MYCN is a member of the group of MYC-box genes, and its product is a bHLH protein (Schwab et al. 2003). MYCN is transiently expressed during normal neural development and defines the direction of neuronal differentiation. MYCN is frequently amplified in advanced-stage neuroblastoma (Schwab et al. 1983,1984; Brodeur et al. 1984; Seeger et al. 1985), and the biology of high-risk neuroblastoma is influenced by the subsequent overexpression of MYCN oncopro-tein and its targets including telomerase and those functioning in ribosome biogenesis and protein synthesis (Mac et al. 2000; Boon et al. 2001).
5.2 Molecular Bases of Differentiation and Programmed Cell Death
It is well known that some subsets of neuroblastoma can regress spontaneously. One of the most important hints to understand the mechanism of spontaneous regression is age of the patient at the onset of neuroblastoma. Regression rarely occurs when the tumor is found in patients over 1 year of age. The dramatic regression of the stage 4s tumor after its rapid growth usually occurs within 6 months after birth; therefore, it is plausible that epigenetic regulations, timed with the development of sympathetic neurons, might also control neuroblastoma regression. It is well known that massive death of sympathetic neurons is induced during the perinatal period - a process called developmentally regulated neuronal programmed cell death following deprivation of tar get tissue-derived neurotrophins (Oppenheim 1991). This same death mechanism appears to be conserved in primary neuroblastomas found in infants, leading to the induction of their spontaneous regression (Nakagawara 1998b).
5.2.2 Neurotrophic Factors and Their Receptors
188.8.131.52 Neurotrophins and Their Receptors in Neuroblastoma
The neurotrophin family of growth factors consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3),and neurotrophin-4/5 (NT-4/5; Huang and Reichardt 2003). The corresponding high-affinity neurotrophin receptors with tyrosine kinase activity have been identified as TrkA, TrkB, and TrkC (Snider 1994) (Fig. 5.5 a,b). TrkA is a preferred receptor for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3. All of the neurotrophins also bind similarly to a lower-affinity neurotrophin receptor p75NTR, a member of the tumor necrosis factor receptor (TNFR)/Fas family (Snider 1994). The targeted disruption of neu-rotrophins and their receptors has demonstrated that NGF/TrkA signaling supports the survival and differentiation of sympathetic and sensory neurons responsive to temperature and pain, while BDNF/TrkB, NT-4/TrkB, and NT-3/TrkC signaling supports those of sensory neurons responsive to tactile stimuli and motor and sensory neurons responsive to limb movement and position, respectively (Klein 1994). These results suggest that neural development and maintenance of the neural network are spatiotemporally controlled by neurotrophin signaling with or without some redundancy in both peripheral and central nervous systems.
In neuroblastoma, high levels of TrkA are expressed in subsets of tumors with good prognosis, often showing spontaneous regression (Nakagawara et al. 1992,1993; Suzuki et al. 1993; Kogner et al. 1993). Such tumors usually occur in patients under 1 year of age, and their DNA ploidy is aneuploid. A very limited amount of NGF may be supplied from stromal cells, e.g., Schwannian cells and fibroblasts, which at least partly regulate the differentiation and pro grammed cell death of neuroblastoma cells (Naka-gawara 1998a). On the other hand, TrkA expression is strongly downregulated in tumors with aggressive behavior that usually possess amplification of the MYCN oncogene and allelic loss of chromosome 1p36 (Nakagawara et al. 1992,1993). TrkB is preferentially expressed in aggressive neuroblastomas together with its preferred ligands BDNF and NT-4/5 which stimulate in an autocrine/paracrine manner, conferring an enhanced malignant phenotype to the tumor cells (Nakagawara et al. 1994; Matsumoto et al. 1995). TrkC is expressed in favorable neuroblastomas at variable levels (Yamashiro et al. 1996), but its preferred ligand, NT-3,is nearly undetectable by RT-PCR in primary neuroblastomas (Nakagawara 1998a); thus, in regressing neuroblastomas, tumor cells expressing the TrkA receptor may be dependent on a limited amount of NGF supplied from stromal cell. In the presence of NGF the cells mature, whereas they will die in the absence of this ligand (Nakagawara 1998a,b); however, in clinically aggressive neuroblastomas, the TrkA is downregulated and the downstream signaling cascades are disturbed, and these cells utilize the BDNF or NT-4/TrkB autocrine system for efficient growth. Neurotrophin signaling may also regulate tumor metastasis (Matsumoto et al. 1995), proliferation (Matsumoto et al. 1995), and angiogen-esis (Canete et al. 2000). The role of p75NTR in neuroblastoma is unclear. The p75NTR receptor is expressed in both neuroblastoma cell lines (Azar et al. 1990) and primary neuroblastomas (Nakagawara et al. 1993). Interestingly, the expression levels of p75NTR mRNA are significantly higher in favorable neuroblastomas (stages 1, 2 and 4s) as compared with the advanced stage tumors, especially those with MYCN amplification (Nakagawara et al. 1993).
