**Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma**

Pierdomenico Ruggeri, Antonietta R. Farina, Lucia Cappabianca, Natalia Di Ianni, Marzia Ragone, Stefania Merolle, Alberto Gulino and Andrew R. Mackay

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55536

**1. Introduction**

Neuroblastoma (NB) is an embryonic tumour that originates from cells of the neural crest (NC) arrested in their differentiation at different stages along the sympatho-adrenal lineage and, less frequently, from precursors of sensory neurons [1, 2]. As a consequence, NB can occur throughout the sympathetic chain from thoracic, abdominal and pelvic sites to the adrenal medulla, which accounts for the majority of NBs. Consistent with this, NBs exhibit a high degree of genetic heterogeneity and biological variability, including differences in catechola‐ mine expression, according to their differentiation state along the sympathoadrenal lineage, with a small number of primitive midline and spinal NBs that do not secrete catecholamines considered to be of dorsal root sensory origin [1, 2].

Sympathetic nervous system development is orchestrated by neurotrophins (NT) and their respective neurotrophin receptors (NTR), which exhibit subtle temporal and spatial changes in expression that are critical for the delamination, migration, proliferation, survival, differ‐ entiation and apoptotic programs of NC lineages that form the fully differentiated and functional sympathetic nervous system. Not surprisingly NBs, consistent with their origin and particular differentiation state at the time of transformation, exhibit a variety of different patterns of NT and NTR expression. A great deal of research has focussed on characterising and exploiting these different patterns of expression for potential prognostic and therapeutic benefit. Recent studies have led to exciting new developments in understanding how block‐ ages in sympathetic differentiation promote NB and how NBs utilise different patterns of NT

© 2013 Ruggeri et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and NTR expression to select a more malignant, stress-resistant, invasive, genetically unstable, stem cell-like phenotype. Furthermore, they have also identified novel potential therapeutic targets and characterised patterns of NT/NTR expression of value in prognosis and therapeutic choice. In this chapter therefore, we will review the origins of NB during neural crest migation and sympathetic nervous system development, introduce NTs and NTRs and describe their roles NC and sympathetic nervous system development, examine patterns of NT/NTR expression in NB, review their potential roles in regulating spontaneous NB regression and metastatic NB progression, and discuss potential therapeutic ways to target the NT/NTR system in NB.

each new somite forming on the caudal side of an existing somite. Somites further differen‐ tiate into dermomyotome and sclerotome structures that will eventually provide the cells for skin, muscle and skeletal formation. Contemporarily, the embryonic neuroectoderm undergoes progressive indentation to form the neural groove, neural folds and neural plate. This neurulation process causes the fusion of opposing neural folds at the future upper cervical level, which progresses in both rostral and caudal directions, eventually resulting in continuity between neural and squamous surface ectoderm. This event separates the presumptive epidermis from the neural plate, which in turn forms the distinct and sepa‐ rate columnar cellular structure of the Neural Tube. Interaction between the neural plate and presumptive epidermis is regulated by Wnts, BMPs and FGFs and results in mesenchymal transformation of the epithelial cells that line the margins of the neural fold. These cells organise between the epidermis and neural tube to form the transient Neural Crest (NC)

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

49

NC cells (NCC) delaminate from the NC and migrate initially in a ventrolateral manner and later in a dorsolateral direction, relative to the somites. Ventrolateral NCC migration occurs in chain-like manner [7] between the somites and neural tube and the rostral half of each somite [8]. NCC initially migrate through the inter-somitic boundary before switching to a sclerotome pathway controlled by semaphorin and its receptor neuropilin, with the entire dermomytome

Dorsolateral NCC migration occurs between the developing dermis and the dorsal dermo‐ myotome boundary [8, 10]. During NCC migration cells receive signals from adjacent struc‐ tures that initiate a series of differentiation processes that will eventually lead to differentiation-commitment and specific cell fates at different locations. This process provides a wide variety of differentiated tissues, including: epidermal pigment cells (melanocytes); dorsal root, sympathetic and parasympathetic ganglia, neurons and plexuses; neuroglial and Schwann cells; endocrine/paracrine cells of the adrenal medulla, carotid body and organ of Zuckerland; cartilage and bones of the facial and ventral skull; corneal endothelium and stroma; tooth papillae; dermis, smooth muscle and adipose tissue of the head and neck;

embryonic structure [2, 6] (Fig. 1).

repulsing neuropilin positive trunk NCC [9] (Fig. 2).

**Figure 2.** Neural Crest cell ventrolateral and dorsolateral migration

## **2. Formation of the neural crest, neural crest cell delamination and migration**

NBs originate from NC cells (NCC) during sympathetic nervous system development. In this section therefore, we will briefly describe the natural history of neural crest, sensory dorsal root and sympathetic nervous system development, focussing attention on the sympathoadrenal neuroblast lineage, which is responsible for generating neuroendocrine chromaffin tissues, SIF and ganglion cells, and in particular the adrenal medulla within which the majority (40-50%) of NBs develop [2, 3].

**Figure 1.** Formation of the Neural Crest and Neural Crest cell migration

During the 3rd week of human embryonic development the intra-embryonic mesoderm differentiates into paraxial, intermediate and lateral plate portions. The paraxial mesoderm organises into primitive segmented somites and the lateral plate mesoderm splits into somatic (parietal) and splanchnic (visceral) layers. This event occurs in a BMP-induced Notchdependent "clock" and Wnt-dependent "wave" manner in a rostral to caudal gradient of FGF [2, 4-6] and results in the simultaneous formation of Somite pairs either side of the forming neural tube, in a head to tail direction along the entire length of the embryo, with

each new somite forming on the caudal side of an existing somite. Somites further differen‐ tiate into dermomyotome and sclerotome structures that will eventually provide the cells for skin, muscle and skeletal formation. Contemporarily, the embryonic neuroectoderm undergoes progressive indentation to form the neural groove, neural folds and neural plate. This neurulation process causes the fusion of opposing neural folds at the future upper cervical level, which progresses in both rostral and caudal directions, eventually resulting in continuity between neural and squamous surface ectoderm. This event separates the presumptive epidermis from the neural plate, which in turn forms the distinct and sepa‐ rate columnar cellular structure of the Neural Tube. Interaction between the neural plate and presumptive epidermis is regulated by Wnts, BMPs and FGFs and results in mesenchymal transformation of the epithelial cells that line the margins of the neural fold. These cells organise between the epidermis and neural tube to form the transient Neural Crest (NC) embryonic structure [2, 6] (Fig. 1).

NC cells (NCC) delaminate from the NC and migrate initially in a ventrolateral manner and later in a dorsolateral direction, relative to the somites. Ventrolateral NCC migration occurs in chain-like manner [7] between the somites and neural tube and the rostral half of each somite [8]. NCC initially migrate through the inter-somitic boundary before switching to a sclerotome pathway controlled by semaphorin and its receptor neuropilin, with the entire dermomytome repulsing neuropilin positive trunk NCC [9] (Fig. 2).

**Figure 2.** Neural Crest cell ventrolateral and dorsolateral migration

and NTR expression to select a more malignant, stress-resistant, invasive, genetically unstable, stem cell-like phenotype. Furthermore, they have also identified novel potential therapeutic targets and characterised patterns of NT/NTR expression of value in prognosis and therapeutic choice. In this chapter therefore, we will review the origins of NB during neural crest migation and sympathetic nervous system development, introduce NTs and NTRs and describe their roles NC and sympathetic nervous system development, examine patterns of NT/NTR expression in NB, review their potential roles in regulating spontaneous NB regression and metastatic NB progression, and discuss potential therapeutic ways to target the NT/NTR

**2. Formation of the neural crest, neural crest cell delamination and**

NBs originate from NC cells (NCC) during sympathetic nervous system development. In this section therefore, we will briefly describe the natural history of neural crest, sensory dorsal root and sympathetic nervous system development, focussing attention on the sympathoadrenal neuroblast lineage, which is responsible for generating neuroendocrine chromaffin tissues, SIF and ganglion cells, and in particular the adrenal medulla within which the majority

During the 3rd week of human embryonic development the intra-embryonic mesoderm differentiates into paraxial, intermediate and lateral plate portions. The paraxial mesoderm organises into primitive segmented somites and the lateral plate mesoderm splits into somatic (parietal) and splanchnic (visceral) layers. This event occurs in a BMP-induced Notchdependent "clock" and Wnt-dependent "wave" manner in a rostral to caudal gradient of FGF [2, 4-6] and results in the simultaneous formation of Somite pairs either side of the forming neural tube, in a head to tail direction along the entire length of the embryo, with

system in NB.

48 Neuroblastoma

**migration**

(40-50%) of NBs develop [2, 3].

**Figure 1.** Formation of the Neural Crest and Neural Crest cell migration

Dorsolateral NCC migration occurs between the developing dermis and the dorsal dermo‐ myotome boundary [8, 10]. During NCC migration cells receive signals from adjacent struc‐ tures that initiate a series of differentiation processes that will eventually lead to differentiation-commitment and specific cell fates at different locations. This process provides a wide variety of differentiated tissues, including: epidermal pigment cells (melanocytes); dorsal root, sympathetic and parasympathetic ganglia, neurons and plexuses; neuroglial and Schwann cells; endocrine/paracrine cells of the adrenal medulla, carotid body and organ of Zuckerland; cartilage and bones of the facial and ventral skull; corneal endothelium and stroma; tooth papillae; dermis, smooth muscle and adipose tissue of the head and neck; connective tissue of the salivary, lachrymal, thymus, thyroid and pituitary glands and connective tissue and smooth muscle in arteries of aortic origin (Fig. 3).

Schwann cells, small intensely fluorescent (SIF) cells and chromaffin cells of the adrenal medulla and extra adrenal paraganglia (Fig. 4). Together, these components form the neuro‐ endocrine SNS, which consists of preganglionic neurones that exit from spinal chord ventral routes of the 12 thoracic and 3 lumbar spinal segments that synapse with neurons of the sympathetic ganglia or specialised chromaffin cells of adrenal medulla and paraganglia. Sympathetic ganglia include paravertebral and prevertebral ganglia, with pairs of paraverte‐ bral ganglia each side of the vertebra interconnected to form the sympathetic chain. Normally, there are 21 to 22 pairs of paravertebral sympathetic ganglia, 3 cervical, 10-11 thoracic, 4 lumbar, 4 sacral and a single ganglion impar in front of the coccyx. Cervical superior, middle and stellar ganglia innervate viscera of the head and neck, thoracic ganglia innervate viscera of the trunk, and lumbar/sacral ganglia innervate the pelvic floor and lower limbs. Sympathetic ganglia also innervate blood vessels, muscle, skin, erector pilli and sweat glands [11, 13].

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

51

**Figure 4.** Cell types generated from differentiated sympathoadrenal neuroblast progentitors

dilation and pili-erection, in preparation for imminent danger [11, 13].

at this point.

In general, preganglionic neurons utilise acetylcholine as the major neurotransmitter, whereas post-ganglionic neurons are noradrenergic and utilise noradrenalin as the major neurotrans‐ mitter, combined with specific neuropeptide transmitters (e.g. neuropeptide Y, somatostatin, vasointestinal peptide and calcitonin related peptide), utilised in an organ-specific manner. Under normal conditions the sympathetic nervous system provides local adjustments (e.g. sweating) and relax adjustment to the cardiovascular system. Under conditions of stress, the entire SNS can activate to induce the "fight or flight" response, during which adrenalin released from the adrenal gland leads to rapid increases in heart rate, cardiac output, skeletal muscle vasodilation, cutaneous and gastrointestinal vasoconstriction, pupil dilation, bronchial

**3.1. Sympatho-adrenal progenitors, SIF and Chromaffin cells of the neuroendocrine SNS**

The vast majority (40-50%) of NBs arise from neuroblastic NCCs within the developing adrenal gland [2, 3]. Therefore, a description of normal adrenal gland development is also warranted

The fully developed functional adrenal gland is composed of cortex and medulla. The adrenal medulla is composed of neuroendocrine-differentiated chromaffin, SIF and ganglion cells, which are also present in extra-adrenal paraganglia of the carotid body and organ of Zucker‐

**Figure 3.** Neural crest cell destinations during embryonic development

#### **3. The sympathetic nervous system**

The vertebrate nervous system is composed of the central (CNS) and peripheral (PNS) nervous systems, the former comprised of the brain and spinal cord and the latter com‐ prised of ganglia and associated plexuses that innervate and connect visceral organs and other tissues to the CNS.

The PNS is divided into the somatic and autonomic nervous systems, the former responsible for skeletal muscle function and the latter for innervation of visceral organs [11, 12]. The autonomic nervous system is further subdivided into sympathetic (SNS) and parasympathetic (PSNS) nervous systems, which are often antagonistic. Motor outflow from both systems is formed by serially connected neurons that initiate with pre-ganglionic neurons of the brain stem or spinal chord, which synapse with ganglia and post ganglion neurones outside the CNS. Parasympathetic ganglia lie close to or within the organs they innervate, whereas sympathetic ganglia lie at some distance from their target organ. Both have sensory fibres that feedback information concerning organ function to the central nervous system [11, 13].

The NC is fundamental for SNS formation. Pluripotent migratory NCC progenitors delaminate from the NC and migrate in a vetrolateral direction through the rostral half of each somite. NCC remaining within somites coalesce to form paraspinal dorsal root ganglia, which contain the nerve bodies of afferent spinal nerves responsible for relaying sensory information into the CNS. NCC that exit somites ventrolaterally initially lose segmental organisation, mix adjacent to the dorsal aorta then re-segregate to form sympathetic ganglia, helping to explain sympa‐ thetic ganglia heterogeneity [7]. At this point cells initiate differentiation that is responsible for the eventual formation of sympathetic ganglia, associated sympathetic neurones and Schwann cells, small intensely fluorescent (SIF) cells and chromaffin cells of the adrenal medulla and extra adrenal paraganglia (Fig. 4). Together, these components form the neuro‐ endocrine SNS, which consists of preganglionic neurones that exit from spinal chord ventral routes of the 12 thoracic and 3 lumbar spinal segments that synapse with neurons of the sympathetic ganglia or specialised chromaffin cells of adrenal medulla and paraganglia. Sympathetic ganglia include paravertebral and prevertebral ganglia, with pairs of paraverte‐ bral ganglia each side of the vertebra interconnected to form the sympathetic chain. Normally, there are 21 to 22 pairs of paravertebral sympathetic ganglia, 3 cervical, 10-11 thoracic, 4 lumbar, 4 sacral and a single ganglion impar in front of the coccyx. Cervical superior, middle and stellar ganglia innervate viscera of the head and neck, thoracic ganglia innervate viscera of the trunk, and lumbar/sacral ganglia innervate the pelvic floor and lower limbs. Sympathetic ganglia also innervate blood vessels, muscle, skin, erector pilli and sweat glands [11, 13].

connective tissue of the salivary, lachrymal, thymus, thyroid and pituitary glands and

The vertebrate nervous system is composed of the central (CNS) and peripheral (PNS) nervous systems, the former comprised of the brain and spinal cord and the latter com‐ prised of ganglia and associated plexuses that innervate and connect visceral organs and

The PNS is divided into the somatic and autonomic nervous systems, the former responsible for skeletal muscle function and the latter for innervation of visceral organs [11, 12]. The autonomic nervous system is further subdivided into sympathetic (SNS) and parasympathetic (PSNS) nervous systems, which are often antagonistic. Motor outflow from both systems is formed by serially connected neurons that initiate with pre-ganglionic neurons of the brain stem or spinal chord, which synapse with ganglia and post ganglion neurones outside the CNS. Parasympathetic ganglia lie close to or within the organs they innervate, whereas sympathetic ganglia lie at some distance from their target organ. Both have sensory fibres that feedback information concerning organ function to the central nervous system [11, 13].

The NC is fundamental for SNS formation. Pluripotent migratory NCC progenitors delaminate from the NC and migrate in a vetrolateral direction through the rostral half of each somite. NCC remaining within somites coalesce to form paraspinal dorsal root ganglia, which contain the nerve bodies of afferent spinal nerves responsible for relaying sensory information into the CNS. NCC that exit somites ventrolaterally initially lose segmental organisation, mix adjacent to the dorsal aorta then re-segregate to form sympathetic ganglia, helping to explain sympa‐ thetic ganglia heterogeneity [7]. At this point cells initiate differentiation that is responsible for the eventual formation of sympathetic ganglia, associated sympathetic neurones and

connective tissue and smooth muscle in arteries of aortic origin (Fig. 3).

**Figure 3.** Neural crest cell destinations during embryonic development

**3. The sympathetic nervous system**

other tissues to the CNS.

50 Neuroblastoma

**Figure 4.** Cell types generated from differentiated sympathoadrenal neuroblast progentitors

In general, preganglionic neurons utilise acetylcholine as the major neurotransmitter, whereas post-ganglionic neurons are noradrenergic and utilise noradrenalin as the major neurotrans‐ mitter, combined with specific neuropeptide transmitters (e.g. neuropeptide Y, somatostatin, vasointestinal peptide and calcitonin related peptide), utilised in an organ-specific manner. Under normal conditions the sympathetic nervous system provides local adjustments (e.g. sweating) and relax adjustment to the cardiovascular system. Under conditions of stress, the entire SNS can activate to induce the "fight or flight" response, during which adrenalin released from the adrenal gland leads to rapid increases in heart rate, cardiac output, skeletal muscle vasodilation, cutaneous and gastrointestinal vasoconstriction, pupil dilation, bronchial dilation and pili-erection, in preparation for imminent danger [11, 13].

#### **3.1. Sympatho-adrenal progenitors, SIF and Chromaffin cells of the neuroendocrine SNS**

The vast majority (40-50%) of NBs arise from neuroblastic NCCs within the developing adrenal gland [2, 3]. Therefore, a description of normal adrenal gland development is also warranted at this point.

The fully developed functional adrenal gland is composed of cortex and medulla. The adrenal medulla is composed of neuroendocrine-differentiated chromaffin, SIF and ganglion cells, which are also present in extra-adrenal paraganglia of the carotid body and organ of Zucker‐ land [14]. Chromaffin and SIF cells, characterised by their affinity from chromium salts, are closely related to sympathetic neurons and, like sympathetic neurons, synthesise, store, uptake and release catecholamines and express enzymes for noradrenalin synthesis including tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH). Unlike sympathetic neurons, chro‐ maffin cells also synthesise, store and release adrenalin and retain their capacity proliferate but do not produce axons or dendrites [15]. Adrenal and extra-adrenal chromaffin tissues, like sympathetic ganglia, are innervated by pre-ganglionic neurones originating from the spinal chord [16]. Chromaffin, SIF and sympathetic neurons exemplify the wide spectrum of sympathoadrenal cell types that originate from NCC [17].

adrenal medulla exhibit heterogeneity, and consist of SOX10/Phox2B/p75NTR, SOX10/

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

53

Chromaffin and sympathetic neuron differentiation is regulated by BMP-induced transcrip‐ tion factors Phox2B, Mash-1, Insm1, Hand2 and Gata 2/3 [31]. Knockout technology has identified a fundamental role for Phox2B in chromaffin and sympathetic neuronal differen‐ tiation [18], with Phox2B knockout increasing neuron but not chromaffin precursor death. This not only relates to specific cell traits but also differences in environment and migra‐ tion [32, 33], and confirms that adrenal anlage are colonised by undifferentiated NCC progenitors. Knockout technology has also characterised a role for Mash-1 as an accelera‐ tor of sympathetic neuronal and chromaffin differentiation [34, 35], a role for Insm-1 as a regulator of catecholamine synthesising enzyme expression and, therefore, endocrine differentiation [36, 37], a role for Hand2 in the induction and maintenance of noradrener‐ gic differentiation [38, 39] and a role for Gata3 in the differentiation of both sympathetic

It has now been confirmed that adrenal cortex glucocorticoids are not responsible for chro‐ maffin cell differentiation [41] but they do, however, regulate postnatal chromaffin cell survival and phenyl ethanolamine N-methyl transferase expression [30, 42]. The adrenal cortex is also dispensable for chromaffin differentiation, which is also found in extra-adrenal neuroendocrine tissue, but may regulate adrenal chromaffin cell numbers and associated vascularity [30, 43]. Within the adrenal gland, hypoxia has recently been shown to promote

**4. Neurotrophins and neurotrophin receptors in neural crest, sympathetic**

Neurotrophins (NTs) and NT receptors (NTRs) are critical for the development and mainte‐ nance of the vertebral CNS and PNS [47-50], NTs and NTRs are also expressed by human NBs and have been implicated in both NB regression and malignant progression. In this section, therefore, we will introduce NTs and NTRs and describe their potential involvement in normal

NTs are a family of growth, differentiation, survival and apoptosis-inducing factors that are involved in many aspects of nervous system development, maintenance and function. They comprise four structurally related basic 115-130 amino acid containing polypeptides, nerve growth factor (NGF), brain-derived growth factor (BDNF), and the neurotrophins 3 (NT-3) and 4/5. NGF was first NT to be described and purified from the mouse salivary gland [51]. This was followed by the discovery of BDNF, NT-3 and NT-4/5 some 30 years later [52-54].

p75NTR and PHox2B/p75NTR sub-populations [22].

ganglia and chromaffin cells [31, 40].

SNS and adrenal development.

**4.1. Neurotrophins (NTs)**

chromaffin/SIF cell differentiation from neuroblasts [44-46].

