1 Frontiers in Oncology 2012 Vol: 2. DOI: 10.3389/fonc.2012.00173

p53, SKP2, and DKK3 as MYCN Target Genes and Their Potential Therapeutic Significance

Neuroblastoma is the most common extra-cranial solid tumor of childhood. Despite significant advances, it currently still remains one of the most difficult childhood cancers to cure, with less than 40% of patients with high-risk disease being long-term survivors. MYCN is a proto-oncogene implicated to be directly involved in neuroblastoma development. Amplification of MYCN is associated with rapid tumor progression and poor prognosis. Novel therapeutic strategies which can improve the survival rates whilst reducing the toxicity in these patients are therefore required. Here we discuss genes regulated by MYCN in neuroblastoma, with particular reference to p53, SKP2, and DKK3 and strategies that may be employed to target them.

Mentions
Figures
Figure 1: MYC proteins and the p53/MDM2/p14ARF pathway. In response to cellular stresses p53 can mediate the expression of genes involved in various cellular responses such as apoptosis (e.g., BAX, NOXA, and PUMA), cell cycle arrest (e.g., p21CIP1), differentiation, DNA repair or senescence. p53, MDM2 and p14ARF form an autoregulatory feedback loop to tightly regulate p53 expression and activity. p14ARF can be activated in response to aberrant oncogenic factors such as c-MYC, and possibly MYCN (as indicated by the dashed line). p14ARF can also exhibit p53-independent tumor suppressor activity by directly binding and inhibiting the activity of c-MYC and MYCN. Both p53 and MDM2 are direct target genes of MYCN. MDM2 can regulate MYCN mRNA stability and translation, thereby forming a positive feedback loop. Figure 2: Summary of the potential mechanisms by which MYCN can both positively and negatively regulate p53 activity and function. Figure 3: MYC proteins and SKP2. SKP2 is an oncoprotein which can be upregulated by MYC proteins to drive tumorigenesis. SKP2 is a direct target gene of c-MYC, and can regulate the stability of c-MYC and be a co-factor for c-MYC mediated transcriptional activation of target genes. Due to the homology between c-MYC and MYCN, it is possible that SKP2 is also a direct target gene of MYCN and plays a similar role in regulating MYCN stability and transactivation of MYCN target genes (as indicated by the dashed lines). In neuroblastoma, MYCN can indirectly upregulate SKP2 via CDK4. In addition to upregulation by oncogenic MYC proteins, several signaling pathways closely linked to carcinogenesis such as PI3K/AKT and mTOR have been shown to influence SKP2 expression, stability and SCFSKP2 ligase activity. SKP2 is a component of the SCFSKP2 complex which mediates the degradation of several substrates including CDK inhibitors p21CIP1, p27KIP1, and p57KIP2. Independently of SCF complex formation, SKP2 can bind to p300 and attenuate p53 function. Interestingly, p300 is able to reciprocally regulate SKP2 activity. SKP2B, an alternatively spliced variant of SKP2, can perturb both p53 and pRB pathways via degradation of Prohibitin. It is possible that SKP2 has other functions which may promote tumorigenesis. Figure 4: MYCN mediated downregulation of DKK proteins in neuroblastoma. DKK1 and DKK3 can inhibit the proliferation of neuroblastoma cells, therefore downregulation of DKK proteins by MYCN will favor neuroblastoma tumorigenesis. MYCN mediated downregulation of DKK3 can occur via upregulation of the oncogenic miR-17-92 cluster, and due to the homology between DKK family members, this may also be a potential mechanism for MYCN mediated downregulation of DKK1 (as indicated by the dashed line). DKK1 inhibits the proliferation of neuroblastoma cells via upregulation of SYNPO2, and although the mechanism for DKK3 remains unclear, due to the homology between DKK family members this may also occur via SYNPO2 (as indicated by the dashed line).
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References
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    • . . . The E3 ubiquitin ligase APCCdh1 complex mediates the ubiquitination and subsequent degradation of SKP2, primarily in early G1 (Bashir et al., 2004; Wei et . . .
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    • . . . Indirect target genes of MYCN are genes which are altered as a consequence of other genes or pathways that are directly regulated by MYCN (Bell et al., 2010). . . .
    • . . . Furthermore due to the homology between the Myc family members, the candidate gene approach is often used to determine whether previously known c-MYC target genes are also MYCN target genes (Bell et al., 2010) . . .
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    • . . . Lower expression was observed in the presence of ectopic MYCN and in MYCN amplified tumors (Bell et al., 2007b; Koppen et al., 2007; Chen et al., 2010) . . .
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    • . . . In neuroblastoma AURKA has been found to be expressed at high levels in MYCN amplified tumors and required for the growth of MYCN amplified cells (Berwanger et al., 2002; Otto et al., 2009) . . .
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    • . . . This is predominantly regulated by post-translational modifications conferred on the p53 protein, such as phosphorylation, ubiquitylation, acetylation, and sumoylation (Bode and Dong, 2004) . . .
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    • . . . To date several mechanisms for p53-mediated transcriptional repression have been identified (reviewed by Wang et al., 2010a; Bohlig and Rother, 2011) . . .
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    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
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    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et al., 2005), c-MYC (Bretones et al., 2011), STAT3 (Huang et al., 2012), and NOR1 (Gizard et . . .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
    • . . . In particular, as p53 has previously been shown to be a direct target gene of MYCN, and a mechanism for MYCN mediated apoptosis in neuroblastoma, it is plausible that MYCN directly upregulates SKP2 to attenuate p53-mediated apoptosis (Kitagawa et al., 2008; Chen et al., 2010), as has previously been reported for c-MYC (Bretones et al., 2011; Figure 3). . . .
    • . . . However, as c-MYC and MYCN are known to share some common target genes, and c-MYC has recently been shown to directly bind to two high affinity E-Box motifs (CATGCG and CACGCG) mapping to −756 and −389 bp upstream of the transcriptional start site within the SKP2 promoter and drive transcription (Bretones et al., 2011), it is therefore possible that MYCN may also directly regulate SKP2 expression (Figure 3). . . .
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    • . . . Of interest, although not directly linked to SKP2, bortezomib is shown to induce apoptosis and inhibit cell growth, migration, angiogenesis, and metastasis both in vitro and in murine models of chemosensitive and chemoresistant neuroblastoma (Brignole et al., 2006; Michaelis et . . .
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    • . . . The majority are missense mutations resulting in single amino acid substitutions and map within the DBD of p53 (Brosh and Rotter, 2009) . . .
    • . . . This has been shown for malignancies of the breast, head and neck, colorectum, and hematopoietic system (Brosh and Rotter, 2009; Olivier and Taniere, 2011). . . .
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    • . . . The crucial role p53 plays in tumor suppression is emphasized by observations that the p53 pathway is abrogated in around half of all cancers due to an inactivating p53 mutation, and the rest have impaired upstream or downstream p53 pathways (Brown et al., 2009) . . .
    • . . . In addition to p53 mutations, amplification, and/or overexpression of MDM2 or MDMX, as well as p14ARF mutation, deletion, or methylation can also lead to abrogation of the p53 pathway (reviewed by Brown et al., 2009) . . .
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    • . . . Very recently, as an additional mechanism for BMI-1 mediated p53 inactivation, BMI-1 was found to directly bind to p53 in a complex with other Polycomb complex proteins in neuroblastoma cells leading to increased p53 ubiquitination and degradation (Calao et al., 2012; Figure 2). . . .
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    • . . . The frequency of p53 mutations varies from 10 to 70% across different cancers types, and are more common in solid tumors compared with hematological malignancies (Calin et al., 1999; Soussi et al., 2000) . . .
  42. S. Cantilena; F. Pastorino; A. Pezzolo; O. Chayka; V. Pistoia; M. Ponzoni Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas Oncotarget 2, 976-983 (2011) .
    • . . . Additionally, the expression of FZD6 is reported to predict poor survival in neuroblastoma patients, and marks rare and highly tumorigenic neuroblastoma stem cells (Cantilena et al., 2011) . . .
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    • . . . In support of this, studies in neuroblastoma have observed that MYCN functionally cooperates with Twist-1 or BMI-1 to induce neuroblastoma tumorigenesis, where overexpression of Twist-1 or BMI-1 is necessary for tumor growth both in vitro and in vivo (Valsesia-Wittmann et al., 2004; Cui et al., 2007; Huang et al., 2011b) . . .
  44. H. Caren; J. Erichsen; L. Olsson; C. Enerback; R. M. Sjoberg; J. Abrahamsson High-resolution array copy number analyses for detection of deletion, gain, amplification and copy-neutral LOH in primary neuroblastoma tumors: four cases of homozygous deletions of the CDKN2A gene BMC Genomics 9, 353 (2008) .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010; Wolf et al., 2010) . . .
    • . . . In contrast, the proportion of neuroblastoma tumors with aberrations in the p53/MDM2/p14ARF pathway which are MYCN amplified are lower than in cell lines (Corvi et al., 1995; Gonzalez-Gomez et al., 2003; Su et al., 2004; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010), therefore it is possible that these abnormalities are selected for during the in vitro establishment and/or maintenance of these cell lines . . .
  45. H. Carol; I. Boehm; C. P. Reynolds; M. H. Kang; J. M. Maris; C. L. Morton Efficacy and pharmacokinetic/pharmacodynamic evaluation of the aurora kinase A inhibitor MLN8237 against preclinical models of pediatric cancer Cancer Chemother. Pharmacol. 68, 1291-1304 (2011) .
    • . . . Preclinical evaluations of a second generation AURKA inhibitor MLN8237 in pediatric cancers including neuroblastoma have been promising (Maris et al., 2010; Carol et al., 2011) . . .
  46. J. Carr; E. Bell; A. D. Pearson; U. R. Kees; H. Beris; J. Lunec Increased frequency of aberrations in the p53/MDM2/p14(ARF) pathway in neuroblastoma cell lines established at relapse Cancer Res. 66, 2138-2145 (2006) .
  47. A. C. Carrano; E. Eytan; A. Hershko; M. Pagano SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27 Nat. Cell Biol. 1, 193-199 (1999) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et . . .
  48. A. C. Carrano; M. Pagano Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression J. Cell Biol. 153, 1381-1390 (2001) .
    • . . . Overexpression of SKP2 can drive quiescent cells to enter the cell cycle (Sutterluty et al., 1999), and promote adhesion-independent growth of cancer cells (Carrano and Pagano, 2001; Signoretti et al., 2002) . . .
  49. J. Carr-Wilkinson; R. Griffiths; R. Elston; L. D. Gamble; B. Goranov; C. P. Redfern Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression Cell Cycle 10, 3778-3787 (2011) .
    • . . . In addition siRNA mediated inhibition of p53 in neuroblastoma cell lines led to morphological evidence of differentiation (Carr-Wilkinson et al., 2011) . . .
  50. J. Carr-Wilkinson; K. O’Toole; K. M. Wood; C. C. Challen; A. G. Baker; J. R. Board High frequency of p53/MDM2/p14ARF pathway abnormalities in relapsed neuroblastoma Clin. Cancer Res. 16, 1108-1118 (2010) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et al., 1997; Omura-Minamisawa et al., 2001; Tweddle et al., 2001b; Carr-Wilkinson et al., 2010) . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et al., 1997; Omura-Minamisawa et al., 2001; Tweddle et al., 2001b; Carr-Wilkinson et al., 2010) . . .
    • . . . In line with these observations, we recently reported that the presence of a p53 mutation was independently prognostic for overall survival in neuroblastoma patients (Carr-Wilkinson et al., 2010). . . .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010; Wolf et . . .
    • . . . Analysis of neuroblastoma cell lines reported to date with aberrations in the p53/MDM2/p14ARF pathway demonstrates that 31/40 (78%) of these cell lines are MYCN amplified and predominantly established following previous cytotoxic therapy at relapse (Table 1), when abnormalities of the p53 pathway in neuroblastoma tumors have also been previously reported (reviewed by Tweddle et al., 2003; Carr-Wilkinson et al., 2010) . . .
  51. J. S. Castresana; M. J. Bello; J. A. Rey; P. Nebreda; A. Queizan; P. Garcia-Miguel No TP53 mutations in neuroblastomas detected by PCR-SSCP analysis Genes Chromosomes Cancer 10, 136-138 (1994) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et . . .
  52. V. D. Cataldo; J. Cortes; A. Quintas-Cardama Azacitidine for the treatment of myelodysplastic syndrome Expert Rev. Anticancer Ther. 9, 875-884 (2009) .
    • . . . Demethylating agents azacitidine and decitabine have been approved in the treatment of myelodysplastic syndromes (Cataldo et al., 2009; Santos et al., 2010), however this method is not gene specific and could alter the epigenetic patterns of the entire genome . . .
  53. S. Cattelani; R. Defferrari; S. Marsilio; R. Bussolari; O. Candini; F. Corradini Impact of a single nucleotide polymorphism in the MDM2 gene on neuroblastoma development and aggressiveness: results of a pilot study on 239 patients Clin. Cancer Res. 14, 3248-3253 (2008) .
    • . . . A single-nucleotide polymorphism (SNP) in the MDM2 promoter (SNP309T to G) leading to high levels of MDM2 expression has been found in some tumors and is associated with a poor prognostic outcome, including neuroblastoma (Cattelani et al., 2008; Perfumo et . . .
    • . . . MDM2 is a direct target gene of MYCN (Slack et al., 2005; Westermann et al., 2008) and non-syntenic co-amplification of MDM2 and MYCN has been reported in neuroblastoma (Corvi et al., 1995) . . .
  54. S. Cattelani; G. Ferrari-Amorotti; S. Galavotti; R. Defferrari; B. Tanno; S. Cialfi The p53 Codon 72 Pro/Pro genotype identifies poor-prognosis neuroblastoma patients: correlation with reduced apoptosis and enhanced senescence by the p53-72P Isoform Neoplasia 14, 634-643 (2012) .
    • . . . Very recently, analysis of the p53 codon 72 Arg/Pro polymorphism identified the Pro/Pro phenotype as an independent marker of poor prognosis in neuroblastoma patients, and in vitro led to reduced levels of apoptosis in response to chemotherapy and irradiation (Cattelani et al., 2012). . . .
  55. C. H. Chan; S. W. Lee; C. F. Li; J. Wang; W. L. Yang; C. Y. Wu Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis Nat. Cell Biol. 12, 457-467 (2010a) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
    • . . . Targeting the expression or stability of SKP2 is an appealing option due to the oncogenic functions of SKP2 which are independent of SCFSKP2 ligase formation and activity (Ji et al., 2006; Kitagawa et al., 2008; Chan et al., 2010a). . . .
  56. C. H. Chan; S. W. Lee; J. Wang; H. K. Lin Regulation of Skp2 expression and activity and its role in cancer progression ScientificWorldJournal 10, 1001-1015 (2010b) .
    • . . . More recently, studies have also identified the involvement of SKP2 in several processes closely linked to tumorigenesis such as cell survival, apoptosis, migration, and metastasis (reviewed by Chan et al., 2010b). . . .
    • . . . Furthermore, Pim-1 has also been shown to phosphorylate Cdh1, impairing its association with CDC27 and inhibiting APCCdh1 activity, thereby protecting SKP2 from degradation (reviewed by Chan et al., 2010b) . . .
    • . . . Studies have demonstrated that PI3K/AKT signaling pathway and Cyclin D positively regulate SCFSKP2 complex formation and ligase activity, potentially through neddylation of CUL1 (reviewed by Chan et al., 2010b). . . .
  57. C. H. Chan; C. F. Li; W. L. Yang; Y. Gao; S. W. Lee; Z. Feng The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis Cell 149, 1098-1111 (2012) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  58. H. Chander; M. Halpern; L. Resnick-Silverman; J. J. Manfredi; D. Germain Skp2B attenuates p53 function by inhibiting prohibitin EMBO Rep. 11, 220-225 (2010) .
    • . . . Taken together, these observations suggest that amplification of the SKP2 locus would represent a powerful mechanism to attenuate p53 function in cancer (Chander et al., 2010; Figure 3). . . .
    • . . . Furthermore, with the discovery of the role of SKP2 and SKP2B in attenuating p53-mediated apoptosis and transcriptional activity, and that SKP2 deficiency triggers a potent ARF-p53-independent cellular senescence in the presence of oncogenic conditions (such as inactivation of TSGs/overexpression of proto-oncogenes), has implicated the wide applicability of targeting SKP2 as a strategy to reactivate p53 and as pro-senescence therapy (Kitagawa et al., 2008; Chander et al., 2010; Lin et . . .
  59. T.-C. Chang; E. A. Wentzel; O. A. Kent; K. Ramachandran; M. Mullendore; H. Lee Kwang Transactivation of miR-34a by p53 Broadly influences gene expression and promotes apoptosis Mol. Cell 26, 745-752 (2007) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
  60. S. Charrasse; I. Carena; V. Brondani; K. H. Klempnauer; S. Ferrari Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway Oncogene 19, 2986-2995 (2000) .
    • . . . SKP2 protein is approximately 45 kDa, consisting of an N-terminal F-Box domain which mediates the interaction between SKP2 and SKP1, thereby tethering SKP2 to the SCF complex, and C-terminal leucine-rich repeats (LRR) which enable SKP2 to directly bind to target substrates (Bai et al., 1996; Skowyra et al., 1997; Schulman et al., 2000; Zheng et al., 2002) . . .
  61. D. Chen; M. Frezza; S. Schmitt; J. Kanwar; Q. P. Dou Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives Curr. Cancer Drug Targets 11, 239-253 (2011) .
    • . . . Despite the overall success of bortezomib, severe side-effects have been reported in patients, and may be attributable to the broad ranging functions and targets of the UPS (Chen et al., 2011) . . .
  62. L. Chen An Investigation of the Relationship Between p53 Function, Differentiation and MYCN in Neuroblastoma , (2009) .
    • . . . These included downregulation of additional Wnt signaling antagonists SFRP1 and APC together with the upregulated expression of positive regulators FRAT2, CSNK2A1, and RUVBL1 (Chen, 2009; Chen et al., 2010) . . .
  63. L. Chen; N. Iraci; S. Gherardi; L. D. Gamble; K. M. Wood; G. Perini p53 Is a direct transcriptional target of MYCN in neuroblastoma Cancer Res. 70, 1377-1388 (2010) .
  64. L. Chen; A. J. Malcolm; K. M. Wood; M. Cole; S. Variend; C. Cullinane p53 is nuclear and functional in both undifferentiated and differentiated neuroblastoma Cell Cycle 6, 2685-2696 (2007) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik, 1996) . . .
  65. Q. Chen; W. Xie; D. J. Kuhn; P. M. Voorhees; A. Lopez-Girona; D. Mendy Targeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy Blood 111, 4690-4699 (2008) .
    • . . . Using high-throughput screening, Cpd A and SMIP004 were identified (Chen et al., 2008; Rico-Bautista et al., 2010) . . .
  66. Z. Chen; Y. Lin; E. Barbieri; S. Burlingame; J. Hicks; A. Ludwig Mdm2 deficiency suppresses MYCN-driven neuroblastoma tumorigenesis in vivo Neoplasia 11, 753-762 (2009) .
    • . . . These observations thereby demonstrate the necessity of MYCN to overcome p53-mediated tumor suppression during neuroblastoma tumorigenesis either via direct inhibition of p53 by MDM2 or suppression of the p14ARF/p53 pathway (Chen et al., 2009). . . .
    • . . . Several lines of evidence from published literature lend support to the notion that during the process of neuroblastoma progression there is evasion of p53-mediated tumor suppression via inactivation of the p53/MDM2/p14ARF pathway (reviewed by Van Maerken et al., 2009b) as well as a requirement for MYCN amplified neuroblastoma to circumvent MYCN driven apoptosis (reviewed by Hogarty, 2003) . . .
    • . . . Consistent with observations that Mdm2 haploinsufficiency inhibits c-MYC induced lymphomagenesis (Alt et al., 2003), MDM2 has been shown to be necessary for MYCN to overcome p53-mediated tumor suppression for MYCN directed centrosome amplification and genomic instability (Slack et al., 2007), and also during MYCN driven neuroblastoma tumorigenesis (Chen et al., 2009; Figure 2) . . .
  67. Z. Chen; D. Zhang; F. Yue; M. Zheng; Z. Kovacevic; D. R. Richardson The iron chelators Dp44mT and DFO inhibit TGF-beta-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1) J. Biol. Chem. 287, 17016-17028 (2012) .
    • . . . Very recently, NDRG1 has been shown to upregulate NEDD4L, PTEN, and SMAD4 and inhibit the PI3K and Ras signaling pathways, thereby implicating its involvement in regulating key oncogenic pathways (Kovacevic et al., 2012) . . .
  68. C. F. Cheok; C. S. Verma; J. Baselga; D. P. Lane Translating p53 into the clinic Nat. Rev. Clin. Oncol. 8, 25-37 (2011) .
    • . . . Overall, MDM2-p53 antagonists have been shown to activate the p53 pathway, inducing p53-dependent apoptosis and sensitizing tumor cells to cytotoxic and other molecular targeted therapies whilst inducing a reversible cell cycle arrest in normal cells (reviewed by (Van Maerken et al., 2009a); (Vassilev, 2004; Shangary et al., 2008; Korotchkina et al., 2009; Cheok et al., 2011)) . . .
  69. L. Chesler; D. D. Goldenberg; R. Collins; M. Grimmer; G. E. Kim; T. Tihan Chemotherapy-induced apoptosis in a transgenic model of neuroblastoma proceeds through p53 induction Neoplasia 10, 1268-1274 (2008) .
    • . . . Initial studies in neuroblastoma showed that MYCN amplified tumors expressed significantly higher levels of p53 mRNA in comparison with non-amplified tumors (Raschella et al., 1991; Berwanger et al., 2002; Westermann et al., 2008), and higher p53 protein expression in the presence of ectopic MYCN in cell lines (Cui et al., 2005; Bell et al., 2006; Sugihara et al., 2006) . . .
    • . . . MYCN driven tumor formation had higher penetrance and reduced latency in p53 haploinsufficient mice, and chemotherapy induced apoptosis was shown to be p53-dependent, in which apoptosis was significantly reduced in TH-MYCN p53 +/- tumors compared with TH-MYCN p53 + / + tumors (Chesler et al., 2008) . . .
    • . . . In vivo studies have demonstrated the importance of the p53 pathway in neuroblastoma (Chesler et al., 2008; Chen et al., 2009) . . .
  70. L. Chesler; C. Schlieve; D. D. Goldenberg; A. Kenney; G. Kim; A. McMillan Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma Cancer Res. 66, 8139-8146 (2006) .
    • . . . In support of this, PI3K inhibitor LY294002 and Rapamycin have been shown to decrease SKP2 expression (Motti et al., 2005; Shapira et al., 2006) . . .
  71. C. H. Cheung; M. S. Coumar; J. Y. Chang; H. P. Hsieh Aurora kinase inhibitor patents and agents in clinical testing: an update (2009–10) Expert Opin. Ther. Pat. 21, 857-884 (2011) .
