gpr82 is an orphan G protein-coupled receptor of unknown function (Lee et al., 2001). Smg1 is known to be required for embryogenesis since using a gene-trap model of Smg1 deficiency it has been showed that its loss is lethal at embryonic day 8.5 (McIlwain et al., 2010). H19 is a gene encoding for an imprinted maternally expressed transcript (non-protein coding), located downstream the growth-promoting insulin-like growth factor 2 (Igf2), with which shares a common imprinting mechanism; in fact, their variable imprinting has been observed in fetal cerebellum and MB (Albrecht et al., 1996). Growth in the developing mouse embryo is largely governed by Igf2 and Shh can transcriptionally activate Igf2 (Chao and D'Amore, 2008). H19 has been reported both as oncogene and tumor suppressor but also in fetal growth syndromes in humans (Guo et al., 2014;Matouk et al., 2014;Park et al., 2014). H19 is also a microRNA precursor whose expression results in the post-transcriptional down-regulation of specific mRNAs during vertebrate development (Cai and Cullen, 2007) and inhibits cell proliferation (Keniry et al., 2012) (H19 is down-regulated in Set A). Furthermore, H19 has been shown in mice to act as trans-regulator of a group of co-expressed genes belonging to the imprinted gene network controlling fetal and early postnatal growth (Gabory et al., 2010). The Mettl14 gene is instead a modulator of RNA stability in embryonic stem cells, since through its activity of RNA methylation (epitranscriptomics) plays an important role in RNA processing and metabolism, by destabilizing the mRNAs encoding of developmental regulators (m 6 A methylation is inversely correlated with mRNA stability and gene expression) (Lin and Gregory, 2014;Wang et al., 2014). Concerning the gene Fat4, a decrease of its expression in the mouse embryonic neuroepithelium has been correlated with an increase of the number of cortical progenitor cells and decrease of their differentiation into neurons; such effect is counteracted by the activation of the Hippo signaling pathway, which implicates Fat4 as key regulator of the mammalian neurogenesis (Cappello et al., 2013). Sema4b is mainly expressed in glial cells of the developing cerebellum (Maier et al., 2011). Semaphorin-plexin signaling is activated from the binding of Semaphorin-4B to Plexin-B2 (Humbert and Godde, 2015), an event which in non-small lung cancer cells seems to promote the tumor invasion (Jian et al., 2014a;Jian et al., 2014b). The Serine/threonine-protein kinase Lats2, by negatively regulating YAP1 in the Hippo signaling pathway, known to act in MBs in concert with the Shh pathway (Roussel and Hatten, 2011), prevents DNA damage-induced apoptosis (Reuven et al., 2013) and controls the TGFβ-SMAD pathway (Varelas et al., 2010).
Among the up-regulated genes in Set A, the Rgs5 encodes for an endogenous repressor of Shh signaling whose therapeutic role has been discussed in the main text (paragraph 2.2.8). 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., 2013). Sgsm2 product functions as a modulator of the RAP and RAB subfamily members of the vesicle transportation small G protein superfamily (Yang et al., 2007). Emd gene encodes for the Emerin protein that has been proposed to block or attenuate the nuclear accumulation of at least three signaling proteins: ERK1/2, Lmo7 and β-catenin (Berk et al., 2013). Concerning Rab18, interestingly, loss-of-function mutations of this gene have been found to cause the Warburg Micro syndrome which is associated with cerebellar and cerebellar vermis hypoplasia; moreover, it is also known a correlation in human healthy adults of the Rab18 gene polymorphism (rs3765133) with cerebellar development and in particular with its volume (Bem et al., 2011;Cheng et al., 2014). The Vps35 gene product is known to regulate the Wnt signaling through its cargo activity, since the loss of Vps35 function prevents the endosome-to-Golgi recycling of Wntless, a protein essential for secretion of Wnt ligands (Berwick and Harvey, 2014). Nlk encodes for a negative regulator of Wnt/β-Catenin signaling, which is activated by the non-canonical Wnt-5a/Ca 2+ pathway (Ishitani et al., 2003) This latter has been described as involved in the pathogenesis of MB group C, or 3 (Northcott et al., 2011;Chen et al., 2013) and, remarkably, Wnt-5a is heavily down-regulated by the ablation of Tis21 (Set A) but is up-regulated in conditions of heterozygosity of Ptch1 (in Set B and D). This fact may imply that the ablation of Tis21 increases MB tumorigenesis by modulating genes implied in group 3 tumors. Other genes modified in Set A (although not significantly) and belonging to group 3 are Ppp2r2b and Raf1 ( (Kool et al., 2008;Gibson et al., 2010;Northcott et al., 2011;Northcott et al., 2012;Taylor et al., 2012;Hooper et al., 2014) see Conclusions). Concerning the Gigyf2 gene, its protein interacts with