**nervous system and adrenal development**

**3.2. Transcriptional regulation of sympathoadrenal differentiation**

Chromaffin and SIF cells differentiate from pluripotent NCC progenitors that delaminate from the NC at the "adreno-medullary" somite level (somites 18-24 in avian development) [18]. These cells migrate ventrolaterally initially between Somite dermomyotome and sclerotome then through the rostral sclerotome mesenchyme to arrive at para-aortic sites [18-20]. At these sites, NCC mix, re-segregate and coalesce to form sympathetic ganglia. At the same time, NCC derived from the "adreo-medullar" somite region coalesce adjacent to the adrenal cortex anlage then invade the anlage in considerable numbers, initially in a nerve fibre-independent then nerve fibre-dependent manner [21], in a Sox transcription factor-dependent manner [22]. Once within the adrenal primordium, NCCs form rosettes, nests and nodules along nerve fibres, proliferate and initiate pheo-chromoblast differentiation. This process continues throughout foetal development and into the neonatal period, providing differentiated Chromaffin and SIF adrenal medulla cell populations. In humans, the gestational period between 17 and 20 weeks is critical for adrenal sympathetic component development, with neuroblastic NCC proliferation peaking during this period in terms of maximal nodule size and number, waning thereafter. Neuroblastic nodules tend to disappear during the third trimester and are usually absent at birth. However, nodules that continue to grow and persist into neonatal life are not infrequent and have been classified as *in situ* NB. A sizeable number of these NBs spontaneously regress and are likely, therefore, to represent delayed differentia‐ tion in addition to neoplastic transformation [23].

Chromaffin cells, SIF cells and sympathetic neurons develop from catecholaminergic sympa‐ thoadrenal (SA) progenitors [18, 24, 25] and their formation involves BMP signalling [18, 26-28]. However, the classical concept that a common SA lineage acquires neuronal and catecholaminergic traits prior to migration to secondary sympathetic ganglia and adrenal sites [24, 25] has now been discounted, as chromaffin cells undergo catecholaminergic differentia‐ tion within the adrenal anlage and not within primary sympathetic ganglia, and do not express neuronal markers at the onset or even following induction of TH expression [29, 30]. Therefore, sympathetic neuronal and chromaffin lineages must separate upstream prior to catecholami‐ nergic differentiation, despite evidence of sympathoadrenal marker expression in some migrating cells [22, 24, 30], and enter the adrenal primordium as undifferentiated Sox10 expressing NCC [22, 30]. Indeed, chromaffin and sympathetic neurones originate at the same axial level from common NC progenitors but differ in the time of catecholaminergic differen‐ tiation [18]. Furthermore, NCC populations migrating to the adrenal anlage and within the adrenal medulla exhibit heterogeneity, and consist of SOX10/Phox2B/p75NTR, SOX10/ p75NTR and PHox2B/p75NTR sub-populations [22].

#### **3.2. Transcriptional regulation of sympathoadrenal differentiation**

Chromaffin and sympathetic neuron differentiation is regulated by BMP-induced transcrip‐ tion factors Phox2B, Mash-1, Insm1, Hand2 and Gata 2/3 [31]. Knockout technology has identified a fundamental role for Phox2B in chromaffin and sympathetic neuronal differen‐ tiation [18], with Phox2B knockout increasing neuron but not chromaffin precursor death. This not only relates to specific cell traits but also differences in environment and migra‐ tion [32, 33], and confirms that adrenal anlage are colonised by undifferentiated NCC progenitors. Knockout technology has also characterised a role for Mash-1 as an accelera‐ tor of sympathetic neuronal and chromaffin differentiation [34, 35], a role for Insm-1 as a regulator of catecholamine synthesising enzyme expression and, therefore, endocrine differentiation [36, 37], a role for Hand2 in the induction and maintenance of noradrener‐ gic differentiation [38, 39] and a role for Gata3 in the differentiation of both sympathetic ganglia and chromaffin cells [31, 40].

It has now been confirmed that adrenal cortex glucocorticoids are not responsible for chro‐ maffin cell differentiation [41] but they do, however, regulate postnatal chromaffin cell survival and phenyl ethanolamine N-methyl transferase expression [30, 42]. The adrenal cortex is also dispensable for chromaffin differentiation, which is also found in extra-adrenal neuroendocrine tissue, but may regulate adrenal chromaffin cell numbers and associated vascularity [30, 43]. Within the adrenal gland, hypoxia has recently been shown to promote chromaffin/SIF cell differentiation from neuroblasts [44-46].

## **4. Neurotrophins and neurotrophin receptors in neural crest, sympathetic nervous system and adrenal development**

Neurotrophins (NTs) and NT receptors (NTRs) are critical for the development and mainte‐ nance of the vertebral CNS and PNS [47-50], NTs and NTRs are also expressed by human NBs and have been implicated in both NB regression and malignant progression. In this section, therefore, we will introduce NTs and NTRs and describe their potential involvement in normal SNS and adrenal development.

#### **4.1. Neurotrophins (NTs)**

land [14]. Chromaffin and SIF cells, characterised by their affinity from chromium salts, are closely related to sympathetic neurons and, like sympathetic neurons, synthesise, store, uptake and release catecholamines and express enzymes for noradrenalin synthesis including tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH). Unlike sympathetic neurons, chro‐ maffin cells also synthesise, store and release adrenalin and retain their capacity proliferate but do not produce axons or dendrites [15]. Adrenal and extra-adrenal chromaffin tissues, like sympathetic ganglia, are innervated by pre-ganglionic neurones originating from the spinal chord [16]. Chromaffin, SIF and sympathetic neurons exemplify the wide spectrum of

Chromaffin and SIF cells differentiate from pluripotent NCC progenitors that delaminate from the NC at the "adreno-medullary" somite level (somites 18-24 in avian development) [18]. These cells migrate ventrolaterally initially between Somite dermomyotome and sclerotome then through the rostral sclerotome mesenchyme to arrive at para-aortic sites [18-20]. At these sites, NCC mix, re-segregate and coalesce to form sympathetic ganglia. At the same time, NCC derived from the "adreo-medullar" somite region coalesce adjacent to the adrenal cortex anlage then invade the anlage in considerable numbers, initially in a nerve fibre-independent then nerve fibre-dependent manner [21], in a Sox transcription factor-dependent manner [22]. Once within the adrenal primordium, NCCs form rosettes, nests and nodules along nerve fibres, proliferate and initiate pheo-chromoblast differentiation. This process continues throughout foetal development and into the neonatal period, providing differentiated Chromaffin and SIF adrenal medulla cell populations. In humans, the gestational period between 17 and 20 weeks is critical for adrenal sympathetic component development, with neuroblastic NCC proliferation peaking during this period in terms of maximal nodule size and number, waning thereafter. Neuroblastic nodules tend to disappear during the third trimester and are usually absent at birth. However, nodules that continue to grow and persist into neonatal life are not infrequent and have been classified as *in situ* NB. A sizeable number of these NBs spontaneously regress and are likely, therefore, to represent delayed differentia‐

Chromaffin cells, SIF cells and sympathetic neurons develop from catecholaminergic sympa‐ thoadrenal (SA) progenitors [18, 24, 25] and their formation involves BMP signalling [18, 26-28]. However, the classical concept that a common SA lineage acquires neuronal and catecholaminergic traits prior to migration to secondary sympathetic ganglia and adrenal sites [24, 25] has now been discounted, as chromaffin cells undergo catecholaminergic differentia‐ tion within the adrenal anlage and not within primary sympathetic ganglia, and do not express neuronal markers at the onset or even following induction of TH expression [29, 30]. Therefore, sympathetic neuronal and chromaffin lineages must separate upstream prior to catecholami‐ nergic differentiation, despite evidence of sympathoadrenal marker expression in some migrating cells [22, 24, 30], and enter the adrenal primordium as undifferentiated Sox10 expressing NCC [22, 30]. Indeed, chromaffin and sympathetic neurones originate at the same axial level from common NC progenitors but differ in the time of catecholaminergic differen‐ tiation [18]. Furthermore, NCC populations migrating to the adrenal anlage and within the

sympathoadrenal cell types that originate from NCC [17].

52 Neuroblastoma

tion in addition to neoplastic transformation [23].

NTs are a family of growth, differentiation, survival and apoptosis-inducing factors that are involved in many aspects of nervous system development, maintenance and function. They comprise four structurally related basic 115-130 amino acid containing polypeptides, nerve growth factor (NGF), brain-derived growth factor (BDNF), and the neurotrophins 3 (NT-3) and 4/5. NGF was first NT to be described and purified from the mouse salivary gland [51]. This was followed by the discovery of BDNF, NT-3 and NT-4/5 some 30 years later [52-54]. NTs exhibit close structural homology, with the exception of NT4/5 that exhibits only 50% homology to the others NTs, and all contain six conserved cysteines that form structurally important disulphide bridges [55, 56]. NTs are expressed by both neuronal and non-neuronal cells as pre-NTs and are converted to pro-NTs upon signal peptide removal. This can occur within the endoplasmic reticulum (ER), in which NTs are converted to mature-NTs by furins. Alternatively, NTs are transported to the cell surface and released following signal peptide removal as pro-NTs. Secreted pro-NTs, which also exhibit biological activity, are converted to mature NTs by enzymes including plasmin and the matrix metalloproteinases MMP-7 and MMP-9 [56-58]. Within the extracellular environment, pro- and mature NTs form homo-dimers and bind specific receptors to induce an array of biological activities, including cell migration, proliferation, survival, differentiation, apoptosis and neuronal synapse/junction plasticity, depending upon the cell population, receptor expression and activation status [57, 60]. The human *NGF* gene localises to chromosome 1p13.1 [61], the human *BDNF* gene localises to chromosome 11p13 [62], the human *NT-3* gene localises to chromosome 12p13 [62] and the human *NT4/5* gene localises to chromosome 19q13.3 [63]. Since the discovery of NTs, their respective receptors have been identified and many of their roles in nervous system develop‐ ment and function have been elucidated.

of amino acids 192-284 that encode the D4 extracellular immunoglobulin-like domain, several functional N-glycosylation sites and introduces a valine substitution at the novel exon 5/8 splice junction [73]. In addition to being expressed by primary human NBs, TrkAIII is also developmentally regulated and is detected from stages E13-E18 of mouse embryonic devel‐ opment and is also expressed by immature thymocytes within the developing thymus [73, 77]. Unlike fully spliced TrkA receptors, TrkAIII is not expressed at the cell surface but is retained within intracellular membranes of the endoplasmic reticulum (ER), GN and ER/GN intermediate compartment (ERGIC) [73, 78, 79], within which it exhibits interphase-restricted

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

55

TrkB is the preferred receptor of BDNF but also binds NT-4/5 [65, 80-82]. The 590kb human *TrkB* gene maps to chromosome 9q22 and contains 24 exons [70, 83]. In addition to fully spliced gp145kDa TrkB, eight TrkB variant isoforms have been described, including a gp95kDa Cterminal truncated receptor that lacks the tyrosine kinase and Shc binding domains; a Cterminal truncated receptor that lacks the tyrosine kinase domain but retains the Shc binding site; a C-terminal truncated receptor that lacks exons 23 and 24 but retains tyrosine kinase activity and four N-terminal truncated receptors that exclude combinations of exons 1-5 and upstream signal sequence [75, 83-85]. The *TrkB* gene has also been reported to encode up to 100 different transcripts ranging from 0.7-9kb, at least 36 of which can be translated into functional TrkB proteins [85-87]. Both full length and C-terminal truncated TrkB receptors are expressed in the brain and share 100% extracellular domain homology, consisting of 5 highly

TrkC binds NT-3 and no other NT [88]. The 387kb human *TrkC* gene maps to chromosome 15q25 and is organised into 18 exons [70] and six TrkC isoforms have been described. In addition to the fully spliced gp145kDa receptor, these isoforms include C14/K2, C25/K3 and C39 variants which contain 14, 25 and 39 additional amino acid insertions between kinase subdomains VII and VIII, downstream of the TDYYR motif of the putative Trk receptor family autophosphorylation site [89] and NC1/T1 and NC-2/T2 non-catalytic variants truncated in the tyrosine kinase domain by short C-terminal sequences [90-92]. Full-length TrkC receptors are expressed during development, whereas truncated receptors predominate in later life in post mitotic cerebellar granule neurons and young stem cell-derived differentiated neurons but not in proliferating neural stem cells. TrkC NC1/T1 and NC2/T2 variants do not support NT-induced neuritogenesis, suggesting that TrkC variants could exert different roles during

The p75 neurotrophin receptor (CD271/p75NTR) is a member of the tumour necrosis factor (TNFR)/FAS receptor superfamily and binds all NTs in pro-form with high affinity and mature NGF with low affinity [94, 95]. The 3.4kb *CD271/p75NTR* gene is organised into 5 exons and maps to chromosome 17q21-q22 [96]. In addition to the fully spliced 75kDa CD271/p75NTR receptor, a truncated alternative s-p75NTR splice variant has been described that is devoid of exon III. S-p75NTR lacks the NT binding domain, does not bind NTs and is expressed by several neural tissues [97]. The fully spliced CD271/p75NTR extracellular-domain contains four 40-

spontaneous ligand-independent activation [73, 78, 79].

glycosylated extracellular binding domains (D1-5) [75, 85, 86].

nervous system development [90, 93].

*4.2.2. CD271/p75 neurotrophin receptor*

#### **4.2. NT receptors**

#### *4.2.1. Tropomyosin-related kinases TrkA, TrkB and TrkC*

The family of NT receptors includes the tropomyosin-related tyrosine kinases TrkA, TrkB and TrkC [64]. TrkA is the preferred receptor of mature NGF but also binds the mature neurotro‐ phin NT-3 [64, 65]. Identified following the discovery of the first tumour-associated TrkA oncogene [64, 66, 67], the 25kb human *TrkA* gene maps to chromosome 1q21-22 and is organised into 17 exons [68-70]. TrkA proteins are expressed either as the fully spliced gp140kDa TrkAII receptor, alternatively spliced TrkA L0 and L1 variants that exhibit differ‐ ential exon 2-4 use [71], the TrkAI variant that exhibits exon 9 skipping [72] or the TrkAIII variant, which exhibits in-frame skipping of exons 6 and 7 combined with exon 9 omission [73]. TrkA L0 (exons 2, 3 and 4 alternatively spliced) and TrkA L1 (exons 2 and 3 alternatively spliced) are expressed during rat development [71] as truncated receptors with in-frame deletions of leucine-rich sequences encoded within exons 2-4 [68]. Since, TrkA leucine rich sequences may modulate ligand binding [74], these variants may exhibit altered ligandbinding activity similar to analogous alternative TrkB splice variants [75]. TrkAI (exon 9 exclusion) and TrkAII (exon 9 inclusion) splice variants [72] are expressed as cell surface transmembrane receptors and exon 9 omission does not result in ligand-independent receptor activation. TrkAI and TrkAII variants bind NGF and NT3 [72, 76] but TrkAII exhibits higher levels of NT-3-mediated activation when co-expressed with the low affinity neurotrophin receptor CD271/p75NTR [76]. TrkAII is predominantly expressed within the nervous system, whereas TrkAI expression predominates in the thymus [72].

TrkAIII was identified as an unexpected RT-PCR product in primary human NBs [73]. This variant exhibits exon 6 and 7 skipping plus exon 9 omission, resulting in the in-frame deletion of amino acids 192-284 that encode the D4 extracellular immunoglobulin-like domain, several functional N-glycosylation sites and introduces a valine substitution at the novel exon 5/8 splice junction [73]. In addition to being expressed by primary human NBs, TrkAIII is also developmentally regulated and is detected from stages E13-E18 of mouse embryonic devel‐ opment and is also expressed by immature thymocytes within the developing thymus [73, 77]. Unlike fully spliced TrkA receptors, TrkAIII is not expressed at the cell surface but is retained within intracellular membranes of the endoplasmic reticulum (ER), GN and ER/GN intermediate compartment (ERGIC) [73, 78, 79], within which it exhibits interphase-restricted spontaneous ligand-independent activation [73, 78, 79].

TrkB is the preferred receptor of BDNF but also binds NT-4/5 [65, 80-82]. The 590kb human *TrkB* gene maps to chromosome 9q22 and contains 24 exons [70, 83]. In addition to fully spliced gp145kDa TrkB, eight TrkB variant isoforms have been described, including a gp95kDa Cterminal truncated receptor that lacks the tyrosine kinase and Shc binding domains; a Cterminal truncated receptor that lacks the tyrosine kinase domain but retains the Shc binding site; a C-terminal truncated receptor that lacks exons 23 and 24 but retains tyrosine kinase activity and four N-terminal truncated receptors that exclude combinations of exons 1-5 and upstream signal sequence [75, 83-85]. The *TrkB* gene has also been reported to encode up to 100 different transcripts ranging from 0.7-9kb, at least 36 of which can be translated into functional TrkB proteins [85-87]. Both full length and C-terminal truncated TrkB receptors are expressed in the brain and share 100% extracellular domain homology, consisting of 5 highly glycosylated extracellular binding domains (D1-5) [75, 85, 86].

TrkC binds NT-3 and no other NT [88]. The 387kb human *TrkC* gene maps to chromosome 15q25 and is organised into 18 exons [70] and six TrkC isoforms have been described. In addition to the fully spliced gp145kDa receptor, these isoforms include C14/K2, C25/K3 and C39 variants which contain 14, 25 and 39 additional amino acid insertions between kinase subdomains VII and VIII, downstream of the TDYYR motif of the putative Trk receptor family autophosphorylation site [89] and NC1/T1 and NC-2/T2 non-catalytic variants truncated in the tyrosine kinase domain by short C-terminal sequences [90-92]. Full-length TrkC receptors are expressed during development, whereas truncated receptors predominate in later life in post mitotic cerebellar granule neurons and young stem cell-derived differentiated neurons but not in proliferating neural stem cells. TrkC NC1/T1 and NC2/T2 variants do not support NT-induced neuritogenesis, suggesting that TrkC variants could exert different roles during nervous system development [90, 93].

#### *4.2.2. CD271/p75 neurotrophin receptor*

NTs exhibit close structural homology, with the exception of NT4/5 that exhibits only 50% homology to the others NTs, and all contain six conserved cysteines that form structurally important disulphide bridges [55, 56]. NTs are expressed by both neuronal and non-neuronal cells as pre-NTs and are converted to pro-NTs upon signal peptide removal. This can occur within the endoplasmic reticulum (ER), in which NTs are converted to mature-NTs by furins. Alternatively, NTs are transported to the cell surface and released following signal peptide removal as pro-NTs. Secreted pro-NTs, which also exhibit biological activity, are converted to mature NTs by enzymes including plasmin and the matrix metalloproteinases MMP-7 and MMP-9 [56-58]. Within the extracellular environment, pro- and mature NTs form homo-dimers and bind specific receptors to induce an array of biological activities, including cell migration, proliferation, survival, differentiation, apoptosis and neuronal synapse/junction plasticity, depending upon the cell population, receptor expression and activation status [57, 60]. The human *NGF* gene localises to chromosome 1p13.1 [61], the human *BDNF* gene localises to chromosome 11p13 [62], the human *NT-3* gene localises to chromosome 12p13 [62] and the human *NT4/5* gene localises to chromosome 19q13.3 [63]. Since the discovery of NTs, their respective receptors have been identified and many of their roles in nervous system develop‐

The family of NT receptors includes the tropomyosin-related tyrosine kinases TrkA, TrkB and TrkC [64]. TrkA is the preferred receptor of mature NGF but also binds the mature neurotro‐ phin NT-3 [64, 65]. Identified following the discovery of the first tumour-associated TrkA oncogene [64, 66, 67], the 25kb human *TrkA* gene maps to chromosome 1q21-22 and is organised into 17 exons [68-70]. TrkA proteins are expressed either as the fully spliced gp140kDa TrkAII receptor, alternatively spliced TrkA L0 and L1 variants that exhibit differ‐ ential exon 2-4 use [71], the TrkAI variant that exhibits exon 9 skipping [72] or the TrkAIII variant, which exhibits in-frame skipping of exons 6 and 7 combined with exon 9 omission [73]. TrkA L0 (exons 2, 3 and 4 alternatively spliced) and TrkA L1 (exons 2 and 3 alternatively spliced) are expressed during rat development [71] as truncated receptors with in-frame deletions of leucine-rich sequences encoded within exons 2-4 [68]. Since, TrkA leucine rich sequences may modulate ligand binding [74], these variants may exhibit altered ligandbinding activity similar to analogous alternative TrkB splice variants [75]. TrkAI (exon 9 exclusion) and TrkAII (exon 9 inclusion) splice variants [72] are expressed as cell surface transmembrane receptors and exon 9 omission does not result in ligand-independent receptor activation. TrkAI and TrkAII variants bind NGF and NT3 [72, 76] but TrkAII exhibits higher levels of NT-3-mediated activation when co-expressed with the low affinity neurotrophin receptor CD271/p75NTR [76]. TrkAII is predominantly expressed within the nervous system,

TrkAIII was identified as an unexpected RT-PCR product in primary human NBs [73]. This variant exhibits exon 6 and 7 skipping plus exon 9 omission, resulting in the in-frame deletion

ment and function have been elucidated.

*4.2.1. Tropomyosin-related kinases TrkA, TrkB and TrkC*

whereas TrkAI expression predominates in the thymus [72].