    • . . . Preclinical evaluations of a second generation AURKA inhibitor MLN8237 in pediatric cancers including neuroblastoma have been promising (Maris et al., 2010; Carol et al., 2011) . . .
  72. C. H. Cho; M. Seo; Y. I. Lee; S. Y. Kim; H. D. Youn; Y. S. Juhnn Dibutyryl cAMP stimulates the proliferation of SH-SY5Y human neuroblastoma cells by up-regulating Skp2 protein J. Cancer Res. Clin. Oncol. 133, 135-144 (2007) .
    • . . . In line with observations that SKP2 drives cellular proliferation, cAMP induces proliferation of neuroblastoma cells by upregulating SKP2 (Cho et al., 2007), while growth arrest and differentiation of neuroblastoma cell lines induced by retinoic acid, BMP2, and the HDAC inhibitor Helminthosporium carbonum toxin is accompanied by a decrease in SKP2 levels (Nakamura et al., 2003a,b; Cuende et al., 2008; Deubzer et al., 2008a) . . .
  73. I. M. Chu; L. Hengst; J. M. Slingerland The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy Nat. Rev. Cancer 8, 253-267 (2008) .
    • . . . In the vast majority of cases, SKP2 overexpression inversely correlates with p27KIP1 expression (Nakayama and Nakayama, 2006; Frescas and Pagano, 2008), which is consistent p27KIP1 being a key target of SKP2 and to be rarely mutated in cancer (Chu et al., 2008) . . .
  74. S. L. Cohn; N. Ikegaki; G. M. Brodeur; T. Sawada; Y. Tsuchida; P. A. Voûte “Expression of MYCN mRNA and Protein,” Neuroblastoma , 137-146 (2000) .
    • . . . In contrast to c-MYC, which is expressed in a wide variety of embryonic and adult tissues, MYCN expression is limited to the developing nervous system and selected other sites (Cohn and Ikegaki, 2000). . . .
  75. S. L. Cohn; D. A. Tweddle MYCN amplification remains prognostically strong 20 years after its “clinical debut.” Eur. J. Cancer 40, 2639-2642 (2004) .
    • . . . A typical feature of high-risk disease is MYCN amplification which occurs in ~25% of neuroblastoma, associating with rapid tumor progression and a poor prognosis (reviewed by Cohn and Tweddle, 2004) . . .
  76. K. A. Cole; E. F. Attiyeh; Y. P. Mosse; M. J. Laquaglia; S. J. Diskin; G. M. Brodeur A Functional Screen Identifies miR-34a as a candidate neuroblastoma tumor suppressor gene Mol. Cancer Res. 6, 735-742 (2008) .
    • . . . The 3′-UTR of MYCN has been identified as a direct target of miR-34a (Wei et al., 2008), a miRNA which is directly upregulated by p53 and mediates several tumor suppressive functions of p53 (reviewed by Hermeking, 2007) . . .
  77. R. Corvi; L. Savelyeva; S. Breit; A. Wenzel; R. Handgretinger; J. Barak Non-syntenic amplification of MDM2 and MYCN in human neuroblastoma Oncogene 10, 1081-1086 (1995) .
  78. J. Cuende; S. Moreno; J. P. Bolanos; A. Almeida Retinoic acid downregulates Rae1 leading to APC(Cdh1) activation and neuroblastoma SH-SY5Y differentiation Oncogene 27, 3339-3344 (2008) .
    • . . . In line with observations that SKP2 drives cellular proliferation, cAMP induces proliferation of neuroblastoma cells by upregulating SKP2 (Cho et al., 2007), while growth arrest and differentiation of neuroblastoma cell lines induced by retinoic acid, BMP2, and the HDAC inhibitor Helminthosporium carbonum toxin is accompanied by a decrease in SKP2 levels (Nakamura et al., 2003a,b; Cuende et al., 2008; Deubzer et . . .
    • . . . Using high-throughput screening, Cpd A and SMIP004 were identified (Chen et al., 2008; Rico-Bautista et al., 2010) . . .
  79. H. Cui; B. Hu; T. Li; J. Ma; G. Alam; W. T. Gunning Bmi-1 is essential for the tumorigenicity of neuroblastoma cells Am. J. Pathol. 170, 1370-1378 (2007) .
    • . . . In neuroblastoma, BMI-1 is shown to be essential for neuroblastoma tumorigenesis in vitro and in vivo, cooperating with MYCN via inhibition of MYCN driven apoptosis and downregulation of p53 expression (Cui et al., 2007; Huang et al., 2011b) . . .
    • . . . In support of this, studies in neuroblastoma have observed that MYCN functionally cooperates with Twist-1 or BMI-1 to induce neuroblastoma tumorigenesis, where overexpression of Twist-1 or BMI-1 is necessary for tumor growth both in vitro and in vivo (Valsesia-Wittmann et al., 2004; Cui et al., 2007; Huang et al., 2011b) . . .
  80. H. Cui; T. Li; H. F. Ding Linking of N-Myc to death receptor machinery in neuroblastoma cells J. Biol. Chem. 280, 9474-9481 (2005) .
    • . . . Initial studies in neuroblastoma showed that MYCN amplified tumors expressed significantly higher levels of p53 mRNA in comparison with non-amplified tumors (Raschella et al., 1991; Berwanger et al., 2002; Westermann et al., 2008), and higher p53 protein expression in the presence of ectopic MYCN in cell lines (Cui et al., 2005; Bell et al., 2006; Sugihara et al., 2006) . . .
  81. H. Cui; A. Schroering; H. F. Ding p53 mediates DNA damaging drug-induced apoptosis through a caspase-9-dependent pathway in SH-SY5Y neuroblastoma cells Mol. Cancer Ther. 1, 679-686 (2002) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et al., 1998; McKenzie et al., 1999; Smart et al., 1999; Keshelava et al., 2000, 2001; Tweddle et al., 2001a,b; Cui et al., 2002; Goldschneider et al., 2004; Chen et al., 2007, 2010; Xue et al., 2007; Kurata et al., 2008; Van Maerken et al., 2011; Gamble et al., 2012) . . .
  82. C. V. Dang c-Myc target genes involved in cell growth, apoptosis, and metabolism Mol. Cell. Biol. 19, 1-11 (1999) .
    • . . . Alternatively, the inference approach is used and is based on identifying putative target genes by the presence of MYCN/MAX binding sites within their gene regulatory regions (Dang, 1999) . . .
  83. M. K. Danks; D. O. Whipple; C. R. Mcpake; D. Lu; L. C. Harris Differences in epitope accessibility of p53 monoclonal antibodies suggest at least three conformations or states of protein binding of p53 protein in human tumor cell lines Cell Death Differ. 5, 678-686 (1998) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et . . .
  84. A. A. Dar; A. Zaika; M. B. Piazuelo; P. Correa; T. Koyama; A. Belkhiri Frequent overexpression of aurora kinase A in upper gastrointestinal adenocarcinomas correlates with potent antiapoptotic functions Cancer 112, 1688-1698 (2008) .
    • . . . There is additional evidence to suggest that AURKA can also inhibit p53 via the AKT/MDM2 axis in gastric cancer cells, however this remains to be shown in neuroblastoma (Dar et al., 2008) . . .
  85. A. M. Davidoff; J. C. Pence; N. A. Shorter; J. D. Iglehart; J. R. Marks Expression of p53 in human neuroblastoma- and neuroepithelioma-derived cell lines Oncogene 7, 127-133 (1992) .
  86. S. Davidovich; O. Ben-Izhak; M. Shapira; B. Futerman; D. D. Hershko Over-expression of Skp2 is associated with resistance to preoperative doxorubicin-based chemotherapy in primary breast cancer Breast Cancer Res. 10, R63 (2008) .
    • . . . In the vast majority of cases, SKP2 overexpression inversely correlates with p27KIP1 expression (Nakayama and Nakayama, 2006; Frescas and Pagano, 2008), which is consistent p27KIP1 being a key target of SKP2 and to be rarely mutated in cancer (Chu et al., 2008) . . .
  87. S. De Brouwer; P. Mestdagh; I. Lambertz; F. Pattyn; A. De Paepe; F. Westermann Dickkopf-3 is regulated by the MYCN-induced miR-17-92 cluster in neuroblastoma Int. J. Cancer 130, 2591-2598 (2012) .
  88. J. De Calisto; C. Araya; L. Marchant; C. F. Riaz; R. Mayor Essential role of non-canonical Wnt signalling in neural crest migration Development 132, 2587-2597 (2005) .
    • . . . Interestingly, these non-canonical Wnt pathways have previously been shown to control neural crest migration (De Calisto et al., 2005). . . .
  89. T. H. Dellinger; K. Planutis; D. D. Jandial; R. N. Eskander; M. E. Martinez; X. Zi Expression of the Wnt antagonist Dickkopf-3 is associated with prognostic clinicopathologic characteristics and impairs proliferation and invasion in endometrial cancer Gynecol. Oncol. 126, 259-267 (2012) .
    • . . . In contrast, others have however confirmed DKK3 as an inhibitor of the canonical pathway (Yue et al., 2008; Lee et al., 2009; Dellinger et al., 2012). . . .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et al., 2011; Ueno et al., 2011; Dellinger et al., 2012; Yang et . . .
  90. R. J. Deshaies SCF and cullin/ring H2-based ubiquitin ligases Annu. Rev. Cell Dev. Biol. 15, 435-467 (1999) .
    • . . . The invariable core components of SCF complexes are SKP1, CUL1, and RBX1 (Skowyra et al., 1997; Deshaies, 1999; Nakayama and Nakayama, 2005) . . .
  91. H. E. Deubzer; V. Ehemann; A. E. Kulozik; F. Westermann; L. Savelyeva; A. Kopp-Schneider Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro Cancer Lett. 264, 21-28 (2008a) .
    • . . . In line with observations that SKP2 drives cellular proliferation, cAMP induces proliferation of neuroblastoma cells by upregulating SKP2 (Cho et al., 2007), while growth arrest and differentiation of neuroblastoma cell lines induced by retinoic acid, BMP2, and the HDAC inhibitor Helminthosporium carbonum toxin is accompanied by a decrease in SKP2 levels (Nakamura et al., 2003a,b; Cuende et al., 2008; Deubzer et al., 2008a) . . .
  92. H. E. Deubzer; V. Ehemann; F. Westermann; R. Heinrich; G. Mechtersheimer; A. E. Kulozik Histone deacetylase inhibitor helminthosporium carbonum (HC)-toxin suppresses the malignant phenotype of neuroblastoma cells Int. J. Cancer 122, 1891-1900 (2008b) .
    • . . . This decrease has been attributed to retinoic acid induced downregulation of Rae1 which leads to increased APCCdh1 mediated degradation of SKP2, or HDAC inhibitor induced activation of the Rb pathway (Cuende et al., 2008; Deubzer et al., 2008b). . . .
  93. L. A. Donehower The p53-deficient mouse: a model for basic and applied cancer studies Semin. Cancer Biol. 7, 269-278 (1996) .
    • . . . Additionally, p53 null mice are reported to develop a range of spontaneous tumors, most commonly T-cell lymphomas (Donehower, 1996). . . .
  94. P. Dumont; J. I. Leu; A. C. Della Pietra; D. L. George; M. Murphy The codon 72 polymorphic variants of p53 have markedly different apoptotic potential Nat. Genet. 33, 357-365 (2003) .
    • . . . In p53, a SNP at codon 72 which leads to an Arg > Pro substitution has previously been identified, and the Arg72 variant has been shown to exhibit enhanced apoptotic capability compared with the Pro72 variant (Dumont et al., 2003) . . .
  95. J. S. Duncan; D. W. Litchfield Too much of a good thing: the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2 Biochim. Biophys. Acta 1784, 33-47 (2008) .
    • . . . CSNK2A1 and RUVBL1 have been reported to play multiple roles in driving malignant transformation (Duncan and Litchfield, 2008; Huber et al., 2008), and RUVBL1 expression was identified to be associated with poor overall survival in neuroblastoma patients (Westermann et al., 2008) . . .
  96. K. Edamura; Y. Nasu; M. Takaishi; T. Kobayashi; F. Abarzua; M. Sakaguchi Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model Cancer Gene Ther. 14, 765-772 (2007) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et . . .
    • . . . Ad-REIC/DKK3 has been used with success in preclinical studies demonstrating robust anti-tumor effects including induction of cancer cell specific apoptosis and inhibition of metastatic disease (Abarzua et al., 2005; Edamura et al., 2007; Kawasaki et al., 2009; Sakaguchi et al., 2009; Than et al., 2011), and is currently in clinical trials for use in prostate cancer (see footnote 3; NCT01197209) . . .
  97. O. Eizenberg; A. Faber-Elman; E. Gottlieb; M. Oren; V. Rotter; M. Schwartz p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells Mol. Cell. Biol. 16, 5178-5185 (1996) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik, 1996) . . .
  98. W. S. el-Deiry; S. E. Kern; J. A. Pietenpol; K. W. Kinzler; B. Vogelstein Definition of a consensus binding site for p53 Nat. Genet. 1, 45-49 (1992) .
    • . . . p53 functions as an active tetramer to directly bind to a consensus p53 DNA binding sequence consisting of two adjacent 10 bp half-sites 5′-RRRCWWGYYY-3′ (R = A or G, Y = C or T, W = A or T), separated by 0–13 bp, located within the promoters of downstream target genes (el-Deiry et al., 1992) . . .
  99. T. P. Ellen; Q. Ke; P. Zhang; M. Costa NDRG1, a growth and cancer related gene: regulation of gene expression and function in normal and disease states Carcinogenesis 29, 2-8 (2008) .
    • . . . NDRG1 has been shown to be upregulated in response to differentiation and suppress metastasis, and there is growing interest in using NDRG1 as a biomarker of disease progression (reviewed by Ellen et al., 2008) . . .
  100. S. C. Evans; G. Lozano The Li-Fraumeni syndrome: an inherited susceptibility to cancer Mol. Med. Today 3, 390-395 (1997) .
    • . . . This is further supported by the severe predisposition to cancers observed in individuals with Li-Fraumeni syndrome (Evans and Lozano, 1997) . . .
  101. A. Faisal; L. Vaughan; V. Bavetsias; C. Sun; B. Atrash; S. Avery The aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo Mol. Cancer Ther. 10, 2115-2123 (2011) .
    • . . . Preclinical evaluations of a second generation AURKA inhibitor MLN8237 in pediatric cancers including neuroblastoma have been promising (Maris et al., 2010; Carol et al., 2011) . . .
  102. G. Feinberg-Gorenshtein; S. Avigad; M. Jeison; G. Halevy-Berco; J. Mardoukh; D. Luria Reduced levels of miR-34a in neuroblastoma are not caused by mutations in the TP53 binding site Genes Chromosomes Cancer 48, 539-543 (2009) .
    • . . . Consistent with this, reduced levels of miR-34 have been observed in both tumors and cell lines, including neuroblastoma (Bommer et al., 2007; Chang et al., 2007; Tarasov et al., 2007; Welch et al., 2007; Feinberg-Gorenshtein et al., 2009) . . .
    • . . . Consistent with this, lower expression levels of miR-34a have been reported to correlate with 1p36 LOH in neuroblastoma (Welch et al., 2007; Cole et al., 2008; Feinberg-Gorenshtein et al., 2009; Figure 2) . . .
  103. P. C. Fernandez; S. R. Frank; L. Wang; M. Schroeder; S. Liu; J. Greene Genomic targets of the human c-Myc protein Genes Dev. 17, 1115-1129 (2003) .
    • . . . The precise mechanism of MYCN mediated upregulation of SKP2 remains unclear, as SKP2 is a direct target gene of E2F (Zhang and Wang, 2006), E2F is a direct target of c-MYC (Fernandez et al., 2003) and higher E2F expression levels are observed in the presence of MYCN (Mac et al., 2000; Woo et al., 2008) . . .
  104. A. Ferreira; K. S. Kosik Accelerated neuronal differentiation induced by p53 suppression J. Cell. Sci. 109, 1509-1516 (1996) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik, 1996) . . .
  105. M. Flahaut; R. Meier; A. Coulon; K. A. Nardou; F. K. Niggli; D. Martinet The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway Oncogene 28, 2245-2256 (2009) .
    • . . . To date, studies into Wnt signaling in neuroblastoma have shown that siRNA mediated inhibition of the Wnt1/β-catenin pathway leads to apoptosis of SHSY5Y cells (Zhang et al., 2009), and deregulated canonical and non-canonical Wnt signaling in chemoresistant and high-risk disease (Blanc et al., 2005; Liu et al., 2008b; Flahaut et . . .
  106. S. A. Forbes; G. Bhamra; S. Bamford; E. Dawson; C. Kok; J. Clements The catalogue of somatic mutations in cancer (COSMIC) Curr. Protoc. Hum. Genet. 10, (2008) .
  107. D. Frescas; M. Pagano Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer Nat. Rev. Cancer 8, 438-449 (2008) .
    • . . . The binding and recognition of the substrate by SKP2 is dependent on prior phosphorylation of the target substrate, and in some cases also requires the activity of a co-factor protein, CKS1 (Cyclin-kinase-subunit 1; Ganoth et al., 2001; Spruck et al., 2001; Frescas and Pagano, 2008). . . .
    • . . . SKP2 overexpression at the mRNA and/or protein level have been detected in a number of human tumors and cell lines including prostate, breast, pancreatic, gastric, colorectal, ovarian, melanoma, lymphoma, and leukemia (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
    • . . . Furthermore, high SKP2 expression was observed to correlate with low p27KIP1 expression levels (Westermann et al., 2007), consistent with studies in other cancer types (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
    • . . . Finally, the availability of crystallographic maps of the structure of SKP2 and SCFSKP2 complex components and their interaction with substrates, together with biochemical data provides the opportunity to develop novel SKP2 inhibitors (Frescas and Pagano, 2008). . . .
  108. T. Fujita; J. Igarashi; E. R. Okawa; T. Gotoh; J. Manne; V. Kolla CHD5, a tumor suppressor gene deleted from 1p36.31 in neuroblastomas J. Natl. Cancer Inst. 100, 940-949 (2008) .
    • . . . Low levels of CHD5 expression have been found in neuroblastoma cell lines, as well as correlating with MYCN amplification and poor prognosis in neuroblastoma tumors (Thompson et al., 2003; Fujita et al., 2008; Koyama et al., 2012). . . .
    • . . . Similar to miR-34a, CHD5 is also located at 1p36, and is frequently deleted and/or methylated in several human cancers including neuroblastoma. 1p deletion and epigenetic silencing of CHD5 have been suggested to account for the low expression observed in both neuroblastoma tumors and cell lines, as homozygous deletions or mutations were reported to be infrequent (Fujita et al., 2008; Koyama et al., 2012) . . .
  109. S. Fulda; W. Lutz; M. Schwab; K. M. Debatin MycN sensitizes neuroblastoma cells for drug-induced apoptosis Oncogene 18, 1479-1486 (1999) .
    • . . . This is consistent with, and may help to explain why human MYCN amplified and TH-MYCN transgenic mouse neuroblastoma tumors have high levels of apoptosis, and MYCN amplified and Tet21N MYCN+ neuroblastoma cells undergo higher levels of apoptosis in response to chemotherapeutic agents (Fulda et al., 1999, 2000; Paffhausen et al., 2007; Chesler et al., 2008), irradiation (Bell et al., 2006), and MDM2-p53 antagonists (Gamble et al., 2012) . . .
  110. S. Fulda; W. Lutz; M. Schwab; K. M. Debatin MycN sensitizes neuroblastoma cells for drug-triggered apoptosis Med. Pediatr. Oncol. 35, 582-584 (2000) .
    • . . . This is consistent with, and may help to explain why human MYCN amplified and TH-MYCN transgenic mouse neuroblastoma tumors have high levels of apoptosis, and MYCN amplified and Tet21N MYCN+ neuroblastoma cells undergo higher levels of apoptosis in response to chemotherapeutic agents (Fulda et al., 1999, 2000; Paffhausen et al., 2007; Chesler et al., 2008), irradiation (Bell et al., 2006), and MDM2-p53 antagonists (Gamble et al., 2012) . . .
  111. L. D. Gamble; U. R. Kees; D. A. Tweddle; J. Lunec MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63 Oncogene 31, 752-763 (2012) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et al., 1998; McKenzie et al., 1999; Smart et al., 1999; Keshelava et al., 2000, 2001; Tweddle et al., 2001a,b; Cui et al., 2002; Goldschneider et al., 2004; Chen et al., 2007, 2010; Xue et al., 2007; Kurata et al., 2008; Van Maerken et al., 2011; Gamble et al., 2012) . . .
    • . . . This is consistent with, and may help to explain why human MYCN amplified and TH-MYCN transgenic mouse neuroblastoma tumors have high levels of apoptosis, and MYCN amplified and Tet21N MYCN+ neuroblastoma cells undergo higher levels of apoptosis in response to chemotherapeutic agents (Fulda et al., 1999, 2000; Paffhausen et al., 2007; Chesler et al., 2008), irradiation (Bell et al., 2006), and MDM2-p53 antagonists (Gamble et al., 2012) . . .
    • . . . Evaluation of Nutlin-3 and other MDM2-p53 antagonists in preclinical models of neuroblastoma have reported potent anti-tumor effects such as induction of growth arrest, senescence, differentiation and apoptosis, and inhibition of tumor cell proliferation and metastasis (Barbieri et al., 2006; Van Maerken et al., 2006, 2009a, 2011; Hardcastle et al., 2011; Patterson et al., 2011; Gamble et al., 2012) . . .
  112. D. Ganoth; G. Bornstein; T. K. Ko; B. Larsen; M. Tyers; M. Pagano The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27 Nat. Cell Biol. 3, 321-324 (2001) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et al., 2006), Cyclins D (Yu et al., 1998) and E (Yeh et al., 2001), as well as oncogenes c-MYC (Kim et al., 2003; von der Lehr et al., 2003) and MYB (Charrasse et al., 2000) . . .
  113. S. A. Georges; M. C. Biery; S. Y. Kim; J. M. Schelter; J. Guo; A. N. Chang Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215 Cancer Res. 68, 10105-10112 (2008) .
    • . . . Additionally, p53 has also been reported to induce the expression of miR-192, miR-215, miR-145, and miR-107, of which miR-145 was shown to inhibit c-MYC expression (Braun et al., 2008; Georges et . . .
  114. F. Gizard; Y. Zhao; H. M. Findeisen; H. Qing; D. Cohn; E. B. Heywood Transcriptional regulation of S phase kinase-associated protein 2 by NR4A orphan nuclear receptor NOR1 in vascular smooth muscle cells J. Biol. Chem. 286, 35485-35493 (2011) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et al., 2005), c-MYC (Bretones et al., 2011), STAT3 (Huang et al., 2012), and NOR1 (Gizard et . . .
  115. S. C. Goldman; C. Y. Chen; T. J. Lansing; T. M. Gilmer; M. B. Kastan The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplasmic localization Am. J. Pathol. 148, 1381-1385 (1996) .
