Ploidy

According to their DNA index, neuroblastomas can be divided into a group with a near-diploid nuclear DNA content (about 45% of neuroblastomas) and those with a near-triploid DNA content (about 55%). DNA index is a prognostic marker for patients younger than 2 years who have disseminated disease . This genetic subtype of tumor is frequently seen in patients of less than 1 year of age, where tumors are localized and have a good prognosis .

It was suggested that a near-triploid DNA content, which is found more often in localized or metastatic neuroblastomas, was because these tumors have a fundamental defect in mitosis leading to gains or losses of whole chromosomes. On the other hand, locoregional or metastatic tumors with a near-diploid DNA content have a fundamental defect in genome stability leading to chromosomal rearrangements such as unbalanced translocations .

Many neuroblastoma tumors display DNA diploid status and bear partial gains, losses, amplification, or other structural chromosome aberrations. Recurrent structural chromosomal alterations commonly associated with advanced stage of disease and poor outcome in neuroblastoma include MYCN amplification, deletion of chromosome arms 1p, 3p, 4p, and 11q, and gain of chromosome arm 17q .

MYCN

MYCN oncogene plays a major role in neuroblastoma tumorigenesis and defines an aggressive subset of tumors. MYCN oncogene (located on chromosome 2p24; ‘N′ stands for neuroblastoma-derived) was found to be amplified in 20%–25% of neuroblastomas, and is usually present in the form of double-minutes (chromosome fragments) or homogeneously staining regions . MYCN gene encodes a transcription factor that forms heterodimers with the MAX protein . Evidence for a direct involvement in the development of neuroblastoma was obtained through the construction of a mouse neuroblastoma model in which a human MYCN cDNA was placed under the control of a tyrosine hydroxylase promoter, leading to the development of this tumor . So, overexpression of MYCN alone was sufficient to initiate neuroblastoma formation in mice.

This transcription factor both activates and represses genetic targets (e.g., mRNA, miRNAs, lncRNAs) through direct DNA binding as well as indirect protein/protein interaction mechanisms . Both MYCN and MYCC (C-MYC) have well-described anti-p53, pro-proliferative functions, and pro-epithelial-mesenchymal transition (EMT) functions . During normal embryogenesis and neural crest development, MYCN is transiently expressed in the ventral-lateral migrating crest cells destined to become sympathetic ganglia . Thus, it is not surprising to find high levels of MYCN in a subset of poorly differentiated aggressive neuroblastomas . Numerous studies focusing on identifying the signaling pathways influenced by MYCN have established that high level of it enhances the expression of several genes involved in cell proliferation, and also represses expression of differentiation- and apoptosis-related genes either in a direct or indirect fashion . Targets directly induced by MYCN include the high mobility group A ( HMGA1 ) , the minichromosome maintenance complex component 7 , the Mdm2-p53 binding protein homolog ( MDM2 ) , p53 , and the multidrug resistance-associated protein ( MRP1 ) .

Despite this, MYCN status cannot predict all cases of poor survival in neuroblastoma, and 80% of neuroblastomas do not display MYCN amplification . Interestingly, high MYCN target gene expression is not restricted to MYCN amplified neuroblastomas but is also apparent in high-stage MYCN non-amplified tumors, indicating that common pathways are altered in high-stage tumors . A functional 157-gene signature in neuroblastoma consisting of relevant genes that are regulated by MYCN and predictive of outcome was revealed. Interestingly, a subgroup of the tumors displaying this signature and the poor outcome did not have MYCN amplification or high MYCN mRNA levels, but high nuclear MYCN protein levels . It suggests that the aggressive phenotype of MYCN might not only be associated with MYCN c opy numbers, but with other signals that regulate MYCN expressions, such as RNA binding proteins and microRNAs. However, many high-risk cases have a minimal MYCN expression, suggesting additional mechanisms for tumorigenesis independent of MYCN deregulation .