**4.2. NT receptors**

54 Neuroblastoma

The p75 neurotrophin receptor (CD271/p75NTR) is a member of the tumour necrosis factor (TNFR)/FAS receptor superfamily and binds all NTs in pro-form with high affinity and mature NGF with low affinity [94, 95]. The 3.4kb *CD271/p75NTR* gene is organised into 5 exons and maps to chromosome 17q21-q22 [96]. In addition to the fully spliced 75kDa CD271/p75NTR receptor, a truncated alternative s-p75NTR splice variant has been described that is devoid of exon III. S-p75NTR lacks the NT binding domain, does not bind NTs and is expressed by several neural tissues [97]. The fully spliced CD271/p75NTR extracellular-domain contains four 40amino acid repeats with 6 cysteine residues at conserved positions that are required for NT binding, a serine/threonine-rich region, a single transmembrane domain and a 155-amino acid cytoplasmic domain, which does not exhibit catalytic activity. CD271/p75NTR acts either as an independent NT receptor, a NT receptor complex with Sortilin or a co-receptor for TrkA, and is involved in regulating death, differentiation or survival signals [94, 98]. The CD271/P75NTR receptor is devoid of intrinsic catalytic activity, indicating that signalling from this receptor must depend upon intracellular interactors [99, 100].

and prevent spontaneous receptor oligomerisation and activation. Deletions, chimeric receptors and point mutations that disrupt the structure of the first (D4) and second (D5) immunoglobulin-like domains result in ligand-independent spontaneous receptor activation

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

57

CD271/p75NTR receptors modulate the affinity and enhance the specificity of TrkA for NGF, and TrkB for BDNF, with optimal affinity reflecting the ratio of Trk to CD271/p75NTR receptors [116-118]. In contrast, CD271/p75NTR reduces TrkAI activity in response to NT-3 and TrkB activity in response to NT-3 and NT-4/5 [76, 119, 120]. The CD271/p75NTR receptor analogue neurotrophin-related homolog-2 (NRH2) that is expressed by neural cells, also interacts with

In the absence of ligand, Trk receptors are maintained as inactive oligomers [120], concentrated within caveolin and cholesterol-containing cell membrane caveolae invaginations, which also contain components of the Ras signalling pathway [122]. Receptor oligomers are maintained in an inactive state by mature extracellular domain N-glycosylation, intact D4 and D5 domains and by receptor-associated protein tyrosine phosphatases (PTPases) [110, 123-126]. Upon ligand binding, oligomeric Trk receptors dimerize, alter their conformation and acquire tyrosine kinase activity, facilitated by temporary inactivation of receptor-associated PTPases, which results in auto- and trans-phosphorylation of receptor tyrosine residues Y490, Y670, Y674/675, Y751 and Y785, in TrkA and their equivalents in TrkB and TrkC. These tyrosines act as phosphorylation-dependent binding sites for a variety of signalling proteins, including the adapters Shc and FRS-2; Grb-2 and SOS; the IP3K subunit p85α and PLCγ. These interactions, which are modulated by CD271/p75NTR, provide avenues for signal transduction through RAS/ MAPK, IP3K/Akt/NF-κB and PKC pathways that mediate NT effects upon migration, prolif‐ eration, survival, differentiation and apoptosis [73, 111, 127-141]. Cell surface Trk localisation and NT-mediated Trk activation also involves interaction with the heat shock protein chap‐

and the acquisition of oncogenic activity [73, 110, 115] (Fig. 5).

TrkA to promote high affinity NGF binding [121].

**4.4. NT receptor signalling**

erone Hsp90 [78].

**Figure 6.** Trk receptor signalling and outcome

#### *4.2.3. Sortilin*

Sortilin is a member of the Vps10p domain-containing transmembrane proteins that binds both mature NGF and the neurotrophins NGF, BDNF and NT-3 in pro-form [98, 101, 102]. The 7kb human *Sortilin* gene localises to chromosome 1p13.3 and is expressed as a gp95-100kda glycoprotein [103, 104]. Sortilin co-expression with CD271/p75NTR results in the formation of a co-receptor complex that augments affinity for proNGF and acts principally as an inducer of apoptosis [105].

#### **4.3. NT receptor structure and ligand binding**

All three Trk receptors share significant sequence homology and a conserved domain organ‐ ization. This organization comprises from N-terminus to C-terminus of five extracellular domains, a transmembrane region and the intracellular kinase domain.

**Figure 5.** NT receptor structure and ligand binding domains

The first three extracellular domains consist of a leucine-rich region (D-2) flanked by two cysteine-rich regions (D-1 and D-3), and domains 4 and 5 are immunoglobulin-like domains. Studies on TrkB and TrkC have shown that D-5 is sufficient for the binding of ligands and is responsible for binding specificity [106-109], although the D-4 domain, leucines and cysteine clusters may regulate ligand binding [55, 73]. Receptor transmembrane and juxta-membrane regions are critical for signal internalisation and transduction. The intracellular tyrosine-rich carboxyl terminal cytoplasmic domain exhibits tyrosine kinase activity upon ligand-mediated activation and is responsible for propagating post-receptor signal transduction [74, 107, 110-114]. The immunoglobulin-like D4 and D5 domains stabilise receptors in monomeric form and prevent spontaneous receptor oligomerisation and activation. Deletions, chimeric receptors and point mutations that disrupt the structure of the first (D4) and second (D5) immunoglobulin-like domains result in ligand-independent spontaneous receptor activation and the acquisition of oncogenic activity [73, 110, 115] (Fig. 5).

CD271/p75NTR receptors modulate the affinity and enhance the specificity of TrkA for NGF, and TrkB for BDNF, with optimal affinity reflecting the ratio of Trk to CD271/p75NTR receptors [116-118]. In contrast, CD271/p75NTR reduces TrkAI activity in response to NT-3 and TrkB activity in response to NT-3 and NT-4/5 [76, 119, 120]. The CD271/p75NTR receptor analogue neurotrophin-related homolog-2 (NRH2) that is expressed by neural cells, also interacts with TrkA to promote high affinity NGF binding [121].

#### **4.4. NT receptor signalling**

amino acid repeats with 6 cysteine residues at conserved positions that are required for NT binding, a serine/threonine-rich region, a single transmembrane domain and a 155-amino acid cytoplasmic domain, which does not exhibit catalytic activity. CD271/p75NTR acts either as an independent NT receptor, a NT receptor complex with Sortilin or a co-receptor for TrkA, and is involved in regulating death, differentiation or survival signals [94, 98]. The CD271/P75NTR receptor is devoid of intrinsic catalytic activity, indicating that signalling from this receptor

Sortilin is a member of the Vps10p domain-containing transmembrane proteins that binds both mature NGF and the neurotrophins NGF, BDNF and NT-3 in pro-form [98, 101, 102]. The 7kb human *Sortilin* gene localises to chromosome 1p13.3 and is expressed as a gp95-100kda glycoprotein [103, 104]. Sortilin co-expression with CD271/p75NTR results in the formation of a co-receptor complex that augments affinity for proNGF and acts principally as an inducer of

All three Trk receptors share significant sequence homology and a conserved domain organ‐ ization. This organization comprises from N-terminus to C-terminus of five extracellular

The first three extracellular domains consist of a leucine-rich region (D-2) flanked by two cysteine-rich regions (D-1 and D-3), and domains 4 and 5 are immunoglobulin-like domains. Studies on TrkB and TrkC have shown that D-5 is sufficient for the binding of ligands and is responsible for binding specificity [106-109], although the D-4 domain, leucines and cysteine clusters may regulate ligand binding [55, 73]. Receptor transmembrane and juxta-membrane regions are critical for signal internalisation and transduction. The intracellular tyrosine-rich carboxyl terminal cytoplasmic domain exhibits tyrosine kinase activity upon ligand-mediated activation and is responsible for propagating post-receptor signal transduction [74, 107, 110-114]. The immunoglobulin-like D4 and D5 domains stabilise receptors in monomeric form

domains, a transmembrane region and the intracellular kinase domain.

must depend upon intracellular interactors [99, 100].

**4.3. NT receptor structure and ligand binding**

**Figure 5.** NT receptor structure and ligand binding domains

*4.2.3. Sortilin*

56 Neuroblastoma

apoptosis [105].

In the absence of ligand, Trk receptors are maintained as inactive oligomers [120], concentrated within caveolin and cholesterol-containing cell membrane caveolae invaginations, which also contain components of the Ras signalling pathway [122]. Receptor oligomers are maintained in an inactive state by mature extracellular domain N-glycosylation, intact D4 and D5 domains and by receptor-associated protein tyrosine phosphatases (PTPases) [110, 123-126]. Upon ligand binding, oligomeric Trk receptors dimerize, alter their conformation and acquire tyrosine kinase activity, facilitated by temporary inactivation of receptor-associated PTPases, which results in auto- and trans-phosphorylation of receptor tyrosine residues Y490, Y670, Y674/675, Y751 and Y785, in TrkA and their equivalents in TrkB and TrkC. These tyrosines act as phosphorylation-dependent binding sites for a variety of signalling proteins, including the adapters Shc and FRS-2; Grb-2 and SOS; the IP3K subunit p85α and PLCγ. These interactions, which are modulated by CD271/p75NTR, provide avenues for signal transduction through RAS/ MAPK, IP3K/Akt/NF-κB and PKC pathways that mediate NT effects upon migration, prolif‐ eration, survival, differentiation and apoptosis [73, 111, 127-141]. Cell surface Trk localisation and NT-mediated Trk activation also involves interaction with the heat shock protein chap‐ erone Hsp90 [78].

**Figure 6.** Trk receptor signalling and outcome

Trk receptors activated by NTs use two main pathways to activate MAPKs. The first pathway involves Shc, Grb-2, SOS, Ras and Raf, and the second pathway involves CrkL, Rap and Raf [142, 143]. Trk activation of MAPK is now considered to depend not only upon the phos‐ phorylated Trk Y490 tyrosine residue [144, 145] but also the ankyrin repeat-rich membrane spanning protein ARMS, acting through CrkL [146, 147]. MAPKs activate CREB transcription factors to promote differentiation and survival [148-150]. Trk activation of PI3K/Akt signalling occurs through Shc/Grb-2 and Gab-1 and induces pro-survival signals [73, 151, 152], resulting from the phosphorylation of Bad and activates the pro-survival transcription factor NF-κB [73, 153, 154]. PLCγ is activated as a consequence of being recruited to the phosphorylated Trk tyrosine Y785 [73, 132] and provides additional differentiation and survival signals that involve MAPK [155] (Fig. 6).

The alterative TrkAIII splice variant, in contrast to other Trk receptors (see above), is not expressed at the cell surface but accumulates within intracellular membranes. Intracellular TrkAIII does not bind extracellular NTs and is prone to spontaneous ligand-independent intracellular activation [73, 78, 79]. In contrast to ligand activated cell surface TrkA signalling, spontaneously active TrkAIII signals through PI3K/Akt/NF-κB but not Ras/MAPK, resulting in increased survival and the induction/maintenance of a stem cell-like undifferentiated phenotype [73, 78, 79, 156] (Fig. 6).

 expression, which is exemplified by the shift from NT-3/TrkC to NGF/TrkA dependence observed during SNS development [164]. CD271/p75NTR may also influence Trk signalling by binding of the Shc adapter, which also binds to activated Trk, to augment or inhibit Trk signalling [165, 166], and in Trk-complexed form may result in different signalling to that from Trk dimers alone [147], resulting in differences in capacity to complete differentiation pro‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

59

As a pro-apoptotic receptor CD271/p75NTR also exhibits Trk-independent activity. The cytoplasmic tail of CD271/p75NTR contains death domains and its role in apoptosis has been clearly demonstrated in CD271/p75NTR exon 3 knockout mice [168]. CD271/p75NTR exon 3 knockout mice combined with TrkA knockout mice have highlighted the dual function for CD271/p75NTR in refining innervation and eliminating neuronal excess during early develop‐ ment and later in neuronal survival [169, 170]. Apoptosis induced by CD271/p75NTR involves JNK, phosphorylated c-jun, p53, Bad, Bim and activated caspases [168, 169, 171-174]. Apoptosis induced by CD271/p75NTR may also involve β-secretase-mediated release of the intra-cyto‐ plasmic domain, its subsequent nuclear transport and potential involvement in transcriptional regulation, together with TRAF6, NRAGE, NADE, NRIF and SC-1. TRAF6 interaction with NRIF has been implicated in the generation of death signals through the activation of JNK [169, 175]. NRAGE interaction with CD271/p75NTR is involved in inducing cell death through JNK and caspase activation, and is blocked by TrkA [176]. A role for NADE in CD271/p75NTR– mediated apoptosis, involving NGF but not BDNF or NT-3, has been reported [177], whereas CD271/p75NTR interaction with SC-1 has been implicated in cell cycle arrest via transcriptional repression of cyclins [178] (Fig. 7). Further advances in the understanding of this effect have come with the observation that inactive pro-form NT precursors bind CD271/p75NTR receptors with high affinity and trigger apoptosis at far lower concentrations than active counterparts, which bind with low affinity (Lee et al., 2001). Up to 60% of NTs released by cells are proform [56]. Indeed proNGF induces death in CD271/p75NTR expressing cells, highlighting an opposite effect to activated NGF in cells, including sympathetic neurones [56]. The capacity of proNGF to activate CD271/p75NTR but not TrkA is now known to depend upon Sortilin, a 95kDa member

grams [167] (Fig. 7).

**Figure 7.** CD271/p75NTR receptor signalling and outcome

An additional feature of TrkA receptors is retrograde transport signalling within the cell. This depends upon receptor/ligand interaction, internalisation and retrograde transport of activat‐ ed receptors, resulting in signal transduction within the cell body. Sympathetic neurons most dramatically illustrate this activity, with retrograde transport of NGF-activated TrkA occur‐ ring along the axonal length to the neuronal cell body. This phenomenon involves ubiquitin mediated receptor internalisation through interaction with CD271/p75NTR and TRAF6, receptor endocytosis within clatherin-coated vesicles and receptor endocytosis facilitated by the endocytosis inducing protein EHD4/Pincher [157-159]. In addition, immature Trk receptors also localise to intracellular membranes of the Golgi Network (GN) and can be trans-activated by agonists of the G-protein linked A2A adenosine receptors, potentially through the nonreceptor tyrosine kinase Src [160, 161], providing evidence for intracellular neurotrophinindependent Trk activation. Post receptor signal transduction from GN-associated TrkA differs from cell surface-activated TrkA, by signalling through IP3K/Akt but not RAS/MAPK, which results in NF-κB transcription factor activation, inducing a more stress-resistant phenotype, not dissimilar to that induced by the intracellular alternative TrkAIII splice variant [73, 124, 160]. TrkA localisation to the GN may not only reflect transient passage of de-novo synthesised receptors but also alterations in receptor extracellular domain N-glycosylation and folding.

CD271/p75NTR receptors regulate cell survival, apoptosis, differentiation and proliferation. CD271/p75NTR is a positive modulator of Trk-mediated survival, and within this context, it is likely that CD271/p75NTR does not directly bind NTs in competition with Trks [162] but acts as a co-receptor, interacting with Trk dimers ligated to active NTs, refining receptor specificity (e.g. increasing specificity for NGF, while restricting NT-3 binding) [163]. This may be responsible for shifting NT dependence during development coincident with CD271/p75NTR Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma http://dx.doi.org/10.5772/55536 59

**Figure 7.** CD271/p75NTR receptor signalling and outcome

Trk receptors activated by NTs use two main pathways to activate MAPKs. The first pathway involves Shc, Grb-2, SOS, Ras and Raf, and the second pathway involves CrkL, Rap and Raf [142, 143]. Trk activation of MAPK is now considered to depend not only upon the phos‐ phorylated Trk Y490 tyrosine residue [144, 145] but also the ankyrin repeat-rich membrane spanning protein ARMS, acting through CrkL [146, 147]. MAPKs activate CREB transcription factors to promote differentiation and survival [148-150]. Trk activation of PI3K/Akt signalling occurs through Shc/Grb-2 and Gab-1 and induces pro-survival signals [73, 151, 152], resulting from the phosphorylation of Bad and activates the pro-survival transcription factor NF-κB [73, 153, 154]. PLCγ is activated as a consequence of being recruited to the phosphorylated Trk tyrosine Y785 [73, 132] and provides additional differentiation and survival signals that

The alterative TrkAIII splice variant, in contrast to other Trk receptors (see above), is not expressed at the cell surface but accumulates within intracellular membranes. Intracellular TrkAIII does not bind extracellular NTs and is prone to spontaneous ligand-independent intracellular activation [73, 78, 79]. In contrast to ligand activated cell surface TrkA signalling, spontaneously active TrkAIII signals through PI3K/Akt/NF-κB but not Ras/MAPK, resulting in increased survival and the induction/maintenance of a stem cell-like undifferentiated

An additional feature of TrkA receptors is retrograde transport signalling within the cell. This depends upon receptor/ligand interaction, internalisation and retrograde transport of activat‐ ed receptors, resulting in signal transduction within the cell body. Sympathetic neurons most dramatically illustrate this activity, with retrograde transport of NGF-activated TrkA occur‐ ring along the axonal length to the neuronal cell body. This phenomenon involves ubiquitin mediated receptor internalisation through interaction with CD271/p75NTR and TRAF6, receptor endocytosis within clatherin-coated vesicles and receptor endocytosis facilitated by the endocytosis inducing protein EHD4/Pincher [157-159]. In addition, immature Trk receptors also localise to intracellular membranes of the Golgi Network (GN) and can be trans-activated by agonists of the G-protein linked A2A adenosine receptors, potentially through the nonreceptor tyrosine kinase Src [160, 161], providing evidence for intracellular neurotrophinindependent Trk activation. Post receptor signal transduction from GN-associated TrkA differs from cell surface-activated TrkA, by signalling through IP3K/Akt but not RAS/MAPK, which results in NF-κB transcription factor activation, inducing a more stress-resistant phenotype, not dissimilar to that induced by the intracellular alternative TrkAIII splice variant [73, 124, 160]. TrkA localisation to the GN may not only reflect transient passage of de-novo synthesised receptors but also alterations in receptor extracellular domain N-glycosylation and

CD271/p75NTR receptors regulate cell survival, apoptosis, differentiation and proliferation. CD271/p75NTR is a positive modulator of Trk-mediated survival, and within this context, it is likely that CD271/p75NTR does not directly bind NTs in competition with Trks [162] but acts as a co-receptor, interacting with Trk dimers ligated to active NTs, refining receptor specificity (e.g. increasing specificity for NGF, while restricting NT-3 binding) [163]. This may be responsible for shifting NT dependence during development coincident with CD271/p75NTR

involve MAPK [155] (Fig. 6).

58 Neuroblastoma

phenotype [73, 78, 79, 156] (Fig. 6).

folding.

 expression, which is exemplified by the shift from NT-3/TrkC to NGF/TrkA dependence observed during SNS development [164]. CD271/p75NTR may also influence Trk signalling by binding of the Shc adapter, which also binds to activated Trk, to augment or inhibit Trk signalling [165, 166], and in Trk-complexed form may result in different signalling to that from Trk dimers alone [147], resulting in differences in capacity to complete differentiation pro‐ grams [167] (Fig. 7).

As a pro-apoptotic receptor CD271/p75NTR also exhibits Trk-independent activity. The cytoplasmic tail of CD271/p75NTR contains death domains and its role in apoptosis has been clearly demonstrated in CD271/p75NTR exon 3 knockout mice [168]. CD271/p75NTR exon 3 knockout mice combined with TrkA knockout mice have highlighted the dual function for CD271/p75NTR in refining innervation and eliminating neuronal excess during early develop‐ ment and later in neuronal survival [169, 170]. Apoptosis induced by CD271/p75NTR involves JNK, phosphorylated c-jun, p53, Bad, Bim and activated caspases [168, 169, 171-174]. Apoptosis induced by CD271/p75NTR may also involve β-secretase-mediated release of the intra-cyto‐ plasmic domain, its subsequent nuclear transport and potential involvement in transcriptional regulation, together with TRAF6, NRAGE, NADE, NRIF and SC-1. TRAF6 interaction with NRIF has been implicated in the generation of death signals through the activation of JNK [169, 175]. NRAGE interaction with CD271/p75NTR is involved in inducing cell death through JNK and caspase activation, and is blocked by TrkA [176]. A role for NADE in CD271/p75NTR– mediated apoptosis, involving NGF but not BDNF or NT-3, has been reported [177], whereas CD271/p75NTR interaction with SC-1 has been implicated in cell cycle arrest via transcriptional repression of cyclins [178] (Fig. 7). Further advances in the understanding of this effect have come with the observation that inactive pro-form NT precursors bind CD271/p75NTR receptors with high affinity and trigger apoptosis at far lower concentrations than active counterparts, which bind with low affinity (Lee et al., 2001). Up to 60% of NTs released by cells are proform [56]. Indeed proNGF induces death in CD271/p75NTR expressing cells, highlighting an opposite effect to activated NGF in cells, including sympathetic neurones [56]. The capacity of proNGF to activate CD271/p75NTR but not TrkA is now known to depend upon Sortilin, a 95kDa member of the Vps10p-domain receptor family [98, 101]. In this interaction, complexes between CD271/ p75NTR and Sortilin are augmented by proNGF, which simultaneously binds both receptors to induce apoptosis. Thus Sortilin acts as an essential co-receptor capable of switching cells that co-express TrkA and CD271/p75NTR from survival to apoptosis.

In a separate study, TrkA and TrkC but not TrkB induced apoptosis in neurons differentiated from stable transfected embryonic stem cells and promoted loss of all TrkA and TrkC but not TrkB transfected cells with associated loss of nervous system at E13.5 during mouse embryonic development, through a CD271/p75NTR -mediated mechanism, in which CD271/p75NTR is recruited as a "hired killer" [192]. Therefore in the absence of ligand, TrkC acts directly as a death receptor and TrkA death receptor activity appears to depend upon CD271/p75NTR,

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

61

TrkC is the only NT receptor expressed during early embryogenesis. During avian develop‐ ment TrkC expression coincides with neurulation and is detected in both neural tube and neural plate anlage [193, 194]. TrkC is also expressed in hindbrain rhombomeres 3 and 5. This, however, does not associate with lateral NCC migration, suggesting that either TrkC positive NCC cells die prior to NCC migration or that they migrate away from these regions. NT-3 expression is low at this time and the recent characterisation of TrkC as pro-apoptotic de‐ pendency receptor, supports the former hypothesis [191, 192]. Neither TrkC nor NT-3 knock‐ out prevent neurulation but do result in neuronal loss from sympathetic ganglia [195-197], indicating that TrkC/NT-3 interactions are not required for neurulation but are required for later stages of SNS development. Consistent with this, the NT-3 protein is detected at later developmental stages. There have been no reports concerning the expression of alternative

During PNS formation, TrkC is expressed by neurogenic pre-migratory and migrating NCC subsets [194, 198, 199] and TrkC/NT-3 interactions are required prior to NCC arrival at destination [200]. Indeed, NT-3 acts as a NCC survival factor and promotes NCC proliferation in the presence of somites [201]. Furthermore, somites express NT-3 during this period [198, 202], sympathetic neuroblasts and neurons also express TrkC and NT-3, NT-3 is expressed by non-neuronal sympathetic cells [194, 199, 203], and NT-3 and TrkC expression during this time is stimulated by neuroregulin, PDGF and CNTF [204]. NT-3 in sympathetic tissues increases mature neuron numbers by promoting the survival of proliferating neuroblast and their subsequent differentiation, without directly effecting proliferation [205]. This temporary effect subsequently declines with a switch to NGF-dependence [206], associated with reduced TrkC expression and the induction of TrkA and later CD271/p75NTR expression [204, 207]. NT-3 continues to be expressed by both sympathetic neural and non-neural cells [198, 199, 204], by adult non-neural cells [208] and TrkA expression is regulated in part by NT-3 [207]. Therefore, NT-3 acts as both a survival and differentiating factor through TrkA, eventually rendering differentiating post-mitotic neurons dependent upon NGF produced by effector tissues. NT-3, at this stage, acts as an autocrine interim and not peripherally derived paracrine factor, corroborated by the lack of target innervation at this time [207]. In support of this, NT-3 knockout mice exhibit sympathectomy [106, 197, 209-212] caused principally by neuroblast

whereas TrkB does not exhibit death receptor activity (Fig. 8)

**5.1. TrkC and NT–3**

TrkC isoforms during early development.