  116. D. Goldschneider; E. Blanc; G. Raguenez; M. Barrois; A. Legrand; G. Le Roux Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line J. Cell. Sci. 117, 293-301 (2004) .
  117. D. Goldschneider; E. Horvilleur; L.-F. Plassa; M. Guillaud-Bataille; K. Million; E. Wittmer-Dupret Expression of C-terminal deleted p53 isoforms in neuroblastoma Nucleic Acids Res. 34, 5603-5612 (2006) .
  118. P. Gonzalez-Gomez; M. J. Bello; J. Lomas; D. Arjona; M. E. Alonso; C. Aminoso Aberrant methylation of multiple genes in neuroblastic tumours. Relationship with MYCN amplification and allelic status at 1p Eur. J. Cancer 39, 1478-1485 (2003) .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010; Wolf et al., 2010) . . .
    • . . . Analysis of neuroblastoma cell lines reported to date with aberrations in the p53/MDM2/p14ARF pathway demonstrates that 31/40 (78%) of these cell lines are MYCN amplified and predominantly established following previous cytotoxic therapy at relapse (Table 1), when abnormalities of the p53 pathway in neuroblastoma tumors have also been previously reported (reviewed by Tweddle et al., 2003; Carr-Wilkinson et al., 2010) . . .
  119. S. Goto; S. Umehara; R. B. Gerbing; D. O. Stram; G. M. Brodeur; R. C. Seeger Histopathology (international neuroblastoma pathology classification) and MYCN status in patients with peripheral neuroblastic tumors: a report from the children’s cancer group Cancer 92, 2699-2708 (2001) .
    • . . . This paradox is observed histologically by a high mitosis-karyorrhexis index, a combined index of both proliferation and apoptosis, in both human MYCN amplified neuroblastoma tumors (Shimada et al., 1995, 1999; Goto et al., 2001; Altungoz et al., 2007) and TH-MYCN transgenic mouse neuroblastoma tumors (Moore et al., 2008). . . .
  120. C. Grandori; S. M. Cowley; L. P. James; R. N. Eisenman The Myc/Max/Mad network and the transcriptional control of cell behavior Annu. Rev. Cell Dev. Biol. 16, 653-699 (2000) .
    • . . . In contrast, MAX is stable and constitutively expressed, and normally present in stoichiometric excess to MYC, which suggests that the abundance of active heterodimers is dependent on the levels of MYC proteins (reviewed by Grandori et al., 2000). . . .
  121. M. R. Grimmer; W. A. Weiss Childhood tumors of the nervous system as disorders of normal development Curr. Opin. Pediatr. 18, 634-638 (2006) .
    • . . . Additionally, embryonal malignancies of childhood such as neuroblastoma have been suggested to be disorders of normal development (reviewed by Grimmer and Weiss, 2006; Johnsen et al., 2009), therefore it is not surprising that deregulated Wnt pathway signaling may be involved in neuroblastoma development. . . .
  122. L. Gu; H. Zhang; J. He; J. Li; M. Huang; M. Zhou MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells Oncogene 31, 1342-1353 (2012) .
    • . . . Very recently, MDM2 was reported to play a p53-independent role by interacting directly with MYCN mRNA and regulating its stabilization and translation, thereby forming a positive feedback loop, critical for MYCN amplified neuroblastoma tumor cell growth and survival (Gu et al., 2012; Figures 1 and 2). . . .
  123. Y. M. Gu; Y. H. Ma; W. G. Zhao; J. Chen Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro World J. Gastroenterol. 17, 3810-3817 (2011) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et . . .
  124. W. C. Gustafson; W. A. Weiss Myc proteins as therapeutic targets Oncogene 29, 1249-1259 (2010) .
    • . . . Direct inhibition of MYCN has not yet been clinically successful (Gustafson and Weiss, 2010), consequently there is a focus on developing therapeutic strategies directed at destabilizing MYCN protein, and at the downstream targets or pathways which mediate the oncogenic functions of MYCN, and drive the aggressive behavior and progression of MYCN amplified tumors . . .
  125. Y. Hamamori; V. Sartorelli; V. Ogryzko; P. L. Puri; H. Y. Wu; J. Y. Wang Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A Cell 96, 405-413 (1999) .
    • . . . Additionally, it interferes with p53 stabilization and activity via inhibition of the p53/p14ARF pathway by reducing p14ARF levels (Maestro et al., 1999), and inhibition of Ser-20 phosphorylation in response to DNA damage (Stasinopoulos et al., 2005) . . .
  126. J. B. Hamner; P. V. Dickson; T. L. Sims; J. Zhou; Y. Spence; C. Y. Ng Bortezomib inhibits angiogenesis and reduces tumor burden in a murine model of neuroblastoma Surgery 142, 185-191 (2007) .
    • . . . Of interest, although not directly linked to SKP2, bortezomib is shown to induce apoptosis and inhibit cell growth, migration, angiogenesis, and metastasis both in vitro and in murine models of chemosensitive and chemoresistant neuroblastoma (Brignole et al., 2006; Michaelis et al., 2006; Hamner et al., 2007; Valentiner et al., 2009) . . .
  127. W. Hanel; U. M. Moll Links between mutant p53 and genomic instability J. Cell. Biochem. 113, 433-439 (2012) .
    • . . . These new gain-of-function mutants have been reported to play a role in promoting tumorigenesis including increased metastasis and genomic instability, and resistance to anti-cancer therapies (reviewed by Xu, 2008; Brosh and Rotter, 2009; Oren and Rotter, 2010; Hanel and Moll, 2012) . . .
  128. I. R. Hardcastle; J. Liu; E. Valeur; A. Watson; S. U. Ahmed; T. J. Blackburn Isoindolinone inhibitors of the murine double minute 2 (MDM2)-p53 protein-protein interaction: structure-activity studies leading to improved potency J. Med. Chem. 54, 1233-1243 (2011) .
    • . . . This class includes cis-imidazolines (e.g., Nutlins), spiro-oxindoles (MI compounds), benzodiazepinediones, isoindolinones, isoquinolinones, and thiophenes (RITA; reviewed by (Yuan et al., 2011); (Hardcastle et . . .
    • . . . Evaluation of Nutlin-3 and other MDM2-p53 antagonists in preclinical models of neuroblastoma have reported potent anti-tumor effects such as induction of growth arrest, senescence, differentiation and apoptosis, and inhibition of tumor cell proliferation and metastasis (Barbieri et al., 2006; Van Maerken et al., 2006, 2009a, 2011; Hardcastle et al., 2011; Patterson et . . .
  129. K. Harms; S. Nozell; X. Chen The common and distinct target genes of the p53 family transcription factors Cell. Mol. Life Sci. 61, 822-842 (2004) .
    • . . . Furthermore, the identification of non-consensus p53 binding sites also contributes to the ever expanding repertoire of p53 target genes (reviewed by Menendez et al., 2009). p53 target genes have been previously reviewed (Mirza et al., 2003; Harms et al., 2004; Nakamura, 2004; Riley et al., 2008; Wang et al., 2010a). . . .
  130. B. H. Haug; J. R. Henriksen; J. Buechner; D. Geerts; E. Tomte; P. Kogner MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma Carcinogenesis 32, 1005-1012 (2011) .
  131. L. He; X. He; L. P. Lim; E. De Stanchina; Z. Xuan; Y. Liang A microRNA component of the p53 tumour suppressor network Nature 447, 1130-1134 (2007) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
  132. H. Hermeking p53 enters the microRNA world Cancer Cell 12, 414-418 (2007) .
    • . . . The 3′-UTR of MYCN has been identified as a direct target of miR-34a (Wei et al., 2008), a miRNA which is directly upregulated by p53 and mediates several tumor suppressive functions of p53 (reviewed by Hermeking, 2007) . . .
  133. H. Hermeking; D. Eick Mediation of c-Myc-induced apoptosis by p53 Science 265, 2091-2093 (1994) .
    • . . . p53 has long been known to be a direct target gene of c-MYC, and mediate c-MYC induced apoptosis (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994; Zeller et al., 2003) . . .
  134. D. D. Hershko Oncogenic properties and prognostic implications of the ubiquitin ligase Skp2 in cancer Cancer 112, 1415-1424 (2008) .
    • . . . SKP2 overexpression at the mRNA and/or protein level have been detected in a number of human tumors and cell lines including prostate, breast, pancreatic, gastric, colorectal, ovarian, melanoma, lymphoma, and leukemia (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
    • . . . Furthermore, high SKP2 expression was observed to correlate with low p27KIP1 expression levels (Westermann et al., 2007), consistent with studies in other cancer types (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
  135. Y. Hiramatsu; K. Kitagawa; T. Suzuki; C. Uchida; T. Hattori; H. Kikuchi Degradation of Tob1 mediated by SCFSkp2-dependent ubiquitination Cancer Res. 66, 8477-8483 (2006) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et . . .
  136. T. Hirata; M. Watanabe; H. Kaku; Y. Kobayashi; H. Yamada; M. Sakaguchi REIC/Dkk-3-encoding adenoviral vector as a potentially effective therapeutic agent for bladder cancer Int. J. Oncol. 41, 559-564 (2012) .
    • . . . In addition, DKK3 also appears to play a role in chemoresistance, and Ad-REIC/DKK3 mediated downregulation of MDR-1 expression through JNK activation sensitized previously chemoresistant tumor cells to chemotherapy, which supports the applicability of using Ad-REIC/DKK3 in combination with convention chemotherapies in the treatment of drug-resistant cancers (Kawasaki et al., 2009; Hirata et al., 2012) . . .
  137. J. C. Hoehner; C. Gestblom; L. Olsen; S. Pahlman Spatial association of apoptosis-related gene expression and cellular death in clinical neuroblastoma Br. J. Cancer 75, 1185-1194 (1997) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et . . .
  138. M. D. Hogarty The requirement for evasion of programmed cell death in neuroblastomas with MYCN amplification Cancer Lett. 197, 173-179 (2003) .
    • . . . Several lines of evidence from published literature lend support to the notion that during the process of neuroblastoma progression there is evasion of p53-mediated tumor suppression via inactivation of the p53/MDM2/p14ARF pathway (reviewed by Van Maerken et al., 2009b) as well as a requirement for MYCN amplified neuroblastoma to circumvent MYCN driven apoptosis (reviewed by Hogarty, 2003) . . .
  139. R. Honda; H. Tanaka; H. Yasuda Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 FEBS Lett. 420, 25-27 (1997) .
    • . . . Under normal cellular conditions, p53 is maintained at low levels mainly due to MDM2, an E3 ubiquitin ligase and the critical negative regulator of p53 (Honda et al., 1997) . . .
  140. R. Honda; H. Yasuda Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53 EMBO J. 18, 22-27 (1999) .
    • . . . Studies have shown that p14ARF promotes p53 stability and activity by inhibiting MDM2-mediated degradation of p53 via direct interaction with MDM2 and inhibiting its E3 ligase activity (Honda and Yasuda, 1999), preventing MDM2 and p53 nuclear export (Tao and Levine, 1999b), sequestering MDM2 within the nucleolus (Weber et al., 1999), and also by promoting MDM2 degradation (Zhang et al., 1998) . . .
  141. G. Hosoi; J. Hara; T. Okamura; Y. Osugi; S. Ishihara; M. Fukuzawa Low frequency of the p53 gene mutations in neuroblastoma Cancer 73, 3087-3093 (1994) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et . . .
  142. S. Y. Hsieh; P. S. Hsieh; C. T. Chiu; W. Y. Chen Dickkopf-3/REIC functions as a suppressor gene of tumor growth Oncogene 23, 9183-9189 (2004) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et . . .
  143. D. Hu; W. Liu; G. Wu; Y. Wan Nuclear translocation of Skp2 facilitates its destruction in response to TGFbeta signaling Cell Cycle 10, 285-292 (2011) .
    • . . . In contrast, studies have shown that pRb interacts with Cdh1 and promotes SKP2 degradation (Binne et al., 2007), and TGFβ signaling induces nuclear translocation of SKP2 which facilitates its ubiquitylation by APCCdh1 and subsequent degradation (Hu et al., 2011). . . .
  144. J. Hu; Y. Xu; J. Hao; S. Wang; C. Li; S. Meng MiR-122 in hepatic function and liver diseases Protein Cell 3, 364-371 (2012) .
    • . . . The restoration of miR-34a and inhibition of miR-380-5p have been shown to reactivate the p53 pathway and inhibit MYCN expression, as well as inhibiting tumor growth in cell lines and orthotopic murine models of neuroblastoma (Wei et al., 2008; Swarbrick et al., 2010; Tivnan et al., 2012) . . .
    • . . . Certainly, targeting miRNAs as a new class of therapeutics has been gaining increased interest in recent years, and Miravirsen, which inhibits miR-122 is currently in phase II trials for the treatment of patients with Hepatitis C virus (Hu et al., 2012). . . .
  145. H. Huang; W. Zhao; D. Yang Stat3 induces oncogenic Skp2 expression in human cervical carcinoma cells Biochem. Biophys. Res. Commun. 418, 186-190 (2012) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et al., 2005), c-MYC (Bretones et al., 2011), STAT3 (Huang et al., 2012), and NOR1 (Gizard et al., 2011) . . .
  146. H. C. Huang; C. L. Lin; J. K. Lin 1,2,3,4,6-penta-O-galloyl-beta-d-glucose, quercetin, curcumin and lycopene induce cell-cycle arrest in MDA-MB-231 and BT474 cells through downregulation of Skp2 protein J. Agric. Food Chem. 59, 6765-6775 (2011a) .
    • . . . In addition to the above SMIs, several natural compounds have also been shown to downregulate SKP2 activity, including retinoic acid, silibinin, curcumin, quercetin, lycopene, epigallocatechin-3-gallate (Nakamura et al., 2003a; Roy et al., 2007; Cuende et al., 2008; Huang et al., 2011a) . . .
  147. R. Huang; N. K. Cheung; J. Vider; I. Y. Cheung; W. L. Gerald; S. K. Tickoo MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas FASEB J. 25, 4138-4149 (2011b) .
    • . . . BMI-1 is a direct transcriptional target of c-MYC and MYCN and is overexpressed in ~90% of neuroblastoma, correlating with MYCN expression (Ochiai et al., 2010; Huang et al., 2011b) . . .
    • . . . In support of this, studies in neuroblastoma have observed that MYCN functionally cooperates with Twist-1 or BMI-1 to induce neuroblastoma tumorigenesis, where overexpression of Twist-1 or BMI-1 is necessary for tumor growth both in vitro and in vivo (Valsesia-Wittmann et al., 2004; Cui et al., 2007; Huang et al., 2011b) . . .
  148. O. Huber; L. Menard; V. Haurie; A. Nicou; D. Taras; J. Rosenbaum Pontin and reptin, two related ATPases with multiple roles in cancer Cancer Res. 68, 6873-6876 (2008) .
    • . . . To date, studies into Wnt signaling in neuroblastoma have shown that siRNA mediated inhibition of the Wnt1/β-catenin pathway leads to apoptosis of SHSY5Y cells (Zhang et al., 2009), and deregulated canonical and non-canonical Wnt signaling in chemoresistant and high-risk disease (Blanc et al., 2005; Liu et al., 2008b; Flahaut et al., 2009) . . .
  149. F. Ille; L. Sommer Wnt signaling: multiple functions in neural development Cell. Mol. Life Sci. 62, 1100-1108 (2005) .
    • . . . Certainly, the Wnt pathway is involved in early formation of the neural crest and the subsequent development, migration, and terminal differentiation of neural crest cells (Barembaum and Bronner-Fraser, 2005; Ille and Sommer, 2005), and Wnt/β-catenin signaling has been shown to directly induce MYCN expression (Kuwahara et al., 2010) . . .
  150. H. Imaki; K. Nakayama; S. Delehouzee; H. Handa; M. Kitagawa; T. Kamura Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein Cancer Res. 63, 4607-4613 (2003) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et al., 2005), c-MYC (Bretones et al., 2011), STAT3 (Huang et al., 2012), and NOR1 (Gizard et al., 2011) . . .
  151. J. Imamura; C. R. Bartram; F. Berthold; D. Harms; H. Nakamura; H. P. Koeffler Mutation of the p53 gene in neuroblastoma and its relationship with N-myc amplification Cancer Res. 53, 4053-4058 (1993) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et . . .
  152. H. Inuzuka; D. Gao; L. W. Finley; W. Yang; L. Wan; H. Fukushima Acetylation-dependent regulation of skp2 function Cell 150, 179-193 (2012) .
    • . . . In part the latter was reported to be achieved through cytoplasmic SKP2 mediated increased cellular migration via ubiquitination and destruction of E-cadherin (Inuzuka et al., 2012). . . .
  153. J. S. Isaacs; R. Hardman; T. A. Carman; J. C. Barrett; B. E. Weissman Differential subcellular p53 localization and function in N- and S-type neuroblastoma cell lines Cell Growth Differ. 9, 545-555 (1998) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et . . .
  154. T. Ishii; T. Matsuse; M. Masuda; S. Teramoto The effects of S-phase kinase-associated protein 2 (SKP2) on cell cycle status, viability, and chemoresistance in A549 lung adenocarcinoma cells Exp. Lung Res. 30, 687-703 (2004) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  155. P. Ji; L. Goldin; H. Ren; D. Sun; D. Guardavaccaro; M. Pagano Skp2 contains a novel cyclin A binding domain that directly protects cyclin A from inhibition by p27Kip1 J. Biol. Chem. 281, 24058-24069 (2006) .
    • . . . In support of this, PI3K inhibitor LY294002 and Rapamycin have been shown to decrease SKP2 expression (Motti et al., 2005; Shapira et al., 2006) . . .
  156. F. Jiang; N. P. Caraway; R. Li; R. L. Katz RNA silencing of S-phase kinase-interacting protein 2 inhibits proliferation and centrosome amplification in lung cancer cells Oncogene 24, 3409-3418 (2005) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et . . .
  157. G. S. Jimenez; S. H. Khan; J. M. Stommel; G. M. Wahl p53 regulation by post-translational modification and nuclear retention in response to diverse stresses Oncogene 18, 7656-7665 (1999) .
    • . . . The type of response can be dependent on several factors that are both extrinsic and intrinsic to the cell, such as cell type, cellular environment, oncogenic lesions present in the cell, and also stress type (Jimenez et al., 1999; Vousden and Lu, 2002). . . .
  158. J. I. Johnsen; P. Kogner; A. Albihn; M. A. Henriksson Embryonal neural tumours and cell death Apoptosis 14, 424-438 (2009) .
    • . . . Additionally, embryonal malignancies of childhood such as neuroblastoma have been suggested to be disorders of normal development (reviewed by Grimmer and Weiss, 2006; Johnsen et al., 2009), therefore it is not surprising that deregulated Wnt pathway signaling may be involved in neuroblastoma development. . . .
  159. S. N. Jones; A. R. Hancock; H. Vogel; L. A. Donehower; A. Bradley Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis Proc. Natl. Acad. Sci. U.S.A. 95, 15608-15612 (1998) .
    • . . . Similar to p53 null mice, mice deficient in p14ARF, or overexpressing MDM2 or MDMX also developed spontaneous tumors, albeit at a slower rate (Jones et al., 1998; Kamijo et al., 1999; Xiong et al., 2010) . . .
  160. S. N. Jones; A. E. Roe; L. A. Donehower; A. Bradley Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53 Nature 378, 206-208 (1995) .
    • . . . This is supported by the observed embryonic lethality of MDM2 knockout mice and their rescue by the concomitant deletion of p53 (Jones et al., 1995; Montes de Oca Luna et . . .
  161. M. R. Junttila; G. I. Evan p53 – a Jack of all trades but master of none Nat. Rev. Cancer 9, 821-829 (2009) .
    • . . . Although it is not clear why certain mechanisms of p53 pathway inactivation are favored in some tumors but not others, it is likely to be influenced by the various selective pressures acting upon the cancer (Junttila and Evan, 2009). . . .
  162. M. Kaghad; H. Bonnet; A. Yang; L. Creancier; J. C. Biscan; A. Valent Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers Cell 90, 809-819 (1997) .
  163. T. Kamijo; S. Bodner; E. Van De Kamp; D. H. Randle; C. J. Sherr Tumor spectrum in ARF-deficient mice Cancer Res. 59, 2217-2222 (1999) .
    • . . . Similar to p53 null mice, mice deficient in p14ARF, or overexpressing MDM2 or MDMX also developed spontaneous tumors, albeit at a slower rate (Jones et al., 1998; Kamijo et al., 1999; Xiong et al., 2010) . . .
  164. T. Kamura; T. Hara; S. Kotoshiba; M. Yada; N. Ishida; H. Imaki Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation Proc. Natl. Acad. Sci. U.S.A. 100, 10231-10236 (2003) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et . . .
  165. Y. Kashiwakura; K. Ochiai; M. Watanabe; F. Abarzua; M. Sakaguchi; M. Takaoka Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and Smad regulates REIC/Dickkopf-3-induced apoptosis Cancer Res. 68, 8333-8341 (2008) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et . . .
  166. Y. Katagiri; Y. Hozumi; S. Kondo Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo J. Dermatol. Sci. 42, 215-224 (2006) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
  167. H. Katayama; K. Sasai; H. Kawai; Z. M. Yuan; J. Bondaruk; F. Suzuki Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53 Nat. Genet. 36, 55-62 (2004) .
    • . . . AURKA has been shown to directly phosphorylate p53 at Ser-215 and Ser-315 which abrogates p53 DNA binding and transactivation of target genes (Liu et al., 2004), and promotes MDM2-mediated destabilization and inhibition of p53 (Katayama et . . .
    • . . . Studies have shown that it suppresses p53 transcriptional activity as well as promoting increased MDM2-mediated p53 degradation (Katayama et al., 2004; Liu et . . .
  168. K. Kawasaki; M. Watanabe; M. Sakaguchi; Y. Ogasawara; K. Ochiai; Y. Nasu REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer Cancer Gene Ther. 16, 65-72 (2009) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et al., 2011; Ueno et al., 2011; Dellinger et al., 2012; Yang et al., 2012) . . .
    • . . . Demethylating agents azacitidine and decitabine have been approved in the treatment of myelodysplastic syndromes (Cataldo et al., 2009; Santos et al., 2010), however this method is not gene specific and could alter the epigenetic patterns of the entire genome . . .
  169. N. Keshelava; J. J. Zuo; P. Chen; S. N. Waidyaratne; M. C. Luna; C. J. Gomer Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines Cancer Res. 61, 6185-6193 (2001) .
  170. N. Keshelava; J. J. Zuo; N. S. Waidyaratne; T. J. Triche; C. P. Reynolds p53 mutations and loss of p53 function confer multidrug resistance in neuroblastoma Med. Pediatr. Oncol. 35, 563-568 (2000) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et al., 1998; McKenzie et al., 1999; Smart et al., 1999; Keshelava et al., 2000, 2001; Tweddle et al., 2001a,b; Cui et al., 2002; Goldschneider et al., 2004; Chen et al., 2007, 2010; Xue et al., 2007; Kurata et al., 2008; Van Maerken et al., 2011; Gamble et al., 2012) . . .