ALK

Familial neuroblastoma is a rare event, as it only accounts for 1%–2% of cases. Inheritance seems to follow an autosomal dominant pattern with incomplete penetrance . Inherited mutations in the homeodomain transcription factor, paired-like homeobox 2B ( PHOX2B ) and the anaplastic lymphoma kinase ( ALK ), have been reported to predispose to familial neuroblastoma .

Activating mutations of ALK are also implicated as oncogenic drivers of neuroblastoma . Mutations are found in almost all cases of familial neuroblastoma (<1% of total neuroblastoma cases) and between 6% and 10% of spontaneous cases . DNA amplification and protein overexpression, as well as activating point mutations of ALK , have been described in neuroblastomas . ALK is linked to sympathetic neuron development and survival of migratory neural crest cells . This gene is an important regulator of stem cell functions, including STAT3 dependent self-renewal, and as a transcriptional target of MYCN, high expression predicts poor outcome .

A recent study demonstrated that ALK F1174L, which is the most frequent and aggressive ALK mutation, was sufficient to promote neuroblastoma development in mice. In addition, when ALK F1174L and MYCN were coexpressed, a synergic effect was displayed in tumor development. Interestingly, these tumors had minimal chromosomal aberrations, suggesting that these two genes were sufficient to drive neuroblastoma formation .

PHOX2B

Germ line mutations of PHOX2B are found in a subset of familial neuroblastoma and about 4% of sporadic cases . PHOX2B and PHOX2A drive differentiation of neural crest precursors toward sympathetic neurons . Missense or frame-shift mutations in the homeodomain of PHOX2B were described in a rare subset of neuroblastomas with congenital central hypoventilation syndrome (CCHS). Recently, PHOX2B loss-of-function mutations have been shown to block neuroblastoma differentiation by disrupting calcium regulation . PHOX2B may also inhibit ALK expression in neuroblastoma ; although PHOX2B could give selective advantages to tumor cells, it is likely not sufficient to drive neuroblastoma pathogenesis.

Chromosome 1p Loss

Deletion of the short arm of chromosome 1 has been detected in approximately 25%–35% of primary neuroblastomas . This aberration is frequently associated with amplification of MYCN , is found in approximately 70% of aggressive neuroblastomas , and it has been reported that 1p LOH is independently associated with poor outcome in patients with localized tumors .

This finding suggests the presence of one or more tumor suppressor gene(s) in this chromosome region. In the past, many attempts were made to delineate the shortest region of deletion on 1p36 . It is currently defined to 1p36.31 (a region of approximately 2 Mb) . One of the most promising candidate neuroblastoma suppressor genes in this region is Chromodomain Helicase DNA binding protein 5 ( CHD5 ). This gene is a member of the chromatin remodeling family and is expressed mostly in the nervous system . The mouse Chd5 gene was found to control proliferation, senescence, and apoptosis through the p19Arf-p53 pathway and was therefore validated as a mouse tumor suppressor gene . Fujita et al. found that human CHD5 expression was low in neuroblastoma cell lines and in tumors harboring a chromosome 1p deletion. In addition, their data strongly suggested that inactivation of the second allele of CHD5 in neuroblastoma occurs by means of epigenetic silencing by CHD5 promoter hypermethylation. Based on the positive correlation between 1p LOH and MYCN amplification found in neuroblastoma tumors, the authors suggested that CHD5 promoter methylation could be an MYCN -mediated effect .

Recently, CAMTA1, a transcription factor mapping to 1p36, was also identified as a tumor suppressor gene in neuroblastoma . Expression of CAMTA1 induces the transcription of genes involved in neuronal differentiation and inhibits genes related to cell proliferation. In addition, subcutaneous inoculation of athymic nude mice with CAMTA1-inducible neuroblastoma cells resulted in a significant reduction of the tumors, demonstrating a role for CAMTA1 as a tumor suppressor in an in vivo model .