**5. NTs and NTRs in sympathetic nervous system development**

CD271/p75NTR receptors, therefore, promote either survival or death in response to NTs, depending upon NT status and the cellular context. Survival through CD271/p75NTR receptors involves NF-κB activated through TRFA6, p62, Interleukin-1 receptor-associated kinase IRAK and receptor interacting protein RIP2 [179]. CD271/p75NTR promotion of axon growth involves neurotrophin-mediated dissociation of axonal growth inhibitory complexes between CD271/p75NTR and the G-protein Rho [180]. Furthermore, the proteolytic shedding of cell surface CD271/p75NTR releases an intracellular domain that moves to the nucleus and may act as a transcription factor [181].

#### **4.5. Trks A and C are dependence receptors**

A classical concept is that NT activation of Trk receptors inhibits default apoptotic programs to promote NT-dependent survival [134]. This concept is considered to involve PI3K/Akt/NFκB signalling and induction of Bcl-2 inhibitor of mitochondrial apoptosis. In this mechanism, NT depletion results in the turning-off of PI3K/Akt signalling, which reduces Bad phosphor‐ ylation and releases it from the chaperone 14-3-3. This results in Bcl-2 and Bcl-XL sequestration [182, 183], reduces FOX03A phosphorylation resulting in nuclear translocation, induces proapoptotic FAS, Trail, Puma and BIM transcription [184-188], abrogates CREB and NF-κB survival signals [189, 190] and activates pro-apoptotic JNK, inducing BIM expression [188], which together trigger apoptosis (Fig. 8).

**Figure 8.** TrkA and TrkC receptors and Apoptosis

Recently, however, both TrkA and TrkC have also been characterised as true dependence receptors. In one study, TrkC but not TrkA or TrkB triggered apoptosis in the absence of NT-3 in a variety of cell lines by an activated caspase-dependent cleavage mechanism, releasing a pro-apoptotic intracellular TrkC domain capable of inducing caspase-9 dependent death [191]. In a separate study, TrkA and TrkC but not TrkB induced apoptosis in neurons differentiated from stable transfected embryonic stem cells and promoted loss of all TrkA and TrkC but not TrkB transfected cells with associated loss of nervous system at E13.5 during mouse embryonic development, through a CD271/p75NTR -mediated mechanism, in which CD271/p75NTR is recruited as a "hired killer" [192]. Therefore in the absence of ligand, TrkC acts directly as a death receptor and TrkA death receptor activity appears to depend upon CD271/p75NTR, whereas TrkB does not exhibit death receptor activity (Fig. 8)

#### **5. NTs and NTRs in sympathetic nervous system development**

#### **5.1. TrkC and NT–3**

of the Vps10p-domain receptor family [98, 101]. In this interaction, complexes between CD271/ p75NTR and Sortilin are augmented by proNGF, which simultaneously binds both receptors to induce apoptosis. Thus Sortilin acts as an essential co-receptor capable of switching cells that

CD271/p75NTR receptors, therefore, promote either survival or death in response to NTs, depending upon NT status and the cellular context. Survival through CD271/p75NTR receptors involves NF-κB activated through TRFA6, p62, Interleukin-1 receptor-associated kinase IRAK and receptor interacting protein RIP2 [179]. CD271/p75NTR promotion of axon growth involves neurotrophin-mediated dissociation of axonal growth inhibitory complexes between CD271/p75NTR and the G-protein Rho [180]. Furthermore, the proteolytic shedding of cell surface CD271/p75NTR releases an intracellular domain that moves to the nucleus and may act

A classical concept is that NT activation of Trk receptors inhibits default apoptotic programs to promote NT-dependent survival [134]. This concept is considered to involve PI3K/Akt/NFκB signalling and induction of Bcl-2 inhibitor of mitochondrial apoptosis. In this mechanism, NT depletion results in the turning-off of PI3K/Akt signalling, which reduces Bad phosphor‐ ylation and releases it from the chaperone 14-3-3. This results in Bcl-2 and Bcl-XL sequestration [182, 183], reduces FOX03A phosphorylation resulting in nuclear translocation, induces proapoptotic FAS, Trail, Puma and BIM transcription [184-188], abrogates CREB and NF-κB survival signals [189, 190] and activates pro-apoptotic JNK, inducing BIM expression [188],

Recently, however, both TrkA and TrkC have also been characterised as true dependence receptors. In one study, TrkC but not TrkA or TrkB triggered apoptosis in the absence of NT-3 in a variety of cell lines by an activated caspase-dependent cleavage mechanism, releasing a pro-apoptotic intracellular TrkC domain capable of inducing caspase-9 dependent death [191].

co-express TrkA and CD271/p75NTR from survival to apoptosis.

as a transcription factor [181].

60 Neuroblastoma

**4.5. Trks A and C are dependence receptors**

which together trigger apoptosis (Fig. 8).

**Figure 8.** TrkA and TrkC receptors and Apoptosis

TrkC is the only NT receptor expressed during early embryogenesis. During avian develop‐ ment TrkC expression coincides with neurulation and is detected in both neural tube and neural plate anlage [193, 194]. TrkC is also expressed in hindbrain rhombomeres 3 and 5. This, however, does not associate with lateral NCC migration, suggesting that either TrkC positive NCC cells die prior to NCC migration or that they migrate away from these regions. NT-3 expression is low at this time and the recent characterisation of TrkC as pro-apoptotic de‐ pendency receptor, supports the former hypothesis [191, 192]. Neither TrkC nor NT-3 knock‐ out prevent neurulation but do result in neuronal loss from sympathetic ganglia [195-197], indicating that TrkC/NT-3 interactions are not required for neurulation but are required for later stages of SNS development. Consistent with this, the NT-3 protein is detected at later developmental stages. There have been no reports concerning the expression of alternative TrkC isoforms during early development.

During PNS formation, TrkC is expressed by neurogenic pre-migratory and migrating NCC subsets [194, 198, 199] and TrkC/NT-3 interactions are required prior to NCC arrival at destination [200]. Indeed, NT-3 acts as a NCC survival factor and promotes NCC proliferation in the presence of somites [201]. Furthermore, somites express NT-3 during this period [198, 202], sympathetic neuroblasts and neurons also express TrkC and NT-3, NT-3 is expressed by non-neuronal sympathetic cells [194, 199, 203], and NT-3 and TrkC expression during this time is stimulated by neuroregulin, PDGF and CNTF [204]. NT-3 in sympathetic tissues increases mature neuron numbers by promoting the survival of proliferating neuroblast and their subsequent differentiation, without directly effecting proliferation [205]. This temporary effect subsequently declines with a switch to NGF-dependence [206], associated with reduced TrkC expression and the induction of TrkA and later CD271/p75NTR expression [204, 207]. NT-3 continues to be expressed by both sympathetic neural and non-neural cells [198, 199, 204], by adult non-neural cells [208] and TrkA expression is regulated in part by NT-3 [207]. Therefore, NT-3 acts as both a survival and differentiating factor through TrkA, eventually rendering differentiating post-mitotic neurons dependent upon NGF produced by effector tissues. NT-3, at this stage, acts as an autocrine interim and not peripherally derived paracrine factor, corroborated by the lack of target innervation at this time [207]. In support of this, NT-3 knockout mice exhibit sympathectomy [106, 197, 209-212] caused principally by neuroblast apoptosis, which is partially rescued by exogenous NGF [212]. In the adult, NT-3 continues to be expressed by a wide range of tissues [202, 203, 213, 214] and, together with NGF, continues to be important for post-natal sympathetic neuron survival [214, 215]. Consistent with this, exogenous NT-3 promotes target organ sympathetic innervation in NT-3 knockout animals [211, 212], suggesting that the switch from NT-3 to NGF dependence observed in sympathetic neurons *in vitro* [216] does not actually occur *in vivo.* This may relate to environmental differences, corroborated by the mitogenic effect of NT-3 on neuroblasts *in vitro* [201] but not *in vivo* [217], the susceptibility of TrkC transcription to environmental factors and also by the capacity of NT-3 to bind and activate TrkA receptors, and in particular TrkAII [76]. This helps to explain how NT-3 rescues NGF-dependent neurons from NGF depletion and *vice versa* and is consistent with the characterisation of TrkA as a functional NT-3 receptor *in vivo.* However, one difference between these two NTs is that exogenous NGF but not NT-3 induces sympa‐ thetic ganglia hyperplasia [218].

is expressed by both the developing and adult adrenal gland [229]. This suggests that TrkA/ NGF interactions are of transient importance in adrenal gland development. Consistent with this, both TrkA and NGF knockout mice exhibit a relatively normal adrenal medulla chromaffin cell content, although cholinergic innervation of pre-ganglionic origin is lost in TrkA knockout animals [230], and both chromaffin and SIF cells express TrkA but do not depend upon NGF for survival. Normal NCC progenitors entering the developing adrenal glands express TrkC and begin to express TrkA upon seeding under the influence of the adrenal environment. This event may depend upon NT-3 and/or NT4/5 expressed by the adrenal cortex anlage, with subsequent chromaffin/SIF differentiation and survival regulat‐ ed by these NTs. In contrast, sympathetic neurones in paravertebral sympathetic ganglia, despite their common origin, express TrkA and require NGF for their development, differentiation and survival [229-231]. In support of this, NGF neutralising antibodies do not delay adrenal development nor induce chromaffin cell degeneration [232]. Differences between human and rodent adrenal development include observations that the adult rat adrenal cortex but not medulla express TrkB or TrkC [229, 230], whereas TrkA immunoreac‐ tivity is restricted to the adrenal cortex and TrkC immunoreactivity to the adrenal medulla with no TrkB immunoreactivity detected in the human adult adrenal glands [233]. Interest‐ ingly, stress induces a massive release of NGF from salivary glands, which targets adrenal chromaffin cells inducing marked adrenal medullary hyperplasia and catecholamine synthesis through enhanced TH and BDH expression [234-236]. In chromaffin tissues, sympathoadrenal cells of the carotid body express NGF and TrkA, providing an autocrine/ paracrine mechanism [237]. Pre-natal and post-natal differentiating and differentiated chromaffin cells express TrkA mRNA within the adrenal medulla [238], which increases with development, at times when NGF expression is all but absent [229]. TrkA knockout elimi‐ nates the acetylcholine positive component but does not influence chromaffin content of the adrenal medulla [230], indicating that chromaffin cells, unlike their sympathetic neuronal cousins, do not depend upon NGF/TrkA interactions for survival [232, 239]. Chromaffin cells do, however, respond to NGF with acute hyperplasia [235] and eventual neuronal differen‐ tiation [240, 241]. In rodents, immature sympathoblasts within sympathetic ganglia cells express TrkA from E14 onwards and express CD271/p75NTR from E16 to birth, in associa‐ tion with acquisition of NGF-responsiveness [242]. Differentiated neurons within sympathet‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

63

TrkB, like TrkA, is also not expressed during neurulation but is expressed by motor progenitors in hindbrain rhombomere 2 at later stages 9-10 and 12, during avian development, either side of the floor plate in the caudal midbrain, extending through the hindbrain and into the spinal chord [193]. Alternative TrkB splice variant expression has not been assessed during early

Following neurulation, TrkB expression is detected within motor neuron progenitors of the ventral neural tube and corresponds to BDNF expression by elements within dorsal neural tube, which coordinate motor neuron development [243]. Consistent with this, both TrkB and

ic ganglia express TrkA but not NGF [208].

**5.3. TrkB, BDNF and NT4/5**

development.

NT-3 released from effector tissues and acting through TrkA also promotes sympathetic innervation of target organs [202, 213, 214, 219, 220]. In support of this, effector tissue elimi‐ nation induces the death of innervating neurons, which cannot be completely reversed by exogenous NGF alone, and adult sympathetic neurons expressing TrkA are immunoreactive for both NGF and NT-3 [49, 221, 222]. Therefore, NT-3 plays an important role throughout sympathetic neuron life-cycle from neuroblast to neuron, acting initially through the TrkC receptor as an autocrine/paracrine factor stimulator of migration and survival in proliferating sympathetic neuroblasts and later as a paracrine promoter of sympathetic neuron differentia‐ tion, survival and target organ innervation acting through the TrkA receptor. CD271/p75NTR is also required for optimal neurotrophin sensitivity since CD271/p75NTR deficient dorsal root and sympathetic neurons exhibit reduced sensitivity to NGF [223].

During sympathoadrenal development, progenitors switch from being dependent upon NT3 and TrkC to dependence upon NGF and TrkA, through an intermediate stage of combined TrkA and TrkC expression. In murine thoracic sympathetic ganglia TrkC expression alone is detected at E14-15, whereas both TrkA and TrkC expression are detected at E16.5-17 and only TrkA at E19.5 [224]. Interestingly, sympathetic chromaffin tissues of the adrenal medulla and paraganglia, which form in parallel to sympathetic ganglia, exhibit differences in NT receptor expression consistent with upstream progenitor separation. This difference is characterised by the expression of TrkC but not TrkA by NCCs migrating into the adrenal anlage, at times when TrkC expression is lost in associated with the induction of TrkA expression by NCCs within sympathetic ganglia [225].

#### **5.2. TrkA and NGF**

Unlike TrkC, TrkA is not expressed during neurulation, NC development or NCC dorsolat‐ eral or vetrolateral migration. In rodent development, TrkA is detected at E12.5 within sensory cranial and spinal dorsal root ganglia and subsequently in the paravertebral sympathetic ganglia [226]. NGF is expressed during the mid-stage of development initiating within CNS structures then within PNS structures at later stages of development [227, 228]. Within the developing adrenal gland NGF exhibits a brief period of post-natal expression, whereas NT-3 is expressed by both the developing and adult adrenal gland [229]. This suggests that TrkA/ NGF interactions are of transient importance in adrenal gland development. Consistent with this, both TrkA and NGF knockout mice exhibit a relatively normal adrenal medulla chromaffin cell content, although cholinergic innervation of pre-ganglionic origin is lost in TrkA knockout animals [230], and both chromaffin and SIF cells express TrkA but do not depend upon NGF for survival. Normal NCC progenitors entering the developing adrenal glands express TrkC and begin to express TrkA upon seeding under the influence of the adrenal environment. This event may depend upon NT-3 and/or NT4/5 expressed by the adrenal cortex anlage, with subsequent chromaffin/SIF differentiation and survival regulat‐ ed by these NTs. In contrast, sympathetic neurones in paravertebral sympathetic ganglia, despite their common origin, express TrkA and require NGF for their development, differentiation and survival [229-231]. In support of this, NGF neutralising antibodies do not delay adrenal development nor induce chromaffin cell degeneration [232]. Differences between human and rodent adrenal development include observations that the adult rat adrenal cortex but not medulla express TrkB or TrkC [229, 230], whereas TrkA immunoreac‐ tivity is restricted to the adrenal cortex and TrkC immunoreactivity to the adrenal medulla with no TrkB immunoreactivity detected in the human adult adrenal glands [233]. Interest‐ ingly, stress induces a massive release of NGF from salivary glands, which targets adrenal chromaffin cells inducing marked adrenal medullary hyperplasia and catecholamine synthesis through enhanced TH and BDH expression [234-236]. In chromaffin tissues, sympathoadrenal cells of the carotid body express NGF and TrkA, providing an autocrine/ paracrine mechanism [237]. Pre-natal and post-natal differentiating and differentiated chromaffin cells express TrkA mRNA within the adrenal medulla [238], which increases with development, at times when NGF expression is all but absent [229]. TrkA knockout elimi‐ nates the acetylcholine positive component but does not influence chromaffin content of the adrenal medulla [230], indicating that chromaffin cells, unlike their sympathetic neuronal cousins, do not depend upon NGF/TrkA interactions for survival [232, 239]. Chromaffin cells do, however, respond to NGF with acute hyperplasia [235] and eventual neuronal differen‐ tiation [240, 241]. In rodents, immature sympathoblasts within sympathetic ganglia cells express TrkA from E14 onwards and express CD271/p75NTR from E16 to birth, in associa‐ tion with acquisition of NGF-responsiveness [242]. Differentiated neurons within sympathet‐ ic ganglia express TrkA but not NGF [208].

#### **5.3. TrkB, BDNF and NT4/5**

apoptosis, which is partially rescued by exogenous NGF [212]. In the adult, NT-3 continues to be expressed by a wide range of tissues [202, 203, 213, 214] and, together with NGF, continues to be important for post-natal sympathetic neuron survival [214, 215]. Consistent with this, exogenous NT-3 promotes target organ sympathetic innervation in NT-3 knockout animals [211, 212], suggesting that the switch from NT-3 to NGF dependence observed in sympathetic neurons *in vitro* [216] does not actually occur *in vivo.* This may relate to environmental differences, corroborated by the mitogenic effect of NT-3 on neuroblasts *in vitro* [201] but not *in vivo* [217], the susceptibility of TrkC transcription to environmental factors and also by the capacity of NT-3 to bind and activate TrkA receptors, and in particular TrkAII [76]. This helps to explain how NT-3 rescues NGF-dependent neurons from NGF depletion and *vice versa* and is consistent with the characterisation of TrkA as a functional NT-3 receptor *in vivo.* However, one difference between these two NTs is that exogenous NGF but not NT-3 induces sympa‐

NT-3 released from effector tissues and acting through TrkA also promotes sympathetic innervation of target organs [202, 213, 214, 219, 220]. In support of this, effector tissue elimi‐ nation induces the death of innervating neurons, which cannot be completely reversed by exogenous NGF alone, and adult sympathetic neurons expressing TrkA are immunoreactive for both NGF and NT-3 [49, 221, 222]. Therefore, NT-3 plays an important role throughout sympathetic neuron life-cycle from neuroblast to neuron, acting initially through the TrkC receptor as an autocrine/paracrine factor stimulator of migration and survival in proliferating sympathetic neuroblasts and later as a paracrine promoter of sympathetic neuron differentia‐ tion, survival and target organ innervation acting through the TrkA receptor. CD271/p75NTR is also required for optimal neurotrophin sensitivity since CD271/p75NTR deficient dorsal root

During sympathoadrenal development, progenitors switch from being dependent upon NT3 and TrkC to dependence upon NGF and TrkA, through an intermediate stage of combined TrkA and TrkC expression. In murine thoracic sympathetic ganglia TrkC expression alone is detected at E14-15, whereas both TrkA and TrkC expression are detected at E16.5-17 and only TrkA at E19.5 [224]. Interestingly, sympathetic chromaffin tissues of the adrenal medulla and paraganglia, which form in parallel to sympathetic ganglia, exhibit differences in NT receptor expression consistent with upstream progenitor separation. This difference is characterised by the expression of TrkC but not TrkA by NCCs migrating into the adrenal anlage, at times when TrkC expression is lost in associated with the induction of TrkA expression by NCCs within

Unlike TrkC, TrkA is not expressed during neurulation, NC development or NCC dorsolat‐ eral or vetrolateral migration. In rodent development, TrkA is detected at E12.5 within sensory cranial and spinal dorsal root ganglia and subsequently in the paravertebral sympathetic ganglia [226]. NGF is expressed during the mid-stage of development initiating within CNS structures then within PNS structures at later stages of development [227, 228]. Within the developing adrenal gland NGF exhibits a brief period of post-natal expression, whereas NT-3

and sympathetic neurons exhibit reduced sensitivity to NGF [223].

thetic ganglia hyperplasia [218].

62 Neuroblastoma

sympathetic ganglia [225].

**5.2. TrkA and NGF**

TrkB, like TrkA, is also not expressed during neurulation but is expressed by motor progenitors in hindbrain rhombomere 2 at later stages 9-10 and 12, during avian development, either side of the floor plate in the caudal midbrain, extending through the hindbrain and into the spinal chord [193]. Alternative TrkB splice variant expression has not been assessed during early development.

