Oncogene (2014) 33, 4709–4721 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW Oncoprotein stabilization in brain tumors

S-M Hede1, V Savov1, H Weishaupt1, O Sangfelt2 and FJ Swartling1

Proteins involved in promoting cell proliferation and viability need to be timely expressed and carefully controlled for the proper development of the brain but also efficiently degraded in order to prevent cells from becoming brain cancer cells. A major pathway for targeted protein degradation in cells is the ubiquitin–proteasome system (UPS). Oncoproteins that drive tumor development and tumor maintenance are often deregulated and stabilized in malignant cells. This can occur when oncoproteins escape degradation by the UPS because of mutations in either the oncoprotein itself or in the UPS components responsible for recognition and ubiquitylation of the oncoprotein. As the pathogenic accumulation of an oncoprotein can lead to effectively sustained cell growth, viability and tumor progression, it is an indisputable target for cancer treatment. The most common types of malignant brain tumors in children and adults are medulloblastoma and glioma, respectively. Here, we review different ways of how deregulated proteolysis of oncoproteins involved in major signaling cancer pathways contributes to medulloblastoma and glioma development. We also describe means of targeting relevant oncoproteins in brain tumors with treatments affecting their stability or therapeutic strategies directed against the UPS itself.

Oncogene (2014) 33, 4709–4721; doi:10.1038/onc.2013.445; published online 28 October 2013 Keywords: glioma; medulloblastoma; ubiquitin–proteasome system; brain tumor development; E3 ligases

INTRODUCTION system itself may be attractive candidates for brain cancer Medulloblastoma is the most common malignant brain tumor in therapy. children. Around 70% of patients are cured after surgery and radiotherapy; however, survivors often succumb from serious side effects following these treatments.1 Glioblastoma (GBM) is one of MEDULLOBLASTOMA BIOLOGY the most lethal of all cancers and also the most common type of Recent genomic efforts have divided medulloblastoma into four primary malignant brain tumors (glioma) in adults.2 GBM is a WHO main molecular subgroups: WNT, SHH (Sonic Hedgehog), Group 3 grade IV astrocytoma and can either develop de novo without and Group 4.5 For all molecular subgroups, there are key previous diagnosis (primary GBM) or derive and progress into a medulloblastoma proteins that are characterized as target secondary GBM from an astrocytoma of a lower grade. Other proteins for the UPS. For example, the WNT (Wingless) pathway less frequent types of glioma include oligodendroglioma and substrate b-catenin is regulated by the UPS through the beta- ependymoma. transducin repeat-containing E3 Ub protein ligase (beta-TrCP).6 In recent years, great strides have been made in characterizing The beta-TrCP recognition site of the b-catenin gene CTNNB1 is changes in gene expression or genetic and epigenetic alterations often mutated, leading to b-catenin stabilization in WNT tumors.5,7 associated with the genesis and/or progression of various types of The SHH tumors show alterations in proteins that lead to SHH brain tumors. However, less is known about how alterations in pathway activation. For example, the beta-TrCP substrate, GLI protein level and protein activity contribute to brain tumor family zinc-finger 2 (GLI2),8 is often elevated and is a marker of development. One specific type of post-translational modification poor prognosis for SHH tumors.9 Furthermore, inactivation of is the covalent attachment of a small (8 kDa) and highly conserved Patched (PTCH)10,11 can result in stabilization of the downstream protein called ubiquitin (Ub), in a process known as ubiquityla- MYCN oncoprotein.12 Finally, Group 3 and Group 4 tumors often tion.3 Ubiquitylation typically involves (1) the covalent attachment present amplifications in MYC and MYCN, respectively,13,14 of Ub to a specific protein substrate and (2) the degradation of the although MYC protein levels can also be elevated in other polyubiquitylated substrate in a large multisubunit protease tumors without amplifications in MYC genes.15 complex known as the 26S proteasome.4 The ubiquitin–proteasome system (UPS) controls diverse cellular processes involving proliferation, maturation, differentia- GLIOBLASTOMA BIOLOGY tion and cell death. A wealth of recent experimental evidence GBMs have recently been divided into 3–6 molecular subgroups further demonstrates that deregulation of the Ub system has a depending on the classification scheme and clustering.16,17 The causative role in cancer development and progression. In this most commonly used scheme is provided by The Cancer Genome review, we describe important UPS substrates involved in brain Atlas currently classifying GBMs into four molecular subtypes: tumor development and the potential premise that protein Proneural, Neural, Classical and Mesenchymal groups.18 GBMs are stabilization can be targeted or that components of the Ub genetically diverse; however, generally speaking, 70–80% of GBMs

1Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden and 2Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden. Correspondence: Dr F Swartling, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SE-751 85, Sweden. E-mail: [email protected] Received 29 June 2013; revised 11 September 2013; accepted 12 September 2013; published online 28 October 2013 Oncoprotein stabilization S-M Hede et al 4710 are frequently characterized by loss of chromosome 10 and gain repair, among others.27 Ubiquitylation is counteracted by specific of chromosome 7. Several proteins driving GBM development and isopeptidases called deubiquitinating enzymes or DUBs, which progression are direct or indirect targets of the UPS. Receptor catalyze the hydrolysis of the peptide bond between the Ub tyrosine kinases (RTKs) that are commonly overexpressed or molecule and the substrate, highlighting the reversibility of the Ub amplified in GBMs including epidermal growth factor receptors system (Figure 1).28 (EGFRs), platelet-derived growth factor (PDGF) receptors and MET (encoding the hepatocyte growth factor receptor) are normally targeted for degradation by the CBL proto-oncogene and E3 Ub E3 Ub enzymes protein ligase (CBL), and these RTKs often find means to avoid CBL In the Ub system, substrate specificity is achieved through the use recognition during cancer development.19 Important downstream of a large number of different E3s that physically interact with kinases including phosphatidylinositol 3’-kinase (PI3K) and the specific target substrates, linking them to the E2 Ub-conjugating 29 mammalian target of rapamycin (mTOR) are also targets of the enzyme. E3 ligases are encoded by at least 600 human genes, 30 UPS. For instance, PI3K is regulated by the Ub ligases CBL-B20 and thus exceeding the number of protein kinase genes. The F-box and leucine-rich repeat protein 2 (FBXL2),21 whereas the majority of E3s contain a RING (Really-Interesting New Gene)- F-box and WD repeat domain containing 7 (FBW7), E3 Ub protein finger homology domain, which binds to the E2 enzyme and ligase promotes proteasomal degradation of mTOR.22 The E3 Ub directly mediates the transfer of Ub to the substrate. In contrast, ligase mouse double minute 2 (MDM2) is a well-established HECT (Homologous to E6-AP C-Terminus) domain E3s form a oncoprotein and negative regulator of p53 protein expression.23 transient covalent linkage between Ub and a cysteine residue on Several other proteins regulated by the UPS that are involved in the E3 prior to transferring the Ub molecule to the bound 31 brain tumor development and progression will be further substrate. RING E3 ligases can be further categorized into single reviewed below. subunit E3s (one protein contains both a RING domain and a substrate-binding domain—for example, MDM2) or multisubunit E3s (for example, Cullin-RING ligases (CRLs)). The CRL is the largest THE UPS family of E3s and includes the well-described SCF (SKP1-Cullin1-F- box) and APC/C (anaphase-promoting complex/cyclosome) Ub Breakdown of proteins (proteolysis) occurs constantly inside cells ligase complexes that have central roles in cell cycle regulation, as the production of new proteins must be balanced by an equal checkpoint control and genome stability.32 amount of protein degradation for cells to survive. The molecular The APC/C is composed of at least 13 protein subunits and machinery catalyzing the majority of intracellular protein degrada- 24,25 targets numerous cell cycle regulators for proteolysis in mitosis tion is known as the UPS. and in G1. The SCF Ub ligases consist of the following four subunits: the RING protein retinoblastoma (RB)X1, the scaffold The Ub enzymatic cascade protein Cullin1, the adapter protein SKP1 and a variable substrate- binding component known as the F-box protein.32 SCF ligases are The decoration of protein substrates with chains of Ub molecules B is the result of multiple rounds of Ub-conjugation reactions carried active throughout the cell cycle, and to date, 70 different F-box proteins have been identified in humans, each likely targeting a out by a three-step enzymatic cascade. First, an E1 enzyme 33 mediates the ATP-dependent activation of Ub. Next, Ub is unique set of substrates for degradation. Not surprisingly, translocated to an E2-conjugating enzyme, and finally, an E3 Ub deregulation of Ub ligases or their target substrates has a ligase facilitates the transfer of Ub to a specific lysine (K) residue causative role in cancer development and progression. on the target substrate (Figure 1).3 The 76-amino-acid Ub polypeptide has seven internal lysines, and Ub chains may be assembled through any of these lysine residues (in addition to the PROTEIN STABILIZATION AND REGULATION BY THE UPS IN amino group in the N-terminus of Ub). Substrates tagged with K48 BRAIN CANCER or K11-linked polyubiquitin chains are usually destined for The central nervous system arises from the neural plate, and the proteolytic destruction by the 26S proteasome,26 whereas brain and the spinal cord later develop from the neural tube of the substrates conjugated with a single Ub (monoubiquitin) or embryo. Proteins that are involved in early regulation of neuronal polyubiquitin chains linked through other lysines in Ub (for embryogenesis include bone morphogenetic proteins, SHH example, K63) have non-proteolytic functions modulating various proteins and fibroblast growth factors (FGFs).34 Other factors biological processes, such as transcription, DNA replication and essential for normal brain development include the NOTCH family,