    • . . . Other studies have also correlated loss of p53 function with drug resistance in neuroblastoma cell lines, and found that transfection of wt p53 drug sensitive cell lines with E6 vectors led to chemoresistance (Keshelava et al., 2000, 2001) . . .
  171. S. Y. Kim; A. Herbst; K. A. Tworkowski; S. E. Salghetti; W. P. Tansey Skp2 regulates Myc protein stability and activity Mol. Cell 11, 1177-1188 (2003) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et . . .
    • . . . SKP2 has previously been shown to regulate the stability of c-MYC and to be a co-factor for c-MYC mediated transcriptional activation of target genes (Kim et al., 2003; von der Lehr et . . .
  172. A. F. Kisselev; W. A. Van Der Linden; H. S. Overkleeft Proteasome inhibitors: an expanding army attacking a unique target Chem. Biol. 19, 99-115 (2012) .
    • . . . Inhibitors targeting the ubiquitin proteasome system (UPS) have been developed and used with success in preclinical and/or clinical studies thereby highlighting these inhibitors as a new class of cancer therapeutics (Kisselev et al., 2012) . . .
  173. M. Kitagawa; S. H. Lee; F. Mccormick Skp2 suppresses p53-dependent apoptosis by inhibiting p300 Mol. Cell 29, 217-231 (2008) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
    • . . . Initially, Kitagawa et al. (2008) identified that SKP2 is able to suppress p53-mediated apoptosis by inhibiting the p53 transcriptional co-activator, p300 . . .
    • . . . As proof-of-concept, the study demonstrated that inhibition of SKP2 combined with DNA damaging agents or Nutlin-3, led to greater induction of apoptosis (Kitagawa et al., 2008) . . .
    • . . . In particular, as p53 has previously been shown to be a direct target gene of MYCN, and a mechanism for MYCN mediated apoptosis in neuroblastoma, it is plausible that MYCN directly upregulates SKP2 to attenuate p53-mediated apoptosis (Kitagawa et al., 2008; Chen et al., 2010), as has previously been reported for c-MYC (Bretones et al., 2011; Figure 3). . . .
    • . . . Furthermore, with the discovery of the role of SKP2 and SKP2B in attenuating p53-mediated apoptosis and transcriptional activity, and that SKP2 deficiency triggers a potent ARF-p53-independent cellular senescence in the presence of oncogenic conditions (such as inactivation of TSGs/overexpression of proto-oncogenes), has implicated the wide applicability of targeting SKP2 as a strategy to reactivate p53 and as pro-senescence therapy (Kitagawa et al., 2008; Chander et al., 2010; Lin et al., 2010) . . .
    • . . . Targeting the expression or stability of SKP2 is an appealing option due to the oncogenic functions of SKP2 which are independent of SCFSKP2 ligase formation and activity (Ji et al., 2006; Kitagawa et al., 2008; Chan et al., 2010a). . . .
  174. A. Klaus; W. Birchmeier Wnt signalling and its impact on development and cancer Nat. Rev. Cancer 8, 387-398 (2008) .
    • . . . Deregulated activation of this pathway has been implicated in several malignancies (Klaus and Birchmeier, 2008) . . .
  175. T. Kobayashi; M. Sakaguchi; R. Tanimoto; F. Abarzua; M. Takaishi; H. Kaku Mechanistic analysis of resistance to REIC/Dkk-3-induced apoptosis in human bladder cancer cells Acta Med. Okayama 62, 393-401 (2008) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et . . .
  176. H. Koga; M. Harada; M. Ohtsubo; S. Shishido; H. Kumemura; S. Hanada Troglitazone induces p27Kip1-associated cell-cycle arrest through down-regulating Skp2 in human hepatoma cells Hepatology 37, 1086-1096 (2003) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et . . .
  177. K. Kojima; M. Konopleva; T. Tsao; H. Nakakuma; M. Andreeff Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia Blood 112, 2886-2895 (2008) .
    • . . . Interestingly, the concomitant inhibition of the MDM2-p53 interaction and Aurora kinases has been shown to act synergistically to induce apoptosis in leukemia cells (Kojima et al., 2008), and should be assessed in neuroblastoma. . . .
  178. H. Komuro; Y. Hayashi; M. Kawamura; K. Hayashi; Y. Kaneko; S. Kamoshita Mutations of the p53 gene are involved in Ewing’s sarcomas but not in neuroblastomas Cancer Res. 53, 5284-5288 (1993) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et . . .
  179. A. Koppen; R. Ait-Aissa; S. Hopman; J. Koster; F. Haneveld; R. Versteeg Dickkopf-1 is down-regulated by MYCN and inhibits neuroblastoma cell proliferation Cancer Lett. 256, 218-228 (2007) .
    • . . . Similar to DKK3, DKK1 is also reported to be secreted in neuroblastoma cell lines, and downregulated indirectly by MYCN (Koppen et al., 2007) . . .
  180. A. Koppen; R. Ait-Aissa; J. Koster; I. Ora; J. Bras; P. G. Van Sluis Dickkopf-3 expression is a marker for neuroblastic tumor maturation and is down-regulated by MYCN Int. J. Cancer 122, 1455-1464 (2008) .
  181. L. G. Korotchkina; Z. N. Demidenko; A. V. Gudkov; M. V. Blagosklonny Cellular quiescence caused by the Mdm2 inhibitor nutlin-3A Cell Cycle 8, 3777-3781 (2009) .
    • . . . Overall, MDM2-p53 antagonists have been shown to activate the p53 pathway, inducing p53-dependent apoptosis and sensitizing tumor cells to cytotoxic and other molecular targeted therapies whilst inducing a reversible cell cycle arrest in normal cells (reviewed by (Van Maerken et al., 2009a); (Vassilev, 2004; Shangary et al., 2008; Korotchkina et . . .
  182. Z. Kovacevic; S. Chikhani; G. Y. Lui; S. Sivagurunathan; D. R. Richardson The Iron-Regulated metastasis suppressor NDRG1 targets NEDD4L, PTEN, and SMAD4 and Inhibits the PI3K and Ras signaling pathways Antioxid Redox Signal , (2012) .
    • . . . Very recently, NDRG1 has been shown to upregulate NEDD4L, PTEN, and SMAD4 and inhibit the PI3K and Ras signaling pathways, thereby implicating its involvement in regulating key oncogenic pathways (Kovacevic et al., 2012) . . .
  183. H. Koyama; T. Zhuang; J. E. Light; V. Kolla; M. Higashi; P. W. McGrady Mechanisms of CHD5 Inactivation in neuroblastomas Clin. Cancer Res. 18, 1588-1597 (2012) .
    • . . . Low levels of CHD5 expression have been found in neuroblastoma cell lines, as well as correlating with MYCN amplification and poor prognosis in neuroblastoma tumors (Thompson et al., 2003; Fujita et al., 2008; Koyama et al., 2012). . . .
    • . . . Similar to miR-34a, CHD5 is also located at 1p36, and is frequently deleted and/or methylated in several human cancers including neuroblastoma. 1p deletion and epigenetic silencing of CHD5 have been suggested to account for the low expression observed in both neuroblastoma tumors and cell lines, as homozygous deletions or mutations were reported to be infrequent (Fujita et al., 2008; Koyama et al., 2012) . . .
  184. J. P. Kruse; W. Gu Modes of p53 regulation Cell 137, 609-622 (2009) .
    • . . . It has been shown to enhance MDM2-mediated ubiquitination and degradation of p53, and repress p53-mediated transcription (reviewed by Marine et al., 2007; Kruse and Gu, 2009) . . .
  185. K. Kurata; R. Yanagisawa; M. Ohira; M. Kitagawa; A. Nakagawara; T. Kamijo Stress via p53 pathway causes apoptosis by mitochondrial Noxa upregulation in doxorubicin-treated neuroblastoma cells Oncogene 27, 741-754 (2008) .
    • . . . In contrast, a number of studies including our own have reported predominantly nuclear localization and/or functional p53 in neuroblastoma (Layfield et al., 1995; Hoehner et al., 1997; Danks et al., 1998; Isaacs et al., 1998; McKenzie et al., 1999; Smart et al., 1999; Keshelava et al., 2000, 2001; Tweddle et al., 2001a,b; Cui et al., 2002; Goldschneider et al., 2004; Chen et al., 2007, 2010; Xue et al., 2007; Kurata et al., 2008; Van Maerken et al., 2011; Gamble et al., 2012) . . .
  186. K. Kurose; M. Sakaguchi; Y. Nasu; S. Ebara; H. Kaku; R. Kariyama Decreased expression of REIC/Dkk-3 in human renal clear cell carcinoma J. Urol. 171, 1314-1318 (2004) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et . . .
  187. T. Kusafuka; M. Fukuzawa; T. Oue; Y. Komoto; A. Yoneda; A. Okada Mutation analysis of p53 gene in childhood malignant solid tumors J. Pediatr. Surg. 32, 1175-1180 (1997) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et . . .
  188. A. Kuwahara; Y. Hirabayashi; P. S. Knoepfler; M. M. Taketo; J. Sakai; T. Kodama Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex Development 137, 1035-1044 (2010) .
    • . . . Certainly, the Wnt pathway is involved in early formation of the neural crest and the subsequent development, migration, and terminal differentiation of neural crest cells (Barembaum and Bronner-Fraser, 2005; Ille and Sommer, 2005), and Wnt/β-catenin signaling has been shown to directly induce MYCN expression (Kuwahara et al., 2010) . . .
  189. D. P. Lane; C. F. Cheok; S. Lain p53-based cancer therapy Cold Spring Harb. Perspect. Biol. 2, a001222 (2010) .
    • . . . The importance of p53 in human cancer has led to vast efforts in the development of p53-based cancer therapeutics (reviewed by Lane et al., 2010) . . .
  190. D. P. Lane; L. V. Crawford T antigen is bound to a host protein in SV40-transformed cells Nature 278, 261-263 (1979) .
    • . . . p53 was discovered over three decades ago as one of the first tumor suppressors (Lane and Crawford, 1979; Linzer and Levine, 1979) and has since been shown to be the most frequently mutated gene in human cancer. p53 is involved in the regulation of several processes that contribute to its central role in maintaining genomic stability and tumor suppression, including cell cycle arrest, apoptosis, senescence, differentiation, autophagy, DNA repair, angiogenesis, cell migration, metabolism, and the immune response . . .
  191. G. A. Lang; T. Iwakuma; Y. A. Suh; G. Liu; V. A. Rao; J. M. Parant Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome Cell 119, 861-872 (2004) .
    • . . . Studies using murine models have observed that mutant p53 knock-in mice develop more aggressive and metastatic tumors in comparison to p53 null mice, and that the different p53 mutants are associated with distinct tumor patterns (Lang et al., 2004; Olive et . . .
  192. E. Latres; R. Chiarle; B. A. Schulman; N. P. Pavletich; A. Pellicer; G. Inghirami Role of the F-box protein Skp2 in lymphomagenesis Proc. Natl. Acad. Sci. U.S.A. 98, 2515-2520 (2001) .
    • . . . In transgenic mouse models, SKP2 cooperates with N-Ras to drive lymphomagenesis (Latres et al., 2001), and tissue targeted expression of SKP2 results in hyperplasia, dysplasia, and low-grade carcinoma of the prostate gland (Shim et al., 2003) . . .
  193. L. M. Lau; J. K. Nugent; X. Zhao; M. S. Irwin HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function Oncogene 27, 997-1003 (2008) .
    • . . . Some studies to date have reported several p53-independent functions of Nutlin-3, including disruption of the MDM2-p73 interaction, which leads to enhanced p73 activity, suppression of tumor cell growth, and induction of apoptosis in p53 deficient neuroblastoma cells (Lau et al., 2008) . . .
  194. E. Laurenti; B. Varnum-Finney; A. Wilson; I. Ferrero; W. E. Blanco-Bose; A. Ehninger Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity Cell Stem Cell 3, 611-624 (2008) .
    • . . . More recent studies have reported significant overlap between c-MYC and MYCN-regulated gene sets (Laurenti et al., 2008; Westermann et . . .
  195. L. J. Layfield; J. K. Thompson; R. K. Dodge; B. J. Kerns Prognostic indicators for neuroblastoma: stage, grade, DNA ploidy, MIB-1-proliferation index, p53, HER-2/neu and EGFr – a survival study J. Surg. Oncol. 59, 21-27 (1995) .
    • . . . Early studies reported cytoplasmic sequestration of wt p53 as a non-mutational mechanism for p53 inactivation and accumulation in neuroblastoma (Moll et al., 1995, 1996) . . .
  196. E. J. Lee; M. Jo; S. B. Rho; K. Park; Y. N. Yoo; J. Park Dkk3, downregulated in cervical cancer, functions as a negative regulator of beta-catenin Int. J. Cancer 124, 287-297 (2009) .
    • . . . In contrast, others have however confirmed DKK3 as an inhibitor of the canonical pathway (Yue et al., 2008; Lee et al., 2009; Dellinger et al., 2012). . . .
  197. S. H. Lee; F. McCormick Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells J. Mol. Med. 83, 296-307 (2005) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
  198. A. J. Levine p53, the cellular gatekeeper for growth and division Cell 88, 323-331 (1997) .
    • . . . An important mechanism for chemo- and radioresistance is inactivation of the p53 pathway (reviewed by Levine, 1997) . . .
  199. M. Levrero; V. De Laurenzi; A. Costanzo; J. Gong; J. Y. Wang; G. Melino The p53/p63/p73 family of transcription factors: overlapping and distinct functions J. Cell. Sci. 113, 1661-1670 (2000) .
    • . . . Identified more recently, p63 and p73 are two homologs which share structural and functional similarity to p53 and belong to the p53 family (reviewed by Levrero et al., 2000). . . .
  200. C. F. Li; J. M. Wang; H. Y. Kang; C. K. Huang; J. W. Wang; F. M. Fang Characterization of gene amplification-driven SKP2 overexpression in myxofibrosarcoma: potential implications in tumor progression and therapeutics Clin. Cancer Res. 18, 1598-1610 (2012a) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  201. H. Li; W. Xu; Y. Huang; X. Huang; L. Xu; Z. Lv Genistein demethylates the promoter of CHD5 and inhibits neuroblastoma growth in vivo Int. J. Mol. Med. 30, 1081-1086 (2012b) .
    • . . . Very recently, genistein has been reported to demethylate the CHD5 promoter, enhance the expression of CHD5 and p53, and inhibit neuroblastoma growth in vivo (Li et al., 2012b). . . .
  202. J. Li; L. Kretzner The growth-inhibitory Ndrg1 gene is a Myc negative target in human neuroblastomas and other cell types with overexpressed N- or c-myc Mol. Cell. Biochem. 250, 91-105 (2003) .
    • . . . NDRG1 and NDRG2 were originally identified as a genes downregulated by MYCN (Shimono et al., 1999; Li and Kretzner, 2003; Zhang et al., 2006, 2008) . . .
  203. H. K. Lin; Z. Chen; G. Wang; C. Nardella; S. W. Lee; C. H. Chan Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence Nature 464, 374-379 (2010) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
    • . . . Furthermore, with the discovery of the role of SKP2 and SKP2B in attenuating p53-mediated apoptosis and transcriptional activity, and that SKP2 deficiency triggers a potent ARF-p53-independent cellular senescence in the presence of oncogenic conditions (such as inactivation of TSGs/overexpression of proto-oncogenes), has implicated the wide applicability of targeting SKP2 as a strategy to reactivate p53 and as pro-senescence therapy (Kitagawa et al., 2008; Chander et al., 2010; Lin et . . .
    • . . . Using high-throughput screening, Cpd A and SMIP004 were identified (Chen et al., 2008; Rico-Bautista et al., 2010) . . .
  204. D. I. Linzer; A. J. Levine Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells Cell 17, 43-52 (1979) .
    • . . . p53 was discovered over three decades ago as one of the first tumor suppressors (Lane and Crawford, 1979; Linzer and Levine, 1979) and has since been shown to be the most frequently mutated gene in human cancer. p53 is involved in the regulation of several processes that contribute to its central role in maintaining genomic stability and tumor suppression, including cell cycle arrest, apoptosis, senescence, differentiation, autophagy, DNA repair, angiogenesis, cell migration, metabolism, and the immune response . . .
  205. N. Liu; L. Wang; X. Li; Q. Yang; X. Liu; J. Zhang N-Myc downstream-regulated gene 2 is involved in p53-mediated apoptosis Nucleic Acids Res. 36, 5335-5349 (2008a) .
    • . . . Similar results have been reported for NDRG2 (Liu et al., 2008a) . . .
  206. X. Liu; P. Mazanek; V. Dam; Q. Wang; H. Zhao; R. Guo Deregulated Wnt/beta-catenin program in high-risk neuroblastomas without MYCN amplification Oncogene 27, 1478-1488 (2008b) .
    • . . . To date, studies into Wnt signaling in neuroblastoma have shown that siRNA mediated inhibition of the Wnt1/β-catenin pathway leads to apoptosis of SHSY5Y cells (Zhang et al., 2009), and deregulated canonical and non-canonical Wnt signaling in chemoresistant and high-risk disease (Blanc et al., 2005; Liu et al., 2008b; Flahaut et al., 2009) . . .
  207. Q. Liu; S. Kaneko; L. Yang; R. I. Feldman; S. V. Nicosia; J. Chen Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215 J. Biol. Chem. 279, 52175-52182 (2004) .
    • . . . AURKA has been shown to directly phosphorylate p53 at Ser-215 and Ser-315 which abrogates p53 DNA binding and transactivation of target genes (Liu et al., 2004), and promotes MDM2-mediated destabilization and inhibition of p53 (Katayama et . . .
    • . . . Studies have shown that it suppresses p53 transcriptional activity as well as promoting increased MDM2-mediated p53 degradation (Katayama et al., 2004; Liu et . . .
  208. G. Lozano The oncogenic roles of p53 mutants in mouse models Curr. Opin. Genet. Dev. 17, 66-70 (2007) .
    • . . . Studies using murine models have observed that mutant p53 knock-in mice develop more aggressive and metastatic tumors in comparison to p53 null mice, and that the different p53 mutants are associated with distinct tumor patterns (Lang et al., 2004; Olive et al., 2004; Lozano, 2007; Song et al., 2007; Oren and Rotter, 2010). . . .
  209. A. Lujambio; S. W. Lowe The microcosmos of cancer Nature 482, 347-355 (2012) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et al., 2007; He et al., 2007; Raver-Shapira et al., 2007; Tarasov et al., 2007) . . .
  210. S. M. Mac; C. A. D’Cunha; P. J. Farnham Direct recruitment of N-myc to target gene promoters Mol. Carcinog. 29, 76-86 (2000) .
    • . . . The precise mechanism of MYCN mediated upregulation of SKP2 remains unclear, as SKP2 is a direct target gene of E2F (Zhang and Wang, 2006), E2F is a direct target of c-MYC (Fernandez et al., 2003) and higher E2F expression levels are observed in the presence of MYCN (Mac et al., 2000; Woo et al., 2008) . . .
  211. R. Maestro; A. P. Dei Tos; Y. Hamamori; S. Krasnokutsky; V. Sartorelli; L. Kedes Twist is a potential oncogene that inhibits apoptosis Genes Dev. 13, 2207-2217 (1999) .
    • . . . Additionally, it interferes with p53 stabilization and activity via inhibition of the p53/p14ARF pathway by reducing p14ARF levels (Maestro et al., 1999), and inhibition of Ser-20 phosphorylation in response to DNA damage (Stasinopoulos et al., 2005) . . .
  212. R. Manhani; L. M. Cristofani; V. Odone Filho; I. Bendit Concomitant p53 mutation and MYCN amplification in neuroblastoma Med. Pediatr. Oncol. 29, 206-207 (1997) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et . . .
  213. B. Mao; C. Niehrs Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling Gene 302, 179-183 (2003) .
    • . . . The co-binding of DKK proteins to KRM1/2 receptors potentiates the ability of DKK proteins to inhibit Wnt signaling, through the formation of a ternary complex that leads to endocytosis and degradation of LRP receptors (Mao et al., 2002; Mao and Niehrs, 2003) . . .
  214. B. Mao; W. Wu; G. Davidson; J. Marhold; M. Li; B. M. Mechler Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling Nature 417, 664-667 (2002) .
    • . . . The co-binding of DKK proteins to KRM1/2 receptors potentiates the ability of DKK proteins to inhibit Wnt signaling, through the formation of a ternary complex that leads to endocytosis and degradation of LRP receptors (Mao et al., 2002; Mao and Niehrs, 2003) . . .
  215. J. C. Marine; M. A. Dyer; A. G. Jochemsen MDMX: from bench to bedside J. Cell. Sci. 120, 371-378 (2007) .
    • . . . It has been shown to enhance MDM2-mediated ubiquitination and degradation of p53, and repress p53-mediated transcription (reviewed by Marine et al., 2007; Kruse and Gu, 2009) . . .
  216. J. M. Maris Unholy matrimony: aurora A and N-Myc as malignant partners in neuroblastoma Cancer Cell 15, 5-6 (2009) .
    • . . . In particular, AURKA has been shown to have oncogenic properties and is amplified and/or overexpressed in a range of human cancers (Maris, 2009) . . .
  217. J. M. Maris; C. L. Morton; R. Gorlick; E. A. Kolb; R. Lock; H. Carol Initial testing of the aurora kinase A inhibitor MLN8237 by the pediatric preclinical testing program (PPTP) Pediatr. Blood Cancer 55, 26-34 (2010) .
    • . . . Preclinical evaluations of a second generation AURKA inhibitor MLN8237 in pediatric cancers including neuroblastoma have been promising (Maris et al., 2010; Carol et al., 2011) . . .
  218. P. P. McKenzie; S. M. Guichard; D. S. Middlemas; R. A. Ashmun; M. K. Danks; L. C. Harris Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated Clin. Cancer Res. 5, 4199-4207 (1999) .
  219. C. R. McPake; D. M. Tillman; C. A. Poquette; E. O. George; J. A. Houghton; L. C. Harris Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status Oncol. Res. 10, 235-244 (1998) .
  220. D. Menendez; A. Inga; M. A. Resnick The expanding universe of p53 targets Nat. Rev. Cancer 9, 724-737 (2009) .
    • . . . Furthermore, the identification of non-consensus p53 binding sites also contributes to the ever expanding repertoire of p53 target genes (reviewed by Menendez et al., 2009). p53 target genes have been previously reviewed (Mirza et al., 2003; Harms et al., 2004; Nakamura, 2004; Riley et al., 2008; Wang et al., 2010a). . . .
  221. X. Mergui; F. Leteurtre; M. Lipinski; J. Benard; M. Amor-Gueret Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma Biochimie 90, 1656-1666 (2008) .