In a rat pheochromocytoma cell line PC12, differentiation signals by NGF may be mediated through the tyrosine phosphorylation of the Trk receptor and through the subsequent activation of Shc/Grb2/SOS, Ras, Raf, MEK, and ERKs,while survival signals in the same cells may be transduced through the direct p75NTR
Figure 5.5 a,b
Neurotrophins and their receptors. a TrkA is a preferred high-affinity receptor for NGF, TrkB for BDNF, and NT-4/5,and TrkC for NT-3. All of the neurotrophins also bind similarly to a lower affinity neurotrophin receptor p75NTR. b The structures of neurotrophin family receptors. The extracellular domains of TrkA, TrkB, and TrkC have high structural similarity.The intracellular domain of Trks possesses tyrosine kinase activity.TrkB and TrkC receptors have truncated forms which lack the tyrosine kinase domain.The low-affinity receptor, p75NTR, has a short intracellular region containing the death domain, and belongs to the Fas/TNFR family of the receptors
activation of PI3-kinase which in turn activates downstream molecules,e.g.,Akt and Bad (Klesse and Parada 1999). On the other hand, in normal sympathetic neurons, the activation of PI3-kinase is mediated not by the tyrosine phosphorylation of the receptor but by the Ras activation which promotes neuronal survival, suggesting that the Trk intracellular signaling pathway might be deregulated in cancer cells. This is also the case in neuroblastoma. In the neuroblastoma cell lines with a single copy of MYCN, NGF can induce differentiation when exogenous TrkA is overexpressed (Eggert et al. 2000). In the cell lines with MYCN amplification, however, the NGF-stimulated TrkA receptors which were overexpressed cannot normally activate downstream signaling molecules, resulting in unresponsiveness to the ligand. Furthermore, it is surprising that BDNF/TrkB signaling appears to be functioning in the same cells by promoting survival (Nakagawara et al. 1994; Hishiki et al. 1998), although the signaling pathway might be different from that of sympathetic neurons (Klesse and Parada 1999).
Neurotrophic factors of the glial cell line-derived neurotrophic factor (GDNF) family, which include GDNF, artemin and neurturin, are secreted by neu-roblastoma cells as well as stromal cells and activate their receptor complex composed of Ret tyrosine kinase and the GFRa co-receptors expressed in neuroblastoma cells (Hishiki et al. 1998; Ichikawa et al. 2004). In contrast to NGF/TrkA and BDNF/TrkB, however, the GDNF/Ret/GFRa autocrine system is functioning in both favorable and unfavorable neu-roblastomas to enhance the survival and differentiation of tumor cells (Hishiki et al. 1998).
Neuroblastoma cells express other growth factors and receptors. Both pleiotrophin (PTN) and midkine (MK) are factors in the same family with neurotrophic function (Kadomatsu et al. 1990; Li et al. 1990; Kadomatsu and Muramatsu 2004). PTN is expressed significantly at high levels in favorable neuroblas tomas, while MK is highly expressed in almost all neuroblastomas with a tendency to be expressed at high levels in tumors in advanced stages (Nakagawara et al. 1995). Neuroblastoma also expresses many other receptors, e.g., fibroblast growth factor receptor (FGFR; Schweigerer et al. 1991), insulin-like growth factor (IGFR; El-Badry et al. 1991), DCC (deleted in colon cancer) (Reale et al. 1996), and neuronal leucine-rich repeat receptors (NLRRs; Hamano et al. 2004), as well as a novel plasma membrane enzyme ECEL1, which is significantly highly expressed in favorable neuroblastomas (Kawamoto et al. 2003). The biological significance of these factors and receptors in neuroblastoma are not currently known.
Recent lines of evidence suggest that both the p53 tumor suppressor protein and its related protein p73 are involved in the induction of programmed cell death and growth arrest in neuronal cells (Pozniak et al. 2000). p73 is a recently identified candidate tumor suppressor gene mapped to chromosome 1p36.2, a frequently deleted region in many human cancers including neuroblastoma and oligodendroglioma (Ichimiya et al. 1999; Billon et al. 2004). In cultured neonatal sympathetic neurons, p53 protein levels are increased in response to NGF withdrawal as well as p75NTR activation, and it functions downstream of c-Jun NH2-terminal kinase (JNK) and upstream of Bax to induce apoptosis (Aloyz et al. 1998) (Fig. 5.6). Indeed, in p53/- mice, naturally occurring sympathetic neuron death is inhibited. Pozniak et al. (2000) have also reported that p73 is primarily present in developing neurons as ANp73, an NH2-terminally truncated isoform, whose level is decreased when sympathetic neurons undergo apoptosis after NGF withdrawal, and that p53 becomes activated to be pro-apoptotic. In contrast to the truncated form of p73, full-length p73 has induced neuronal differentiation in a mouse neuroblastoma cell line N1E115 (Laurenzi et al. 2000). These data suggest that the neuronal apoptosis induced by NGF withdrawal is at least partly regulated by a reciprocal balance between levels of pro-apoptotic p53 and anti-apoptotic ANp73.
A model of signaling pathway for survival and death in sympathetic neurons regulated by NGF. NGF depletion may induce activation of JNK/p53 pathway which could be modified by p73/ANp73 regulatory system. p75NTR activation, which sends signals of both survival and death, may also regulate downstream p53/p73/ANp73 pathway
A possible signaling pathway regulating growth, differentiation and survival in neuroblas-toma cells or sympathetic neurons. The NGF-triggered auto-phosphorylation of TrkA tyrosine kinase receptor induces activation of Ras/MAPK pathway,which in turn regulates nuclear pRB and Mdm2. In some poor-outcome neuroblastomas, p53, which is shuttling between cytosol and nucleus, is trapped in the cytosol by Parc, an anchoring protein of p53.MYCN induces expression of Id-2 whose protein product in turn inhibits pRB.E2F1 negatively regulated by pRB directly induces expression of p73. p73 is regulated by ANp73 in a negative autoregulatory manner (Naka-gawa et al.2002),and ANp73 also inhibits p53
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