the GRB10 adapter that in turn modulates the IGF-I receptor signaling (Giovannone et al., 2003); Gigyf2 is also deregulated in Set B and Set D (see fig.3). The K-potassium channel tetramerization domain protein encoded by Kctd5 has been identified as a substrate-specific adaptor for cullin3-based E3 ligases (Bayon et al., 2008;Balasco et al., 2014), whose Cul3-mediated ubiquitination has different actions. Namely, the control of different cell-cycle phases (Singer et al., 1999;Sumara et al., 2007;Maerki et al., 2009;Beck et al., 2013), the regulation of intracellular trafficking, in particular secretion and endosome maturation (Hubner and Peter, 2012;Huotari et al., 2012). Moreover, Cul3-mediated ubiquitination is involved in ubiquitination and proteasomal degradation of different proteins, including GLI2 and GLI3 in complex with the substrate-binding adaptor Spop (Wang et al., 2010). As mentioned in the main text, many proteins belonging to the ubiquitin-dependent degradation within the GCPs are deregulated in Set B and Set D, meaning that the ablation of Tis21 has different effects whether it occurs in a wildtype background or heterozygous for Ptch1 (see fig.3). The functional product of Ankrd11 is a chromatin regulator controlling histone acetylation and gene expression during neural development; in fact, knockdown of Ankrd11 in developing murine or human cortical neural precursors has been shown to cause decreased proliferation, reduced neurogenesis and aberrant neuronal positioning (Gallagher et al., 2015). The chemokine Cxcl12, encoded by the Cxcl12 gene, is known to play a central role in normal cerebellar development by influencing both the migration and proliferation of cerebellar granule cells (Klein et al., 2001), and consequently it plays an important role in MB pathogenesis (Ozawa et al., 2014). In particular, a new molecular subgroup of MB characterized by the coactivation of the SHH and CXCL12/CXCR4 pathways has been identified in human youngest patients in association with desmoplastic histology (Sengupta et al., 2012). This is of particular relevance if we consider that Cxcl12 expression is heavily increased by the ablation of Tis21 in GCPs. Finally, the Pdgfd gene encodes for a protein known to have an important role in the regulation of physiological and pathological cell growth (LaRochelle et al., 2002;Heldin, 2013); its function in MB migration together with the CXCL12/CXCR4 signaling has been discussed in the section treating the migration of the GCPs (Yuan et al., 2013).

Epigenetic modulation.
Hist2h2bb (down-regulated in Set A) and Hist3h2ba (up-regulated in Set A) are described as pseudogenes in human but have their histone functional products in mouse (Marzluff et al., 2002;Gonzalez-Romero et al., 2010) and are known as replication-dependent histone genes (Marzluff et al., 2002).
Among the down-regulated genes in Set A, we also detected the Cbx3 gene, which encodes for the Heterochromatin protein 1. This protein has been primarily identified as a reader, able to recognize and bind methylated histone H3 at Lys9, leading to the epigenetic repression of differentiation (Arney and Fisher, 2004). Moreover, it has been shown to be responsible for the histone H4 K20 trimethylation, through which epigenetically controls both cell differentiation and cancer development, suggesting its importance as cancer therapeutic target (Takanashi et al., 2009). Padi4 (peptidylarginine deiminase 4) encodes for a protein that mediates gene expression by demethylating histones, i.e., by converting methyl-Arg residues of histones H3, H4 as well as H1 to citrulline and releasing methylamine (Wang et al., 2004). Notably, through H1 citrullination, Padi4 activates pluripotency of pluripotent cells in the early mouse embryo, since citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation (Christophorou et al., 2014). Recently, this protein has shown to citrullinate also the DNA (cytosine-5)-methyltransferase 3A, regulating its DNA methyltransferases activity (Deplus et al., 2014). Compared to benign and non-tumor diseases, many malignant tumor types exhibit increased peptidylarginine deiminase 4 levels in tumorous cells, highlighting its importance in the promotion of tumorigenesis (Chang et al., 2009). In particular, it has been shown to regulate tumor suppressor gene expression acting as a corepressor of p53 to regulate SESN2-mTORC1 autophagy pathway . Interestingly, the citrullination of H4R3 and Lamin C has been negatively correlated with p53 protein expression and with tumor size in nonsmall cell lung cancer tissues, suggesting that peptidylarginine deiminase 4 could function as a tumor suppressor that mediates the apoptotic process of damaged cells (Tanikawa et al., 2012). In analogy, the decrease of Padi4 observed in SetA, might be associated with the enhancement of tumorigenicity occurring in Tis21-null GCPs.