Ub Ub Ubiquitin Ub Conjugation DUBs Ub E3 Ub Substrate Ub Substrate E1 E2 Substrate ATP

ATP Specific E3 Degradation Ub Targeting Ub Ub Ub Ub Substrate 26S Proteasome E1 E2 Ub

Figure 1. Schematic overview of the UPS. Ubiquitylation, or Ubiquitin (Ub) conjugations of protein substrates, is an ATP-dependent reaction that involves a concerted effort of an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin ligase (E3). In the ubiquitylation process, E3 ligases are the primary sources of substrate specificity, resulting in ubiquitin conjugation to specific lysine (K) residue(s) on the protein substrate. Proteins with K48-linked polyubiquitin chains are degraded by the 26S proteasome complex in an ATP- dependent manner, whereas other types of ubiquitin chains (for example, K63) have non-proteolytic functions regulating protein activity. The ubiquitylation process is counteracted by DUBs enzymes that hydrolyze ubiquitin–protein peptide bonds and recycle ubiquitin molecules.

Oncogene (2014) 4709 – 4721 & 2014 Macmillan Publishers Limited Oncoprotein stabilization S-M Hede et al 4711 Table 1. Oncoprotein stabilization in brain tumor models and molecular targets for putative therapies

Cancer protein/s/ stabilized or altered Brain tumor model/cell system Potential destabilizing treatment strategy

Medulloblastoma CTNNB1 (exon 3) with conditional Floxed exon 3 stabilizes b-catenin generating WNT Targeting downstream TCF-dependent p53 loss medulloblastoma45 transcription MYCWT/MYCT58A with p53 loss A mutationally stabilized MYC protein drives Targeting MYC by inhibiting CDKs, Aurora Kinases, Group 3 medulloblastoma114,115 BRDs or PI3K/mTOR MYCNT58A A mutationally stabilized MYCN protein drives Targeting MYCN by inhibiting CDKs, Aurora SHH or Group 4 medulloblastoma117 Kinases, BRDs or PI3K/mTOR MYCN via PTCH1 loss Patched1 loss increases MYCN protein stability as Targeting SMO or GLI proteins a mechanism of medulloblastoma initiation12

Glioma MYCNT58A Stabilized MYCN protein drives MG-PNET or Targeting MYCN by inhibiting CDKs, Aurora pediatric glioma117 Kinases, BRDs or PI3K/mTOR PDGFB Post-translationally modified PDGFB lacking UTR Targeting PDGF ligand binding or PDGF receptors parts drives more aggressive gliomas68 MYC in PTEN À / À /Ink4a/Arf À / À NSCs FBW7 loss raises MYC levels in NSCs to generate Targeting MYC by inhibiting CDKs, Aurora Kinases, and Fbxw7 knockdown GBMs187 BRDs or PI3K/mTOR Cyclin E Knockdown of PARK2 accumulates Cyclin E levels Inhibiting CDKs that bind with Cyclin E in glioma cells137 TRIM11 TRIM11 promotes accumulation of EGFR and Inhibitors of EGFR or MEK MAPK activation199 Abbreviations: BRDs, BET Bromodomain proteins; CDK, Cyclin-dependent kinase; EGFR, epidermal growth factor receptor; GLI, GLI family zinc finger; MG-PNET, malignant glioma with PNET-like morphologies; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; NSC, neural stem cell; PARK2, Parkinson protein 2, E3 ubiquitin protein ligase (parkin); PI3K, phosphatidylinositol 3’-kinase; PDGF, platelet- derived growth factor; PTCH1, Patched1; PTEN, phosphatase and tensin homolog; SHH, Sonic Hedgehog; SMO, Smoothened; TCF, transcription factor; TRIM11, tripartite motif-containing protein 11; UTR, untranslated region.