The zinc-finger transcription factor CASZ1, located on 1p36.22, has also been suggested to play a role in cell differentiation. Consistently, restoration of CASZ1 in neuroblastoma cell lines induced the cell differentiation, enhanced cell adhesion, and suppressed cell growth . Subsequent studies demonstrated that silencing of the second allele of CASZ1 was mediated by the aberrant upregulation of the polycomb protein histone methyltransferase EZH2 (enhancer of zeste homolog 2), which regulates differentiation in many tissues .

Chromosome 17q Gain

The gain of chromosome 17q is the most common genetic aberration, occurring in approximately 80% of neuroblastomas . Frequently, this aberration is caused by unbalanced translocations of segment 17q21-qter and the distal part of chromosomes 1p or 11q , although other chromosomes can also be involved in 17q gains . The involvement of chromosome 1 and chromosome 17 in the etiology of neuroblastoma was further reinforced by the identification of a constitutionally balanced t(1; 17) translocation in a patient with neuroblastoma . The chromosome 17q breakpoints are not confined to one cytogenetic band but are scattered throughout the long arm of chromosome 17 . Based on these findings, the presence of one or more dosage-sensitive genes on 17q implicated in neuroblastoma seems the most plausible hypothesis. Near-triploid tumors have often gained an extra whole chromosome 17, whereas unbalanced 17q gain is characteristic for near-diploid tumors.

Chromosome 17q gain was found to be the parameter for poor outcome, whereas whole chromosome 17 gain was associated with good prognosis . Numerous studies have reported that 17q gain is significantly associated with advanced stage of disease, increased patient age, 1p LOH, 11q LOH, and MYCN amplification . However, the independence of 17q gain as a prognostic factor is controversial. Whether an independent prognostic factor or merely a modifying factor, identifying the gene aberrations caused by 17q unbalance will be crucial for fully understanding the neuroblastoma progression.

Chromosome 11q Loss

Another common structural chromosome aberration associated with aggressive clinical behavior is 11q LOH, occurring in approximately 40%–45% of neuroblastoma cases . Although inversely correlated with MYCN amplification, a small subset of tumors display both an 11q LOH and MYCN amplification . Numerous studies suggest that MYCN amplified and 11q LOH represent two distinct subtypes of neuroblastoma tumors, both of which can be associated with poor clinical outcome . As a result, in 2009, aberrations of chromosome 11q were included in the INRG classification system .

Therefore, identifying genes on the 11q chromosome that contributes to neuroblastoma aggressiveness is crucial for understanding the pathways deregulated in these tumors. However, in spite of the intensive effort, only a few genes have been identified to date. In tumors with 11q LOH, the frequency of segmental aberrations is significantly higher than in MYCN amplified tumors . This fact was explained in part by the loss of the H2AFX gene, located in the 11q23.3 deleted region. This gene has been shown to play a role in genomic stability modification, and enhanced susceptibility to cancer in mice .

Other studies identified the cell adhesion molecule 1 ( CADM1 ), which transcribes a cellular adhesion protein involved in neural cell development, as a candidate tumor suppressor gene in the 11q23 deleted region. CADM1 was significantly downregulated in tumors with 11q LOH relative to 11q diploid tumors and significantly associated with the advanced stage of disease and poor survival . Overexpression of CADM1 revealed a significant inhibition of cell proliferation and colony forming ability in a panel of four different cell lines, demonstrating that CADM1 expression attenuates the malignant phenotype in cultured cells. No evidence for inactivating mutations of CADM1 or hypermethylation of its promoter was found, suggesting that other mechanisms such as haplosufficiency or posttranscriptional regulators of gene expression may be involved in the modulation of CADM1 expression .

p53/MDM2 Interaction

p53 is a tumor suppressor protein that plays a major role in the protection of genomic stability and prevention of tumor development by directly activating several genes, including miRNAs, which promote DNA repair, cell cycle arrest, and apoptosis. Inactivating mutations or deletions in the p53 gene are found in >50% of adult human cancers . However, in neuroblastoma, this gene is seldom mutated, occurring in <2% of cases at diagnosis, and ∼15% at relapse .