  222. J. A. Mertz; A. R. Conery; B. M. Bryant; P. Sandy; S. Balasubramanian; D. A. Mele Targeting MYC dependence in cancer by inhibiting BET bromodomains Proc. Natl. Acad. Sci. U.S.A. 108, 16669-16674 (2011) .
    • . . . Alternative strategies more specific to neuroblastoma may involve the direct targeting of the oncogenic miR-17-92 cluster to re-establish DKK3 expression in MYCN amplified neuroblastoma, or the use of agents which directly affect MYCN expression in neuroblastoma cells such as the BET inhibitor, JQ1 (Mertz et al., 2011) . . .
  223. M. Michaelis; I. Fichtner; D. Behrens; W. Haider; F. Rothweiler; A. Mack Anti-cancer effects of bortezomib against chemoresistant neuroblastoma cell lines in vitro and in vivo Int. J. Oncol. 28, 439-446 (2006) .
    • . . . Of interest, although not directly linked to SKP2, bortezomib is shown to induce apoptosis and inhibit cell growth, migration, angiogenesis, and metastasis both in vitro and in murine models of chemosensitive and chemoresistant neuroblastoma (Brignole et al., 2006; Michaelis et . . .
  224. M. Michaelis; F. Rothweiler; S. Barth; J. Cinatl; M. Van Rikxoort; N. Loschmann Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells Cell Death Dis. 2, e243 (2011) .
    • . . . Consequently these results suggest that patients treated with MDM2-p53 antagonists should be closely monitored for the development of p53 mutations and/or that MDM2-p53 antagonists should be given in combination with other agents to try to prevent the development of p53 mutations (Michaelis et al., 2011). . . .
  225. M. Michaelis; F. Rothweiler; D. Klassert; A. Von Deimling; K. Weber; B. Fehse Reversal of P-glycoprotein-mediated multidrug resistance by the murine double minute 2 antagonist nutlin-3 Cancer Res. 69, 416-421 (2009) .
    • . . . In addition, Nutlin-3 has been shown to sensitize neuroblastoma cells to chemotherapy induced apoptosis (Barbieri et al., 2006; Michaelis et al., 2009; Peirce and Findley, 2009a; Patterson et al., 2011) . . .
    • . . . Additionally, Nutlin-3 has also been shown to sensitize p53 deficient chemoresistant neuroblastoma cells to chemotherapy induced apoptosis via upregulation of TAp73 and E2F1 (Ambrosini et al., 2007; Peirce and Findley, 2009b), and inhibition of P-glycoprotein (Michaelis et al., 2009). . . .
  226. A. Mirza; Q. Wu; L. Wang; T. Mcclanahan; W. R. Bishop; F. Gheyas Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression Oncogene 22, 3645-3654 (2003) .
    • . . . In contrast, most genes which are repressed by p53 tend to lack p53 DNA binding sites within their promoters (Mirza et al., 2003) . . .
  227. Y. Mizobuchi; K. Matsuzaki; K. Kuwayama; K. Kitazato; H. Mure; T. Kageji REIC/Dkk-3 induces cell death in human malignant glioma Neuro oncol. 10, 244-253 (2008) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et . . .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et . . .
  228. U. M. Moll; M. Laquaglia; J. Benard; G. Riou Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors Proc. Natl. Acad. Sci. U.S.A. 92, 4407-4411 (1995) .
    • . . . Early studies reported cytoplasmic sequestration of wt p53 as a non-mutational mechanism for p53 inactivation and accumulation in neuroblastoma (Moll et al., 1995, 1996) . . .
  229. U. M. Moll; A. G. Ostermeyer; R. Haladay; B. Winkfield; M. Frazier; G. Zambetti Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage Mol. Cell. Biol. 16, 1126-1137 (1996) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik . . .
  230. U. M. Moll; N. Slade p63 and p73: roles in development and tumor formation Mol. Cancer Res. 2, 371-386 (2004) .
    • . . . Interestingly in contrast to p53, p63, and p73 are rarely mutated in human cancers and neither p63 nor p73 knockout mice exhibit an increased susceptibility to developing spontaneous tumors (Moll and Slade, 2004). . . .
  231. U. M. Moll; S. Wolff; D. Speidel; W. Deppert Transcription-independent pro-apoptotic functions of p53 Curr. Opin. Cell Biol. 17, 631-636 (2005) .
    • . . . p53 is also able to exert functions via protein-protein interactions, and several proteins involved in cell cycle control, DNA repair, gene transcription, and apoptosis have been shown to bind to p53 (Moll et al., 2005; Braithwaite et al., 2006; Speidel et al., 2006) . . .
  232. J. Momand; G. P. Zambetti; D. C. Olson; D. George; A. J. Levine The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation Cell 69, 1237-1245 (1992) .
    • . . . MDM2 directly binds to the N-terminal TAD of p53 to inhibit p53 transcriptional activity (Momand et al., 1992), as well as promoting nuclear export and targeting p53 for ubiquitin mediated proteasome degradation (Honda et al., 1997; Tao and Levine, 1999a). p14ARF is a tumor suppressor and the negative regulator of MDM2 . . .
  233. R. Montes de Oca Luna; D. S. Wagner; G. Lozano Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53 Nature 378, 203-206 (1995) .
    • . . . This is supported by the observed embryonic lethality of MDM2 knockout mice and their rescue by the concomitant deletion of p53 (Jones et al., 1995; Montes de Oca Luna et . . .
  234. H. C. Moore; K. M. Wood; M. S. Jackson; M. A. Lastowska; D. Hall; H. Imrie Histological profile of tumours from MYCN transgenic mice J. Clin. Pathol. 61, 1098-1103 (2008) .
    • . . . This paradox is observed histologically by a high mitosis-karyorrhexis index, a combined index of both proliferation and apoptosis, in both human MYCN amplified neuroblastoma tumors (Shimada et al., 1995, 1999; Goto et al., 2001; Altungoz et al., 2007) and TH-MYCN transgenic mouse neuroblastoma tumors (Moore et al., 2008). . . .
  235. L. Moro; A. A. Arbini; E. Marra; M. Greco Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation J. Biol. Chem. 281, 22100-22107 (2006) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et . . .
  236. M. L. Motti; D. Califano; G. Troncone; C. De Marco; I. Migliaccio; E. Palmieri Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization Am. J. Pathol. 166, 737-749 (2005) .
    • . . . In support of this, PI3K inhibitor LY294002 and Rapamycin have been shown to decrease SKP2 expression (Motti et al., 2005; Shapira et al., 2006) . . .
  237. D. Muth; S. Ghazaryan; I. Eckerle; E. Beckett; C. Pohler; J. Batzler Transcriptional repression of SKP2 is impaired in MYCN-amplified neuroblastoma Cancer Res. 70, 3791-3802 (2010) .
  238. M. Nakamura; T. Matsuo; J. Stauffer; L. Neckers; C. J. Thiele Retinoic acid decreases targeting of p27 for degradation via an N-myc-dependent decrease in p27 phosphorylation and an N-myc-independent decrease in Skp2 Cell Death Differ. 10, 230-239 (2003a) .
    • . . . In line with observations that SKP2 drives cellular proliferation, cAMP induces proliferation of neuroblastoma cells by upregulating SKP2 (Cho et al., 2007), while growth arrest and differentiation of neuroblastoma cell lines induced by retinoic acid, BMP2, and the HDAC inhibitor Helminthosporium carbonum toxin is accompanied by a decrease in SKP2 levels (Nakamura et al., 2003a,b; Cuende et al., 2008; Deubzer et al., 2008a) . . .
    • . . . In addition to the above SMIs, several natural compounds have also been shown to downregulate SKP2 activity, including retinoic acid, silibinin, curcumin, quercetin, lycopene, epigallocatechin-3-gallate (Nakamura et al., 2003a; Roy et al., 2007; Cuende et al., 2008; Huang et al., 2011a) . . .
  239. Y. Nakamura; T. Ozaki; H. Koseki; A. Nakagawara; S. Sakiyama Accumulation of p27 KIP1 is associated with BMP2-induced growth arrest and neuronal differentiation of human neuroblastoma-derived cell lines Biochem. Biophys. Res. Commun. 307, 206-213 (2003b) .
    • . . . In line with observations that SKP2 drives cellular proliferation, cAMP induces proliferation of neuroblastoma cells by upregulating SKP2 (Cho et al., 2007), while growth arrest and differentiation of neuroblastoma cell lines induced by retinoic acid, BMP2, and the HDAC inhibitor Helminthosporium carbonum toxin is accompanied by a decrease in SKP2 levels (Nakamura et al., 2003a,b; Cuende et al., 2008; Deubzer et al., 2008a) . . .
  240. R. E. Nakamura; A. S. Hackam Analysis of Dickkopf3 interactions with Wnt signaling receptors Growth Factors 28, 232-242 (2010) .
    • . . . In some studies, DKK3 is not shown to bind to LRP5/6 at the cell surface membrane, and whether it binds to KRM1/2 remains controversial (Mao et al., 2002; Nakamura and Hackam, 2010) . . .
  241. Y. Nakamura Isolation of p53-target genes and their functional analysis Cancer Sci. 95, 7-11 (2004) .
    • . . . Furthermore, the identification of non-consensus p53 binding sites also contributes to the ever expanding repertoire of p53 target genes (reviewed by Menendez et al., 2009). p53 target genes have been previously reviewed (Mirza et al., 2003; Harms et al., 2004; Nakamura, 2004; Riley et al., 2008; Wang et al., 2010a). . . .
  242. Y. Nakamura; T. Ozaki; H. Niizuma; M. Ohira; T. Kamijo; A. Nakagawara Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line Biochem. Biophys. Res. Commun. 354, 892-898 (2007) .
  243. K. Nakayama; H. Nagahama; Y. A. Minamishima; M. Matsumoto; I. Nakamichi; K. Kitagawa Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication EMBO J. 19, 2069-2081 (2000) .
    • . . . In addition, the prognostic significance and overexpression of SKP2 in a variety of human cancers, combined with observations that SKP2 knockout mice are both viable and fertile (Nakayama et al., 2000), further strengthens its appeal . . .
  244. K. I. Nakayama; K. Nakayama Regulation of the cell cycle by SCF-type ubiquitin ligases Semin. Cell Dev. Biol. 16, 323-333 (2005) .
    • . . . The invariable core components of SCF complexes are SKP1, CUL1, and RBX1 (Skowyra et al., 1997; Deshaies, 1999; Nakayama and Nakayama, 2005) . . .
  245. K. I. Nakayama; K. Nakayama Ubiquitin ligases: cell-cycle control and cancer Nat. Rev. Cancer 6, 369-381 (2006) .
    • . . . SKP2 overexpression at the mRNA and/or protein level have been detected in a number of human tumors and cell lines including prostate, breast, pancreatic, gastric, colorectal, ovarian, melanoma, lymphoma, and leukemia (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
    • . . . Furthermore, high SKP2 expression was observed to correlate with low p27KIP1 expression levels (Westermann et al., 2007), consistent with studies in other cancer types (reviewed by Nakayama and Nakayama, 2006; Frescas and Pagano, 2008; Hershko, 2008) . . .
  246. C. Niehrs Function and biological roles of the Dickkopf family of Wnt modulators Oncogene 25, 7469-7481 (2006) .
    • . . . DKK1, −2 and −4, are well-established antagonists of the canonical Wnt/β-catenin pathway through direct high affinity binding to Wnt co-receptors lipoprotein receptor-related protein 5 and 6 (LRP5/6), and also DKK receptors Kremen 1 and 2 (KRM1/2; reviewed by (Niehrs, 2006)) . . .
  247. A. Y. Nikolaev; W. Gu PARC: a potential target for cancer therapy Cell Cycle 2, 169-171 (2003) .
    • . . . To date several mechanisms for cytoplasmic sequestration of p53 in neuroblastoma have been proposed, including masking of the p53 C-terminal nuclear localization signal (Ostermeyer et al., 1996), hyperactive nuclear export (Stommel et al., 1999), binding to the cytoplasmic anchor, Parc (Nikolaev and Gu, 2003; Nikolaev et al., 2003), aberrant hyperubiquitylation of p53 (Becker et al., 2007), and MDMX and MDM2-mediated cytoplasmic tethering (Ohtsubo et al., 2009) . . .
  248. A. Y. Nikolaev; M. Li; N. Puskas; J. Qin; W. Gu Parc: a cytoplasmic anchor for p53 Cell 112, 29-40 (2003) .
    • . . . To date several mechanisms for cytoplasmic sequestration of p53 in neuroblastoma have been proposed, including masking of the p53 C-terminal nuclear localization signal (Ostermeyer et al., 1996), hyperactive nuclear export (Stommel et al., 1999), binding to the cytoplasmic anchor, Parc (Nikolaev and Gu, 2003; Nikolaev et al., 2003), aberrant hyperubiquitylation of p53 (Becker et al., 2007), and MDMX and MDM2-mediated cytoplasmic tethering (Ohtsubo et al., 2009) . . .
  249. I. Nozaki; T. Tsuji; O. Iijima; Y. Ohmura; A. Andou; M. Miyazaki Reduced expression of REIC/Dkk-3 gene in non-small cell lung cancer Int. J. Oncol. 19, 117-121 (2001) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
  250. H. Ochiai; H. Takenobu; A. Nakagawa; Y. Yamaguchi; M. Kimura; M. Ohira Bmi1 is a MYCN target gene that regulates tumorigenesis through repression of KIF1Bbeta and TSLC1 in neuroblastoma Oncogene 29, 2681-2690 (2010) .
    • . . . BMI-1 is a direct transcriptional target of c-MYC and MYCN and is overexpressed in ~90% of neuroblastoma, correlating with MYCN expression (Ochiai et al., 2010; Huang et al., 2011b) . . .
  251. H. Ohgaki; R. H. Eibl; M. Schwab; M. B. Reichel; L. Mariani; M. Gehring Mutations of the p53 tumor suppressor gene in neoplasms of the human nervous system Mol. Carcinog. 8, 74-80 (1993) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et . . .
  252. C. Ohtsubo; D. Shiokawa; M. Kodama; C. Gaiddon; H. Nakagama; A. G. Jochemsen Cytoplasmic tethering is involved in synergistic inhibition of p53 by Mdmx and Mdm2 Cancer Sci. 100, 1291-1299 (2009) .
    • . . . To date several mechanisms for cytoplasmic sequestration of p53 in neuroblastoma have been proposed, including masking of the p53 C-terminal nuclear localization signal (Ostermeyer et al., 1996), hyperactive nuclear export (Stommel et al., 1999), binding to the cytoplasmic anchor, Parc (Nikolaev and Gu, 2003; Nikolaev et al., 2003), aberrant hyperubiquitylation of p53 (Becker et al., 2007), and MDMX and MDM2-mediated cytoplasmic tethering (Ohtsubo et al., 2009) . . .
  253. K. P. Olive; D. A. Tuveson; Z. C. Ruhe; B. Yin; N. A. Willis; R. T. Bronson Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome Cell 119, 847-860 (2004) .
    • . . . Studies using murine models have observed that mutant p53 knock-in mice develop more aggressive and metastatic tumors in comparison to p53 null mice, and that the different p53 mutants are associated with distinct tumor patterns (Lang et al., 2004; Olive et . . .
  254. M. Olivier; P. Taniere Somatic mutations in cancer prognosis and prediction: lessons from TP53 and EGFR genes Curr. Opin. Oncol. 23, 88-92 (2011) .
    • . . . This has been shown for malignancies of the breast, head and neck, colorectum, and hematopoietic system (Brosh and Rotter, 2009; Olivier and Taniere, 2011). . . .
  255. M. Omura-Minamisawa; M. B. Diccianni; R. C. Chang; A. Batova; L. J. Bridgeman; J. Schiff p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease Clin. Cancer Res. 7, 3481-3490 (2001) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et al., 1993; Ohgaki et al., 1993; Vogan et al., 1993; Castresana et al., 1994; Hosoi et al., 1994; Kusafuka et al., 1997; Manhani et al., 1997; Omura-Minamisawa et al., 2001; Tweddle et . . .
    • . . . Consequently, gaining a greater understanding into the mechanisms behind resistance to cytotoxic therapies could lead to the development and use of novel therapies in progressing or relapsed disease (Tweddle et al., 2001a) . . .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et . . .
  256. M. Oren; V. Rotter Mutant p53 gain-of-function in cancer Cold Spring Harb. Perspect. Biol. 2, a001107 (2010) .
    • . . . These new gain-of-function mutants have been reported to play a role in promoting tumorigenesis including increased metastasis and genomic instability, and resistance to anti-cancer therapies (reviewed by Xu, 2008; Brosh and Rotter, 2009; Oren and Rotter, 2010; Hanel and Moll, 2012) . . .
  257. A. G. Ostermeyer; E. Runko; B. Winkfield; B. Ahn; U. M. Moll Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide Proc. Natl. Acad. Sci. U.S.A. 93, 15190-15194 (1996) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik, 1996) . . .
  258. T. Otto; S. Horn; M. Brockmann; U. Eilers; L. Schuttrumpf; N. Popov Stabilization of N-Myc is a critical function of Aurora A in human neuroblastoma Cancer Cell 15, 67-78 (2009) .
    • . . . Interestingly, AURKA has also been reported to stabilize MYCN, which would further promote MYCN driven tumorigenesis (Otto et al., 2009; Figure 2). . . .
    • . . . In neuroblastoma AURKA has been found to be expressed at high levels in MYCN amplified tumors and required for the growth of MYCN amplified cells (Berwanger et al., 2002; Otto et al., 2009) . . .
  259. T. Paffhausen; M. Schwab; F. Westermann Targeted MYCN expression affects cytotoxic potential of chemotherapeutic drugs in neuroblastoma cells Cancer Lett. 250, 17-24 (2007) .
    • . . . This is consistent with, and may help to explain why human MYCN amplified and TH-MYCN transgenic mouse neuroblastoma tumors have high levels of apoptosis, and MYCN amplified and Tet21N MYCN+ neuroblastoma cells undergo higher levels of apoptosis in response to chemotherapeutic agents (Fulda et al., 1999, 2000; Paffhausen et al., 2007; Chesler et al., 2008), irradiation (Bell et al., 2006), and MDM2-p53 antagonists (Gamble et al., 2012) . . .
  260. Y. Pan; J. Chen MDM2 promotes ubiquitination and degradation of MDMX Mol. Cell. Biol. 23, 5113-5121 (2003) .
    • . . . Interestingly, MDM2 can promote ubiquitination and degradation of MDMX, an effect which is stimulated by p14ARF and which correlates with the ability of p14ARF to bind MDM2 (Pan and Chen, 2003; Figure 1). . . .
  261. J. R. Park; A. Eggert; H. Caron Neuroblastoma: biology, prognosis, and treatment Hematol. Oncol. Clin. North Am. 24, 65-86 (2010) .
    • . . . It accounts for 8–10% of all pediatric cancers and 15% of childhood cancer mortality (Park et al., 2010) . . .
  262. D. M. Patterson; D. Gao; D. N. Trahan; B. A. Johnson; A. Ludwig; E. Barbieri Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma Angiogenesis 14, 255-266 (2011) .
    • . . . Evaluation of Nutlin-3 and other MDM2-p53 antagonists in preclinical models of neuroblastoma have reported potent anti-tumor effects such as induction of growth arrest, senescence, differentiation and apoptosis, and inhibition of tumor cell proliferation and metastasis (Barbieri et al., 2006; Van Maerken et al., 2006, 2009a, 2011; Hardcastle et al., 2011; Patterson et . . .
  263. S. K. Peirce; H. W. Findley High level MycN expression in non-MYCN amplified neuroblastoma is induced by the combination treatment nutlin-3 and doxorubicin and enhances chemosensitivity Oncol. Rep. 22, 1443-1449 (2009a) .
    • . . . In addition, Nutlin-3 has been shown to sensitize neuroblastoma cells to chemotherapy induced apoptosis (Barbieri et al., 2006; Michaelis et al., 2009; Peirce and Findley, 2009a; Patterson et al., 2011) . . .
  264. S. K. Peirce; H. W. Findley The MDM2 antagonist nutlin-3 sensitizes p53-null neuroblastoma cells to doxorubicin via E2F1 and TAp73 Int. J. Oncol. 34, 1395-1402 (2009b) .
    • . . . Additionally, Nutlin-3 has also been shown to sensitize p53 deficient chemoresistant neuroblastoma cells to chemotherapy induced apoptosis via upregulation of TAp73 and E2F1 (Ambrosini et al., 2007; Peirce and Findley, 2009b), and inhibition of P-glycoprotein (Michaelis et al., 2009). . . .
  265. C. Perfumo; S. Parodi; K. Mazzocco; R. Defferrari; A. Inga; R. Haupt Impact of MDM2 SNP309 genotype on progression and survival of stage 4 neuroblastoma Eur. J. Cancer 44, 2634-2639 (2008) .
    • . . . A single-nucleotide polymorphism (SNP) in the MDM2 promoter (SNP309T to G) leading to high levels of MDM2 expression has been found in some tumors and is associated with a poor prognostic outcome, including neuroblastoma (Cattelani et al., 2008; Perfumo et . . .
    • . . . MDM2 is a direct target gene of MYCN (Slack et al., 2005; Westermann et al., 2008) and non-syntenic co-amplification of MDM2 and MYCN has been reported in neuroblastoma (Corvi et al., 1995) . . .
  266. C. Perfumo; S. Parodi; K. Mazzocco; R. Defferrari; A. Inga; G. B. Scarra MDM2 SNP309 genotype influences survival of metastatic but not of localized neuroblastoma Pediatr. Blood Cancer 53, 576-583 (2009) .
    • . . . Consistent with observations that Mdm2 haploinsufficiency inhibits c-MYC induced lymphomagenesis (Alt et al., 2003), MDM2 has been shown to be necessary for MYCN to overcome p53-mediated tumor suppression for MYCN directed centrosome amplification and genomic instability (Slack et al., 2007), and also during MYCN driven neuroblastoma tumorigenesis (Chen et al., 2009; Figure 2) . . .
  267. S. Piccinin; E. Tonin; S. Sessa; S. Demontis; S. Rossi; L. Pecciarini A “twist box” code of p53 inactivation: twist box:p53 interaction promotes p53 degradation Cancer Cell 22, 404-415 (2012) .
    • . . . Twist-1 which belongs to the bHLH transcription factor family, and BMI-1, a polycomb ring finger oncogene, are both overexpressed in several human cancers, and have been shown to be involved in epithelial-mesenchymal transition and cancer stemness which have clinical implications of cancer metastasis, drug resistance, and survival (Wu et al., 2012a) . . .
  268. J. J. Qin; S. Nag; S. Voruganti; W. Wang; R. Zhang Natural Product MDM2 Inhibitors: anticancer Activity and Mechanisms of Action Curr. Med. Chem , (2012) .
    • . . . In recent years, there has been increased interest in the use of MDM2-p53 antagonists in a cyclotherapeutic setting to protect normal cells from the harmful effects of chemotherapy (reviewed by Cheok et al., 2011; van Leeuwen, 2012) and also in the identification of natural MDM2 inhibitors (Qin et al., 2012) . . .