WNT proteins as well as various kinase receptors and their like SCFb-TrCP further regulates downstream signaling of the SHH downstream targets, including transcription factors and cell cycle pathway, as GLI2 is also targeted by SCFb-TrCP and then degraded.8 regulators. Several of these proteins are intimately involved in Gorlin’s syndrome is a rare inherited condition with germline brain tumor formation and cancer progression. Important mutations in the PTCH gene. It is characterized by an increased mechanisms that control oncoprotein stabilization during tumor incidence of tumors including basal cell carcinoma and development have further been discovered when using various medulloblastoma.10 Interestingly, step-wise loss of PTCH1 during animal models of brain tumors (Table 1). In the following sections, tumor formation and progression in mice leads to stabilization of we will review the recent literature of how the UPS regulates the MYCN protein12 (Table 1). proteins involved in brain tumor biology. The WNT pathway. There are two different WNT pathways: the Developmental pathways canonical or the WNT/b-catenin pathway controlling cell-fate determination and the non-canonical pathway regulating cell The SHH pathway. The Hedgehog pathway is a well-conserved movement and tissue polarity.41 SCFb-TrCP negatively regulates the developmental pathway. Hedgehog ligands, such as SHH, act as WNT/b-catenin signaling pathway by targeting b-catenin for morphogens during embryogenesis and regulate cell fate and 35 ubiquitylation following GSK3b-mediated phosphorylation on brain patterning. SHH signaling depends on a functional primary specific serine/threonine residues in b-catenin at a conserved cilium—a non-motile microtubule organelle that forms an 6 36 beta-TrCP-binding motif (degron). These residues are commonly antenna-like structure on the cell membrane. When SHH binds mutated on CTNNB1 in the WNT group of medulloblastoma,7,42 to the inhibitory receptor PTCH it releases smoothened, leading to 43 37 resulting in defective degradation of b-catenin because of the the activation of GLI family (GLI1-3) transcription factors. failure of beta-TrCP to interact with b-catenin.44 Similarly, Whereas GLI1 works as an activator of the SHH pathway, both mutationally stabilized CTNNB1 has been valuable in describing GLI2 and GLI3 can be processed into transcription repressor forms. how WNT-driven medulloblastoma could be derived from distinct A speckle-type POZ protein—cullin 3 Ub ligase—promotes developing brain stem regions (of the lower rhombic lip) in ubiquitylation and degradation of GLI2 and GLI3 full-length 45 38 transgenic animals (Table 1). Mutations in another member of activators. GLI1 is constitutively active and cannot convert the WNT pathway, the adenomatous polyposis coli (APC) gene, between activator and repressor forms. GLI1 interacts with itchy can also lead to stabilization of b-catenin in the development of E3 Ub protein ligase (ITCH)—a member of the HECT family of E3 medulloblastoma in Turcot’s syndrome.46 enzymes.39 ITCH binds GLI1 and promotes its polyubiqitylation in collaboration with numb homolog (Drosophila) (NUMB)—an adaptor protein that enhances ITCH activity and GLI1 The NOTCH pathway. NOTCH signaling is involved in the degradation.39 Interestingly, NUMB expression is attenuated in communication between neighboring cells during brain develop- the SHH group of medulloblastoma consistent with its role in ment.47 Interaction with ligands, DELTA or JAGGED, results in promoting GLI1 degradation. In the absence of SHH, GLI3 is sequential cleavage of NOTCH receptors at the membrane and phosphorylated at multiple sites by v-akt murine thymoma viral translocation of the NOTCH intracellular domains into the nucleus, oncogene homolog 1 (AKT), glycogen synthase kinase 3 beta where they serve as transcription factors regulating transcription (GSK3b) and casein kinase 1 (CK1). This leads to direct binding and of various target genes including HES (hairy and enhancer of split) ubiquitylation by the SCF Ub ligase, SCFb-TrCP and subsequent or HES-related (HESR/HEY) transcription factors.48 The NOTCH processing of GLI3 into its transcription-repressor form.40 It seems pathway is tightly controlled by the UPS system at several levels.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4709 – 4721 Oncoprotein stabilization S-M Hede et al 4712 NOTCH receptors are regulated by multiple E3s including ITCH, The tyrosine kinase receptor MET is amplified in a smaller DELTEX and NEDD4,49 and turnover of the unstable NOTCH fraction of GBMs.64 In addition, MET is overexpressed in many intracellular domains is mediated by the Ub ligase FBW7 (also GBMs75 in which expression correlates with increasing malignancy known as SEL-10, Ago and CDC4).50,51 FBW7 preferentially targets grade of the brain tumor. The degradation of activated RTKs can NOTCH intracellular domain phosphorylated by Cyclin C-CDK8 for be altered on many levels in human tumors. The activity of CBL is degradation in the nucleus.52 The activated NOTCH1 intracellular regulated by the tyrosine kinase SRC. Phosphorylation by SRC domain induces SHH group medullobastoma when overexpressed leads to enhanced autoubiquitylating activity and proteasomal in p53-deficient animals.53 Surprisingly, NOTCH2 rather than degradation of both CBL and SRC.76 An alternative degradation NOTCH1 levels are elevated in tumors generated from this mechanism was shown for EGFR in clear cell renal carcinoma, in model. As compared with NOTCH1 levels, NOTCH2 levels are which polyubiquitylation of the activated RTK by von Hippel– also elevated in primary human medulloblastoma.54 NOTCH1 Lindau tumor suppressor, E3 Ub protein ligase leads to nuclear staining further correlates with improved outcomes of proteasomal degradation.77 Cancer cells have different ways of proneural high-grade glioma.55 These data may suggest that escaping CBL recognition and avoiding RTK degradation by the NOTCH1 activity regulated by UPS is hypothetically necessary for UPS. For instance, CBL-binding sites on RTK genes can be mutated, tumor initiation but not for tumor progression. CBL recruitment could become inefficient, degradation of CBL itself could increase or mutations could generate an RTK that will form a fusion protein that escapes proteasomal degradation.19 Kinase receptors and downstream pathways Receptor tyrosine kinases. RTKs are transmembrane proteins that, The transforming growth factor beta (TGF-b) pathway upon ligand-binding, dimerize and activate intracellular signaling The TGF-b family of growth factors signals through TGFR1 and pathways controlling cell proliferation, survival, migration and TGFR2, activating downstream signaling pathways via phosphor- differentiation.56 Signaling via EGFRs (ERBB1-4) is important in ylation of the SMAD family members (SMADs) that when activated controlling cell proliferation and fate during neurogenesis. function as transcription factors together with various cofactors. Signaling via PDGFRA and PDGFRB has important roles both Activation of TGF-b signaling results in differential responses, during embryogenesis and in adulthood (reviewed in Andrae depending on the cellular context.78 Ubiquitylation of the TGF-b et al.57). PDGFRA signaling controls neural stem cell (NSC) fate, receptors and downstream SMAD effector proteins is regulated by inducing the formation of oligodendrocytic rather than neuronal different E3 ligases such as the HECT-type SMAD-specific E3 Ub lineage.58,59 protein ligase (SMURF) repressing TGF-b signaling and RING-finger RTK activity is terminated both by the action of tyrosine domain E3 ligase Arkadia (also known as RNF111) enhancing the phosphatases and by internalization and degradation of the TGF-b signal.79 Indeed, dysregulation of both SMURFs and Arkadia activated receptor. RTK signaling leads to phosphorylation and have been found in several types of cancer.79 recruitment of the RING finger E3 Ub ligase CBL60 (See also Altered TGF-b signaling is found in many diseases and TGF-b is Figure 2). CBL binds to phosphorylated tyrosine residues on the known to promote epithelial–mesenchymal transition in cancer.80 activated RTK either directly via its tyrosine kinase-binding domain Normally, TGF-b signaling blocks proliferation by inducing or indirectly through interaction with adapter proteins such as expression of p21 via SMADs and FOXO transcription factors. GRB2. CBL ubiquitylates the intracellular part of the activated RTK, This is overcome in glioma cells where FOXG1 blocks FOXO and and monoubiquitylation at multiple sites serves as a signal for SMAD, and activation of the PI3 kinase pathway leads to receptor internalization via clathrin-coated pits followed by phosphorylation of FOXO.81 TGF-b signaling has also been lysosomal degradation.61 CBL functions as a negative regulator shown to promote proliferation of glioma cells in cases where of many RTKs, including EGFR (ERBB1), ERBB2, PDGFRA and the PDGFB gene is unmethylated. The hyperactive TGFb-SMAD PDGFRB, MET and FGFR (reviewed in Marmor and Yardon60). pathway here induces PDGFB levels, which correlate to poor Activation of oncogenic RTK signaling pathways in glioma can prognosis in glioma patients.82 be caused by elevated levels of ligand or increased abundance The DUB USP15 promotes TGFBR1 stability in GBMs by binding and activity of receptors. Both PDGF ligands and receptors are to the SMAD7-SMURF2 complex and removing Ubs from TGF-b found to be overexpressed in glioma62 often in combination with receptors. Expression of USP15 correlates with high TGF-b activity. the loss of p53.63 The PDGFRA gene is found to be amplified in Amplifications of USP15 further correlate with poor prognosis in 13% of GBM.64 Animal models have indicated that GBM tumors several tumor types including GBMs.83 may form by overexpression of PDGFB alone or in combination with the loss of p53.65–67 Interestingly, excessive expression of PDGFB ligand causes the formation of brain tumors in mice in a The PI3K/PTEN/AKT pathway and mTOR proteins dose-dependent way.68 In this study, certain untranslated regions PI3Ks are activated by several different types of membrane are responsible for post-translational modifications of PDGF, and receptors, including RTKs, T-cell receptors, integrins, cytokine removing these leads to PDGFB protein stabilization (Table 1). receptors and G-protein-coupled receptors. The class I PI3K is the The EGFR is found to be overexpressed and amplified in human best characterized subclass of PI3Ks and is composed of gliomas, and amplification of the EGFR gene is found in B40% of regulatory (p85) and catalytic (p110) subunits. The phosphatase GBM.69 The most common EGFR mutant in GBM is EGFRvIII, which and tensin homolog (PTEN) acts as a phosphatase for phospha- lacks the extracellular ligand-binding domain.70 EGFRvIII is tidylinositol (3,4,5)-triphosphate (PIP3), thereby negatively regulat- constitutively activated, has a reduced capacity of recruiting CBL ing downstream AKT signaling.84 The mTOR is a serine/threonine and shows reduced receptor internalization and degradation.71 kinase that is present in two specific complexes, mTOR complexes Overexpression of ERBB2 is found in many tumors, and the ERBB2 1 and 2 (mTORC1 and mTORC2), which act downstream or gene is found to be mutated in 8% of GBM.64 Studies in breast upstream of AKT, respectively. The mTOR pathway couples tumors have shown that the ERBB2 receptor stabilizes EGFR and nutrient and energy availability to the execution of various inhibits CBL-mediated receptor ubiquitylation by blocking biological processes including cell growth and proliferation, phosphorylation of CBL- and GRB2-binding sites in EGFR.72 survival, motility, protein synthesis and transcription.85 Interestingly, v-erbB, the transforming viral homolog of EGFR, The p85 regulatory subunit of PI3K is a substrate for the Ub has been found to lack the CBL-binding site,73 and overexpressing ligase CBL-B.86 However, when Cbl-b is knocked out in mouse T v-erbB from the S100b promoter has been shown to induce cells, ubiquitylation of p85 is not abrogated. Instead, CBL-B loss led oligodendrogliomas in mice.74 to enhanced ubiquitylation of PTEN upon T-cell receptor