Unlike many adult malignancies, most neuroblastomas do initially respond to chemotherapy, and it is likely that the presence of functionally active p53 is at least partly involved in this initial chemosensitivity. However, over half of previously responsive tumors eventually relapses with the chemoresistant disease and there is evidence for p53 inactivation at this stage in some cases . p53 is a potent transcription factor that positively and negatively modulates a large number of genes involved in apoptosis, metabolism, epigenetics, and cell cycle regulation . p53 is usually expressed at low levels in the cell, where it has a short half-life of only 30 min . Neuroblastoma is a p53 wild-type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Most previous observations confirm that wild-type p53 alleles are present in the vast majority of cases of newly diagnosed neuroblastoma, but that p53/MDM2/ARF responses to chemotherapy are repressed, in part due to unscheduled inhibition of p53 by MDM2. This suggests that downregulation of the p53 axis may underlie the treatment resistance typically seen in high-risk neuroblastoma . The expression, function, and stabilization of p53 are governed by a complex network, which includes its regulators p14ARF and MDM2 .

Originally identified as an amplified gene located on a double minute chromosome in a transformed mouse cell line, MDM2 was later shown to have a critical role in the process of cellular transformation. Thus, deregulation of MDM2 gene expression likely contributes to the pathogenesis of a wide range of human tumors. Amplification and overexpression of MDM2 is found in about 10% of all human tumors. In gliomas, for example, MDM2 amplification identifies a subset of high-risk patients that do not have p53 mutations . In many soft tissue sarcomas, MDM2 amplification and overexpression correlates with poor prognosis.

Additionally, p53 activates the MDM2 gene that encodes a p53 inhibitor forming an autoregulatory feedback loop that tightly controls expression of both p53 and MDM2 . MDM2 helps to target p53 for degradation through its E3 ubiquitin ligase activity.

Recent literature demonstrates that p53 is generally functional, accumulates in the nucleus in response to DNA damage and is efficiently degraded by MDM2 in neuroblastoma. There are reports that p53 function may be compromised as a consequence of aberrant MDM2 expression levels in neuroblastoma. For instance, a study examining etoposide induced p53 activity in a neuroblastoma cell line suggested an important role for elevated MDM2 expression levels in the regulation of p53 translocation .

Also, elevated MDM2 expression and the accompanying loss of p53 function is associated with multidrug resistance in some neuroblastoma cell lines. The activity of MDM2 is critical in neuroblastoma, as recent work demonstrates that MDM2 ubiquitin ligase activity is rate-limiting for p53 degradation in neuroblastoma . Taken together, these data show that the MDM2/p53 interaction is intact in neuroblastoma and suggest that deficiencies in p53 functions may be a consequence of aberrant MDM2 expression.

MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis . A critical negative regulator of the p53 tumor suppressor, MDM2, has been recently characterized in neuroblastoma cell lines as a transcriptional target of MYCN. Targeted inhibition of MYCN results in reduced MDM2 expression levels, with concomitant stabilization of p53 and stimulation of apoptosis in MYCN amplified neuroblastoma cell lines . Moreover, MDM2 has a p53-independent role in the regulation of both MYCN mRNA stabilization and its translation, suggesting that MDM2-mediated MYCN expression is one mechanism associated with the growth of MYCN -associated neuroblastoma and disease progression . In MYCN amplified neuroblastoma raised MDM2 expression had a reinforcing effect on the progression of the disease . MYCN also transcriptionally upregulates p53 expression in neuroblastoma and may be an important mechanism by which MYCN induces apoptosis .

The above evidence, taken together, suggests that inactivation of p53 in neuroblastoma can cause the deregulation of both protein-coding genes and miRNAs, resulting in the deregulation of neuroblastoma related-pathways.