  269. G. Raschella; A. Negroni; C. Giubilei; A. Romeo; S. Ferrari; M. A. Castello Transcription of N-myc and proliferation-related genes is linked in human neuroblastoma Cancer Lett. 56, 45-51 (1991) .
    • . . . Initial studies in neuroblastoma showed that MYCN amplified tumors expressed significantly higher levels of p53 mRNA in comparison with non-amplified tumors (Raschella et al., 1991; Berwanger et al., 2002; Westermann et al., 2008), and higher p53 protein expression in the presence of ectopic MYCN in cell lines (Cui et al., 2005; Bell et al., 2006; Sugihara et al., 2006) . . .
  270. N. Raver-Shapira; E. Marciano; E. Meiri; Y. Spector; N. Rosenfeld; N. Moskovits Transcriptional activation of miR-34a contributes to p53-mediated apoptosis Mol. Cell 26, 731-743 (2007) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
  271. D. Reisman; N. B. Elkind; B. Roy; J. Beamon; V. Rotter c-Myc trans-activates the p53 promoter through a required downstream CACGTG motif Cell Growth Differ. 4, 57-65 (1993) .
    • . . . p53 has long been known to be a direct target gene of c-MYC, and mediate c-MYC induced apoptosis (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994; Zeller et al., 2003) . . .
  272. I. Revet; G. Huizenga; J. Koster; R. Volckmann; P. Van Sluis; R. Versteeg MSX1 induces the Wnt pathway antagonist genes DKK1, DKK2, DKK3, and SFRP1 in neuroblastoma cells, but does not block Wnt3 and Wnt5A signalling to DVL3 Cancer Lett. 289, 195-207 (2010) .
    • . . . In a more recent study of 101 neuroblastic tumors including 88 neuroblastoma tumors, both high DKK3 and DKK2 expression correlated with good prognosis (Revet et al., 2010). . . .
    • . . . These included downregulation of additional Wnt signaling antagonists SFRP1 and APC together with the upregulated expression of positive regulators FRAT2, CSNK2A1, and RUVBL1 (Chen, 2009; Chen et al., 2010) . . .
  273. E. Rico-Bautista; C. C. Yang; L. Lu; G. P. Roth; D. A. Wolf Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells BMC Biol. 8, 153 (2010) .
    • . . . Using high-throughput screening, Cpd A and SMIP004 were identified (Chen et al., 2008; Rico-Bautista et al., 2010) . . .
  274. T. Riley; E. Sontag; P. Chen; A. Levine Transcriptional control of human p53-regulated genes Nat. Rev. Mol. Cell Biol. 9, 402-412 (2008) .
    • . . . Furthermore, the identification of non-consensus p53 binding sites also contributes to the ever expanding repertoire of p53 target genes (reviewed by Menendez et al., 2009). p53 target genes have been previously reviewed (Mirza et al., 2003; Harms et al., 2004; Nakamura, 2004; Riley et al., 2008; Wang et al., 2010a). . . .
  275. K. D. Robertson; P. A. Jones The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53 Mol. Cell. Biol. 18, 6457-6473 (1998) .
    • . . . Activated p53 can subsequently downregulate the expression of p14ARF (Robertson and Jones, 1998; Stott et al., 1998) . . .
  276. J. Roman-Gomez; A. Jimenez-Velasco; X. Agirre; J. A. Castillejo; G. Navarro; M. Barrios Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia Br. J. Cancer 91, 707-713 (2004) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et . . .
  277. A. E. Rose; G. Wang; D. Hanniford; S. Monni; T. Tu; R. L. Shapiro Clinical relevance of SKP2 alterations in metastatic melanoma Pigment Cell Melanoma Res. 24, 197-206 (2011) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  278. B. Roy; J. Beamon; E. Balint; D. Reisman Transactivation of the human p53 tumor suppressor gene by c-Myc/Max contributes to elevated mutant p53 expression in some tumors Mol. Cell. Biol. 14, 7805-7815 (1994) .
    • . . . p53 has long been known to be a direct target gene of c-MYC, and mediate c-MYC induced apoptosis (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994; Zeller et al., 2003) . . .
  279. S. Roy; M. Kaur; C. Agarwal; M. Tecklenburg; R. A. Sclafani; R. Agarwal p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells Mol. Cancer Ther. 6, 2696-2707 (2007) .
    • . . . In addition to the above SMIs, several natural compounds have also been shown to downregulate SKP2 activity, including retinoic acid, silibinin, curcumin, quercetin, lycopene, epigallocatechin-3-gallate (Nakamura et al., 2003a; Roy et al., 2007; Cuende et al., 2008; Huang et al., 2011a) . . .
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    • . . . Consistent with this, reduced levels of miR-34 have been observed in both tumors and cell lines, including neuroblastoma (Bommer et al., 2007; Chang et al., 2007; Tarasov et al., 2007; Welch et al., 2007; Feinberg-Gorenshtein et al., 2009) . . .
  281. K. Saigusa; N. Hashimoto; H. Tsuda; S. Yokoi; M. Maruno; T. Yoshimine Overexpressed Skp2 within 5p amplification detected by array-based comparative genomic hybridization is associated with poor prognosis of glioblastomas Cancer Sci. 96, 676-683 (2005) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  282. M. Sakaguchi; K. Kataoka; F. Abarzua; R. Tanimoto; M. Watanabe; H. Murata Overexpression of REIC/Dkk-3 in normal fibroblasts suppresses tumor growth via induction of interleukin-7 J. Biol. Chem. 284, 14236-14244 (2009) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et al., 2011; Ueno et al., 2011; Dellinger et al., 2012; Yang et al., 2012) . . .
    • . . . Demethylating agents azacitidine and decitabine have been approved in the treatment of myelodysplastic syndromes (Cataldo et al., 2009; Santos et al., 2010), however this method is not gene specific and could alter the epigenetic patterns of the entire genome . . .
  283. F. P. Santos; H. Kantarjian; G. Garcia-Manero; J. P. Issa; F. Ravandi Decitabine in the treatment of myelodysplastic syndromes Expert Rev Anticancer Ther 10, 9-22 (2010) .
    • . . . Demethylating agents azacitidine and decitabine have been approved in the treatment of myelodysplastic syndromes (Cataldo et al., 2009; Santos et al., 2010), however this method is not gene specific and could alter the epigenetic patterns of the entire genome . . .
  284. L. M. Sarmento; H. Huang; A. Limon; W. Gordon; J. Fernandes; M. J. Tavares Notch1 modulates timing of G1-S progression by inducing SKP2 transcription and p27 Kip1 degradation J. Exp. Med. 202, 157-168 (2005) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et . . .
  285. G. Schneider; D. Saur; J. T. Siveke; R. Fritsch; F. R. Greten; R. M. Schmid IKKalpha controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression EMBO J. 25, 3801-3812 (2006) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et . . .
  286. S. Schuler; S. Diersch; R. Hamacher; R. M. Schmid; D. Saur; G. Schneider SKP2 confers resistance of pancreatic cancer cells towards TRAIL-induced apoptosis Int. J. Oncol. 38, 219-225 (2011) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  287. B. A. Schulman; A. C. Carrano; P. D. Jeffrey; Z. Bowen; E. R. Kinnucan; M. S. Finnin Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex Nature 408, 381-386 (2000) .
    • . . . SKP2 protein is approximately 45 kDa, consisting of an N-terminal F-Box domain which mediates the interaction between SKP2 and SKP1, thereby tethering SKP2 to the SCF complex, and C-terminal leucine-rich repeats (LRR) which enable SKP2 to directly bind to target substrates (Bai et al., 1996; Skowyra et al., 1997; Schulman et al., 2000; Zheng et al., 2002) . . .
  288. J. H. Schulte; S. Horn; T. Otto; B. Samans; L. C. Heukamp; U. C. Eilers MYCN regulates oncogenic MicroRNAs in neuroblastoma Int. J. Cancer 122, 699-704 (2008) .
    • . . . Through ChIP analysis, MYCN was reported to downregulate DKK3 expression through a mechanism independent of direct transcriptional repression (Koppen et al., 2008) . . .
  289. M. Schwab; G. M. Brodeur; T. Sawada; Y. Tsuchida; P. A. Voûte “MYCN amplification in neuroblastoma,” Neuroblastoma , 75-83 (2000) .
    • . . . The MYCN gene located at 2p24 encodes a 64 kDa nuclear phosphoprotein, which contains a transcriptional activation domain at the N-terminal, and a transcriptional regulation domain with a bHLH-LZ motif at the C-terminal (Schwab, 2000) . . .
  290. J. Seoane; H. V. Le; J. Massague Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage Nature 419, 729-734 (2002) .
    • . . . MYC mediated transcriptional repression via Miz-1 has been shown for p15INK4B (Staller et al., 2001) and p21CIP1 (Seoane et al., 2002) . . .
  291. S. Shangary; K. Ding; S. Qiu; Z. Nikolovska-Coleska; J. A. Bauer; M. Liu Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer Mol. Cancer Ther. 7, 1533-1542 (2008) .
    • . . . Overall, MDM2-p53 antagonists have been shown to activate the p53 pathway, inducing p53-dependent apoptosis and sensitizing tumor cells to cytotoxic and other molecular targeted therapies whilst inducing a reversible cell cycle arrest in normal cells (reviewed by (Van Maerken et al., 2009a); (Vassilev, 2004; Shangary et al., 2008; Korotchkina et al., 2009; Cheok et al., 2011)) . . .
  292. M. Shapira; E. Kakiashvili; T. Rosenberg; D. D. Hershko The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells Breast Cancer Res. 8, R46 (2006) .
    • . . . In support of this, PI3K inhibitor LY294002 and Rapamycin have been shown to decrease SKP2 expression (Motti et al., 2005; Shapira et al., 2006) . . .
  293. T. Shibahara; T. Onishi; O. E. Franco; K. Arima; Y. Sugimura Down-regulation of Skp2 is correlated with p27-associated cell cycle arrest induced by phenylacetate in human prostate cancer cells Anticancer Res. 25, 1881-1888 (2005) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et . . .
  294. E. H. Shim; L. Johnson; H. L. Noh; Y. J. Kim; H. Sun; C. Zeiss Expression of the F-box protein SKP2 induces hyperplasia, dysplasia, and low-grade carcinoma in the mouse prostate Cancer Res. 63, 1583-1588 (2003) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et . . .
  295. H. Shimada; I. M. Ambros; L. P. Dehner; J. Hata; V. V. Joshi; B. Roald Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee Cancer 86, 349-363 (1999) .
    • . . . This paradox is observed histologically by a high mitosis-karyorrhexis index, a combined index of both proliferation and apoptosis, in both human MYCN amplified neuroblastoma tumors (Shimada et al., 1995, 1999; Goto et al., 2001; Altungoz et al., 2007) and TH-MYCN transgenic mouse neuroblastoma tumors (Moore et al., 2008). . . .
  296. H. Shimada; D. O. Stram; J. Chatten; V. V. Joshi; Y. Hachitanda; G. M. Brodeur Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis J. Natl. Cancer Inst. 87, 1470-1476 (1995) .
    • . . . This paradox is observed histologically by a high mitosis-karyorrhexis index, a combined index of both proliferation and apoptosis, in both human MYCN amplified neuroblastoma tumors (Shimada et al., 1995, 1999; Goto et al., 2001; Altungoz et al., 2007) and TH-MYCN transgenic mouse neuroblastoma tumors (Moore et al., 2008). . . .
  297. A. Shimono; T. Okuda; H. Kondoh N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant Mech. Dev. 83, 39-52 (1999) .
    • . . . NDRG1 and NDRG2 were originally identified as a genes downregulated by MYCN (Shimono et al., 1999; Li and Kretzner, 2003; Zhang et al., 2006, 2008) . . .
  298. M. Shiota; H. Izumi; T. Onitsuka; N. Miyamoto; E. Kashiwagi; A. Kidani Twist and p53 reciprocally regulate target genes via direct interaction Oncogene 27, 5543-5553 (2008) .
    • . . . Moreover, it is found to directly interact with the DBD of p53, thereby inhibiting p53-mediated transactivation of downstream target genes (Shiota et al., 2008) . . .
  299. N. Sidell; H. P. Koeffler Modulation of Mr 53,000 protein with induction of differentiation of human neuroblastoma cells Cancer Res. 48, 2226-2230 (1988) .
    • . . . Despite this, p53 has been reported to accumulate in neuroblastoma, which has been suggested to be due to the embryonic nature of these tumors, reflecting a failure of precursor cells to mature (Sidell and Koeffler, 1988; Davidoff et al., 1992) . . .
  300. S. Signoretti; L. Di Marcotullio; A. Richardson; S. Ramaswamy; B. Isaac; M. Rue Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer J. Clin. Invest. 110, 633-641 (2002) .
    • . . . Overexpression of SKP2 can drive quiescent cells to enter the cell cycle (Sutterluty et al., 1999), and promote adhesion-independent growth of cancer cells (Carrano and Pagano, 2001; Signoretti et al., 2002) . . .
  301. D. Skowyra; K. L. Craig; M. Tyers; S. J. Elledge; J. W. Harper F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex Cell 91, 209-219 (1997) .
    • . . . The invariable core components of SCF complexes are SKP1, CUL1, and RBX1 (Skowyra et al., 1997; Deshaies, 1999; Nakayama and Nakayama, 2005) . . .
    • . . . SKP2 protein is approximately 45 kDa, consisting of an N-terminal F-Box domain which mediates the interaction between SKP2 and SKP1, thereby tethering SKP2 to the SCF complex, and C-terminal leucine-rich repeats (LRR) which enable SKP2 to directly bind to target substrates (Bai et al., 1996; Skowyra et al., 1997; Schulman et al., 2000; Zheng et al., 2002) . . .
  302. A. Slack; Z. Chen; R. Tonelli; M. Pule; L. Hunt; A. Pession The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma Proc. Natl. Acad. Sci. U.S.A. 102, 731-736 (2005) .
    • . . . MDM2 is a direct target gene of MYCN (Slack et al., 2005; Westermann et al., 2008) and non-syntenic co-amplification of MDM2 and MYCN has been reported in neuroblastoma (Corvi et al., 1995) . . .
  303. A. D. Slack; Z. Chen; A. D. Ludwig; J. Hicks; J. M. Shohet MYCN-directed centrosome amplification requires MDM2-mediated suppression of p53 activity in neuroblastoma cells Cancer Res. 67, 2448-2455 (2007) .
    • . . . Consistent with observations that Mdm2 haploinsufficiency inhibits c-MYC induced lymphomagenesis (Alt et al., 2003), MDM2 has been shown to be necessary for MYCN to overcome p53-mediated tumor suppression for MYCN directed centrosome amplification and genomic instability (Slack et al., 2007), and also during MYCN driven neuroblastoma tumorigenesis (Chen et al., 2009; Figure 2) . . .
  304. P. Smart; E. B. Lane; D. P. Lane; C. Midgley; B. Vojtesek; S. Lain Effects on normal fibroblasts and neuroblastoma cells of the activation of the p53 response by the nuclear export inhibitor leptomycin B Oncogene 18, 7378-7386 (1999) .
    • . . . To date several mechanisms for cytoplasmic sequestration of p53 in neuroblastoma have been proposed, including masking of the p53 C-terminal nuclear localization signal (Ostermeyer et al., 1996), hyperactive nuclear export (Stommel et al., 1999), binding to the cytoplasmic anchor, Parc (Nikolaev and Gu, 2003; Nikolaev et al., 2003), aberrant hyperubiquitylation of p53 (Becker et al., 2007), and MDMX and MDM2-mediated cytoplasmic tethering (Ohtsubo et al., 2009) . . .
  305. M. A. Smith; J. M. Maris; R. Gorlick; E. A. Kolb; R. Lock; H. Carol Initial testing of the investigational NEDD8-activating enzyme inhibitor MLN4924 by the pediatric preclinical testing program Pediatr. Blood Cancer 59, 246-253 (2012) .
    • . . . Specifically in the neuroblastoma panel, MLN4924 had a median relative IC50 of 278nM, and induced intermediate activity (EFS T/C values >2) in 1/4 xenografts (Smith et al., 2012) . . .
  306. H. Song; M. Hollstein; Y. Xu p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM Nat. Cell Biol. 9, 573-580 (2007) .
    • . . . Studies using murine models have observed that mutant p53 knock-in mice develop more aggressive and metastatic tumors in comparison to p53 null mice, and that the different p53 mutants are associated with distinct tumor patterns (Lang et al., 2004; Olive et al., 2004; Lozano, 2007; Song et al., 2007; Oren and Rotter, 2010). . . .
  307. M. S. Song; S. J. Song; S. J. Kim; K. Nakayama; K. I. Nakayama; D. S. Lim Skp2 regulates the antiproliferative function of the tumor suppressor RASSF1A via ubiquitin-mediated degradation at the G1-S transition Oncogene 27, 3176-3185 (2008) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et al., 2006), Cyclins D (Yu et al., 1998) and E (Yeh et al., 2001), as well as oncogenes c-MYC (Kim et al., 2003; von der Lehr et al., 2003) and MYB (Charrasse et al., 2000) . . .
  308. T. A. Soucy; P. G. Smith; M. A. Milhollen; A. J. Berger; J. M. Gavin; S. Adhikari An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer Nature 458, 732-736 (2009) .
    • . . . Preclinically, MLN4924 exhibits potent cytotoxicity against a panel of human cancer cell lines, and suppresses tumor growth in mouse xenograft models (Soucy et al., 2009; Lin et al., 2010) . . .
  309. T. Soussi; K. Dehouche; C. Beroud p53 website and analysis of p53 gene mutations in human cancer: forging a link between epidemiology and carcinogenesis Hum. Mutat. 15, 105-113 (2000) .
    • . . . The frequency of p53 mutations varies from 10 to 70% across different cancers types, and are more common in solid tumors compared with hematological malignancies (Calin et al., 1999; Soussi et al., 2000) . . .
  310. D. Speidel; H. Helmbold; W. Deppert Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress Oncogene 25, 940-953 (2006) .
    • . . . p53 is also able to exert functions via protein-protein interactions, and several proteins involved in cell cycle control, DNA repair, gene transcription, and apoptosis have been shown to bind to p53 (Moll et al., 2005; Braithwaite et al., 2006; Speidel et . . .
  311. R. Spitz; A. Oberthuer; M. Zapatka; B. Brors; B. Hero; K. Ernestus Oligonucleotide array-based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome Genes Chromosomes Cancer 45, 1130-1142 (2006) .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et . . .
    • . . . In contrast, the proportion of neuroblastoma tumors with aberrations in the p53/MDM2/p14ARF pathway which are MYCN amplified are lower than in cell lines (Corvi et al., 1995; Gonzalez-Gomez et al., 2003; Su et al., 2004; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010), therefore it is possible that these abnormalities are selected for during the in vitro establishment and/or maintenance of these cell lines . . .
  312. C. Spruck; H. Strohmaier; M. Watson; A. P. Smith; A. Ryan; T. W. Krek A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1 Mol. Cell 7, 639-650 (2001) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et al., 2006), Cyclins D (Yu et al., 1998) and E (Yeh et al., 2001), as well as oncogenes c-MYC (Kim et al., 2003; von der Lehr et al., 2003) and MYB (Charrasse et al., 2000) . . .
  313. P. Staller; K. Peukert; A. Kiermaier; J. Seoane; J. Lukas; H. Karsunky Repression of p15INK4b expression by Myc through association with Miz-1 Nat. Cell Biol. 3, 392-399 (2001) .
    • . . . MYC mediated transcriptional repression via Miz-1 has been shown for p15INK4B (Staller et al., 2001) and p21CIP1 (Seoane et al., 2002) . . .
  314. I. A. Stasinopoulos; Y. Mironchik; A. Raman; F. Wildes; P. Winnard; V. Raman HOXA5-twist interaction alters p53 homeostasis in breast cancer cells J. Biol. Chem. 280, 2294-2299 (2005) .
    • . . . Twist-1 has been shown to directly interact with HOXA5, a potent transactivator of p53 (Stasinopoulos et al., 2005) . . .
  315. S. Stein; E. K. Thomas; B. Herzog; M. D. Westfall; J. V. Rocheleau; R. S. Jackson NDRG1 is necessary for p53-dependent apoptosis J. Biol. Chem. 279, 48930-48940 (2004) .
    • . . . Furthermore, NDRG1 has been found to suppress metastatic cell growth (Stein et al., 2004) . . .
  316. J. M. Stommel; N. D. Marchenko; G. S. Jimenez; U. M. Moll; T. J. Hope; G. M. Wahl A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking EMBO J. 18, 1660-1672 (1999) .
    • . . . To date several mechanisms for cytoplasmic sequestration of p53 in neuroblastoma have been proposed, including masking of the p53 C-terminal nuclear localization signal (Ostermeyer et al., 1996), hyperactive nuclear export (Stommel et al., 1999), binding to the cytoplasmic anchor, Parc (Nikolaev and Gu, 2003; Nikolaev et al., 2003), aberrant hyperubiquitylation of p53 (Becker et al., 2007), and MDMX and MDM2-mediated cytoplasmic tethering (Ohtsubo et al., 2009) . . .
  317. F. J. Stott; S. Bates; M. C. James; B. B. Mcconnell; M. Starborg; S. Brookes The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2 EMBO J. 17, 5001-5014 (1998) .
    • . . . Studies have shown that p14ARF promotes p53 stability and activity by inhibiting MDM2-mediated degradation of p53 via direct interaction with MDM2 and inhibiting its E3 ligase activity (Honda and Yasuda, 1999), preventing MDM2 and p53 nuclear export (Tao and Levine, 1999b), sequestering MDM2 within the nucleolus (Weber et al., 1999), and also by promoting MDM2 degradation (Zhang et al., 1998) . . .
  318. W. T. Su; M. Alaminos; J. Mora; N. K. Cheung; M. P. La Quaglia; W. L. Gerald Positional gene expression analysis identifies 12q overexpression and amplification in a subset of neuroblastomas Cancer Genet. Cytogenet. 154, 131-137 (2004) .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010; Wolf et al., 2010) . . .
    • . . . In contrast, the proportion of neuroblastoma tumors with aberrations in the p53/MDM2/p14ARF pathway which are MYCN amplified are lower than in cell lines (Corvi et al., 1995; Gonzalez-Gomez et al., 2003; Su et al., 2004; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010), therefore it is possible that these abnormalities are selected for during the in vitro establishment and/or maintenance of these cell lines . . .
  319. E. Sugihara; M. Kanai; S. Saito; T. Nitta; H. Toyoshima; K. Nakayama Suppression of centrosome amplification after DNA damage depends on p27 accumulation Cancer Res. 66, 4020-4029 (2006) .
  320. H. Sugiyama; M. Arita; Z. Min; X. Zhong; I. Iwasaki; K. Hirano A novel dysfunctional p53 mutation in the human neuroblastoma cell line TGW Tohoku J. Exp. Med. 201, 229-237 (2003) .