Oncogene (2014) 4709 – 4721 & 2014 Macmillan Publishers Limited Oncoprotein stabilization S-M Hede et al 4713

RTK

P P

P P P Ub P CBL CBL DUB

Ub

P P Ub CBL ERK P P AKT mTORC1 P P CBL LYSOSOME

GSK3B PROTEASOMAL DEGRADATION

Ub Ub S62 Ub P MYC Ub PP2A P MYC FBW7 Ub MDM2 Ub Ub Ub P UBE4B Ub P p53 p53 T58 MYC MDM2 PIRH2 MDM2 CDKs MDM2 MDM4

Ub Ub DUB Ub P Ub Ub MYC p53 MDM4 Ub Ub MAD P Ub MDM2 MXI1 ATM

MNT P MYC MAX p53 p53 E-box Figure 2. A schematic representation of ubiquitin-mediated protein degradation in the key signaling pathways involved in brain tumor development and progression. RTK signaling pathways modulate MYC stability via phosphorylation and dephosphorylation events required for substrate recognition and ubiquitylation by the E3 ligase FBW7. RTK internalization and lysosomal degradation are mediated by E3 ligase CBL. Alternatively, RTKs can be recirculated to the cell surface via early endosomes. In the nucleus, degradation of p53 is regulated by mono- and polyubiquitylation carried out by various ubiquitin ligases (not shown). The monoubiquitylated form of p53 is exported to the cytosol, where further polyubiquitylation targets the protein for proteasomal degradation. Phosphorylation of p53 by stress-induced kinases such as ATM disrupts ubiquitylation mediated by MDM2. The activities of both p53 and MYC factors are regulated by shuttling in and out of the nucleus, and proteasomal targeting could take place both within the nucleus and in the cytosol. In the figure, ubiquitin ligases are shown as gray ovals. stimulation.87 Recently, SCFFBXL2 has further been shown to target PTEN.22 mTOR and especially the mTORC1/2 complexes are the p85 for proteasomal degradation.21 In GBMs, mutations have been essential brain tumor pathways.92 In response to growth factor found in PIK3R1 and PIK3CA genes that encode regulatory and stimulation, SCFb-TrCP also regulates mTOR activity through the catalytic subunits of PI3K.64 targeted degradation of DEPTOR (DEP domain containing mTOR- The HECT-domain protein NEDD4-1 can regulate PTEN by interacting protein), an inhibitor of both mTORC1 and mTORC2.93 mono- and polyubiquitylation.88 Surprisingly, NEDD4-1 is not required for PTEN degradation, as PTEN stability is unaltered in Nedd4-1 knockout mice.89 Furthermore, a recent study Transcriptional regulators demonstrates that WWP2, a member of the NEDD4-like protein MYC proteins. The MYC family of proteins consists of three family, interacts with PTEN and stimulates Ub-dependent members— MYC, MYCL and MYCN—that are important regulators degradation of PTEN.90 Loss of heterozygosity on chromosome of transcription. MYC proteins are essential for normal brain 10 is a hallmark of many types of tumors including GBMs. PTEN, development and specific disruption of Mycn in nestin-positive being situated on this chromosome, is frequently altered in brain neural stem, and precursor cells (NSCs) present a severely tumor cells.91 diminished forebrain and hindbrain.94 FBW7 binds a specific phosphodegron in mTOR, promoting its MYC proteins are regulated by different post-translational ubiquitylation and proteasomal degradation.22 This targeting by modifications. At least five different E3 Ub ligases, FBW7, beta- FBW7 leads to breast cancer suppression in cooperation with TrCP, transient receptor potential cation channel, subfamily C,