Following neurulation, TrkB expression is detected within motor neuron progenitors of the ventral neural tube and corresponds to BDNF expression by elements within dorsal neural tube, which coordinate motor neuron development [243]. Consistent with this, both TrkB and BDNF knockout mice exhibit the loss of motor and sensory neurons from dorsal root, trige‐ minal, nodose/petrosal, vestibular, and geniculate ganglia [244].

levels and N-myc amplification and expression, with low to no TrkA mRNA expression associated with poor prognosis. This salient study not only implicated Nmyc in the repres‐ sion of TrkA expression but also reported moderate to high TrkA expression in non-Nmyc amplified disease. The inverse relationship between Nmyc amplification and expression, low TrkA expression and advanced stage disease has now been confirmed by many studies, and it is generally accepted that low TrkA expression combined with Nmyc amplification and expression characterises unfavourable NB and carries poor prognosis [156, 257-270]. In support of this, NBs that form in the root ganglia of Nmyc transgenic mice also exhibit reduced TrkA expression [271]. Nmyc amplified NBs, however, also exhibit heterogeneity [272] and a small number of these tumours exhibit high TrkA expression and favourable histology [270], suggesting that the relationship between NMYC and TrkA in NB is not always

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

65

Adding to the observation that moderate to high TrkA levels associate with non-Nmyc amplified NB [257, 258], Shimada and colleagues extended the clinical relationship between TrkA expression in NB to include outcome, prognostic significance, biological relevance and histopathological status. They reported that TrkA expression could not distinguish prognostic groups but could distinguish between Nmyc amplified (low TrkA) and non-Nmyc (high TrkA) amplified NB, between Nmyc amplified NB with favourable (high TrkA) and unfavourable (low TrkA) histology, but could not distinguish between non-Nmyc amplified NB with favourable histology (moderate to high TrkA) and unfavourable histology (moderate to high TrkA) [270]. This contrasts with some reports [257, 258, 264] but not others [263]. Adult NBs are aggressive non-Nmyc amplified tumours that express high TrkA levels and in such bear

Low TrkA expression by Nmyc amplified NBs may relate to an origin along the sympathoa‐ drenal lineage within non-TrkA expressing NCC subpopulations that colonize coalescing sympathetic ganglia, paraganglia and adrenal medulla anlage during development [225]. Alternatively, reduced TrkA expression in Nmyc amplified NBs may occur post transforma‐ tion, since Nmyc represses TrkA transcription by promoting TrkA promoter methylation and

Moderate to high TrkA levels in non Nmyc-amplified NBs may also relate to cellular origin within undifferentiated TrkA expressing NCC subpopulations of the sympathetic chain and adrenal primordia [225, 276], or may also occur post-transformation, regulated by NTs, growth

Despite elevated TrkA expression in advanced stage non-Nmyc amplified and in a small subgroup of Nmyc amplified NBs with favourable histology [270], full length TrkA exhibits a tumour suppressor function in NB models, suggesting that defects in TrkA receptor signalling occur in NB [280]. Consistent with this, TrkA gene transfection in the absence of CD271/ p75NTR restores NGF responsiveness to NB cells, inducing either neuronal differentiation, growth arrest and/or apoptosis in response to NGF [73, 281-284]. Differentiation induced by NGF in TrkA transfected NB cells involves insulin growth factor II [285], RET [286], c-Src [287], protein kinase C-ε [288] and Ras/MAPK/Erk signalling [289, 290], and associates with reduced angiogenic factor expression and angiogenesis resulting in reduced tumorigenic activity [291,

similarity to non-Nmyc amplified paediatric NBs [156, 268, 273].

factors and/or cytokines [277-279].

TrkA promoter methylation is detected in Nmyc amplified NBs [274, 275].

straightforward.

During SNS development, TrkB exhibits expression restricted to sub-populations of preganglionic cells [230, 245], and sympathoblasts within coalescing sympathetic ganglia, which exhibit transient TrkB expression prior to differentiating [225]. Sympathoblasts that express TrkB within coalescing sympathetic ganglia are non-proliferating but do proliferate in response to BDNF *in vitro*, suggesting that the concentration of BDNF within coalescing sympathetic ganglia is sub-threshold at this time [225]. Pre-ganglionic cells respond to BDNF expressed and release by effector tissues, resulting in pre-ganglionic innervation [230]. Within the adrenal gland, chromaffin cells express NT4/5 but not TrkB, which is weekly expressed by the adrenal cortex, providing a neurotrophic source for extra-adrenal TrkB expressing preganglionic neurons located in spinal chord segments T7-T10. Thes cells use adrenal medullary NT-4/5 to project axons into the adrenal medulla in a TrkB-dependent manner [230]. BDNF, on the other hand, is expressed by sympathetic neurones and regulates sympathetic synaptic complexity [246].

The fact that NT4/5 but not TrkB is expressed within the developing adrenal medulla [230, 238, 247] has prompted hypotheses that medullary NT-4/5 may also ligate and activate TrkA receptors expressed by adrenal medullary neuroblasts and chromaffin cells [63, 80, 229, 230]. However, adrenal medullary chromaffin tissues do exhibit rapid stress-induced TrkB expres‐ sion, which facilitates the adrenal catecholamine response to stress-induced elevation of blood bourn BDNF [248].

#### **5.4. CD271/P75NTR**

CD271/p75NTR is a neural crest marker that is expressed by NC crest stem cells during early development, by NC stem cells in peripheral neural tissues during late development after NCC migration has ceased, and by nerve associated post natal and adult NC stem cells [249]. CD271/ p75NTR expressing adult NC stem cells have been identified as a potential origin for adult tumours of the PNS and NC, including adult NB [249, 250]. Within the human foetal adrenal medulla, CD271/p75NTR immunoreactivity is detected in nerve fibres and primitive neuroblast clusters, and in the adult adrenal medulla is detected in nerve fibres, ganglion cells and connective tissue cells of septi but not chromaffin cells [251, 252]. CD271/p75NTR is required for normal sympathetic neuronal death and the death of damaged neurons [253-255]. CD271/ p75NTR knockout alters synapses within sympathetic ganglia and reduces sympathetic target organ innervation, consistent with its function in enhancing NT-responsiveness [223, 256].

#### **6. Neurotrophins and neurotrophin receptors in human neuroblastoma**

#### **6.1. TrkA and NGF expression in NB**

The cloning of the TrkA receptor in 1991 [113] initiated the study of TrkA expression in human NBs [257]. This initial report detected an inverse relationship between TrkA mRNA levels and N-myc amplification and expression, with low to no TrkA mRNA expression associated with poor prognosis. This salient study not only implicated Nmyc in the repres‐ sion of TrkA expression but also reported moderate to high TrkA expression in non-Nmyc amplified disease. The inverse relationship between Nmyc amplification and expression, low TrkA expression and advanced stage disease has now been confirmed by many studies, and it is generally accepted that low TrkA expression combined with Nmyc amplification and expression characterises unfavourable NB and carries poor prognosis [156, 257-270]. In support of this, NBs that form in the root ganglia of Nmyc transgenic mice also exhibit reduced TrkA expression [271]. Nmyc amplified NBs, however, also exhibit heterogeneity [272] and a small number of these tumours exhibit high TrkA expression and favourable histology [270], suggesting that the relationship between NMYC and TrkA in NB is not always straightforward.

BDNF knockout mice exhibit the loss of motor and sensory neurons from dorsal root, trige‐

During SNS development, TrkB exhibits expression restricted to sub-populations of preganglionic cells [230, 245], and sympathoblasts within coalescing sympathetic ganglia, which exhibit transient TrkB expression prior to differentiating [225]. Sympathoblasts that express TrkB within coalescing sympathetic ganglia are non-proliferating but do proliferate in response to BDNF *in vitro*, suggesting that the concentration of BDNF within coalescing sympathetic ganglia is sub-threshold at this time [225]. Pre-ganglionic cells respond to BDNF expressed and release by effector tissues, resulting in pre-ganglionic innervation [230]. Within the adrenal gland, chromaffin cells express NT4/5 but not TrkB, which is weekly expressed by the adrenal cortex, providing a neurotrophic source for extra-adrenal TrkB expressing preganglionic neurons located in spinal chord segments T7-T10. Thes cells use adrenal medullary NT-4/5 to project axons into the adrenal medulla in a TrkB-dependent manner [230]. BDNF, on the other hand, is expressed by sympathetic neurones and regulates sympathetic synaptic

The fact that NT4/5 but not TrkB is expressed within the developing adrenal medulla [230, 238, 247] has prompted hypotheses that medullary NT-4/5 may also ligate and activate TrkA receptors expressed by adrenal medullary neuroblasts and chromaffin cells [63, 80, 229, 230]. However, adrenal medullary chromaffin tissues do exhibit rapid stress-induced TrkB expres‐ sion, which facilitates the adrenal catecholamine response to stress-induced elevation of blood

CD271/p75NTR is a neural crest marker that is expressed by NC crest stem cells during early development, by NC stem cells in peripheral neural tissues during late development after NCC migration has ceased, and by nerve associated post natal and adult NC stem cells [249]. CD271/ p75NTR expressing adult NC stem cells have been identified as a potential origin for adult tumours of the PNS and NC, including adult NB [249, 250]. Within the human foetal adrenal medulla, CD271/p75NTR immunoreactivity is detected in nerve fibres and primitive neuroblast clusters, and in the adult adrenal medulla is detected in nerve fibres, ganglion cells and connective tissue cells of septi but not chromaffin cells [251, 252]. CD271/p75NTR is required for normal sympathetic neuronal death and the death of damaged neurons [253-255]. CD271/ p75NTR knockout alters synapses within sympathetic ganglia and reduces sympathetic target organ innervation, consistent with its function in enhancing NT-responsiveness [223, 256].

**6. Neurotrophins and neurotrophin receptors in human neuroblastoma**

The cloning of the TrkA receptor in 1991 [113] initiated the study of TrkA expression in human NBs [257]. This initial report detected an inverse relationship between TrkA mRNA

minal, nodose/petrosal, vestibular, and geniculate ganglia [244].

complexity [246].

64 Neuroblastoma

bourn BDNF [248].

**5.4. CD271/P75NTR**

**6.1. TrkA and NGF expression in NB**

Adding to the observation that moderate to high TrkA levels associate with non-Nmyc amplified NB [257, 258], Shimada and colleagues extended the clinical relationship between TrkA expression in NB to include outcome, prognostic significance, biological relevance and histopathological status. They reported that TrkA expression could not distinguish prognostic groups but could distinguish between Nmyc amplified (low TrkA) and non-Nmyc (high TrkA) amplified NB, between Nmyc amplified NB with favourable (high TrkA) and unfavourable (low TrkA) histology, but could not distinguish between non-Nmyc amplified NB with favourable histology (moderate to high TrkA) and unfavourable histology (moderate to high TrkA) [270]. This contrasts with some reports [257, 258, 264] but not others [263]. Adult NBs are aggressive non-Nmyc amplified tumours that express high TrkA levels and in such bear similarity to non-Nmyc amplified paediatric NBs [156, 268, 273].

Low TrkA expression by Nmyc amplified NBs may relate to an origin along the sympathoa‐ drenal lineage within non-TrkA expressing NCC subpopulations that colonize coalescing sympathetic ganglia, paraganglia and adrenal medulla anlage during development [225]. Alternatively, reduced TrkA expression in Nmyc amplified NBs may occur post transforma‐ tion, since Nmyc represses TrkA transcription by promoting TrkA promoter methylation and TrkA promoter methylation is detected in Nmyc amplified NBs [274, 275].

Moderate to high TrkA levels in non Nmyc-amplified NBs may also relate to cellular origin within undifferentiated TrkA expressing NCC subpopulations of the sympathetic chain and adrenal primordia [225, 276], or may also occur post-transformation, regulated by NTs, growth factors and/or cytokines [277-279].

Despite elevated TrkA expression in advanced stage non-Nmyc amplified and in a small subgroup of Nmyc amplified NBs with favourable histology [270], full length TrkA exhibits a tumour suppressor function in NB models, suggesting that defects in TrkA receptor signalling occur in NB [280]. Consistent with this, TrkA gene transfection in the absence of CD271/ p75NTR restores NGF responsiveness to NB cells, inducing either neuronal differentiation, growth arrest and/or apoptosis in response to NGF [73, 281-284]. Differentiation induced by NGF in TrkA transfected NB cells involves insulin growth factor II [285], RET [286], c-Src [287], protein kinase C-ε [288] and Ras/MAPK/Erk signalling [289, 290], and associates with reduced angiogenic factor expression and angiogenesis resulting in reduced tumorigenic activity [291, 292]. Furthermore, full length TrkA does not promote genetic instability [73, 293] or invasive behaviour of NB cells [294]. Apoptosis induced by TrkA in NB cells is p53-dependent [295], involves the cerebral cavernous malformation 2 protein, CCM2 [296], ERK and caspase-7, and can also be augmented by NGF [297]. As stated above, TrkA may also acts as a true dependency receptor, recruiting CD271/p75NTR as a hired killer to promote apoptosis in the absence of NGF [192]. TrkA responsiveness and specificity for NTs is optimised by CD271/p75NTR, which in its own right acts as a Fas-like apoptosis receptor in response to pro-NTs, supporting the hypothesis that NBs that coexpress TrkA and CD271/p75NTR are favourable tumours that carry good prognosis [298, 299]. It should be noted, however, that metastatic bone marrow NB infiltrates induced in SCID mice express TrkA [300] and human NB metastatic bone marrow infiltrates express CD271/p75NTR [301].

TrkAIII expression in human NB cells is regulated by hypoxic stress [73] and by agents that promote stress within the endoplasmic reticulum (unpublished observations). TrkAIII signalling through IP3K/Akt but not Ras/MAPK, combined with interference in NGF/TrkA signalling, would permit tumours to override NT-dependence, whilst promoting survival and staminality to provide a selective advantage [73, 78, 79]. Therefore, TrkAIII expression in non-Nmyc amplified NBs may parallel the selective advantage provided by BDNF/TrkB in Nmyc

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

67

It remains to be determined whether TrkAIII can counteract the pro-apoptotic effects of the Sortlin-CD271/p75NTR complex in the presence of pro-NTs or prevent CD271/p75NTR-mediated

The CD271/p75NTR low affinity nerve growth factor receptor is a neural crest stem cell marker [249, 250, 315] and is expressed by neural crest-derived melanoma and NB cancer stem cells [250, 316, 317]. In a model of non-Nmyc amplified NB cancer staminality, self replicating CD133, CD271/p75NTR positive clonogenic stem cells produce both a non-malignant fibromus‐ cular lineage and a malignant neuronal (N)-type cell lineage defective in terminal neuronal differentiation. Although Trk expression in this NB population remains to be determined, CD271/p75NTR positive self-replicating neural stem cells have been shown to express TrkA,

Consistent with a restricted pattern of CD271/p75NTR expression in NB, primary human NBs have been reported to not express CD271/p75NTR [252, 259, 319] or to express variable levels of CD271/p75NTR [251, 319], which either correlate [259] or don't correlate with TrkA expression [259, 264]. Indeed, differences in CD271/p75NTR co-expression with TrkA have been associated with survival, with the co-expression of CD271/p75NTR and TrkA in NB associated with a 100% survival probability, TrkA expression in the absence of CD271/p75NTR with a 62.3% (inter‐ mediate) survival probability and no TrkA or p75NTR expression with a 0% probability of survival [259]. Consistent with this, a lack of CD271/p75NTR expression has been reported in Nmyc amplified and undifferentiated NB [252, 319, 321] and high CD271/p75NTR expression reported in more favourable differentiating NBs, ganglioneuromas and ganglioneuroblasto‐ mas [251, 252, 299, 320]. However, despite the general concept that high level CD271/p75NTR expression associates with favourable NB behaviour and outcome [259, 264, 322], CD271/ p75NTR expression characterises GD2 positive stage IV metastatic bone marrow NB infiltrates

Consistent with a general association with favourable NB, CD271/p75NTR exhibits a tumour suppressor role in NB models, promoting differentiation, apoptosis and reducing tumorigenic activity [299, 324, 325]. Differentiation promoted by CD271/p75NTR depends upon the molec‐ ular context and may involve an IP3K-Akt-mediated BcL-X-dependent survival pathway [326, 327] or a TrkA-dependent pathway, in which CD271/p75NTR plays a subtle but critical role in optimising and prolonging NGF-mediated TrkA activation [328-331]. Indeed, mutation of CD271/p75NTR within a TrkA context results in proliferation and not differentiation in response to NGF [332]. Coexpression studies in NB cells have also indicated that, in response to NTs,

amplified NBs [309-313], and NT-3/TrkC in a subgroup of advanced stage NBs [314].

apoptosis in the absence of NTs.

TrkAIII, TrkB and TrkC [73, 318].

[301] and aggressive adult NBs [268, 323].

**6.3. CD271/ p75NTR expression in NB**

Although, TrkA gene rearrangements have not been described in NB, a c.1810 C>T TrkA polymorphism has been detected in approximately 9% of NB, with potential to predict disease relapse in non-Nmyc amplified NB [302].

#### **6.2. The alternative TrkAIII splice variant in NB**

Anomalies of TrkA expression that do not support an exclusively tumour suppressing role for TrkA in NB, include moderate to high TrkA expression reported in non-Nmyc ampli‐ fied advanced stage, metastatic unfavourable NBs. These reports may be explained by TrkAIII expression [73], an increase in which was originally reported in advanced stage NB [73], and later confirmed [156, 303, 304]. Recently, TrkAIII expression in a cohort of 500 NBs was found to be significantly higher in high TrkA expressing unfavourable NBs compared to high TrkA expressing favourable NBs (p<0.0001) and to correlate with worse prognosis [156]. Further‐ more in the latter study, TrkAIII promoted a cancer stem cell NB phenotype [156], helping to explain high TrkA levels in unfavourable non-Nmyc amplified NB, adult NB and a subset of relapsing NBs [73, 156, 270, 303, 304]. In support of this, gene-based outcome prediction studies focussed on exon-specific expression, have identified a TrkA splicing difference between stage I and stage IV non-Myc amplified NBs [305, 306], and an exon gene array analysis using TrkAI/II specific primers, excluding TrkAIII, reported to provide a signifi‐ cant prognostic and predictive statistical advantage, associating high TrkAI/II expression with better prognosis in NB [307].

TrkAIII represents a developmental and stress-regulated TrkA isoform [73, 77] that exhibits spontaneous ligand-independent activation and oncogenic activity in NB models [73, 78, 79] and promotes a nestin, CD117, CD133 and Sox2 positive NB stem cell phenotype [156]. In contrast to full length TrkA, TrkAIII does not restore NGF responsiveness to NB cells nor induce NB cell differentiation or apoptosis [73, 78, 79] but interfers with NGF/TrkA signalling through Ras/MAPK, augments genetic instability by promoting centrosome amplification [79] and promotes angiogenesis by altering the equilibrium between MMP-9, VEGF and throm‐ bospondin, through IP3K/Atk. Together these phenomena promote NB cell xenograft primary [73] and metastatic tumorigenic activity [308]. Furthermore, TrkAIII increases NB cell resist‐ ance to stress, doxorubicin and geladanamycin-induced cytotoxicity [73, 78, 79].

TrkAIII expression in human NB cells is regulated by hypoxic stress [73] and by agents that promote stress within the endoplasmic reticulum (unpublished observations). TrkAIII signalling through IP3K/Akt but not Ras/MAPK, combined with interference in NGF/TrkA signalling, would permit tumours to override NT-dependence, whilst promoting survival and staminality to provide a selective advantage [73, 78, 79]. Therefore, TrkAIII expression in non-Nmyc amplified NBs may parallel the selective advantage provided by BDNF/TrkB in Nmyc amplified NBs [309-313], and NT-3/TrkC in a subgroup of advanced stage NBs [314].

It remains to be determined whether TrkAIII can counteract the pro-apoptotic effects of the Sortlin-CD271/p75NTR complex in the presence of pro-NTs or prevent CD271/p75NTR-mediated apoptosis in the absence of NTs.

#### **6.3. CD271/ p75NTR expression in NB**

292]. Furthermore, full length TrkA does not promote genetic instability [73, 293] or invasive behaviour of NB cells [294]. Apoptosis induced by TrkA in NB cells is p53-dependent [295], involves the cerebral cavernous malformation 2 protein, CCM2 [296], ERK and caspase-7, and can also be augmented by NGF [297]. As stated above, TrkA may also acts as a true dependency receptor, recruiting CD271/p75NTR as a hired killer to promote apoptosis in the absence of NGF [192]. TrkA responsiveness and specificity for NTs is optimised by CD271/p75NTR, which in its own right acts as a Fas-like apoptosis receptor in response to pro-NTs, supporting the hypothesis that NBs that coexpress TrkA and CD271/p75NTR are favourable tumours that carry good prognosis [298, 299]. It should be noted, however, that metastatic bone marrow NB infiltrates induced in SCID mice express TrkA [300] and human NB metastatic bone marrow

Although, TrkA gene rearrangements have not been described in NB, a c.1810 C>T TrkA polymorphism has been detected in approximately 9% of NB, with potential to predict disease

Anomalies of TrkA expression that do not support an exclusively tumour suppressing role for TrkA in NB, include moderate to high TrkA expression reported in non-Nmyc ampli‐ fied advanced stage, metastatic unfavourable NBs. These reports may be explained by TrkAIII expression [73], an increase in which was originally reported in advanced stage NB [73], and later confirmed [156, 303, 304]. Recently, TrkAIII expression in a cohort of 500 NBs was found to be significantly higher in high TrkA expressing unfavourable NBs compared to high TrkA expressing favourable NBs (p<0.0001) and to correlate with worse prognosis [156]. Further‐ more in the latter study, TrkAIII promoted a cancer stem cell NB phenotype [156], helping to explain high TrkA levels in unfavourable non-Nmyc amplified NB, adult NB and a subset of relapsing NBs [73, 156, 270, 303, 304]. In support of this, gene-based outcome prediction studies focussed on exon-specific expression, have identified a TrkA splicing difference between stage I and stage IV non-Myc amplified NBs [305, 306], and an exon gene array analysis using TrkAI/II specific primers, excluding TrkAIII, reported to provide a signifi‐ cant prognostic and predictive statistical advantage, associating high TrkAI/II expression

TrkAIII represents a developmental and stress-regulated TrkA isoform [73, 77] that exhibits spontaneous ligand-independent activation and oncogenic activity in NB models [73, 78, 79] and promotes a nestin, CD117, CD133 and Sox2 positive NB stem cell phenotype [156]. In contrast to full length TrkA, TrkAIII does not restore NGF responsiveness to NB cells nor induce NB cell differentiation or apoptosis [73, 78, 79] but interfers with NGF/TrkA signalling through Ras/MAPK, augments genetic instability by promoting centrosome amplification [79] and promotes angiogenesis by altering the equilibrium between MMP-9, VEGF and throm‐ bospondin, through IP3K/Atk. Together these phenomena promote NB cell xenograft primary [73] and metastatic tumorigenic activity [308]. Furthermore, TrkAIII increases NB cell resist‐

ance to stress, doxorubicin and geladanamycin-induced cytotoxicity [73, 78, 79].

infiltrates express CD271/p75NTR [301].