  321. H. Sutterluty; E. Chatelain; A. Marti; C. Wirbelauer; M. Senften; U. Muller p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells Nat. Cell Biol. 1, 207-214 (1999) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et . . .
    • . . . Overexpression of SKP2 can drive quiescent cells to enter the cell cycle (Sutterluty et al., 1999), and promote adhesion-independent growth of cancer cells (Carrano and Pagano, 2001; Signoretti et al., 2002) . . .
  322. A. Swarbrick; S. L. Woods; A. Shaw; A. Balakrishnan; Y. Phua; A. Nguyen miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma Nat. Med. 16, 1134-1140 (2010) .
    • . . . Additionally, p53 has also been reported to induce the expression of miR-192, miR-215, miR-145, and miR-107, of which miR-145 was shown to inhibit c-MYC expression (Braun et al., 2008; Georges et al., 2008; Sachdeva et al., 2009; Yamakuchi et al., 2010) . . .
    • . . . Anti-miR-380-5p treatment restored p53 function in p53 wt neuroblastoma cell lines and inhibited the growth of orthotopically transplanted TH-MYCN tumors (Swarbrick et al., 2010). . . .
    • . . . The restoration of miR-34a and inhibition of miR-380-5p have been shown to reactivate the p53 pathway and inhibit MYCN expression, as well as inhibiting tumor growth in cell lines and orthotopic murine models of neuroblastoma (Wei et al., 2008; Swarbrick et al., 2010; Tivnan et al., 2012) . . .
  323. R. Tanimoto; F. Abarzua; M. Sakaguchi; M. Takaishi; Y. Nasu; H. Kumon REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer Int. J. Mol. Med. 19, 363-368 (2007) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et . . .
  324. W. Tao; A. J. Levine Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53 Proc. Natl. Acad. Sci. U.S.A. 96, 3077-3080 (1999a) .
    • . . . MDM2 directly binds to the N-terminal TAD of p53 to inhibit p53 transcriptional activity (Momand et al., 1992), as well as promoting nuclear export and targeting p53 for ubiquitin mediated proteasome degradation (Honda et al., 1997; Tao and Levine, 1999a). p14ARF is a tumor suppressor and the negative regulator of MDM2 . . .
  325. W. Tao; A. J. Levine P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2 Proc. Natl. Acad. Sci. U.S.A. 96, 6937-6941 (1999b) .
    • . . . Studies have shown that p14ARF promotes p53 stability and activity by inhibiting MDM2-mediated degradation of p53 via direct interaction with MDM2 and inhibiting its E3 ligase activity (Honda and Yasuda, 1999), preventing MDM2 and p53 nuclear export (Tao and Levine, 1999b), sequestering MDM2 within the nucleolus (Weber et al., 1999), and also by promoting MDM2 degradation (Zhang et al., 1998) . . .
  326. V. Tarasov; P. Jung; B. Verdoodt; D. Lodygin; A. Epanchintsev; A. Menssen Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest Cell Cycle 6, 1586-1593 (2007) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
  327. D. Tedesco; J. Lukas; S. I. Reed The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2) Genes Dev. 16, 2946-2957 (2002) .
    • . . . SKP2 protein is approximately 45 kDa, consisting of an N-terminal F-Box domain which mediates the interaction between SKP2 and SKP1, thereby tethering SKP2 to the SCF complex, and C-terminal leucine-rich repeats (LRR) which enable SKP2 to directly bind to target substrates (Bai et al., 1996; Skowyra et al., 1997; Schulman et al., 2000; Zheng et al., 2002) . . .
  328. T. Teitz; T. Wei; D. Liu; V. Valentine; M. Valentine; J. Grenet Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN Oncogene 21, 1848-1858 (2002) .
  329. S. S. Than; K. Kataoka; M. Sakaguchi; H. Murata; F. Abarzua; C. Taketa Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma Oncol. Rep. 25, 989-995 (2011) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et . . .
    • . . . In neuroblastoma, demethylating agents are unlikely to be of therapeutic use, as low DKK3 expression was not shown to be due to promoter methylation (Haug et al., 2011) . . .
  330. P. M. Thompson; T. Gotoh; M. Kok; P. S. White; G. M. Brodeur CHD5, a new member of the chromodomain gene family, is preferentially expressed in the nervous system Oncogene 22, 1002-1011 (2003) .
    • . . . Low levels of CHD5 expression have been found in neuroblastoma cell lines, as well as correlating with MYCN amplification and poor prognosis in neuroblastoma tumors (Thompson et al., 2003; Fujita et al., 2008; Koyama et al., 2012). . . .
  331. P. M. Thompson; J. M. Maris; M. D. Hogarty; R. C. Seeger; C. P. Reynolds; G. M. Brodeur Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma Cancer Res. 61, 679-686 (2001) .
  332. A. Tivnan; W. S. Orr; V. Gubala; R. Nooney; D. E. Williams; C. Mcdonagh Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles PLoS ONE 7, e38129 (2012) .
    • . . . The restoration of miR-34a and inhibition of miR-380-5p have been shown to reactivate the p53 pathway and inhibit MYCN expression, as well as inhibiting tumor growth in cell lines and orthotopic murine models of neuroblastoma (Wei et al., 2008; Swarbrick et al., 2010; Tivnan et al., 2012) . . .
  333. H. Totary-Jain; D. Sanoudou; C. N. Dautriche; H. Schneller; L. Zambrana; A. R. Marks Rapamycin resistance is linked to defective regulation of Skp2 Cancer Res. 72, 1836-1843 (2012) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  334. T. Tsuji; M. Miyazaki; M. Sakaguchi; Y. Inoue; M. Namba A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines Biochem. Biophys. Res. Commun. 268, 20-24 (2000) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
  335. T. Tsuji; I. Nozaki; M. Miyazaki; M. Sakaguchi; H. Pu; Y. Hamazaki Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas Biochem. Biophys. Res. Commun. 289, 257-263 (2001) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et al., 2011; Ueno et al., 2011; Dellinger et al., 2012; Yang et al., 2012) . . .
  336. L. M. Tsvetkov; K. H. Yeh; S. J. Lee; H. Sun; H. Zhang p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27 Curr. Biol. 9, 661-664 (1999) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et . . .
  337. D. A. Tweddle; A. J. Malcolm; N. Bown; A. D. Pearson; J. Lunec Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line Cancer Res. 61, 8-13 (2001a) .
  338. D. A. Tweddle; A. J. Malcolm; M. Cole; A. D. Pearson; J. Lunec p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells Am. J. Pathol. 158, 2067-2077 (2001b) .
  339. D. A. Tweddle; A. D. Pearson; M. Haber; M. D. Norris; C. Xue; C. Flemming The p53 pathway and its inactivation in neuroblastoma Cancer Lett. 197, 93-98 (2003) .
    • . . . The initial good response to chemotherapy may be partly due to the presence of functional p53 at diagnosis, and the development of resistance at relapse may be the result of acquired p53 inactivation at a later stage (reviewed by Tweddle et al., 2003) . . .
    • . . . Analysis of neuroblastoma cell lines reported to date with aberrations in the p53/MDM2/p14ARF pathway demonstrates that 31/40 (78%) of these cell lines are MYCN amplified and predominantly established following previous cytotoxic therapy at relapse (Table 1), when abnormalities of the p53 pathway in neuroblastoma tumors have also been previously reported (reviewed by Tweddle et al., 2003; Carr-Wilkinson et al., 2010) . . .
  340. S. Uddin; M. Ahmed; P. Bavi; R. El-Sayed; N. Al-Sanea; A. Abduljabbar Bortezomib (Velcade) induces p27Kip1 expression through S-phase kinase protein 2 degradation in colorectal cancer Cancer Res. 68, 3379-3388 (2008) .
    • . . . Bortezomib (Velcade™) is the first clinically approved proteasome inhibitor, and has been reported to induce p27KIP1 expression through degradation of SKP2 (Uddin et al., 2008, 2009) . . .
  341. S. Uddin; M. Ahmed; A. R. Hussain; Z. Jehan; F. Al-Dayel; A. Munkarah Bortezomib-mediated expression of p27Kip1 through S-phase kinase protein 2 degradation in epithelial ovarian cancer Lab. Invest. 89, 1115-1127 (2009) .
    • . . . Bortezomib (Velcade™) is the first clinically approved proteasome inhibitor, and has been reported to induce p27KIP1 expression through degradation of SKP2 (Uddin et al., 2008, 2009) . . .
  342. K. Ueno; H. Hirata; S. Majid; Y. Chen; M. S. Zaman; Z. L. Tabatabai Wnt antagonist DICKKOPF-3 (Dkk-3) induces apoptosis in human renal cell carcinoma Mol. Carcinog. 50, 449-457 (2011) .
    • . . . Studies into the mechanisms behind reduced DKK3 expression have revealed histone modification in cancers such as renal cell carcinoma (Ueno et al., 2011), and hypermethylation of the DKK3 promoter in a vast number of human cancers including lung, bladder, breast, and leukemia (reviewed by Veeck and Dahl, 2012) . . .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et . . .
  343. U. Valentiner; C. Haane; N. Nehmann; U. Schumacher Effects of bortezomib on human neuroblastoma cells in vitro and in a metastatic xenograft model Anticancer Res. 29, 1219-1225 (2009) .
    • . . . Of interest, although not directly linked to SKP2, bortezomib is shown to induce apoptosis and inhibit cell growth, migration, angiogenesis, and metastasis both in vitro and in murine models of chemosensitive and chemoresistant neuroblastoma (Brignole et al., 2006; Michaelis et al., 2006; Hamner et al., 2007; Valentiner et al., 2009) . . .
  344. S. Valsesia-Wittmann; M. Magdeleine; S. Dupasquier; E. Garin; A. C. Jallas; V. Combaret Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells Cancer Cell 6, 625-630 (2004) .
    • . . . Twist-1 expression has been shown to be consistently overexpressed in MYCN amplified neuroblastoma tumors and cell lines, correlating with MYCN expression (Valsesia-Wittmann et al., 2004) . . .
    • . . . In support of this, studies in neuroblastoma have observed that MYCN functionally cooperates with Twist-1 or BMI-1 to induce neuroblastoma tumorigenesis, where overexpression of Twist-1 or BMI-1 is necessary for tumor growth both in vitro and in vivo (Valsesia-Wittmann et al., 2004; Cui et al., 2007; Huang et al., 2011b) . . .
  345. I. M. van Leeuwen Cyclotherapy: opening a therapeutic window in cancer treatment Oncotarget 3, 596-600 (2012) .
    • . . . In recent years, there has been increased interest in the use of MDM2-p53 antagonists in a cyclotherapeutic setting to protect normal cells from the harmful effects of chemotherapy (reviewed by Cheok et al., 2011; van Leeuwen, 2012) and also in the identification of natural MDM2 inhibitors (Qin et al., 2012) . . .
  346. T. Van Maerken; L. Ferdinande; J. Taildeman; I. Lambertz; N. Yigit; L. Vercruysse Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53 J. Natl. Cancer Inst. 101, 1562-1574 (2009a) .
    • . . . Overall, MDM2-p53 antagonists have been shown to activate the p53 pathway, inducing p53-dependent apoptosis and sensitizing tumor cells to cytotoxic and other molecular targeted therapies whilst inducing a reversible cell cycle arrest in normal cells (reviewed by (Van Maerken et al., 2009a); (Vassilev, 2004; Shangary et al., 2008; Korotchkina et al., 2009; Cheok et al., 2011)) . . .
    • . . . Evaluation of Nutlin-3 and other MDM2-p53 antagonists in preclinical models of neuroblastoma have reported potent anti-tumor effects such as induction of growth arrest, senescence, differentiation and apoptosis, and inhibition of tumor cell proliferation and metastasis (Barbieri et al., 2006; Van Maerken et al., 2006, 2009a, 2011; Hardcastle et al., 2011; Patterson et al., 2011; Gamble et al., 2012) . . .
    • . . . Moreover, observations that inactivation of the p53/MDM2/p14ARF pathway in relapsed neuroblastoma is predominantly due to lesions upstream of p53 combined with the reported therapeutic efficacy of Nutlin-3 in p53 wt multi-drug-resistant preclinical models of neuroblastoma with metastatic burden (Van Maerken et al., 2009a), highly support reactivation of p53 by inhibiting MDM2 as an attractive treatment option for metastatic relapsed neuroblastoma . . .
  347. T. Van Maerken; J. Vandesompele; A. Rihani; A. De Paepe; F. Speleman Escape from p53-mediated tumor surveillance in neuroblastoma: switching off the p14(ARF)-MDM2-p53 axis Cell Death Differ. 16, 1563-1572 (2009b) .
    • . . . Several lines of evidence from published literature lend support to the notion that during the process of neuroblastoma progression there is evasion of p53-mediated tumor suppression via inactivation of the p53/MDM2/p14ARF pathway (reviewed by Van Maerken et al., 2009b) as well as a requirement for MYCN amplified neuroblastoma to circumvent MYCN driven apoptosis (reviewed by Hogarty, 2003) . . .
  348. T. Van Maerken; A. Rihani; D. Dreidax; S. De Clercq; N. Yigit; J. C. Marine Functional analysis of the p53 pathway in neuroblastoma cells using the small-molecule MDM2 antagonist nutlin-3 Mol. Cancer Ther. 10, 983-993 (2011) .
  349. T. Van Maerken; F. Speleman; J. Vermeulen; I. Lambertz; S. De Clercq; E. De Smet Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma Cancer Res. 66, 9646-9655 (2006) .
  350. L. T. Vassilev Small-molecule antagonists of p53-MDM2 binding: research tools and potential therapeutics Cell Cycle 3, 419-421 (2004) .
    • . . . Overall, MDM2-p53 antagonists have been shown to activate the p53 pathway, inducing p53-dependent apoptosis and sensitizing tumor cells to cytotoxic and other molecular targeted therapies whilst inducing a reversible cell cycle arrest in normal cells (reviewed by (Van Maerken et al., 2009a); (Vassilev, 2004; Shangary et al., 2008; Korotchkina et al., 2009; Cheok et al., 2011)) . . .
  351. L. T. Vassilev; B. T. Vu; B. Graves; D. Carvajal; F. Podlaski; Z. Filipovic In vivo activation of the p53 pathway by small-molecule antagonists of MDM2 Science 303, 844-848 (2004) .
    • . . . Nutlins were the first potent and selective inhibitors of the MDM2-p53 interaction (Vassilev et al., 2004), in particular Nutlin-3 has been extensively evaluated in vitro and in vivo in several types of human cancers and the cis-imidazoline RG7112 is currently in phase I clinical trials3 (NCT00559533 and NCT00623870) . . .
  352. J. Veeck; N. Bektas; A. Hartmann; G. Kristiansen; U. Heindrichs; R. Knuchel Wnt signalling in human breast cancer: expression of the putative Wnt inhibitor Dickkopf-3 (DKK3) is frequently suppressed by promoter hypermethylation in mammary tumours Breast Cancer Res. 10, R82 (2008) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et . . .
  353. J. Veeck; E. Dahl Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3 Biochim. Biophys. Acta 1825, 18-28 (2012) .
    • . . . DKK3 has a molecular weight of 38 kDa, and Cys1 and Cys2 are separated by a linker region of 13 amino acids (reviewed by Veeck and Dahl, 2012). . . .
    • . . . Studies into the mechanisms behind reduced DKK3 expression have revealed histone modification in cancers such as renal cell carcinoma (Ueno et al., 2011), and hypermethylation of the DKK3 promoter in a vast number of human cancers including lung, bladder, breast, and leukemia (reviewed by Veeck and Dahl, 2012) . . .
    • . . . It is however important to mention that mechanisms which mediate resistance to DKK3 induced apoptosis have been identified, such as the overexpression of heat shock protein 70/72, an ER-residing chaperone BiP/GRP78 BiP/GRP78 and BCL2 (Veeck and Dahl, 2012) . . .
    • . . . The use of demethylating agents to re-express DKK3 in cancers where promoter hypermethylation leads to gene silencing have been shown in studies using cell lines of several cancers including gastric, prostate, lung, and leukemia (reviewed by Veeck and Dahl, 2012) . . .
  354. J. Veeck; P. J. Wild; T. Fuchs; P. J. Schuffler; A. Hartmann; R. Knuchel Prognostic relevance of Wnt-inhibitory factor-1 (WIF1) and Dickkopf-3 (DKK3) promoter methylation in human breast cancer BMC Cancer 9, 217 (2009) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
  355. C. S. Verissimo; J. J. Molenaar; C. P. Fitzsimons; E. Vreugdenhil Neuroblastoma therapy: what is in the pipeline? Endocr. Relat. Cancer 18, R213-R231 (2011) .
    • . . . Several miRNAs have been suggested to have prognostic significance and therapeutic potential in neuroblastoma including miR-34a and miR-380-5p (reviewed by Verissimo et al., 2011) . . .
  356. K. Vogan; M. Bernstein; J. M. Leclerc; L. Brisson; J. Brossard; G. M. Brodeur Absence of p53 gene mutations in primary neuroblastomas Cancer Res. 53, 5269-5273 (1993) .
    • . . . In contrast to many other human cancers, p53 mutations in neuroblastoma are rare, occurring in ~3% of cases analyzed to date (Imamura et al., 1993; Komuro et . . .
    • . . . Consistent with this, of the few p53 mutations which have been identified in neuroblastoma to date, the majority were in tumors from patients with progressive or relapsed disease and/or post-chemotherapy (Imamura et al., 1993; Komuro et . . .
  357. N. von der Lehr; S. Johansson; S. Wu; F. Bahram; A. Castell; C. Cetinkaya The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription Mol. Cell 11, 1189-1200 (2003) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et . . .
    • . . . SKP2 has previously been shown to regulate the stability of c-MYC and to be a co-factor for c-MYC mediated transcriptional activation of target genes (Kim et al., 2003; von der Lehr et . . .
  358. K. H. Vousden; X. Lu Live or let die: the cell’s response to p53 Nat. Rev. Cancer 2, 594-604 (2002) .
    • . . . The type of response can be dependent on several factors that are both extrinsic and intrinsic to the cell, such as cell type, cellular environment, oncogenic lesions present in the cell, and also stress type (Jimenez et al., 1999; Vousden and Lu, 2002). . . .
    • . . . Some p53 mutants have been shown to exert a dominant negative effect on wild-type (wt) p53 owing to the requirement for p53 to function as an active tetramer (Vousden and Lu, 2002). p53 mutations identified to date can be found within publically available p53 databases such as The International Agency for Research on Cancer (IARC) TP53 Mutation Database1 and The p53 Website2 . . .
  359. M. Wade; G. M. Wahl Targeting Mdm2 and Mdmx in cancer therapy: better living through medicinal chemistry? Mol. Cancer Res. 7, 1-11 (2009) .
    • . . . It is worth mentioning here that despite structural similarities between MDM2 and MDMX, MDM2-p53 antagonists are largely ineffective against MDMX, and in some cases overexpression of MDMX has been reported to confer resistance to MDM2 antagonists (reviewed by Wade and Wahl, 2009). . . .
  360. L. M. Wagner; M. K. Danks New therapeutic targets for the treatment of high-risk neuroblastoma J. Cell. Biochem. 107, 46-57 (2009) .
    • . . . Retinoic acid is of particular relevance to neuroblastoma, as Isotretinoin (13-cis RA) is routinely used in the treatment of high-risk disease following high-dose chemotherapy and stem cell rescue, in a setting of minimal residual disease (Wagner and Danks, 2009). . . .
  361. B. Wang; Z. Xiao; H. L. Ko; E. C. Ren The p53 response element and transcriptional repression Cell Cycle 9, 870-879 (2010a) .
    • . . . To date several mechanisms for p53-mediated transcriptional repression have been identified (reviewed by Wang et al., 2010a; Bohlig and Rother, 2011) . . .
  362. H. Wang; F. Bauzon; P. Ji; X. Xu; D. Sun; J. Locker Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1± mice Nat. Genet. 42, 83-88 (2010b) .
    • . . . Furthermore, work in SKP2 knockout mice demonstrated that SKP2 is necessary for tumor formation induced by PTEN, p19ARF, or Rb deficiency (Lin et al., 2010; Wang et al., 2010b). . . .
  363. S. Wang; J. F. Raven; A. E. Koromilas STAT1 represses Skp2 gene transcription to promote p27Kip1 stabilization in Ras-transformed cells Mol. Cancer Res. 8, 798-805 (2010c) .
    • . . . In addition, Foxp3 (Zuo et al., 2007), FOXO3A (Wu et al., 2012b) and STAT1 (Wang et al., 2010c) have been identified to transcriptionally repress SKP2 expression. . . .
  364. I. C. Wang; Y. J. Chen; D. Hughes; V. Petrovic; M. L. Major; H. J. Park Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase Mol. Cell. Biol. 25, 10875-10894 (2005) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et . . .
  365. W. Wang; W. Zhu; X. Y. Xu; X. C. Nie; X. Yang; Y. N. Xing The clinicopathological significance of REIC expression in colorectal carcinomas Histol. Histopathol. 27, 735-743 (2012a) .
    • . . . In some cases, reduced DKK3 expression and/or DKK3 promoter methylation has been shown to be associated with poor prognostic clinicopathologic characteristics and outcome (Roman-Gomez et al., 2004; Yue et al., 2008; Veeck et al., 2009; Yu et al., 2009; Yang et al., 2010; Dellinger et al., 2012; Wang et al., 2012a). . . .
  366. X. C. Wang; L. L. Tian; J. Tian; X. Y. Jiang Overexpression of SKP2 promotes the radiation resistance of esophageal squamous cell carcinoma Radiat. Res. 177, 52-58 (2012b) .
    • . . . Finally, in recent years, SKP2 overexpression has been shown to mediate resistance to TRAIL induced apoptosis (Schuler et al., 2011), and radio- and chemoresistance of human cancer cells (Ishii et al., 2004; Davidovich et al., 2008; Chan et al., 2012; Totary-Jain et al., 2012; Wang et al., 2012b). . . .
  367. Z. Wang; H. Fukushima; H. Inuzuka; L. Wan; P. Liu; D. Gao Skp2 is a promising therapeutic target in breast cancer Front. Oncol. 1, 57 (2012c) .
    • . . . Numerous studies to date have provided evidence showing the oncogenic potential of SKP2, and its cross-talk with multiple signaling pathways involved in carcinogenesis such as PI3K/AKT, mTOR, ERK, NFkB, Ras/MAPK, and IGF (Wang et al., 2012c) . . .
    • . . . Overall, elevated SKP2 expression has been shown to correlate with a poor prognostic outcome, tumor size, dedifferentiation, advanced grade, and metastasis (reviewed by Wang et al., 2012c) . . .
  368. X. C. Wang; Y. P. Wu; B. Ye; D. C. Lin; Y. B. Feng; Z. Q. Zhang Suppression of anoikis by SKP2 amplification and overexpression promotes metastasis of esophageal squamous cell carcinoma Mol. Cancer Res. 7, 12-22 (2009) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  369. M. Wanzel; S. Herold; M. Eilers Transcriptional repression by Myc Trends Cell Biol. 13, 146-150 (2003) .