& 2014 Macmillan Publishers Limited Oncogene (2014) 4709 – 4721 Oncoprotein stabilization S-M Hede et al 4714 member 4 associated protein (TRUSS), S-phase kinase-associated The MDM2 protein can either homo- or heterodimerize with the protein 2, E3 Ub protein ligase (SKP2) and HECT, UBA and WWE homologous RING finger protein MDM4 (also known as MDMX) domains containing 1, E3 Ub protein ligase (HECTH9/HUWE1) have (Figure 2). MDM4 binds p53; however, it has no intrinsic Ub ligase been shown to regulate MYC.95 Whereas FBW7 and TRUSS act as activity.121 The MDM2/p53 interaction and subsequent ubiquitylation negative regulators of MYC,96–98 Beta-TrCP positively regulates is inhibited by various post-translational modifications including MYC protein stability.99 Interestingly, several other Ub ligases, phosphorylation, acetylation, neddylation and sumoylation.122 including SKP2,100,101 HECTH9/HUWE1102,103 and F-box protein 28 MDM2 can either monoubiquitylate p53, facilitating transport to (FBXO28),104 regulate MYC protein function through non- the cytoplasm or cooperate with MDM4 and other Ub ligases to proteolytic ubiquitylation. polyubiquitylate, and thereby target p53 for degradation by the Regulation of MYC activity and stability is a tightly controlled proteasome.123 Several p53-targeting Ub ligases with process. ERK or CDKs must first phosphorylate MYC on residue complementing functions have been discovered lately, although S62.105 GSK3b needs this priming phosphorylation in order to MDM2 seems to be the main factor regulating p53 levels under phosphorylate T58,96 which can be recognized by FBW7 and normal conditions.124 For example, the E3 ligase PIRH2 (p53-induced degraded first after S62 is dephosphorylated by combined actions protein with RING-H2 domain) targets phosphorylated p53 during of PIN1 prolyl isomerase and the PP2A phosphatase106 (see also DNA damage125 (Figure 2). UBE4B is another Ub ligase that interacts Figure 2). However, FBW7-dependent MYC ubiquitylation appears with both MDM2 and p53. UBE4B is found to be amplified and to be more complex, and MYC stability is also controlled by other overexpressed in brain tumors, and its expression is inversely factors (reviewed in Thomas and Tansley95). correlated to levels of p53.126 The UPS system also controls cellular reprogramming.107 Whereas mutations and loss of heterozygosity of p53 are Depletion of FBW7 results in enhanced reprogramming commonly found in human secondary GBMs, the primary GBM frequency, whereas inactivation of the DUB PSMD14 abrogates more often displays upstream inactivation of the p53 pathway the reprogramming process. The effect of FBW7 loss is designated through amplification and overexpression of MDM2 or deletion of to the stabilization of MYC that is involved in induced pluripotent Ink4a/Arf.69 Recent results from the The Cancer Genome Atlas stem cell generation.108 showed that altered p53 signaling was found in 87% of GBM, with The MYC proteins MYC and MYCN are commonly amplified or amplification of MDM2 in 14% and inactivation of alternate reading overexpressed in brain tumors (for details see the study by frame (ARF) of the CDKN2A gene by deletion or mutation in 49% of Swartling109). Focal high-level amplifications of MYC or MYCN are the cases.64 p53 status is an important risk factor in SHH in most cases mutually exclusive110 and can occur in both GBM64 medulloblastoma subtypes despite the fact that the majority of and medulloblastoma.111 Interestingly, mutations in the histone human medulloblastoma, including Groups 3 and 4, lack p53 H3 gene, H3F3A, are commonly found in pediatric GBMs and cause mutations.127 MDM2canbeinvolvedinregulatingp53levelsin profound upregulation of MYCN.112 MYC or MYCN is further these tumors, as the MDM2 protein is often overexpressed amplified in almost half of all brain cancers categorized as predominantly in medulloblastoma cells where the p53 gene is not malignant glioma with PNET-like morphologies.113 mutated.128 MYCN is downstream of the SHH pathway and is essential for driving SHH-driven brain tumors in various mouse models Cell cycle proteins. The intricate interplay between different E3s is of medulloblastoma. MYCN overexpression can, together of major importance for the timely activation/inactivation of with p53 loss, further drive SHH-dependent medulloblastoma various CDK regulatory proteins (for example, CDK inhibitors and from GPCs following transplantation to the brain.114 By contrast, cyclins). SCF ligases target numerous cell cycle regulatory proteins overexpressed stabilized forms of its sibling MYC for degradation. SCFSKP2 recognizes p27 phosphorylated by Cyclin lead to development and progression of SHH-independent E-CDK2, an interaction which also depends on the co-factor aggressive large cell/anaplastic Group 3 medulloblastoma114,115 CKS1.129 Degradation of p27 results in elevated Cyclin E-CDK2 (Table 1). activity, entry into the S-phase and initiation of DNA replication. As We have recently shown that MYCN can drive aggressive large CDK activity rises in G1, the RB pocket proteins (RB, p107 and cell/anaplastic medulloblastoma when overexpressed from the p130) are inactivated by phosphorylation and E2F factors are Glutamate transporter 1 promoter in an inducible transgenic released. Several different Ub ligases target E2F1 for proteasomal model.116 We further showed that MYCN carrying a stabilizing degradation, including SKP2 at the end of the S-phase130 and APC/ FBW7 degron mutation (T58A) was necessary for brain tumor C in the prometaphase.131 The APC/C ligase controls both positive generation from NSCs117 (Table 1). By transducing forebrain and negative regulators of the cell cycle through association with NSCs with MYCN-T58A, we could mimic malignant glioma its co-activator subunits, CDC20 and CDH1, which determine with PNET-like morphologies113 or pediatric GBMs that are substrate specificity.132 These differences in substrate specificity shown to depend on high levels of MYCN.112 Interestingly, have important roles during neurogenesis, (reviewed by Puram depending on the timing of MYCN induction, both and Bonni133), where, for example, APC/C together with CDH1 SHH-dependent- and -independent medulloblastoma types target ID proteins, regulating cell cycle exit.134 formed from cerebellar NSCs despite using the same stabilized CDKs are often amplified or overexpressed in both glioma and MYCN oncoprotein. medulloblastoma (for a more detailed review on this, see Vlachostergios et al.135). The CDK inhibitor p27 is downregulated p53. The transcription factor p53 is a tightly regulated sensor of and associated with increased glioma grade,136 and SKP2 (that cellular stress and its activation might result in cell cycle arrest, controls p27 degradation) is found at elevated levels in high-grade apoptosis, senescence, DNA repair, altered metabolism or glioma. In addition to FBW7, the Ub ligase PARK2 also targets autophagy.118 Cyclin E for degradation and is commonly found to be mutated in Under normal conditions, protein levels of p53 are held low by GBMs with increased Cyclin E stability137 (Table 1). proteasomal degradation, promoted in part through continuous Almost 80% of GBM tumors display alterations in the RB targeting by the RING finger E3 Ub ligase MDM2.23 Specifically, pathway. The RB gene itself is found to be mutated or deleted in the transcription of MDM2 is upregulated by p53, creating B10% of the cases, whereas CDKN2A and CDKN2B are deleted or a feedback loop where MDM2 targets both p53 and itself mutated in 52 and 47% of GBMs, respectively.64 RB interacts with for proteasomal degradation.119 MDM2 also blocks the APC/C that controls genomic stability by regulating proteins transactivating activity of p53, preventing transcriptional controlling the mitotic spindle formation checkpoint. Several APC/ activation of p53 target genes.120 C targets, including CDC20, polo-like kinase 1, Aurora kinases,