66 Neuroblastoma

relapse in non-Nmyc amplified NB [302].

with better prognosis in NB [307].

**6.2. The alternative TrkAIII splice variant in NB**

The CD271/p75NTR low affinity nerve growth factor receptor is a neural crest stem cell marker [249, 250, 315] and is expressed by neural crest-derived melanoma and NB cancer stem cells [250, 316, 317]. In a model of non-Nmyc amplified NB cancer staminality, self replicating CD133, CD271/p75NTR positive clonogenic stem cells produce both a non-malignant fibromus‐ cular lineage and a malignant neuronal (N)-type cell lineage defective in terminal neuronal differentiation. Although Trk expression in this NB population remains to be determined, CD271/p75NTR positive self-replicating neural stem cells have been shown to express TrkA, TrkAIII, TrkB and TrkC [73, 318].

Consistent with a restricted pattern of CD271/p75NTR expression in NB, primary human NBs have been reported to not express CD271/p75NTR [252, 259, 319] or to express variable levels of CD271/p75NTR [251, 319], which either correlate [259] or don't correlate with TrkA expression [259, 264]. Indeed, differences in CD271/p75NTR co-expression with TrkA have been associated with survival, with the co-expression of CD271/p75NTR and TrkA in NB associated with a 100% survival probability, TrkA expression in the absence of CD271/p75NTR with a 62.3% (inter‐ mediate) survival probability and no TrkA or p75NTR expression with a 0% probability of survival [259]. Consistent with this, a lack of CD271/p75NTR expression has been reported in Nmyc amplified and undifferentiated NB [252, 319, 321] and high CD271/p75NTR expression reported in more favourable differentiating NBs, ganglioneuromas and ganglioneuroblasto‐ mas [251, 252, 299, 320]. However, despite the general concept that high level CD271/p75NTR expression associates with favourable NB behaviour and outcome [259, 264, 322], CD271/ p75NTR expression characterises GD2 positive stage IV metastatic bone marrow NB infiltrates [301] and aggressive adult NBs [268, 323].

Consistent with a general association with favourable NB, CD271/p75NTR exhibits a tumour suppressor role in NB models, promoting differentiation, apoptosis and reducing tumorigenic activity [299, 324, 325]. Differentiation promoted by CD271/p75NTR depends upon the molec‐ ular context and may involve an IP3K-Akt-mediated BcL-X-dependent survival pathway [326, 327] or a TrkA-dependent pathway, in which CD271/p75NTR plays a subtle but critical role in optimising and prolonging NGF-mediated TrkA activation [328-331]. Indeed, mutation of CD271/p75NTR within a TrkA context results in proliferation and not differentiation in response to NGF [332]. Coexpression studies in NB cells have also indicated that, in response to NTs, CD271/p75NTR alone induces mild differentiation, TrkA alone causes a more marked differen‐ tiation and coexpression an even more marked and rapid differentiation [333].

expression in NB exhibits a positive correlation with Nmyc amplification and expression [309, 311, 347, 348, 350]. TrkB expression is stimulated by activated c-erbA in NB cells, unveiling a potential oncogenic receptor tyrosine kinase-mediated mechanism for promoting TrkB expression [351]. Aggressive unfavourable Nmyc amplified NBs also express BDNF, which when coexpressed with TrkB provides an autocrine/paracrine survival mechanism in tissues that do not express NTs [309-313]. Recently, BDNF variants encoding exons 4, 6 and 9 have

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

69

TrkB expression by sympathoblasts subpopulations during SNS development provides a potential origin for TrkB expressing NBs. However, this population proliferates in response to BDNF *in vitro* but does express BDNF *in vivo* [225, 245], suggesting that BDNF expression may be acquired at a later stage. TrkB transcription in NB cells is also up regulated by hypoxia inducible factor-1, providing a potential epigenetic mechanism through which tumour-

In contrast to signals from NGF-activated TrkA, which induces NB cell differentiation and growth arrest [73, 324, 353, 354], BDNF activation of TrkB induces partial differentiation in the absence of growth arrest, through Ret tyrosine kinase [354-357]. BDNF activation of TrkB increases NB cell survival [358], resistance to chemotherapeutic agents [358-363], augments invasive capacity [294] in cooperation with c-Met [364] and galectin-1 [365], promotes angio‐ genesis and angiogenic factor expression [292, 350, 366], augments genetic instability [293] (Schulte et al., 2008) and increases metastatic behaviour by inhibiting anoikis [367]. In contrast, NB cells expressing truncated TrkB lacking the tyrosine kinase domain, display a more differentiated phenotype [311] and this receptor is more frequently detected in ganglioneur‐ oblastomas and ganglioneuromas. Consistent with this, truncated TrkB overexpression in NB cells promotes differentiation suggesting that this receptor variant promotes a more benign phenotype [368]. Oxidative stress up-regulates the expression of full length TrkB relative to the truncated isoform, providing an additional epigenetic mechanism for regulating TrkB

TrkC is expressed by migrating NCC progenitors, sympathoblasts and sympathetic neurons [194, 201, 203], providing many potential origins for TrkC expressing NBs. High level TrkC expression in low stage NBs is associated with favourable outcome (309, 310, 349, 353, 370-372], and is often accompanied by TrkA expression [257, 258, 309]. Recently, however, a subset of advanced stage IV NBs has been identified that exhibit high level NT-3 and TrkC co-expression, providing an autocrine/paracrine survival and proliferation mechanism for selecting these NBs in tissues that do not express NT-3 [314]. This expression pattern bears close similarity to migrating, proliferating NCC sympathoblasts prior to sympathetic neuronal differentiation, which also coexpress NT-3 and TrkC [194, 201, 203], identifying a potential origin for this NB subset. TrkC expression in NB, like that of TrkA, inversely correlates with Nmyc amplification and expression, and Nmyc amplified NBs either do not express TrkC at all, or express truncated TrkC [371, 372]. With the exception of NBs that coexpress NT3 and TrkC [314], the coexpression of TrkC, TrkA and CD271/p75NTR in NB carries the best prognosis and associates

been associated with unfavourable NB outcome [312, 313].

associated hypoxia could augment TrkB expression [352].

involvement in NB [369].

**6.5. TrkC and NT3 in NB**

CD271/p75NTR can acts as either an anti-apoptotic or pro-apoptotic factor, depending upon the molecular context. At low TrkA to CD271/p75NTR ratios the anti-apoptotic activity of NGF requires binding to CD271/p75NTR, whilst at higher TrkA to CD271/p75NTR ratios involves a mechanism independent of CD271/p75NTR binding [334]. Conversely, NGF also induces apoptosis in NB cells with a high CD271/p75NTR to TrkA ratios [335]. In the absence of NTs and TrkA, CD271/p75NTR induces apoptosis and inhibits NB tumorigenic activity [299, 336-338]. It has been reported that apoptosis, induced by non-ligated CD271/p75NTR is inhibited by non-ligated TrkA but this may reflect spontaneous activation of overex‐ pressed TrkA [337]. Furthermore, agents such as prion proteins activate CD271/p75NTR to promote apoptosis in NB cells via NF-κB [339]. In the absence of spontaneous TrkA activation and NT expression, however, the coexpression of CD271/p75NTR and TrkA promotes more marked apoptosis [333]. In the presence of BDNF CD271/p75NTR interaction with TrkB promotes NB cell proliferation and survival, through RAS/MAPK and PI3K/AKT/NF-κB [322]. These reports suggests that CD271/p75NTR is a pivotal regulator of the disparate behaviour of TrkA and TrkB expressing NBs, exhibiting capacity to enhance differentiation and apoptotic responses in TrkA expressing NBs and enhance proliferation and survivalresponses in TrkB expressing NBs, by increasing receptor sensitivity to low NT concentrations and blocking responses to promiscuous NTs.

CD271/p75NTR also interacts with Sortilin and other proteins, complicating potential responses to both pro- and active NTs. The CD271/p75NTR-Sortilin co-receptor complex augments affinity for proNGF and induces apoptosis [105, 340]. Furthermore, CD271/p75NTR also interacts with NRIF, TRAF, NRAGE and MAGE proteins to promote apoptosis [340-342].

With respect to the regulation of CD271/p75NTR expression in NB, Nmyc acts as a transcrip‐ tional repressor of CD271/p75NTR expression by promoting promoter methylation [274]. This effect can be reversed by HDAC inhibitors, resulting in the resoration of NGF-mediated apoptosis [274]. This novel pathway, detected in Nmyc amplified NB, may help to explain the inverse relationship between CD271/p75NTR and Nmyc expression detected in human Nmyc amplified NBs and in root ganglia NBs in Nmyc transgenic mice [271, 343]. The histone methyltransferase EZH2A has also been reported to repress CD271/p75NTR provid‐ ing an additional Nmyc-independent CD271/p75NTR transcriptional repressing mechanism that may contribute to the genesis and maintenance of undifferentiated CD271/p75NTR negative NBs [344].

At the therapeutic level, CD271/p75NTR protects NCC and NB cells from apoptosis induced by antimitotic agents [345], and histone deacetylase inhibitors induce NB cell apoptosis and restore CD271/p75NTR and TrkA expression [274, 346].

#### **6.4. TrkB and BDNF in NB**

Fully spliced TrkB is expressed by a subpopulation of Nmyc amplified NBs [311, 347-349]. Despite observations that Nmyc alone is insufficient to induce TrkB expression [348], TrkB expression in NB exhibits a positive correlation with Nmyc amplification and expression [309, 311, 347, 348, 350]. TrkB expression is stimulated by activated c-erbA in NB cells, unveiling a potential oncogenic receptor tyrosine kinase-mediated mechanism for promoting TrkB expression [351]. Aggressive unfavourable Nmyc amplified NBs also express BDNF, which when coexpressed with TrkB provides an autocrine/paracrine survival mechanism in tissues that do not express NTs [309-313]. Recently, BDNF variants encoding exons 4, 6 and 9 have been associated with unfavourable NB outcome [312, 313].

TrkB expression by sympathoblasts subpopulations during SNS development provides a potential origin for TrkB expressing NBs. However, this population proliferates in response to BDNF *in vitro* but does express BDNF *in vivo* [225, 245], suggesting that BDNF expression may be acquired at a later stage. TrkB transcription in NB cells is also up regulated by hypoxia inducible factor-1, providing a potential epigenetic mechanism through which tumourassociated hypoxia could augment TrkB expression [352].

In contrast to signals from NGF-activated TrkA, which induces NB cell differentiation and growth arrest [73, 324, 353, 354], BDNF activation of TrkB induces partial differentiation in the absence of growth arrest, through Ret tyrosine kinase [354-357]. BDNF activation of TrkB increases NB cell survival [358], resistance to chemotherapeutic agents [358-363], augments invasive capacity [294] in cooperation with c-Met [364] and galectin-1 [365], promotes angio‐ genesis and angiogenic factor expression [292, 350, 366], augments genetic instability [293] (Schulte et al., 2008) and increases metastatic behaviour by inhibiting anoikis [367]. In contrast, NB cells expressing truncated TrkB lacking the tyrosine kinase domain, display a more differentiated phenotype [311] and this receptor is more frequently detected in ganglioneur‐ oblastomas and ganglioneuromas. Consistent with this, truncated TrkB overexpression in NB cells promotes differentiation suggesting that this receptor variant promotes a more benign phenotype [368]. Oxidative stress up-regulates the expression of full length TrkB relative to the truncated isoform, providing an additional epigenetic mechanism for regulating TrkB involvement in NB [369].

#### **6.5. TrkC and NT3 in NB**

CD271/p75NTR alone induces mild differentiation, TrkA alone causes a more marked differen‐

CD271/p75NTR can acts as either an anti-apoptotic or pro-apoptotic factor, depending upon the molecular context. At low TrkA to CD271/p75NTR ratios the anti-apoptotic activity of NGF requires binding to CD271/p75NTR, whilst at higher TrkA to CD271/p75NTR ratios involves a mechanism independent of CD271/p75NTR binding [334]. Conversely, NGF also induces apoptosis in NB cells with a high CD271/p75NTR to TrkA ratios [335]. In the absence of NTs and TrkA, CD271/p75NTR induces apoptosis and inhibits NB tumorigenic activity [299, 336-338]. It has been reported that apoptosis, induced by non-ligated CD271/p75NTR is inhibited by non-ligated TrkA but this may reflect spontaneous activation of overex‐ pressed TrkA [337]. Furthermore, agents such as prion proteins activate CD271/p75NTR to promote apoptosis in NB cells via NF-κB [339]. In the absence of spontaneous TrkA activation and NT expression, however, the coexpression of CD271/p75NTR and TrkA promotes more marked apoptosis [333]. In the presence of BDNF CD271/p75NTR interaction with TrkB promotes NB cell proliferation and survival, through RAS/MAPK and PI3K/AKT/NF-κB [322]. These reports suggests that CD271/p75NTR is a pivotal regulator of the disparate behaviour of TrkA and TrkB expressing NBs, exhibiting capacity to enhance differentiation and apoptotic responses in TrkA expressing NBs and enhance proliferation and survivalresponses in TrkB expressing NBs, by increasing receptor sensitivity to low NT concentrations and

CD271/p75NTR also interacts with Sortilin and other proteins, complicating potential responses to both pro- and active NTs. The CD271/p75NTR-Sortilin co-receptor complex augments affinity for proNGF and induces apoptosis [105, 340]. Furthermore, CD271/p75NTR also interacts with

With respect to the regulation of CD271/p75NTR expression in NB, Nmyc acts as a transcrip‐ tional repressor of CD271/p75NTR expression by promoting promoter methylation [274]. This effect can be reversed by HDAC inhibitors, resulting in the resoration of NGF-mediated apoptosis [274]. This novel pathway, detected in Nmyc amplified NB, may help to explain the inverse relationship between CD271/p75NTR and Nmyc expression detected in human Nmyc amplified NBs and in root ganglia NBs in Nmyc transgenic mice [271, 343]. The histone methyltransferase EZH2A has also been reported to repress CD271/p75NTR provid‐ ing an additional Nmyc-independent CD271/p75NTR transcriptional repressing mechanism that may contribute to the genesis and maintenance of undifferentiated CD271/p75NTR

At the therapeutic level, CD271/p75NTR protects NCC and NB cells from apoptosis induced by antimitotic agents [345], and histone deacetylase inhibitors induce NB cell apoptosis and

Fully spliced TrkB is expressed by a subpopulation of Nmyc amplified NBs [311, 347-349]. Despite observations that Nmyc alone is insufficient to induce TrkB expression [348], TrkB

NRIF, TRAF, NRAGE and MAGE proteins to promote apoptosis [340-342].

tiation and coexpression an even more marked and rapid differentiation [333].

blocking responses to promiscuous NTs.

restore CD271/p75NTR and TrkA expression [274, 346].

negative NBs [344].

68 Neuroblastoma

**6.4. TrkB and BDNF in NB**

TrkC is expressed by migrating NCC progenitors, sympathoblasts and sympathetic neurons [194, 201, 203], providing many potential origins for TrkC expressing NBs. High level TrkC expression in low stage NBs is associated with favourable outcome (309, 310, 349, 353, 370-372], and is often accompanied by TrkA expression [257, 258, 309]. Recently, however, a subset of advanced stage IV NBs has been identified that exhibit high level NT-3 and TrkC co-expression, providing an autocrine/paracrine survival and proliferation mechanism for selecting these NBs in tissues that do not express NT-3 [314]. This expression pattern bears close similarity to migrating, proliferating NCC sympathoblasts prior to sympathetic neuronal differentiation, which also coexpress NT-3 and TrkC [194, 201, 203], identifying a potential origin for this NB subset. TrkC expression in NB, like that of TrkA, inversely correlates with Nmyc amplification and expression, and Nmyc amplified NBs either do not express TrkC at all, or express truncated TrkC [371, 372]. With the exception of NBs that coexpress NT3 and TrkC [314], the coexpression of TrkC, TrkA and CD271/p75NTR in NB carries the best prognosis and associates more frequently with spontaneous regression, differentiation and chemo-responsiveness [100, 258, 259, 333, 370, 371]. The *TrkC* gene, however, encodes multiple NT-3 receptors with distinct biological properties and substrate specificities [89, 373] and, although *TrkC* gene rearrange‐ ments in NB have not been reported, the effect of differential TrkC isoform expression in NB remains to be elucidated.

Association between high TrkC expression and favourable NB outcome, in the absence of NT-3 [333, 371], is consistent with pro-apoptotic TrkC dependency receptor function, which promotes apoptosis in the absence of CD271/p75NTR and NT expression [191, 192]. Further‐ more, NT-3 activation of TrkC induces NB cell differentiation [374] and the co-expression of TrkC with CD271/p75NTR lowers tumorigenic potential and tumour growth [375] but may protect NB cells from doxorubicin and cisplatin cytotoxicity [375].

With respect to the transcriptional regulation of TrkC, Nmyc silencing increases TrkC expres‐ sion in human NB cells [376], corroborating the inverse relationship reported for TrkC expression and Nmyc amplification [371,372]. TrkC expression, furthermore, is abrogated by the activation of c-erbA, providing a potential oncogenic tyrosine kinase-mediated mechanism for repressing TrkC expression in NB [351]. Retinoic acid induces TrkC expression in human NB cells, restoring NT-3-dependent differentiation [152]. Retinoids also induce the expression of microRNAs-9, 125a and 125b that repress truncated kinase domain-deleted TrkC, resulting in altered growth and highlighting a role for the truncated TrkC receptor in the regulation of NB growth and differentiation [377]. MiR-151-3p represses full length TrkC expression, whereas miRs-128, 485-3p, 765 and 768-5p repress truncated TrkC expression in NB cells [378], indicating that full length and truncated TrkC receptors are regulated by different miRs, linking NT-mediated processes to miR expression in NB.

**Figure 9.** Different combinations, potential outcomes and prognosis of NTs and NTRs in NB

Sortilin-CD271/p75NTR complex-mediated apoptosis in response to pro-NTs.

NTR expression in low stage non-Nmyc amplified NB characterised by the coexpression of TrkA and CD271/p75NTR may carry the best prognosis. These tumours may terminally differentiate in response to NGF (TrkAI) or NT-3 (TrkAII), or undergo TrkA and/or CD271/ p75NTR–mediated apoptosis in the absence of NTs, depending upon the CD271/p75NTR to TrkA expression ratio. Furthermore, the coexpression of Sortilin in these tumours would extend apoptotic potential to include pro-NTs (see Sections 4.2.1-4.2.4). NBs that express TrkA but not CD271/p75NTR may have a worse prognosis, as they require higher NT concentrations for TrkA activation and signalling and would also respond to promiscuous NTs potentially with a response of proliferation, survival and/or partial differentiation. In the absence of CD271/ p75NTR, these NBs would neither exhibit TrkA dependency receptor-mediated apoptosis nor

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

71

NBs that co-express TrkC and CD271/p75NTR but not TrkA or TrkB, may have better prognosis with potential to differentiate in response to NT-3 but alternatively could proliferate and survive in response to NT-3, complicating prognosis. NT-3 is rarely expressed in NBs, increasing the potential for TrkC dependency receptor-mediated apoptosis, in the presence or absence of CD271/p75NTR (see Sections 4.5 and 6.3). The coexpression of Sortilin with CD271/p75NTR in these NBs would increase apoptotic potential to include a response to pro-NTs (see Sections 4.2.3 and 6.3). Advanced stage NBs coexpressing TrkC and NT-3 would be expected to carry worse prognosis as a result of this autocrine survival and proliferation

#### **6.6. General considerations on NT and NTR expression patterns in NB**

The concept that different NT and NTR receptor expression profiles characterise NB subsets and that these differences are involved in divergent NB behaviour and therapeutic suscepti‐ bility, continues to evolve with potential to improve prognosis and therapeutic choice, whilst identifying novel potential therapeutic targets.

The hypothesis that high TrkA, high TrkC and/or high CD271/p75NTR expression always associate with low disease stage and better prognosis in NB is clearly not the case. Moderate to high levels of TrkA, TrkC and/or CD271/p75NTR can also characterise advanced stage and relapsing non-Nmyc amplified NBs and a subset of Nmyc amplified NB with favourable histology (see section 6.1). However, high TrkB expression appears to distinguish advanced stage Nmyc amplified from non-Nmyc amplified NB and carries poor prognosis associated with potential therapeutic resistance (see section 6.4). It is also now apparent that NTRs can be expressed as different isoforms with altered biological activity and can interact with one other and with a variety of ancillary proteins to modulate function (see section 6.3), compli‐ cating prognosis and potential therapeutic outcome, as outlined below (Fig. 9).