Among the up-regulated genes in Set A, we detected three genes belonging to the histone modification editors ANKRDs (Plass et al., 2013), i.e. Ankrd11, Ankrd24 and Ankrd26. Ankrd11 has been reported as MB antigen (Behrends et al., 2003), is a recruiter of histone deacetylases to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation (Zhang et al., 2004) and regulates proliferation and neurogenesis in the embryonic brain (Gallagher et al., 2015), while Ankrd26 has been linked to the glucose homeostasis (Raciti et al., 2011). Brwd1 encodes a transcriptional activator containing bromodomains by which binds histone acetyl groups, thus has been classified in the histone modification readers (Arrowsmith et al., 2012;Filippakopoulos and Knapp, 2012;Plass et al., 2013). This nuclear protein is broadly expressed in the mouse embryo and has been associated with a SWI/SNF chromatin remodeling complex component (Huang et al., 2003). Recently, it has been identified as human putative motility modifier, involved in cell morphology and cytoskeleton organization (Bai et al., 2011). Dek is known to be an oncogene, upregulated in group 4 MB (Hooper et al., 2014). Its functional product functions as an ''architectural'' protein in chromatin (Cavellan et al., 2006;Hu et al., 2007), which can be shuttled from the extracellular space to the intracellular (Saha et al., 2013); remarkably, in agreement with its increase observed in Set A, Dek functional product can confer stem cell-like qualities, thus potentially leading to cancer (Privette Vinnedge et al., 2013).
The histone modifier regulators up-regulated in our Set A data were Anp32a, Taf7, Pag2g4, Ipo7 and Emd. Anp32a encodes for a member of the INHAT (inhibitor of histone acetyltransferase) complex that binds to histones and masks accessibility of lysines of histone tails (Seo et al., 2001), and whose depletion promotes neurite outgrowth in vivo, likely by regulating the expression of the Nf-L (a neuron-specific cytoskeletal gene), through binding to its promoter and modulating histone acetylation levels (Kular et al., 2009). Thus, the increase of Anp32a expression would be in line with the decreased differentiation observed in Tis21-null GCPs. This gene has been also described as a tumor suppressor, repressing cell growth through the inhibition of transcription, thanks to its ability to block acetylation and phosphorylation of histone H3 and to initiate its proapoptotic activity (Fan et al., 2006). The Taf7 gene product is a subunit of TFIID, known to inhibit the acetyltransferase activity of the Transcription initiation factor TFIID subunit (Gegonne et al., 2001); in this way Taf7 functional product acts as a transcriptional repressor of the expression of Cyclin D1 and Cyclin A genes, thus acting as cell cycle regulator of G1/S phase (Kloet et al., 2012;Gegonne et al., 2013). Moreover, this protein regulates transcription of both TAF1-dependent and -independent genes, emerging as a critical regulator of transcription initiation and cell proliferation (Gegonne et al., 2013). However, we do not find consistency in the expected action of Taf7 since its large increase in Set A is not matched by a corresponding decrease of cyclin D1 levels, suggesting that further interactions exist. Pa2g4 gene protein, interestingly, represses transcription of some E2F--regulated promoters via its ability to recruit HDAC activity (Zhang et al., 2003). Finally, Emerin (Emd) protein is known to be associated with the core components of the N-CoR complex and to bind directly Histone deacetylase 3 (Berk et al., 2013); in this complex, the functional product of Ipo7 is known to mediate the nuclear import of H1 histone and the core histones H2A, H2B, H3 and H4 (Jakel et al., 1999;Muhlhausser et al., 2001).

RNA Processing and Nonsense-Mediated Decay mechanisms; Ribosome-related mechanisms.