Oncogene (2014) 4709 – 4721 & 2014 Macmillan Publishers Limited Oncoprotein stabilization S-M Hede et al 4715 SKP2, UBE2C and survivin are overexpressed in brain tumors.138 increased protein degradation.156 Surprisingly, in some cancer cells EMI1 is a transcriptional target of E2F1 that inhibits APC/CCdh1 this process is not dependent on GSK-3b phosphorylation that function. Overexpression of EMI1 accelerates S-phase entry and normally regulates b-catenin protein stability. leads to chromosomal instability.139 EMI1 is found to be overexpressed in ependymomas and high-grade gliomas. Loss PI3K, mTOR and MYC proteins. MYCN is indirectly stabilized by the of RB leads to dysregulation of EMI1 and genomic instability in activation of PI3K.157 Blockade of PI3K leads to decreased MYCN human tumors, including oligodendrogliomas.140 EMI1 is further protein levels in neuroblastoma—a childhood tumor with frequent regulated by beta-TrCP and fibroblasts from mice lacking beta- MYCN amplifications.158 mTOR inhibitors are also effective in TrCP show decreased genetic stability and mitotic progression in suppressing MYCN in neuroblastoma159 or inhibiting MYC in part owing to the accumulation of EMI1.141,142 murine medulloblastoma.115 Furthermore, a dual inhibition of PI3K and mTOR shows emergent efficacy when targeting human glioma NFkB. NFkB signaling controls diverse physiological functions cells.160 including cell growth and survival, differentiation, development, Stabilization of MYCN by Aurora A kinase is controlled by FBW7 in immunity and inflammation.143 In unstimulated cells, NFkB neuroblastoma,161 and MYCN protein stabilization is found in a large proteins are generally kept inactive through binding to proteins set of patients who lack MYCN amplifications.15 Small Aurora A known as inhibitors of NFkB (IkBs).144 kinase inhibitors can disrupt the binding of Aurora-A to MYCN and The NFkB pathway is subjected to tight post-translational allow FBW7 to recognize and promote the degradation of MYCN.162 regulation controlled by protein kinases, DUBs145 andUbligases. Therapeutic MYCN depletion totally cures tumors in transgenic Phosphorylation of IkBa by IKK targets this inhibitor for ubiquitylation mouse models of brain tumors,116 and the depletion of all MYC and proteasomal degradation by the SCFb-TrCP Ub ligase, allowing the proteins also cures tumors driven by other oncogenes,163 NFkB protein RelA to translocate to the nucleus and activate gene highlighting MYC proteins as indisputable targets for tumor expression. SCFb-TrCP also contributes to NFkB pathway activation by treatment.164 promoting the formation of specific NFkB protein complexes in the nucleus through ubiquitylation and partial proteolysis of IkBs, such as Targeting UPS components. Inhibition of proteasomal activity p105 and p100. Furthermore, we and others have recently using reversible (Bortezomib) or irreversible (Carfilzomib) protease demonstrated that the SCFFBW7 Ub ligase targets p100 for inhibitors has remarkable efficiency against multiple mye- degradationinaGSK3b-dependent manner.146–148 In this context, loma165,166 but also demonstrates potency for the treatment of NFkB pathway activity is modulated by both SCFFBW7 and SCFb-TrCP. different types of lymphomas and is currently undergoing clinical The IkBa gene NFKB1A is often heterozygously deleted in trials for the treatment of many other malignancies, including nonclassical subtypes of GBM patients,149 suggesting an active role pediatric and adult brain tumors.167,168 for the NFkB pathway in these brain tumors. NFkB activity is higher in Proteasomal inhibitors induce cell cycle arrest and apoptosis. GBMs as compared with the normal brain;150 however, the However, the molecular mechanism(s) is still not clear and various mechanism of NFkB activation in GBMs is largely undefined factors seem to be important in different cell types. Treatment perhaps because of the large heterogeneity of these tumors. with proteasomal inhibitors can shift cellular homeostasis towards However, NFkB could putatively be activated by cytokines such as apoptosis by causing an accumulation of pro-apoptotic proteins TNF-alpha or interleukins that are secreted by activated tumor- such as BID and BAX.169,170 Proteasomal inhibitors can also reduce associated macrophages.151 The regulation of NFkB by the UPS is the expression of anti-apoptotic proteins of the BCL2 or inhibitor further discussed below. of apoptosis families possibly through the inactivation of NFkB transcriptional activity following stabilization of the IkB. In medulloblastoma, Bortezomib has been reported to trigger TARGETING UPS SUBSTRATES OR THE UPS ITSELF IN FUTURE reactive oxidative stress (ROS)-associated apoptosis by stabilizing 171 CANCER THERAPIES the pro-apoptotic protein NOXA. Malignant high-grade brain tumors have a dismal prognosis, and However, as proteasomal inhibitors work non-selectively resistance-associated mechanisms to chemotherapies often pre- blocking the degradation of all proteins passing through the clude the complete eradication of cancer cells resulting in tumor 26S proteasomal machinery, more specific drugs are needed relapse. With this follows the challenge of developing novel (Figure 3). In fact, despite a strong scientific rationale for using Bortezomib as an additional therapy in patients with recurrent therapeutic strategies targeting the key proteins contributing to 167 tumor cell resistance and/or exploring new classes of drug targets. GBM, this combination is clinically ineffective. Therefore, Recently, with the discovery of the proteasome inhibitor Bortezo- targeting the enzymes regulating the ubiquitylation process is mib (PS341/Velcade), the UPS has matured as an important drug an alternative and more specific approach, possibly also with less discovery arena with Ub enzymes as the prime candidate drug unwanted side effects (Figure 3). Inhibitors of E1 and E2 enzymes targets.152 As outlined above, many proteins directly influencing have been reported; however, their potential clinical efficiency brain tumor development and progression are substrates of the remains to be demonstrated. In addition, targeting E1 or E2 UPS. We will now exemplify how such substrates or various enzymes could also result in undesirable effects on normal cells, as components of the UPS could be targeted in brain tumors. they are expected to inhibit most or many E3 ligases. Targeting the E1-activating enzyme (NAE-E1) for the Ub-like protein NEDD8 is a more specific approach because the primary targets for Targeting substrates of the UPS neddylation appears to be CRLs. Neddylation of the cullin Developmental pathways. Long-term treatment with the smooth- component is critical for CRL activity, and inhibitors of NAE-E1 ened inhibitor cyclopamine results in complete disruption of could thus preferentially block CRLs. Indeed, MLN4924—a small glioma stem cell populations. Besides inhibiting smoothened, there molecule inhibitor of the NAE-E1 enzyme—has been developed are now ways to target GLI proteins downstream of SHH and shows promise as an anticancer drug in mouse models and signaling.153 Of the GLIs, GLI2 is supposedly the most important preclinical studies.172,173 therapeutic target in medulloblastoma.154 It is not completely clear how GLI2 stabilization is regulated in tumors. Interestingly, arsenic Targeting specific E3 Ub ligases directly. Higher specificity is treatment has been effective in targeting GLI2 in SHH-dependent achieved if the activity of single E3s or DUBs is blocked. These tumors, as it destabilizes GLI2 and prevents ciliary accumulation.155 enzymes are particularly attractive drug targets, as they are Inhibition of b-catenin by methylseleninic acid is also a result of responsible for mediating ubiquitylation/deubiquitylation of