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma http://dx.doi.org/10.5772/55536 71

**Figure 9.** Different combinations, potential outcomes and prognosis of NTs and NTRs in NB

more frequently with spontaneous regression, differentiation and chemo-responsiveness [100, 258, 259, 333, 370, 371]. The *TrkC* gene, however, encodes multiple NT-3 receptors with distinct biological properties and substrate specificities [89, 373] and, although *TrkC* gene rearrange‐ ments in NB have not been reported, the effect of differential TrkC isoform expression in NB

Association between high TrkC expression and favourable NB outcome, in the absence of NT-3 [333, 371], is consistent with pro-apoptotic TrkC dependency receptor function, which promotes apoptosis in the absence of CD271/p75NTR and NT expression [191, 192]. Further‐ more, NT-3 activation of TrkC induces NB cell differentiation [374] and the co-expression of TrkC with CD271/p75NTR lowers tumorigenic potential and tumour growth [375] but may

With respect to the transcriptional regulation of TrkC, Nmyc silencing increases TrkC expres‐ sion in human NB cells [376], corroborating the inverse relationship reported for TrkC expression and Nmyc amplification [371,372]. TrkC expression, furthermore, is abrogated by the activation of c-erbA, providing a potential oncogenic tyrosine kinase-mediated mechanism for repressing TrkC expression in NB [351]. Retinoic acid induces TrkC expression in human NB cells, restoring NT-3-dependent differentiation [152]. Retinoids also induce the expression of microRNAs-9, 125a and 125b that repress truncated kinase domain-deleted TrkC, resulting in altered growth and highlighting a role for the truncated TrkC receptor in the regulation of NB growth and differentiation [377]. MiR-151-3p represses full length TrkC expression, whereas miRs-128, 485-3p, 765 and 768-5p repress truncated TrkC expression in NB cells [378], indicating that full length and truncated TrkC receptors are regulated by different miRs,

The concept that different NT and NTR receptor expression profiles characterise NB subsets and that these differences are involved in divergent NB behaviour and therapeutic suscepti‐ bility, continues to evolve with potential to improve prognosis and therapeutic choice, whilst

The hypothesis that high TrkA, high TrkC and/or high CD271/p75NTR expression always associate with low disease stage and better prognosis in NB is clearly not the case. Moderate to high levels of TrkA, TrkC and/or CD271/p75NTR can also characterise advanced stage and relapsing non-Nmyc amplified NBs and a subset of Nmyc amplified NB with favourable histology (see section 6.1). However, high TrkB expression appears to distinguish advanced stage Nmyc amplified from non-Nmyc amplified NB and carries poor prognosis associated with potential therapeutic resistance (see section 6.4). It is also now apparent that NTRs can be expressed as different isoforms with altered biological activity and can interact with one other and with a variety of ancillary proteins to modulate function (see section 6.3), compli‐

cating prognosis and potential therapeutic outcome, as outlined below (Fig. 9).

protect NB cells from doxorubicin and cisplatin cytotoxicity [375].

linking NT-mediated processes to miR expression in NB.

identifying novel potential therapeutic targets.

**6.6. General considerations on NT and NTR expression patterns in NB**

remains to be elucidated.

70 Neuroblastoma

NTR expression in low stage non-Nmyc amplified NB characterised by the coexpression of TrkA and CD271/p75NTR may carry the best prognosis. These tumours may terminally differentiate in response to NGF (TrkAI) or NT-3 (TrkAII), or undergo TrkA and/or CD271/ p75NTR–mediated apoptosis in the absence of NTs, depending upon the CD271/p75NTR to TrkA expression ratio. Furthermore, the coexpression of Sortilin in these tumours would extend apoptotic potential to include pro-NTs (see Sections 4.2.1-4.2.4). NBs that express TrkA but not CD271/p75NTR may have a worse prognosis, as they require higher NT concentrations for TrkA activation and signalling and would also respond to promiscuous NTs potentially with a response of proliferation, survival and/or partial differentiation. In the absence of CD271/ p75NTR, these NBs would neither exhibit TrkA dependency receptor-mediated apoptosis nor Sortilin-CD271/p75NTR complex-mediated apoptosis in response to pro-NTs.

NBs that co-express TrkC and CD271/p75NTR but not TrkA or TrkB, may have better prognosis with potential to differentiate in response to NT-3 but alternatively could proliferate and survive in response to NT-3, complicating prognosis. NT-3 is rarely expressed in NBs, increasing the potential for TrkC dependency receptor-mediated apoptosis, in the presence or absence of CD271/p75NTR (see Sections 4.5 and 6.3). The coexpression of Sortilin with CD271/p75NTR in these NBs would increase apoptotic potential to include a response to pro-NTs (see Sections 4.2.3 and 6.3). Advanced stage NBs coexpressing TrkC and NT-3 would be expected to carry worse prognosis as a result of this autocrine survival and proliferation mechanism that may also extend to NBs expressing TrkC but not NT-3 in tissues that express NT-3, and could be further optimised by co-expression of CD271/p75NTR (see Section 6.3).

78,79]. Lestaurtinib inhibits NB growth *in vitro* and *in vivo,* and substantially enhances the efficacy of conventional chemotherapy, such as 13-cis-retinoic acid, ferenteride and bevacizu‐ mab, presumably by inhibiting autocrine TrkB/BDNF [382-384] and/or spontaneous TrkAIII activity [73]. Lestaurtinib is an active metabolite of the Trk kinase inhibitor CEP-751, and is more suitable for clinical trials, as it can be administered orally [384, 385]. These Trk tyrosine kinase inhibitors not only target tumour promoting effects of Trk receptor activation but also Trk-mediated chemotherapeutic resistance, which has been attributed not only to TrkB [348, 358-363] but also to TrkC [375], fully spliced TrkA [375] and TrkAIII [73, 78, 79]. CEP-701

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

73

Nifutimox, a drug used for years to treat Chagas disease, is also currently in clinical trials for refractory or relapsed NB, and has been shown to suppress TrkB-mediated Akt activation and

Tyrosine kinase inhibitors K252a and CEP-701 inhibit TrkAIII tyrosine kinase activity. TrkAIII activity is also inhibited by the Hsp90 inhibitor geladanamycin and its clinically relevant analogues 17-AAG and 17-DMAG, and by the ARF inhibitor Brefeldin A (BFA) [78, 79]. CEP701 inhibits TrkAIII activity and TrkAIII-induced centrosome amplification at nanomolar concen‐ trations, whereas BFA reversibly inhibits spontaneous TrkAIII activation in association with disruption of the Golgi Network and the endoplasmic reticulum/Golgi Network intermediate compartment [78, 78]. Geldanamycin and its analogues reversibly inhibit TrkAIII tyrosine kinase activity and reduce proliferation of TrkAIII expressing NB cells *in vitro* [78]. Inhibitors of TrkAIII activity, however, do not inhibit TrkAIII expression nor promote TrkAIII elimina‐ tion but cause retention within the endoplasmic reticulum, with potential to induce an ER stress response. This may help to explain the high level of resistance to GA-mediated cytotox‐ icity exhibited by TrkAIII but not TrkAI transfected NB cells, despite inhibition of TrkAIII activity [78, 79]. This suggests that, in addition to other off target effects, reversible TrkAIII tyrosine kinase inhibitors may increase stress-resistance by promoting TrkAIII-ER retention and inducing an ER stress response. Consistent with this, geldanamycin selects slow growing TrkAIII expressing NB cells from mixed populations, with TrkAIII re-activation post drugremoval, suggesting a mechanism for potential post therapeutic relapse [78]. To counter this, we have also developed a specific peptide nucleic acid (PNA) inhibitor of TrkAIII expression based upon the novel exon 5/8 splice junction (TrkAIII PNA conjugate (KKAA)4-GGCCGGGA‐ CAC) [78, 79] for use in combination with with TrkAIII tyrosine kinase inhibitors, to maximise

**7.3. Agents that conserve Trk tyrosine phosphorylation and facilitate signal transduction**

TrkA activation and signal transduction is fundamental for NB differentiation and the loss of TrkA expression or defective activation and/or signalling probably contributes to NB patho‐ genesis. Agents that optimise TrkA activation and facilitate subsequent signal transduction may, therefore, overcome defective TrkA signalling and restore differentiation and/or apoptotic responses to NTs. In this context, a novel cyclophane compound CPPy, with low

synergises with retinoids in the treatment of NB by inhibiting TrkB activity [386].

induce caspase-dependent apoptosis of NB cells *in vitro* and *in vivo* [387].

**7.2. TrkAIII inhibitors**

therapeutic efficacy.

High levels of BDNF and TrkB expression in Nmyc-amplified NBs, in the absence of TrkA, TrkC and CD271/p75NTR, carries the worse prognosis as a result of autocrine/paracrine BDNFmediated TrkB activation, which would be expected to promote proliferation, survival and metastatic capacity. Furthermore in the absence of BDNF, TrkB would not be expected to promote apoptosis, as TrkB does not act as a dependence receptor (see Sections 6.4). As with other Trk receptors, TrkB co-expression with CD271/p75NTR would be expected to optimise NT-specificity and responsiveness, which would be expected to further promote aggressive bahaviour in TrkB expressing NBs.

NBs that express CD271/p75NTR but not Trks may carry better prognosis, as they would be expected to respond to active NTs with an apoptotic response and if co-expressed with Sortilin in the absence of Trks, would also be expected to exhibit an apoptotic response to pro-NTs, which comprises up to 50% of secreted NTs (see Sections 4.2.2 and 6.3).

Non-Nmyc amplified NBs that express TrkAIII may carry worse prognosis, as spontaneous TrkAIII activation would override NT-dependency, provide a selective growth advantage in tissues including those that do not express NTs, promote NB cell stamilality, survival, angiogenesis and genetic instability, resulting in a more tumorigenic, metastatic and stressresistant phenotype (see Sections 4.2.1 and 6.2). Although it remains to be elucidated whether TrkAIII may interfere with CD271/p75NTR –mediated apoptosis in the presence or absence of Sortilin, its expression in NB may represent the biological equivalent to BDNF/TrkB expression in Nmyc amplified NB and TrkC/NT3 expresssion in a subset of advanced stage NBs, as an indicator of poor prognosis.

#### **7. Potential therapeutic approaches**

#### **7.1. Trk kinase inhibitors**

Trk kinase inhibitors would be more suitable for use in advanced stage Nmyc amplified TrkB expressing NBs and advanced stage unfavourable non-Nmyc amplified NBs that express the TrkAIII oncogene but may also reduce survival in NBs expressing full length TrkA and TrkC and their corresponding NTs.

Therapeutic Trk kinase inhibitors include the selective Trk kinase inhibitors AZ-23 and AZ623, which inhibit Trk kinase activity at low nanomolar concentrations. AZ-23 has shown efficacy following oral administration in a TrkA-driven mouse allograft NB model [379], whereas AZ623 inhibits BDNF-mediated signalling and NB proliferation, and when combined with topotecan prolongs the inhibition of tumour regrowth and reduces chemo and radio thera‐ peutic resistance [380, 381]. Lestaurtinib (CEP-701) is a small-molecule receptor tyrosine kinase inhibitor that competitively inhibits ATP binding to the Trk kinase domain at nanomolar concentrations. This compound not only inhibits the tyrosine kinase activities of full-length Trk receptors but also inhibits the kinase activity of the alternative TrkAIII splice variant [73, 78,79]. Lestaurtinib inhibits NB growth *in vitro* and *in vivo,* and substantially enhances the efficacy of conventional chemotherapy, such as 13-cis-retinoic acid, ferenteride and bevacizu‐ mab, presumably by inhibiting autocrine TrkB/BDNF [382-384] and/or spontaneous TrkAIII activity [73]. Lestaurtinib is an active metabolite of the Trk kinase inhibitor CEP-751, and is more suitable for clinical trials, as it can be administered orally [384, 385]. These Trk tyrosine kinase inhibitors not only target tumour promoting effects of Trk receptor activation but also Trk-mediated chemotherapeutic resistance, which has been attributed not only to TrkB [348, 358-363] but also to TrkC [375], fully spliced TrkA [375] and TrkAIII [73, 78, 79]. CEP-701 synergises with retinoids in the treatment of NB by inhibiting TrkB activity [386].

Nifutimox, a drug used for years to treat Chagas disease, is also currently in clinical trials for refractory or relapsed NB, and has been shown to suppress TrkB-mediated Akt activation and induce caspase-dependent apoptosis of NB cells *in vitro* and *in vivo* [387].

#### **7.2. TrkAIII inhibitors**

mechanism that may also extend to NBs expressing TrkC but not NT-3 in tissues that express NT-3, and could be further optimised by co-expression of CD271/p75NTR (see Section 6.3).

High levels of BDNF and TrkB expression in Nmyc-amplified NBs, in the absence of TrkA, TrkC and CD271/p75NTR, carries the worse prognosis as a result of autocrine/paracrine BDNFmediated TrkB activation, which would be expected to promote proliferation, survival and metastatic capacity. Furthermore in the absence of BDNF, TrkB would not be expected to promote apoptosis, as TrkB does not act as a dependence receptor (see Sections 6.4). As with other Trk receptors, TrkB co-expression with CD271/p75NTR would be expected to optimise NT-specificity and responsiveness, which would be expected to further promote aggressive

NBs that express CD271/p75NTR but not Trks may carry better prognosis, as they would be expected to respond to active NTs with an apoptotic response and if co-expressed with Sortilin in the absence of Trks, would also be expected to exhibit an apoptotic response to pro-NTs,

Non-Nmyc amplified NBs that express TrkAIII may carry worse prognosis, as spontaneous TrkAIII activation would override NT-dependency, provide a selective growth advantage in tissues including those that do not express NTs, promote NB cell stamilality, survival, angiogenesis and genetic instability, resulting in a more tumorigenic, metastatic and stressresistant phenotype (see Sections 4.2.1 and 6.2). Although it remains to be elucidated whether TrkAIII may interfere with CD271/p75NTR –mediated apoptosis in the presence or absence of Sortilin, its expression in NB may represent the biological equivalent to BDNF/TrkB expression in Nmyc amplified NB and TrkC/NT3 expresssion in a subset of advanced stage NBs, as an

Trk kinase inhibitors would be more suitable for use in advanced stage Nmyc amplified TrkB expressing NBs and advanced stage unfavourable non-Nmyc amplified NBs that express the TrkAIII oncogene but may also reduce survival in NBs expressing full length TrkA and TrkC

Therapeutic Trk kinase inhibitors include the selective Trk kinase inhibitors AZ-23 and AZ623, which inhibit Trk kinase activity at low nanomolar concentrations. AZ-23 has shown efficacy following oral administration in a TrkA-driven mouse allograft NB model [379], whereas AZ623 inhibits BDNF-mediated signalling and NB proliferation, and when combined with topotecan prolongs the inhibition of tumour regrowth and reduces chemo and radio thera‐ peutic resistance [380, 381]. Lestaurtinib (CEP-701) is a small-molecule receptor tyrosine kinase inhibitor that competitively inhibits ATP binding to the Trk kinase domain at nanomolar concentrations. This compound not only inhibits the tyrosine kinase activities of full-length Trk receptors but also inhibits the kinase activity of the alternative TrkAIII splice variant [73,

which comprises up to 50% of secreted NTs (see Sections 4.2.2 and 6.3).

bahaviour in TrkB expressing NBs.

72 Neuroblastoma

indicator of poor prognosis.

**7.1. Trk kinase inhibitors**

and their corresponding NTs.

**7. Potential therapeutic approaches**

Tyrosine kinase inhibitors K252a and CEP-701 inhibit TrkAIII tyrosine kinase activity. TrkAIII activity is also inhibited by the Hsp90 inhibitor geladanamycin and its clinically relevant analogues 17-AAG and 17-DMAG, and by the ARF inhibitor Brefeldin A (BFA) [78, 79]. CEP701 inhibits TrkAIII activity and TrkAIII-induced centrosome amplification at nanomolar concen‐ trations, whereas BFA reversibly inhibits spontaneous TrkAIII activation in association with disruption of the Golgi Network and the endoplasmic reticulum/Golgi Network intermediate compartment [78, 78]. Geldanamycin and its analogues reversibly inhibit TrkAIII tyrosine kinase activity and reduce proliferation of TrkAIII expressing NB cells *in vitro* [78]. Inhibitors of TrkAIII activity, however, do not inhibit TrkAIII expression nor promote TrkAIII elimina‐ tion but cause retention within the endoplasmic reticulum, with potential to induce an ER stress response. This may help to explain the high level of resistance to GA-mediated cytotox‐ icity exhibited by TrkAIII but not TrkAI transfected NB cells, despite inhibition of TrkAIII activity [78, 79]. This suggests that, in addition to other off target effects, reversible TrkAIII tyrosine kinase inhibitors may increase stress-resistance by promoting TrkAIII-ER retention and inducing an ER stress response. Consistent with this, geldanamycin selects slow growing TrkAIII expressing NB cells from mixed populations, with TrkAIII re-activation post drugremoval, suggesting a mechanism for potential post therapeutic relapse [78]. To counter this, we have also developed a specific peptide nucleic acid (PNA) inhibitor of TrkAIII expression based upon the novel exon 5/8 splice junction (TrkAIII PNA conjugate (KKAA)4-GGCCGGGA‐ CAC) [78, 79] for use in combination with with TrkAIII tyrosine kinase inhibitors, to maximise therapeutic efficacy.

#### **7.3. Agents that conserve Trk tyrosine phosphorylation and facilitate signal transduction**

TrkA activation and signal transduction is fundamental for NB differentiation and the loss of TrkA expression or defective activation and/or signalling probably contributes to NB patho‐ genesis. Agents that optimise TrkA activation and facilitate subsequent signal transduction may, therefore, overcome defective TrkA signalling and restore differentiation and/or apoptotic responses to NTs. In this context, a novel cyclophane compound CPPy, with low toxicity, has been shown to facilitate NGF-induced TrkA signal transduction through RAS/ MAPK and to induce NB differentiation [388]. Since, CD271/p75NTR optimizes TrkA responses to NTs and augments NT specificity, agents such as CPPy may be particularly useful in NBs that express TrkA but not CD271/p75NTR.

**Author details**

Marzia Ragone1

**References**

Pierdomenico Ruggeri1

Coppito II, L'Aquila, Italy

3 Neuromed Institute, Pozzilli, Italy

, Antonietta R. Farina1

, Stefania Merolle1

*and Endocrinological Metabolism*, 21, 321-355.

*and Neonatal Medicine*,17,207-215.

ment. *Development*, 129, 863-873.

2639-2648.

*ogy*, 49, 105-116.

*ment*, 132, 235-245.

, Lucia Cappabianca1

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

, Alberto Gulino2,3 and Andrew R. Mackay1

1 Department of Applied Clinical and Biotechnological Science, University of L'Aquila,

[1] Voorhess, M. L. & Gardner, L. S. (1961). Urinary excretion of neurepinephrine and 3 methoxy-4-hydroxy mandelic acid by children with neuroblastoma. *Journal of Clinical*

[2] Jiang, M.; Stanke, J. & Lahti, J. M. (2011). The connections between neural crest devel‐ opment and neuroblastoma. *Current Topics in Developmental Biology*, 94, 77-127.

[3] Fisher, J. P. H. & Tweddle, D. A. (2012). Neonatal neuroblastoma. *Seminars in Fetal*

[4] Endo, Y.; Osumi, N. & Wakamatsu, Y. (2002). Bimodal functions of Notch-mediated signalling are involved in neural crest formation during avian ectoderm develop‐

[5] Cornell, R. A. & Eisen, J. S. (2002). Delta/Notch signaling promotes formation of ze‐ brafish neural crest by repressing Neurogenin 1 function. *Development*, 129,

[6] Kalcheim, C. & Burstyn-Cohen, T. (2000). Early stages of neural crest ontogeny: for‐ mation and regulation of cell delamination. *International Journal of Developmental Biol‐*

[7] Kasemeier-Kulesa, J. C.; Kulesa, P. M. & Lefcort, F. (2005). Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia. *Develop‐*

[8] Gammill, L. S. & Roffers-Agarwal, J. (2010). Division of labour during trunk neural

[9] Schwarz, Q.; Maden, C. H.; Davidson, K. & Ruhrberg, C. (2009). Neuropilin-mediat‐ ed neural crest cell guidance is essential to organise sensory neurons into segmented

crest development. *Developmental Biology*, 344, 555-565.

dorsal root ganglia. *Development*, 136, 1785-1789.

2 Department of Molecular Medicine, University of Rome "La Sapienza", Rome, Italy

, Natalia Di Ianni1

http://dx.doi.org/10.5772/55536

,

75

#### **7.4. DNA methylation and HDACs inhibitors**

Recent reports have identified promoter methylation as an important mechanism in the transcriptional repression of TrkA and CD271/p75NTR in NB [274, 389, 390]. Therapies that reverse or inhibit DNA methylation may, therefore, be useful in malignant NBs to restore the expression of favourable NB genes. In support of this, the DNA methylation inhibitor 5-aza-2' deoxycytidine and histone deacetylase inhibitors 4-phenylbutyrate, trichostatin A and Romidepsin, have been shown to restore TrkA and CD271/p75NTR expression in NB cells, decrease proliferation, reduce tumorigenicity and promote caspase-dependent apoptosis [291, 346, 390]. Romidepsin is presently in clinical trials [346].

#### **7.5. Liposome targeting of TrkB expressing cells**

Considering the importance of TrkB in advanced stage Nmyc amplified NB, a recent report has characterised liposomes that target TrkB expressing cells, providing the opportunity to deliver nanotherapeutic cargos to TrkB expressing cells within NBs [392].

#### **8. Concluding remarks**

The complex nature of NT and NTR expression during normal development of the sympathetic nervous system is reflected in the different patterns of NT and NTR expression exhibited by human NB, which is consistent with their NCC origin at different stages along the differenti‐ ating sympathoadrenal lineage. The different biological potentials of TrkA, TrkB, TrkC, CD271/p75NTR and Sortilin receptors expressed alone or in different combinations, range from promotion of proliferation and/or differentiation to survival and/or apoptosis and to chemo‐ therapeutic resistance. This complexity is increased by the potential of each receptor to be expressed as a functionally altered alternative splice variant, the recent characterisation of TrkA and TrkC as true dependency receptors, and the pro-apoptotic behaviour of the CD271/ p75NTR -Sortilin complex, providing an exciting array of new potential ways to restore and/or modulate Trks, CD271/p75NTR and Sortilin behaviour for therapeutic purposes, based upon accurate characterisation of NT and NTR expression profiles in individual tumours.

#### **Acknowledgements**

This work was supported by grants form PRIN, AIRC and the Maugieri Foundation.