During the data analysis we also noticed an interesting involvement of deregulated genes of Set A that act as regulators or targets of RNA processing as well as of Nonsense-Mediated Decay (NMD) mechanism, or are involved in translation initiation and ribosome-related mechanisms (i.e. biogenesis, processing and transport). In particular, there are two targets of alternative splicing (AS) among the genes discussed in the main text, i.e., Rab11fip4, whose proteic product regulates receptor-mediated endocytosis coupled with cytoskeletal remodeling and microtubule-based vesicle trafficking, and Ehbp1 that has been reported among those affected by alternative splicing in Shhassociated MB (Menghi et al., 2011).
The importance of AS patterns into the genetic determination of diseases, among which cancer, is currently under study but it has already revealed new insights (Xiong et al., 2015). In consideration of this evidence, we have detected the deregulation of many genes of Set A that are involved in AS: i) an AS regulator of apoptotic genes (Bonnal et al., 2008;Fushimi et al., 2008) and of NUMB protein through which affects cancer cell proliferation (Bechara et al., 2013), i.e. the RNA-binding protein 5 encoded by Rbm5 tumor suppressor gene (Mourtada-Maarabouni et al., 2006); ii) the pre-mRNA-splicing regulator WTAP (Ortega et al., 2003) that has been shown to interact with the functional product of Mettl14 as a regulatory subunit of the m6A methyltransferase complex playing a critical role in epitranscriptomic regulation of RNA metabolism and RNA splicing Ping et al., 2014); iii) a target of splicing, i.e. Ehbp1, previously demonstrated to undergo SHHassociated splicing (Menghi et al., 2011) and involved in clathrin-mediated endocytosis linked to cytoskeleton actin reorganization (Guilherme et al., 2004); iv) the Rab11fip4 transcript A (Muto et al., 2007) as well as another target of AS, i.e., v) the RNA-binding protein RALY (Khrebtukova et al., 1999), homolog of a human heterogeneous nuclear ribonucleoprotein that showed to have pleiotropic effects on RNA metabolism and translation (Tenzer et al., 2013); vi) the Srpk2 functional product, , the SRSF protein kinase 2, required for spliceosomal B complex formation and the phosphorylation of the protein DDX23, encoded by Ddx23 gene (up-regulated in Set A) (Mathew et al., 2008); furthermore, Srpk2 interacts with the component of the splicing-dependent multiprotein exon junction complex Acinus in the regulation of Leukemia tumorigenesis (Jang et al., 2008); vii) the splicing associated factor Dek (Le Hir et al., 2000); Viii) the transcription-splicing factor Htatsf1 that regulates, among the others, genes involved in the cell cycle (Miller et al., 2011).
We have also detected the deregulation of two genes involved in the AS-coupled NMD mechanism, i.e. Smg1 and Upf3b that are respectively down and up-regulated in Set A. Their role has been discussed more in detail in the main text (paragraph 2.2.8).
Other evidences in our data reveal a deregulation of the translation activities and other ribosomerelated mechanisms. Three translation initiation factors are up-regulated in Set A, i.e. Eif2c1, Eif3a and Eif3c, known for their role in increased protein synthesis supporting tumor development (Parisi et al., 2011;Hershey, 2014). Rmnd1 functional product is known for its role in mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome (Janer et al., 2012). Concerning the others ribosome-related mechanisms, a certain number of deregulated genes in Set A are involved in ribosome biogenesis (i.e. Rrp1 (Yoshikawa et al., 2011), Gtpbp4 (Lapik et al., 2007)), a 40S ribosomal component (i.e. Rps12), a pre-18S ribosomal RNA processing (i.e. Mphosph10) (Granneman et al., 2003) and a nuclear import of ribosomal proteins (i.e. encoded by Ipo7 (Jakel and Gorlich, 1998)). Notably, the overexpression of genes involved in ribosomal functions, such as Rps20 and Rpl30 that encode for a component of the 60S ribosomal subunit 40S subunit respectively, has been already associated with adverse outcome in medulloblastoma (De Bortoli et al., 2006). Albrecht, S., Waha, A., Koch, A., Kraus, J.A., Goodyer, C.G., and Pietsch, T. (1996). Variable imprinting of H19 and IGF2 in fetal cerebellum and medulloblastoma. J Neuropathol Exp Neurol 55 (12)