& 2014 Macmillan Publishers Limited Oncogene (2014) 4709 – 4721 Oncoprotein stabilization S-M Hede et al 4716 DUB Inhibitors E3 Ligase Inhibitors SKP1 Ub E1 Ub Inhibitors SKP2 Ub Ub p27 p53 MDM2 Ub Substrate Ub Ub E1 E2

ATP CUL1 Ub ATP

NEDD8 Substrate Interaction Neddylation ROC1 E2 Ub Inhibitors Inhibitors Ub Ub 26S Proteasome Ub Ub

E2 Inhibitors

Bortezomib Proteasome Carfilzomib Inhibitors Figure 3. Various ways to target the UPS in brain tumors. Apart from targeting E1 and E2 enzymes directly, there are ways to target the E1- activating enzyme for the ubiquitin-like protein NEDD8, which will specifically inhibit the ubiquitin–ligase activity of Cullin–Ring ubiquitin ligases (CRLs) such as SKP2 as depicted. Even more specific ways to target UPS are by targeting specific DUBs or E3 ligases, for example, by using inhibitors directed against SKP2 or by substrate interaction inhibitors, such as nutlins, that interferes with the p53–MDM2 interaction. A broader therapeutic approach involves using proteasome inhibitors that are currently evaluated in various brain tumor therapies.

protein substrates frequently dysregulated in tumor cells. In mechanism of p53 inactivation, alternative approaches are particular, many E3s/DUBs qualify as drug targets because they are warranted. Prima-1 is one example of a small molecule that overexpressed in human tumors and are often associated with the causes a conformational change in mutated p53, restoring the suppression of multiple tumor-suppressor proteins. Although the wild-type-like conformation and inducing an apoptotic development of DUB inhibitors is still in its infancy, several response.177 compounds are under development.152 Multiple strategies can be envisioned for targeting E3 Ub SKP2. Given that SKP2 is overexpressed in a large number of ligases, including development of small molecules that impair malignancies and, as outlined above, targets preferentially the binding between the E3 ligase and the E2 enzyme, or tumor-suppressor proteins important for cell division control, between the E3 and its specific target substrate(s). In principle, including p27, p21, p57 and p130, makes it an appealing drug compounds that induce a conformational change in the E3 could target.178 The oncogenic function of SKP2 is underscored by the attenuate Ub ligase stability and/or interfere with E3 activity. inverse association with p27 protein level in medulloblastoma179 Nucleic acids (for example, micro RNA, small interfering RNA) that and glioma.136 A schematic overview of SKP2 and its potential reduce the expression of the E3 ligase, or antibodies/peptides substrates in brain tumors is presented in Figure 4. SKP2 mRNA that interfere with E3 subcellular localization or activity, levels are elevated both in medulloblastoma and GBM as represent other possibilities to inhibit oncogenic E3s. Finally, it compared with the normal brain (Figure 4), indicating SKP2 as should be emphasized that there is tight interplay between a promising drug target in these types of tumors. Small molecule protein ubiquitylation and protein phosphorylation, and inhibitors of SCFSKP2 have been discovered, including a understanding how these systems cooperate in controlling the compound that prevents the binding of SKP2 to the core ligase, stability and activity of proteins deregulated in cancer cells are triggering the accumulation of p27 and cell cycle arrest and can important areas of future research. apparently even overcome Bortezomib resistance.180 Recently, inhibitors that selectively target the interaction between SKP2 MDM2. MDM2, the major regulator of p53 protein, has strong and CKS1 as well as its substrate p27 have also been identified181 clinical relevance in brain tumors with a functional p53 gene. and could thus be useful for treating tumors with deregulation of Several compounds have been developed aiming to restore p53 the SKP2–p27 axis. Another report shows how a novel SKP2- activity in tumors by interfering with the p53–MDM2 interaction. specific inhibitor directly binds the F box motif of SKP2,182 Nutlins are one example of low molecular weight compounds that leading to an effective block of SKP2–SKP1 interactions and target the p53-binding pocket of MDM2. By preventing the suppression of SKP2 ligase activity. This novel SKP2 inhibitor interaction of MDM2 with p53, the levels of p53 are increased, effectively inhibits tumor cell proliferation, survival and glycolysis leading to cell cycle arrest and apoptosis in tumor cells with wild- and is involved in triggering p53-independent senescence in type p53.174 An alternative approach involves targeting MDM4 to various animal cancer models. achieve destabilization of MDM2 and induction of a p53 response.175 Efforts have also been made to develop molecules Beta-TrCP. As outlined above, SCFb-TrCP triggers the ubiquityla- directed towards p53. The compound RITA induces apoptosis in tion of several different proteins involved in brain tumor tumor cells by binding the N-terminal domain of p53, which development and/or progression, including GLI2, b-catenin inhibits its interaction with MDM2.176 as well as the NFkB inhibitor, IkBa183 (Figure 4). Thus, targeting However, these drugs will only induce a p53 response in tumor beta-TrCP might be a promising approach in GBM suppression. cells expressing wild-type p53 protein. Depending on the Indeed, an inhibitor that suppresses IkBa ubiquitylation, but not

Oncogene (2014) 4709 – 4721 & 2014 Macmillan Publishers Limited Oncoprotein stabilization S-M Hede et al 4717 SCF ubiquitin ligases Substrates important for Expression of SCF ligases brain tumors in brain tumors

SKP1 p27 Ub SKP2 Ub Ub p21 ATP PP Ub CUL1 Substrate MYC ROC1 E2 Ub Cyclin D1

SKP1 Ub GLI2 bTrCP Ub Ub Ub β-catenin ATP P P

E1 CUL1 Substrate Ub ikBα

ROC1 E2 MYC Ub

MYC SKP1 Ub mTOR FBW7 Ub ATP Ub PP NFkB2 Ub Cyclin E CUL1 Substrate NOTCH1 ROC1 E2 Aurora A Ub

Figure 4. Given their critical functions in regulating the key regulatory proteins, SCF ubiquitin ligases contribute to oncogenesis. (a) SCF ligases trigger ubiquitin-mediated proteolysis of various proteins involved in brain tumorigenesis (red ovals); however, ubiquitylation of others leads to their stabilization (green ovals). Sometimes, the effect is context-dependent (yellow ovals). Both oncoproteins and tumor suppressors are targets of different SCF ligases. Phosphorylation of short conserved sequences on protein substrates (phosphodegrons) allows the F-box protein subunit (black boxes) in SCF ligases to recognize many different protein substrates in a spatial and temporal manner. (b) Comparing gene expression of SKP2, beta-TrCP (BTRC) and FBW7 (FBXW7) between human normal and tumorous fore- and hindbrain, respectively. Left panel: raw expression data for 81 GBM samples195 and 9 forebrain (cerebral cortex) samples196 were preprocessed together using the RMA algorithm197 implementation in the ExpressionFileCreator module on the GenePattern analysis platform.198 Distributions of SKP2 (top), BTRC (middle) and FBXW7 (bottom) expression values in normal and tumor sets, respectively, are depicted as box-and-whisker-plots, with the median of expression represented by horizontal lines inside each box, lower and upper box borders, indicating 25th and 75th percentiles, respectively—regions between whiskers including all non-outlier values and dots representing outliers. Right panel: raw expression data for 62 medulloblastoma samples13 and 9 hindbrain (cerebellum) samples196 were preprocessed and illustrated as above. Po0.01 for all six comparisons using two-tailed Welch’s t-test statistics. Figures were generated in MATLAB.