    • . . . Other candidate proteins which have been proposed to recruit MYC to core promoters include TFII-I, NF-Y, YY-1, and SP1 (reviewed by Wanzel et al., 2003; Adhikary and Eilers, 2005). . . .
  370. J. D. Weber; L. J. Taylor; M. F. Roussel; C. J. Sherr; D. Bar-Sagi Nucleolar Arf sequesters Mdm2 and activates p53 Nat. Cell Biol. 1, 20-26 (1999) .
    • . . . Studies have shown that p14ARF promotes p53 stability and activity by inhibiting MDM2-mediated degradation of p53 via direct interaction with MDM2 and inhibiting its E3 ligase activity (Honda and Yasuda, 1999), preventing MDM2 and p53 nuclear export (Tao and Levine, 1999b), sequestering MDM2 within the nucleolus (Weber et al., 1999), and also by promoting MDM2 degradation (Zhang et al., 1998) . . .
  371. J. S. Wei; Y. K. Song; S. Durinck; Q. R. Chen; A. T. Cheuk; P. Tsang The MYCN oncogene is a direct target of miR-34a Oncogene 27, 5204-5213 (2008) .
    • . . . The 3′-UTR of MYCN has been identified as a direct target of miR-34a (Wei et al., 2008), a miRNA which is directly upregulated by p53 and mediates several tumor suppressive functions of p53 (reviewed by Hermeking, 2007) . . .
    • . . . The restoration of miR-34a and inhibition of miR-380-5p have been shown to reactivate the p53 pathway and inhibit MYCN expression, as well as inhibiting tumor growth in cell lines and orthotopic murine models of neuroblastoma (Wei et al., 2008; Swarbrick et al., 2010; Tivnan et al., 2012) . . .
  372. W. Wei; N. G. Ayad; Y. Wan; G. J. Zhang; M. W. Kirschner; W. G. Kaelin Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex Nature 428, 194-198 (2004) .
    • . . . Levels of SKP2 are low in G0/G1 and late M/early G1, increase during G1/S transition and reach a maximum in S phase (Wirbelauer et al., 2000; Bashir et al., 2004; Wei et . . .
    • . . . The E3 ubiquitin ligase APCCdh1 complex mediates the ubiquitination and subsequent degradation of SKP2, primarily in early G1 (Bashir et al., 2004; Wei et . . .
  373. W. A. Weiss; K. Aldape; G. Mohapatra; B. G. Feuerstein; J. M. Bishop Targeted expression of MYCN causes neuroblastoma in transgenic mice EMBO J. 16, 2985-2995 (1997) .
    • . . . MYCN is a proto-oncogene directly involved in neuroblastoma tumorigenesis, evident by the spontaneous development of neuroblastoma in a MYCN dose-dependent manner in transgenic murine models (Weiss et al., 1997). . . .
  374. C. Welch; Y. Chen; R. L. Stallings MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells Oncogene 26, 5017-5022 (2007) .
    • . . . In recent years, p53 has also been shown to regulate the transcriptional expression and maturation of miRNAs, a class of endogenously expressed small (~18–25 nt) non-coding RNA molecules involved in post-transcriptional regulation of gene expression (Lujambio and Lowe, 2012). p53 has been found to upregulate the expression of the miR-34 cluster which is reported to mediate several tumor suppressive functions of p53 including senescence, cell cycle arrest, and apoptosis (Bommer et al., 2007; Chang et . . .
    • . . . Studies have shown that miR-34a plays a role in inhibiting cellular proliferation and inducing apoptosis in neuroblastoma cells (Welch et al., 2007; Cole et al., 2008) . . .
  375. F. Westermann; K. O. Henrich; J. S. Wei; W. Lutz; M. Fischer; R. Konig High Skp2 expression characterizes high-risk neuroblastomas independent of MYCN status Clin. Cancer Res. 13, 4695-4703 (2007) .
  376. F. Westermann; D. Muth; A. Benner; T. Bauer; K. O. Henrich; A. Oberthur Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas Genome Biol. 9, R150 (2008) .
    • . . . More recent studies have reported significant overlap between c-MYC and MYCN-regulated gene sets (Laurenti et al., 2008; Westermann et . . .
    • . . . Interestingly, a ChIP-chip array study of MYCN/c-MYC target genes in neuroblastoma demonstrated that distinct MYCN/c-MYC target gene expression was associated with overall survival, and independent of well-established markers such as MYCN amplification, disease stage, and age at diagnosis (Westermann et al., 2008). . . .
    • . . . Initial studies in neuroblastoma showed that MYCN amplified tumors expressed significantly higher levels of p53 mRNA in comparison with non-amplified tumors (Raschella et al., 1991; Berwanger et al., 2002; Westermann et al., 2008), and higher p53 protein expression in the presence of ectopic MYCN in cell lines (Cui et al., 2005; Bell et al., 2006; Sugihara et al., 2006) . . .
    • . . . Certainly, a study analyzing MYCN/c-MYC target gene expression and outcome in neuroblastoma has previously suggested p53 as a strong candidate involved in spontaneous regression of 4s tumors (Westermann et al., 2008). . . .
    • . . . MDM2 is a direct target gene of MYCN (Slack et al., 2005; Westermann et al., 2008) and non-syntenic co-amplification of MDM2 and MYCN has been reported in neuroblastoma (Corvi et al., 1995) . . .
    • . . . To date, studies into Wnt signaling in neuroblastoma have shown that siRNA mediated inhibition of the Wnt1/β-catenin pathway leads to apoptosis of SHSY5Y cells (Zhang et al., 2009), and deregulated canonical and non-canonical Wnt signaling in chemoresistant and high-risk disease (Blanc et al., 2005; Liu et al., 2008b; Flahaut et al., 2009) . . .
  377. C. Wirbelauer; H. Sutterluty; M. Blondel; M. Gstaiger; M. Peter; F. Reymond The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts EMBO J. 19, 5362-5375 (2000) .
    • . . . Levels of SKP2 are low in G0/G1 and late M/early G1, increase during G1/S transition and reach a maximum in S phase (Wirbelauer et al., 2000; Bashir et al., 2004; Wei et al., 2004) . . .
  378. M. Wolf; M. Korja; R. Karhu; H. Edgren; S. Kilpinen; K. Ojala Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication BMC Cancer 10, 181 (2010) .
    • . . . In addition to p53 mutations, MDM2 amplification, and p14ARF deletion or methylation have also been reported in neuroblastoma tumors and cell lines, most of which were from patients with progressive or relapsed disease and/or post-chemotherapy (Corvi et al., 1995; Omura-Minamisawa et al., 2001; Thompson et al., 2001; Gonzalez-Gomez et al., 2003; Su et al., 2004; Carr et al., 2006; Spitz et al., 2006; Caren et al., 2008; Carr-Wilkinson et al., 2010; Wolf et . . .
  379. C. W. Woo; F. Tan; H. Cassano; J. Lee; K. C. Lee; C. J. Thiele Use of RNA interference to elucidate the effect of MYCN on cell cycle in neuroblastoma Pediatr. Blood Cancer 50, 208-212 (2008) .
    • . . . The precise mechanism of MYCN mediated upregulation of SKP2 remains unclear, as SKP2 is a direct target gene of E2F (Zhang and Wang, 2006), E2F is a direct target of c-MYC (Fernandez et al., 2003) and higher E2F expression levels are observed in the presence of MYCN (Mac et al., 2000; Woo et al., 2008) . . .
  380. C. Y. Wu; J. J. Hung; K. J. Wu Linkage between Twist1 and Bmi1: molecular mechanism of cancer metastasis/stemness and clinical implications Clin. Exp. Pharmacol. Physiol. 39, 668-673 (2012a) .
    • . . . Twist-1 which belongs to the bHLH transcription factor family, and BMI-1, a polycomb ring finger oncogene, are both overexpressed in several human cancers, and have been shown to be involved in epithelial-mesenchymal transition and cancer stemness which have clinical implications of cancer metastasis, drug resistance, and survival (Wu et al., 2012a) . . .
    • . . . Twist-1 and BMI-1 are involved in inactivation of the p53 pathway and are overexpressed in several cancers including neuroblastoma, often correlating with aggressive disease, and poor prognosis (reviewed by Wu et al., 2012a) . . .
  381. J. Wu; S. W. Lee; X. Zhang; F. Han; S. Y. Kwan; X. Yuan Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex Oncogene , (2012b) .
    • . . . In addition, Foxp3 (Zuo et al., 2007), FOXO3A (Wu et al., 2012b) and STAT1 (Wang et al., 2010c) have been identified to transcriptionally repress SKP2 expression. . . .
  382. J. Wu; L. T. Smith; C. Plass; T. H. Huang ChIP-chip comes of age for genome-wide functional analysis Cancer Res. 66, 6899-6902 (2006) .
    • . . . Direct targets may be further confirmed using quantitative PCR based ChIP analysis (Wu et al., 2006) . . .
  383. J. Xiao; S. Yin; Y. Li; S. Xie; D. Nie; L. Ma SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells Acta Biochim. Biophys. Sin. (Shanghai) 41, 699-708 (2009) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et al., 2003; Jiang et al., 2005; Lee and McCormick, 2005; Shibahara et al., 2005; Katagiri et al., 2006; Kitagawa et al., 2008; Xiao et al., 2009; Chan et al., 2010a; Bretones et al., 2011) . . .
  384. S. Xiong; V. Pant; Y. A. Suh; C. S. Van Pelt; Y. Wang; Y. A. Valentin-Vega Spontaneous tumorigenesis in mice overexpressing the p53-negative regulator Mdm4 Cancer Res. 70, 7148-7154 (2010) .
    • . . . Similar to p53 null mice, mice deficient in p14ARF, or overexpressing MDM2 or MDMX also developed spontaneous tumors, albeit at a slower rate (Jones et al., 1998; Kamijo et al., 1999; Xiong et al., 2010) . . .
  385. Y. Xu Induction of genetic instability by gain-of-function p53 cancer mutants Oncogene 27, 3501-3507 (2008) .
    • . . . These new gain-of-function mutants have been reported to play a role in promoting tumorigenesis including increased metastasis and genomic instability, and resistance to anti-cancer therapies (reviewed by Xu, 2008; Brosh and Rotter, 2009; Oren and Rotter, 2010; Hanel and Moll, 2012) . . .
  386. C. Xue; M. Haber; C. Flemming; G. M. Marshall; R. B. Lock; K. L. Mackenzie p53 determines multidrug sensitivity of childhood neuroblastoma Cancer Res. 67, 10351-10360 (2007) .
    • . . . This is consistent with studies which have shown a decrease in p53 expression following retinoic acid induced in vitro differentiation of neuroblastoma cell lines (Sidell and Koeffler, 1988; Davidoff et al., 1992; Chen et al., 2007), and also during neuronal development/differentiation (Eizenberg et al., 1996; Ferreira and Kosik, 1996) . . .
  387. M. Yamakuchi; C. D. Lotterman; C. Bao; R. H. Hruban; B. Karim; J. T. Mendell P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis Proc. Natl. Acad. Sci. U.S.A. 107, 6334-6339 (2010) .
    • . . . Additionally, p53 has also been reported to induce the expression of miR-192, miR-215, miR-145, and miR-107, of which miR-145 was shown to inhibit c-MYC expression (Braun et al., 2008; Georges et al., 2008; Sachdeva et al., 2009; Yamakuchi et al., 2010) . . .
  388. B. Yang; Z. Du; Y. T. Gao; C. Lou; S. G. Zhang; T. Bai Methylation of Dickkopf-3 as a prognostic factor in cirrhosis-related hepatocellular carcinoma World J. Gastroenterol. 16, 755-763 (2010) .
    • . . . In some cases, reduced DKK3 expression and/or DKK3 promoter methylation has been shown to be associated with poor prognostic clinicopathologic characteristics and outcome (Roman-Gomez et al., 2004; Yue et al., 2008; Veeck et al., 2009; Yu et al., 2009; Yang et al., 2010; Dellinger et al., 2012; Wang et al., 2012a). . . .
  389. Z. R. Yang; W. G. Dong; X. F. Lei; M. Liu; Q. S. Liu Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer World J. Gastroenterol. 18, 1590-1601 (2012) .
    • . . . Overexpression of DKK3 has been shown to mediate potent anti-tumor effects including reduced cell proliferation, anchorage-independent growth, and invasion and metastasis, and induced cancer cell specific apoptosis both in vitro and in murine tumor models (Tsuji et al., 2001; Abarzua et al., 2005; Edamura et al., 2007; Tanimoto et al., 2007; Koppen et al., 2008; Mizobuchi et al., 2008; Kawasaki et al., 2009; Gu et al., 2011; Than et al., 2011; Ueno et al., 2011; Dellinger et al., 2012; Yang et . . .
  390. K. H. Yeh; T. Kondo; J. Zheng; L. M. Tsvetkov; J. Blair; H. Zhang The F-box protein SKP2 binds to the phosphorylated threonine 380 in cyclin E and regulates ubiquitin-dependent degradation of cyclin E Biochem. Biophys. Res. Commun. 281, 884-890 (2001) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et al., 2006), Cyclins D (Yu et al., 1998) and E (Yeh et al., 2001), as well as oncogenes c-MYC (Kim et al., 2003; von der Lehr et al., 2003) and MYB (Charrasse et al., 2000) . . .
  391. S. Yokoi; K. Yasui; T. Iizasa; T. Takahashi; T. Fujisawa; J. Inazawa Down-regulation of SKP2 induces apoptosis in lung-cancer cells Cancer Sci. 94, 344-349 (2003) .
    • . . . Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al., 2003; Yokoi et . . .
  392. S. Yokoi; K. Yasui; M. Mori; T. Iizasa; T. Fujisawa; J. Inazawa Amplification and overexpression of SKP2 are associated with metastasis of non-small-cell lung cancers to lymph nodes Am. J. Pathol. 165, 175-180 (2004) .
    • . . . In addition, amplification of SKP2 at chromosome 5p13 has been reported (Yokoi et al., 2004; Saigusa et al., 2005; Wang et al., 2009; Rose et al., 2011; Li et al., 2012a), and tends to be observed in metastatic tumors whereas overexpression of SKP2 is reported in early cancers (Hershko, 2008) . . .
  393. J. Yu; Q. Tao; Y. Y. Cheng; K. Y. Lee; S. S. Ng; K. F. Cheung Promoter methylation of the Wnt/beta-catenin signaling antagonist Dkk-3 is associated with poor survival in gastric cancer Cancer 115, 49-60 (2009) .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et al., 2008; Yue et al., 2008; Yu et al., 2009; Dellinger et al., 2012) . . .
  394. Z. K. Yu; J. L. Gervais; H. Zhang Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins Proc. Natl. Acad. Sci. U.S.A. 95, 11324-11329 (1998) .
    • . . . Since its discovery, SKP2 has been found to target numerous proteins for ubiquitination and subsequent degradation via the 26S proteasome pathway, including CDK inhibitors p21CIP1 (Yu et al., 1998; Bornstein et al., 2003), p27KIP1 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999), and p57KIP2 (Kamura et al., 2003), Rb family member p130 (Tedesco et al., 2002), apoptosis regulator FOXO1, tumor suppressors BRCA2 (Moro et al., 2006), RASSF1A (Song et al., 2008), and TOB1 (Hiramatsu et al., 2006), Cyclins D (Yu et . . .
  395. Y. Yuan; Y. M. Liao; C. T. Hsueh; H. R. Mirshahidi Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP J. Hematol. Oncol. 4, 16 (2011) .
    • . . . This class includes cis-imidazolines (e.g., Nutlins), spiro-oxindoles (MI compounds), benzodiazepinediones, isoindolinones, isoquinolinones, and thiophenes (RITA; reviewed by (Yuan et al., 2011); (Hardcastle et . . .
  396. W. Yue; Q. Sun; S. Dacic; R. J. Landreneau; J. M. Siegfried; J. Yu Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer Carcinogenesis 29, 84-92 (2008) .
    • . . . In contrast, others have however confirmed DKK3 as an inhibitor of the canonical pathway (Yue et al., 2008; Lee et al., 2009; Dellinger et al., 2012). . . .
    • . . . Originally identified as a novel TSG by Tsuji et al. (2000) using an in vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia (Tsuji et al., 2000; Nozaki et al., 2001; Hsieh et al., 2004; Kurose et al., 2004; Roman-Gomez et al., 2004; Abarzua et al., 2005; Tanimoto et al., 2007; Mizobuchi et al., 2008; Veeck et . . .
  397. Y. Yung; J. L. Walker; J. M. Roberts; R. K. Assoian A Skp2 autoinduction loop and restriction point control J. Cell Biol. 178, 741-747 (2007) .
    • . . . The identification of SKP2 as a direct target gene of E2F (Zhang and Wang, 2006) is of particular importance as Yung et al. (2007) subsequently described the SKP2 autoinduction loop comprising pRB-E2F, SKP2, p27KIP1, and Cyclin E-CDK2, in which SKP2 expression initiates proteolysis of p27KIP1, activation of Cyclin E-CDK2 which feeds back to sustain pRB inactivation, E2F release, and further induction of the SKP2 gene . . .
  398. K. I. Zeller; A. G. Jegga; B. J. Aronow; K. A. O’Donnell; C. V. Dang An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets Genome Biol. 4, R69 (2003) .
    • . . . A vast number of c-MYC target genes have been identified and can be found at http://myccancergene.org/site/mycTargetDB.asp (Zeller et al., 2003), however less is known about the target genes of MYCN . . .
    • . . . p53 has long been known to be a direct target gene of c-MYC, and mediate c-MYC induced apoptosis (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994; Zeller et al., 2003) . . .
  399. H. Zhang; R. Kobayashi; K. Galaktionov; D. Beach p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase Cell 82, 915-925 (1995) .
    • . . . SKP2 was originally discovered as a protein associated with Cyclin A-CDK2, and subsequently shown to play a key role in promoting cell cycle progression, in particular at the G1/S transition (Zhang et al., 1995) . . .
  400. J. Zhang; S. Chen; W. Zhang; J. Zhang; X. Liu; H. Shi Human differentiation-related gene NDRG1 is a Myc downstream-regulated gene that is repressed by Myc on the core promoter region Gene 417, 5-12 (2008) .
    • . . . NDRG1 and NDRG2 were originally identified as a genes downregulated by MYCN (Shimono et al., 1999; Li and Kretzner, 2003; Zhang et al., 2006, 2008) . . .
  401. J. Zhang; F. Li; X. Liu; L. Shen; J. Liu; J. Su The repression of human differentiation-related gene NDRG2 expression by Myc via Miz-1-dependent interaction with the NDRG2 core promoter J. Biol. Chem. 281, 39159-39168 (2006) .
    • . . . NDRG1 and NDRG2 were originally identified as a genes downregulated by MYCN (Shimono et al., 1999; Li and Kretzner, 2003; Zhang et al., 2006, 2008) . . .
  402. L. Zhang; K. Li; Z. Lv; X. Xiao; J. Zheng The effect on cell growth by Wnt1 RNAi in human neuroblastoma SH-SY5Y cell line Pediatr. Surg. Int. 25, 1065-1071 (2009) .
    • . . . To date, studies into Wnt signaling in neuroblastoma have shown that siRNA mediated inhibition of the Wnt1/β-catenin pathway leads to apoptosis of SHSY5Y cells (Zhang et al., 2009), and deregulated canonical and non-canonical Wnt signaling in chemoresistant and high-risk disease (Blanc et al., 2005; Liu et al., 2008b; Flahaut et . . .
  403. L. Zhang; C. Wang F-box protein Skp2: a novel transcriptional target of E2F Oncogene 25, 2615-2627 (2006) .
    • . . . Studies to date have revealed that several transcription factors act directly via the SKP2 promoter to upregulate SKP2 gene expression, such as E2F1 (Zhang and Wang, 2006), NFkB (Schneider et al., 2006), SP1 (Appleman et al., 2006), CBF1 (Sarmento et al., 2005), GABP (Imaki et al., 2003), FOXM1 (Wang et al., 2005), c-MYC (Bretones et al., 2011), STAT3 (Huang et al., 2012), and NOR1 (Gizard et al., 2011) . . .
    • . . . The identification of SKP2 as a direct target gene of E2F (Zhang and Wang, 2006) is of particular importance as Yung et al. (2007) subsequently described the SKP2 autoinduction loop comprising pRB-E2F, SKP2, p27KIP1, and Cyclin E-CDK2, in which SKP2 expression initiates proteolysis of p27KIP1, activation of Cyclin E-CDK2 which feeds back to sustain pRB inactivation, E2F release, and further induction of the SKP2 gene . . .
    • . . . The precise mechanism of MYCN mediated upregulation of SKP2 remains unclear, as SKP2 is a direct target gene of E2F (Zhang and Wang, 2006), E2F is a direct target of c-MYC (Fernandez et al., 2003) and higher E2F expression levels are observed in the presence of MYCN (Mac et al., 2000; Woo et al., 2008) . . .
  404. Y. Zhang; Y. Xiong; W. G. Yarbrough ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways Cell 92, 725-734 (1998) .
    • . . . Studies have shown that p14ARF promotes p53 stability and activity by inhibiting MDM2-mediated degradation of p53 via direct interaction with MDM2 and inhibiting its E3 ligase activity (Honda and Yasuda, 1999), preventing MDM2 and p53 nuclear export (Tao and Levine, 1999b), sequestering MDM2 within the nucleolus (Weber et al., 1999), and also by promoting MDM2 degradation (Zhang et al., 1998) . . .
  405. N. Zheng; B. A. Schulman; L. Song; J. J. Miller; P. D. Jeffrey; P. Wang Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex Nature 416, 703-709 (2002) .
    • . . . SKP2 protein is approximately 45 kDa, consisting of an N-terminal F-Box domain which mediates the interaction between SKP2 and SKP1, thereby tethering SKP2 to the SCF complex, and C-terminal leucine-rich repeats (LRR) which enable SKP2 to directly bind to target substrates (Bai et al., 1996; Skowyra et al., 1997; Schulman et al., 2000; Zheng et al., 2002) . . .
  406. F. Zindy; C. M. Eischen; D. H. Randle; T. Kamijo; J. L. Cleveland; C. J. Sherr Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization Genes Dev. 12, 2424-2433 (1998) .
    • . . . In addition to direct transcriptional upregulation of p53, c-MYC has been previously reported to sensitize cells to increased apoptosis via induction of p14ARF mediated upregulation of p53 expression, stability, and activity (Zindy et al., 1998) . . .
  407. T. Zuo; R. Liu; H. Zhang; X. Chang; Y. Liu; L. Wang FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2 J. Clin. Invest. 117, 3765-3773 (2007) .
    • . . . In addition, Foxp3 (Zuo et al., 2007), FOXO3A (Wu et al., 2012b) and STAT1 (Wang et al., 2010c) have been identified to transcriptionally repress SKP2 expression. . . .
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