phosphorylation, has been identified.184 This could prove NOTCH1 could also enforce cell death by apoptosis.188,189 In fact, promising in restoring IkBa levels, as mutated alleles of NFKB1A loss of FBW7 has been shown to drive normal brain cells into were rarely found in the heterozygously NFKB1A-deleted GBMs.149 apoptosis.190,191 How FBW7-deficient tumor cells evade apoptosis Interestingly, NFKB1A and EGFR amplifications were mutually is still unclear; however, one possibility is that the accumulation of exclusive, suggesting that these events connect in a similar FBW7 substrates with pro-survival functions (for example, MCL1 oncogenic pathway.149 Judging from beta-TrCP mRNA levels, and NFkB2) protects cancer cells from cell death. Indeed, there is SCFb-TrCP is suppressed in GBM patients (Figure 4). Elevation of an intimate association between inactivation of the tumor- SCFb-TrCP expression is instead observed in medulloblastoma, suppressor FBW7 and drug resistance.192 However, whereas where NFKB1A deletions have only been found in Group 4 FBW7-deficient cells are resistant to some antitumor agents, tumors42 (Figure 4). This tumor-specific difference is interesting FBW7 inactivation might sensitize tumor cells to other drugs.193 and could suggest that SCFb-TrCP controls the stability of distinct Interestingly, in chronic myelogenous leukemia-initiating cells,194 oncoproteins in one brain tumor type as compared with another. a sudden FBW7 loss restrains tumorigenesis. There, FBW7 suppression increases the stabilization of MYC, thereby FBW7. FBW7 is an important oncoprotein suppressor and is promoting p53-dependent apoptosis.188 In principle, these frequently inactivated by mutation, deletion and promoter results suggest that specific FBW7 inhibitors could potentially be hypermethylation in multiple malignancies.185 However, the role used to force non-dividing cancer cells to proliferate, thus of FBW7 in brain tumorigenesis is not fully understood. FBW7 is increasing their sensitivity to conventional therapeutic drugs. found to be downregulated in human gliomas186 (Figure 4). Loss Overall, FBW7 levels are low in both MB and GBM (Figure 4) as of FBW7 in p53/PTEN-deficient NSCs can further promote glioma compared with the normal brain, suggesting that FBW7 expres- formation by MYC stabilization187 (Table 1). However, abrupt sion is suppressed (at least to some extent) during development accumulations of FBW7 substrates such as MYC, c-JUN and or progression of specific types of tumors. Whether targeting

& 2014 Macmillan Publishers Limited Oncogene (2014) 4709 – 4721 Oncoprotein stabilization S-M Hede et al 4718 FBW7-deficient brain tumors with drugs directed towards specific 11 Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM et al. Human FBW7 substrates could open up new therapeutic windows homolog of patched, a candidate gene for the basal cell nevus syndrome. remains to be shown. Science 1996; 272: 1668–1671. 12 Thomas WD, Chen J, Gao YR, Cheung B, Koach J, Sekyere E et al. Patched1 deletion increases N-Myc protein stability as a mechanism of medulloblastoma CONCLUSIONS AND FUTURE PERSPECTIVES initiation and progression. Oncogene 2009; 28: 1605–1615. UPS has important functions in normal brain development 13 Kool M, Koster J, Bunt J, Hasselt NE, Lakeman A, van Sluis P et al. Integrated genomics identifies five medulloblastoma subtypes with distinct genetic profiles, controlling cell fate and specification. The accumulation of reports pathway signatures and clinicopathological features. PLoS One 2008; 3:e3088. describing dysregulated UPS components in cancer suggests that 14 Cho YJ, Tsherniak A, Tamayo P, Santagata S, Ligon A, Greulich H et al. Integrative increased protein stabilization of oncoproteins or destabilization genomic analysis of medulloblastoma identifies a molecular subgroup that and degradation of tumor-suppressor proteins is important for drives poor clinical outcome. J Clin Oncol 2011; 29: 1424–1430. brain tumor development. Degradation of stabilized oncoproteins 15 Valentijn LJ, Koster J, Haneveld F, Aissa RA, van Sluis P, Broekmans ME et al. or restoration of destabilized suppressors could prove to be Functional MYCN signature predicts outcome of neuroblastoma irrespective of effective in inhibiting tumor cells. Thus, the UPS regulates many MYCN amplification. Proc Natl Acad Sci USA. 2012; 109: 19190–19195. substrates that partake in brain cancer and is an unquestionable 16 Chen J, McKay RM, Parada LF. Malignant glioma: lessons from genomics, mouse therapeutic target for brain tumor treatment. Notably, brain models, and stem cells. Cell 2012; 149: 36–47. tumors have recently been divided into specific subgroups 17 Sturm D, Witt H, Hovestadt V, Khuong-Quang DA, Jones DT, Konermann C et al. depending on their genetic profiles and their molecular biology, Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 2012; 22: 425–437. implying that individual tumors depend on distinct sets of cancer 18 Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD et al. Integrated proteins. We have described that the most common types of genomic analysis identifies clinically relevant subtypes of glioblastoma char- malignant brain tumors including medulloblastoma and glioma acterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 2010; and their defined subgroups have distinct amplifications or 17: 98–110. deletions in well-known UPS substrates. In summary, it is clear 19 Peschard P, Park M. Escape from Cbl-mediated downregulation: a recurrent that the UPS has an important role in brain tumorigenesis, and theme for oncogenic deregulation of receptor tyrosine kinases. Cancer Cell 2003; specific targeting of this system shows promise for treating these 3: 519–523. devastating brain tumors. 20 Fang D, Wang HY, Fang N, Altman Y, Elly C, Liu YC. Cbl-b, a RING-type E3 ubiquitin ligase, targets phosphatidylinositol 3-kinase for ubiquitination in T cells. J Biol Chem 2001; 276: 4872–4878. CONFLICT OF INTEREST 21 Kuchay S, Duan S, Schenkein E, Peschiaroli A, Saraf A, Florens L et al. FBXL2- and PTPL1-mediated degradation of p110-free p85beta regulatory subunit controls The authors declare no conflict of interest. the PI(3)K signalling cascade. Nat Cell Biol 2013; 15: 472–480. 22 Mao JH, Kim IJ, Wu D, Climent J, Kang HC, DelRosario R et al. FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression. Science ACKNOWLEDGEMENTS 2008; 321: 1499–1502. This work was supported by research from the Swedish Childhood Cancer 23 Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of Foundation (FJS, OS), the Swedish Cancer Society (FJS, OS), the Swedish Research p53. Nature 1997; 387: 296–299. Council (FJS, OS), the Swedish Society of Medicine (FJS), the Swedish Brain 24 Hershko A, Ciechanover A, Heller H, Haas AL, Rose IA. 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