#### **Author details**

toxicity, has been shown to facilitate NGF-induced TrkA signal transduction through RAS/ MAPK and to induce NB differentiation [388]. Since, CD271/p75NTR optimizes TrkA responses to NTs and augments NT specificity, agents such as CPPy may be particularly useful in NBs

Recent reports have identified promoter methylation as an important mechanism in the transcriptional repression of TrkA and CD271/p75NTR in NB [274, 389, 390]. Therapies that reverse or inhibit DNA methylation may, therefore, be useful in malignant NBs to restore the expression of favourable NB genes. In support of this, the DNA methylation inhibitor 5-aza-2' deoxycytidine and histone deacetylase inhibitors 4-phenylbutyrate, trichostatin A and Romidepsin, have been shown to restore TrkA and CD271/p75NTR expression in NB cells, decrease proliferation, reduce tumorigenicity and promote caspase-dependent apoptosis [291,

Considering the importance of TrkB in advanced stage Nmyc amplified NB, a recent report has characterised liposomes that target TrkB expressing cells, providing the opportunity to

The complex nature of NT and NTR expression during normal development of the sympathetic nervous system is reflected in the different patterns of NT and NTR expression exhibited by human NB, which is consistent with their NCC origin at different stages along the differenti‐ ating sympathoadrenal lineage. The different biological potentials of TrkA, TrkB, TrkC, CD271/p75NTR and Sortilin receptors expressed alone or in different combinations, range from promotion of proliferation and/or differentiation to survival and/or apoptosis and to chemo‐ therapeutic resistance. This complexity is increased by the potential of each receptor to be expressed as a functionally altered alternative splice variant, the recent characterisation of TrkA and TrkC as true dependency receptors, and the pro-apoptotic behaviour of the CD271/ p75NTR -Sortilin complex, providing an exciting array of new potential ways to restore and/or modulate Trks, CD271/p75NTR and Sortilin behaviour for therapeutic purposes, based upon

accurate characterisation of NT and NTR expression profiles in individual tumours.

This work was supported by grants form PRIN, AIRC and the Maugieri Foundation.

deliver nanotherapeutic cargos to TrkB expressing cells within NBs [392].

that express TrkA but not CD271/p75NTR.

74 Neuroblastoma

**7.4. DNA methylation and HDACs inhibitors**

346, 390]. Romidepsin is presently in clinical trials [346].

**7.5. Liposome targeting of TrkB expressing cells**

**8. Concluding remarks**

**Acknowledgements**

Pierdomenico Ruggeri1 , Antonietta R. Farina1 , Lucia Cappabianca1 , Natalia Di Ianni1 , Marzia Ragone1 , Stefania Merolle1 , Alberto Gulino2,3 and Andrew R. Mackay1

1 Department of Applied Clinical and Biotechnological Science, University of L'Aquila, Coppito II, L'Aquila, Italy

2 Department of Molecular Medicine, University of Rome "La Sapienza", Rome, Italy

3 Neuromed Institute, Pozzilli, Italy

#### **References**


[10] Anderson, D. J. (2000). Genes, lineages and the neural crest a speculative view. *Philo‐ sophical Transactions of the Royal Society of Biological Sciences*, 355, 953-964.

[24] Anderson, D. J. & Axel, R. (1986). A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. *Cell*, 47, 1079-1090. [25] Anderson, D. J.; Carnahan, J. F.; Michelsohn, A. & Patterson, P. H. (1991). Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in vivo and reveal the timing of commitment to neuronal differentiation in the sym‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

77

[26] Reissmann, E.; Ernsberger, U.; Francis-West, P. H.; Rueger, D.; Brickell, P. M. & Rohr‐ er, H. (1996). Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympa‐

[27] Shah, N. M.; Groves, A. K. & Anderson, D. J. (1996). Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members. *Cell*, 85, 331-343. [28] Bilodeau, M. L.; Boulineau, T.; Greulich, J. D.; Hullinger, R. L. & Andrisani, O. M. (2001). Differential expression of sympathoadrenal lineage determining genes and phenotypic markers in cultured primary neural crest cells. *In Vitro Cellular and Devel‐*

[29] Ernsberger, U.; Esposito, L.; Partimo, S.; Huber, K.; Franke, A.; Bixby, J. L.; Kalcheim, C. & Unsicker, K. (2005). Expression of neuronal markers suggests heterogeneity of chick sympathoadrenal cells prior to invasion of the adrenal anlagen. *Cell and Tissue*

[30] Gut, P.; Huber, K.; Lohr, J.; Bruhl, B.; Oberle, S.; Treier, M.; Ernsberger, U.; Kalcheim, C. & Unsicker, K. (2005). Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation of and differentiation of chromaffin cells. *Development*, 132,

[31] Moriguchi, T.; Lim, K. C. & Engel, J. D. (2007). Transcription factor networks specify sympathetic and adrenal chromaffin cell differentiation. *Functional Developmental Em‐*

[32] Pattyn, A.; Morin, X.; Cremer, H.; Goridis, C. & Brunet, J. F. (1999). The homeobox gene Phox2B is essential for the development of autonomic neural crest derivatives.

[33] Brunet, J. F. & Pattyn, A. (2002). Phox2 genes-from patterning to connectivity. *Current*

[34] Guillemont, F.; Lo, L. C.; Johnson, J. E.; Auerbach, A.; Anderson, D. J. & Joyner, A. L. (1993). Mammalian achaete-scute homolog 1 is required for the early development of

[35] Hirsch, M. R.; Tiveron, M. C.; Guillemont, E.; Brunet, J. F. & Goridis, C. (1998). Con‐ trol of noradrenergic differentiation and Phox2a expression by MASH1 in the central

pathoadrenal lineage. *Journal of Neuroscience*, 11, 3507-3519.

thetic neurons. *Development*, 122, 2079-2088.

*opmental Biology: Animal*, 37, 185-192.

*Research*, 319, 1-13.

*bryology*, 1, 130-135.

*Nature*, 399, 366-370.

*Opinions in Genetic & Development,* 12, 435-440.

olfactory and autonomic neurons. *Cell,* 75, 463-476.

and peripheral nervous system. *Development*, 125, 599-608.

4611-4619.


[24] Anderson, D. J. & Axel, R. (1986). A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. *Cell*, 47, 1079-1090.

[10] Anderson, D. J. (2000). Genes, lineages and the neural crest a speculative view. *Philo‐*

[11] McCorry, L. K. (2007). Physiology of the autonomic nervous system. *American Journal*

[12] Young, H. M.; Cane, K. N. & Anderson, C. R. (2011). Development of the autonomic nervous system: a comparative view. *Autonomic Neuroscience: Basic and Clinical*, 165,

[13] Janig, W. & Habler, H. J. (2000). Specificity in the organisation of the autonomic nerv‐ ous system: a basis for precise neural regulation of homeostatic and protective body

[14] Shepard, D. M. & West, G. B. (1952). Noradrenalin and accessory chromaffin tissue.

[15] Tischler, A. S.; Ruzicka, L. A. & Riseberg, J. C. (1992). Immunocytochemical analysis of chromaffin cell proliferation *in vitro. Journal of Histochemistry and Cytochemistry,* 40,

[16] Schober, A. & Unsicker, K. (2001). Growth and neurotrophic properties regulating development and maintenance of sympathetic preganglionic neurons. *International*

[17] Unsicker, K. & Kriegelstein, K. (1996). Growth factors in chromaffin cells. *Progress in*

[18] Huber, K.; Kalcheim, C. & Unsicker, K. (2009). The development of the chromaffin cell lineage from the neural crest. *Autonomic Neuroscience: Basic and Clinical*, 151,

[19] Loring, J. F. & Erickson, C. A. (1987). Neural crest cell migratory pathways in the

[20] Teillet, M. A.; Kalcheim, C, Le Dourain, N. M. (1987). Formation of the dorsal root ganglia in the avian embyo: segmental origin and migratory behaviour of neural

[21] Waring, H (1935). The development of the adrenal gland of the mouse. *Quarterly*

[22] Reiprich, S.; Stolt, C. C.; Schreiner, S.; Parlato, R. & Wegner, M. (2008). SoxE proteins are differentially required in mouse adrenal gland development. *Molecular and Cellu‐*

[23] Shimada, H. (2005). In situ neuroblastoma: An important concept related to the natu‐ ral history of neural crest tumors. *Pediatric and Developmental Pathology*, 8, 305-306.

trunk of the chick embryo. *Developmental Biology,* 121, 220-236.

crest progenitor cells. *Developmental Biology*, 120, 329-347.

*Journal of Microscopic Science*. Vol. LXXVIII.

*sophical Transactions of the Royal Society of Biological Sciences*, 355, 953-964.

*of Pharmaceutical Education*, 71, 1-11.

functions. *Progress in Brain Research*, 122, 351-367.

10-27.

76 Neuroblastoma

*Nature*, 170, 42-43.

*Review of Cytology,* 205, 37-76.

*Neurobiology,* 48, 307-324.

*lar Biology*, 19, 1575-1586.

1043-1045.

10-16.


[36] Mellitzer, G.; Bonne, S.; Luco, R. F.; Van de Casteele, M.; Lenne-Samuel, N.; Collom‐ bat, P.; Mansouri, A.; Lee, J.; Lan, M.; Pipeleers, D.; Nielsen, F. C.; Ferrer, J.; Grad‐ wohl, G. & Heimberg, H. (2006). 1A1 in NGN-3-dependent and essential for differentiation of the endocrine pancreas. *EMBO Journal*, 25, 1344-1352.

neuro-endocrine secretory protein 55 and other markers of a chromaffin phenotype.

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

79

[47] Bibel, M. & Barde, Y-A. (2000). Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. *Genes and Development*, 14, 2919-2937.

[48] Kaplan, D. R. & Miller, F. D. (2000). Neurotrophin signal transduction in the nervous

[49] Oppenheim, R. W. (1991). Cell death during development of the nervous system. *An‐*

[50] Ernsberger, U. (2009). Role of neurotrophin signalling in the differentiation of neu‐ rons from dorsal root ganglia and sympathetic ganglia. *Cell and Tissue Research*, 336,

[51] Levi-Montalcini, R. & Brooker, B. (1960). Excessive growth of the sympathetic gan‐ glia evoked by a protein isolated from mouse salivary glands. *Proceeding of the Na‐*

[52] Leibrock, J.; Lottspeich, F.; Hohn, A.; Hofer, M.; Hengerer, B.; Masiakowski, P.; Thoe‐ nen, H. & Barde, Y-A. (1989). Molecular cloning and expression of brain-derived neu‐

[53] Ernfors, P. C.; Ibanez, F.; Ebendal, T.; Olson, L. & Persson, H. (1990). Molecular clon‐ ing and neurotrophic activities of a protein with similarities to nerve growth factor: developmental and topographical expression in the brain. *Proceeding of the National*

[54] Hallbook, F.; Ibanez, C. F. & Persson, H. (1991). Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ova‐

[55] McDonald, N. Q.; Lapatto, R.; Murray-Rust, J.; Gunning, J.; Wlodawaer, A. & Blun‐ dell, T. L. (1991). New protein fold revealed by a 2.3-A resolution crystal structure of

[56] Lee, R.; Kermain, P.; Teng, K. K. & Hempstead, B. L. (2001) Regulation of cell surviv‐

[58] Le, A. P. & Friedman, W. J. (2012). Matrix metalloproteinase-7 regulates cleavage of pro- nerve growth factor and is neuroprotective following Kainic acid-induced seiz‐

[59] Lu, B. & Figurov, A. (1997). Role of neurotrophins in synapse development and plas‐

[57] Thoenan, H. (1999). Neurotrophins and neuronal plasticity. *Science*, 270, 593-598.

system. *Current Opinions in Neurobiology*, 10, 381-391.

*nual Reviews in Neuroscience*, 14, 453-501.

*tional Acadamy of Science. USA*, 46, 373-384.

rotrophic factor. *Nature*, 341, 149-152.

*Acadamy of Sciences, USA*, 87, 5454-5458.

nerve growth factor. *Nature,* 354, 411-414.

ures. *Journal of Neuroscience*, 32, 703-712.

ticity. *Reviews in Neuroscience*,8, 1-12

al by secreted neurotrophins. *Science*, 294, 1945-1948.

ry. *Neuron*, 6, 845-858.

*PloS ONE* 5, e12825.

349-384.


neuro-endocrine secretory protein 55 and other markers of a chromaffin phenotype. *PloS ONE* 5, e12825.

[47] Bibel, M. & Barde, Y-A. (2000). Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. *Genes and Development*, 14, 2919-2937.

[36] Mellitzer, G.; Bonne, S.; Luco, R. F.; Van de Casteele, M.; Lenne-Samuel, N.; Collom‐ bat, P.; Mansouri, A.; Lee, J.; Lan, M.; Pipeleers, D.; Nielsen, F. C.; Ferrer, J.; Grad‐ wohl, G. & Heimberg, H. (2006). 1A1 in NGN-3-dependent and essential for

[37] Gierl, M. S.; Karoulias, N.; Wende, H.; Strehle, M. & Birchmeier, C. (2006). The zinc finger factor Insm1 (IA-1) is essential for the development of pancreatic beta cells and

[38] Morikawa, Y.; D'Atreaux, F.; Gershon, M. D. & Cserjesi, P. (2007). Hand2 determines the noradrenergic phenotype in the mouse sympathetic nervous system. *Developmen‐*

[39] Schmidt, M.; Lin, S.; Pape, M.; Ernsberger, U.; Stanke, M.; Kobayashi, K.; Howard, M. J.; and Rohrer, H. (2009). The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons. *De‐*

[40] Tsarovina, K.; Pattyn, A.; Stubbusch, J.; Muller, F.; van der Wees, J.; Schneider, C.; Brunet, J-F. & Rohrer, H. (2004). Essential role of GATA transcription factors in sym‐

[41] Finotto,S.; Kriegelstein, K.; Schober, A.; Diemling, F.; Lindner, K.; Bruhl, B.; Beier, K.; Metz, J.; Garcia-Arraras, J. E.; Roig-Lopez, J. L.; Monighan, P.; Schmid, W.; Cole, T. J.; Kellendonk, C.; Tronche, F.; Schutz, G. & Unsicker, K. (1999). Analysis of mice carry‐ ing targeted mutations of the glucocorticoid receptor gene argues against an essential role for glucocorticoid signalling for generating adrenal chromaffin cells. *Develop‐*

[42] Parlato, R.; Otto, C.; Tuckermann, J.; Stotz, S.; Kaden, S.; Grone, H. J.; Unsicker, K. & Schutz, G. (2009). Conditional inactivation of glucocorticoid receptor gene in dopa‐ mine-beta-hydroxylase cells impairs chromaffin cell survival. *Endocrinology*, 150,

[43] Lohr, J.; Gut, P.; Karch, N.; Unsicker, K. & Huber, K. (2006). Development of adrenal chromaffin cells in Sf1 heterozygous mice. *Cell and Tissue Research*, 325, 437-444.

[44] Wassberg, E.; Hedborg, F.; Skoldenberg, E.; Stridsberg, M. & Christofferson, R. (1999). Inhibition of apoptosis induces chromaffin differentiation and apoptosis in

[45] Hedborg, F.; Ulleras, E.; Grimelius, L.; Wassberg, E.; Maxwell, P. H.; Hero, B.; Bert‐ hold, F.; Schilling, F.; Harms, D.; Sabdstedt, B. & Franklin, G. (2003). Evidence for hy‐ poxia-induced neuronal-chromaffin metaplasia in neuroblastoma. *FASEB Journal,* 17,

[46] Hedborg, F.; Fischer Colbrie, R.; Ostlin, N.; Sandstedt, B.; Tran, M. G. B. & Maxwell, P. H. (2010). Differentiation in neuroblastoma: diffusion-limited hypoxia induces

differentiation of the endocrine pancreas. *EMBO Journal*, 25, 1344-1352.

intestinal endocrine cells. *Genes and Development*, 20, 2465-2478.

pathetic neuron development. *Development,* 131, 4775-4786.

neuroblastoma. *American Journal of Pathology,* 154, 395-403.

*tal Biology*, 307, 114-126.

78 Neuroblastoma

*ment,*126,2935- 2944.

1775-1781.

598-609.

*velopmental Biology*, 15, 191-200.


[60] Lu, B.; Pang, P. T. & Woo, N. H. (2005). The yin and yang of neurotrophin action. *Nature Reviews in Neuroscience*, 6, 603-614.

[72] Barker, P. A.; Lomen-Hoerth, C.; Gensch, E. M.; Meakin, S. O.; Glass, D. J. & Shooter, E. M. (1993). Tissue specific alternative splicing generates two isoforms of the TrkA

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

81

[73] Tacconelli, A., Farina, A. R.; Cappabianca, L.; DeSantis, G.; Tessitore, A.; Vetuschi, A.; Sferra, R.; Rucci, N.; Argenti, B.; Screpanti, I.; Gulino, A. & Mackay, A. R. (2004). TrkA alternative splicing: A regulated tumor-promting switch in human neuroblas‐

[74] Windisch, J. M.; Marksteiner, R. & Schneider, R. (1995). Nerve growth factor binding site on TrkA mapped to a single 24-amino acid rich leucine motif. *Journal of Biological*

[75] Ninkina, N.; Grashchuck, M.; Buchman, V. L. & Davies, A. M. (1997). TrkB variants with deletions in the leucine-rich motifs of the extracellular domain. *Journal of Biologi‐*

[76] Clary, D. O.; & Reichardt, L. F. (1994). An alternative splice form of the nerve growth factor receptor confers an enhanced response to neurotrophin 3. *Proceedings of the Na‐*

[77] Tacconelli, A., Farina, A. R.; Cappabianca, L.; Cea, G.; Panella, S.; Chioda, A.; Gallo, R.; Cinque, B.; Sferra, R.; Vetuschi, A.; Campese, A. F.; Screpanti, I.; Gulino, A. & Mackay, A. R. (2007). TrkAIII expression in the thymus. *Journal of Neuroimmunology*,

[78] Farina, A. R.; Tacconelli, A.; Cappabianca, L.; Cea, G.; Chioda, A.; Romanelli, A.; Pen‐ sato, S.; Pedone, C.; Gulino, A. & Mackay, A. R. (2009). The neuroblastoma tumoursuppressor TrkAI and its oncogenic alternative TrkAIII splice variant exhibit geldanamycin-sensitive interactions with Hsp90 in human neuroblastoma cells. *On‐*

[79] Farina, A. R.; Tacconelli, A.; Cappabianca, L.; Cea, G.; Pannella, S.; Chioda, A.; Roma‐ nelli, A.; Pedone, C.; Gulino, A. & Mackay, A. R. (2009). The TrkAIII splice variant targets the centrosome and promotes genetic instability. *Molecular and Cellular Biolo‐*

[80] Berkemeier, L. R.; Winslow, J. W.; Kaplan, D. R.; Nikolics, K.; Goeddel, D. V. & Rosenthal, A. (1991). Neurotrophin-5: a novel neurotrophic factor that activates Trk

[81] Glass, D. J.; Nye, S. H.; Hantzopoulos, P.; Macchi, M. J.; Squinto, S. P.; Goldfarb, M. & Yancopoulos, G. D. (1991). TrkB mediates BDNF/NT-3-dependent survival and pro‐

[82] Squinto, S. P.; Stitt, T. N.; Aldrich, T. H.; Davis, S.; Bianco, S. M.; Radziejewski, C.; Glass, D. J.; Masiakowski, P.; Furth, M. E.; Venezuela, D. M.; Distefano, P. S. & Yan‐

liferation in fibroblasts lacking low affinity NGF receptor. *Cell*, 66, 405-413.

receptor. *Journal of Biological Chemistry*, 268, 15150-15157.

toma. *Cancer Cell*, 6, 347-360.

*Chemistry,* 270, 28133-28136.

*cal Chemistry*, 272, 13019-13025.

183, 151-161.

*cogene*, 28, 4075-4094.

*gy*, 29, 4812-4830.

and TrkB. *Neuron*, 7, 857-866.

*tional Academy of Science. USA,* 91, 11133-11137.


[72] Barker, P. A.; Lomen-Hoerth, C.; Gensch, E. M.; Meakin, S. O.; Glass, D. J. & Shooter, E. M. (1993). Tissue specific alternative splicing generates two isoforms of the TrkA receptor. *Journal of Biological Chemistry*, 268, 15150-15157.

[60] Lu, B.; Pang, P. T. & Woo, N. H. (2005). The yin and yang of neurotrophin action.

[61] Dracopoli, N. C. & Meisler, M. H. (1990). Mapping the human amylase gene cluster on the proximal short arm of chromosome 1 using a highly informative (CA)n repeat.

[62] Maisonpierre, P. C.; Le Beau, M. M.; Espinosa, R 3rd.; Ip, N. Y.; Belluscio, L.; de la Monte, S. M.; Squinto, S.; Furth, M. E. & Yancopoulos, G. D. (1991). Human and rat brain-derived neurotrophic factor and neurotrophin-3 gene structures, distributions,

[63] Ip, N. Y.; Ibanez, C. F.; Nye, S. H.; McClain, J.; Jones, P. F.; Gles, R.; Belluscio, L.; Le Beau, M. M.; Espinosa, R 3rd. & Squinto, S. P. (1992). Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. *Pro‐*

[64] Patapoutian, A. & Reichardt, L. F. (2001). Trk receptors: mediators of neurotrophin

[65] Klein, R.; Jing, S.; Nanduri, V.; O'Rourke, E. & Barnacid, M. (1991). The trk proto-on‐

[66] Martin-Zanca, D.; Hughes, S. H. & Barbacid, M. (1986). A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequence. *Nature*,

[67] Martin-Zanca, D.; Oskam, R.; Mitra, G.; Copeland, T. & Barbacid, M. (1989). Molecu‐ lar and biochemical characterisation of the human Trk oncogene. *Molecular and Cellu‐*

[68] Greco, A.; Villa, R. & Pierotti, M. A. (1996). Genomic organization of the human

[69] Weier, H-U. G.; Rhein, A. P.; Shadravan, F.; Collins, C. & Polikoff, D. (1995). Rapid physical mapping of the human protoncogene (NTRK1) to human chromsome 1q21-22 by P1 clone selection, fluorescence in situ hybridisation (FISH), and comput‐

[70] Valent, A.; Danglot, G. & Bernheim, A. (1997). Mapping of the tyrosine kinase recep‐ tors trkA (NTRK1), trkB (NTRK2) and trkC (NTRK3) to human chromosomes 1q22, 9q22 and 15q25 by fluorescence in situ hybridization. *Europrean Journal of human ge‐*

[71] Dubus, P.; Parrens, M.; El-Mokhtari, Y.; Ferrer, J.; Groppi, A. & Merlio, J. P. (2000). Identification of novel TrkA variants with deletions in the leucine-rich motifs of the

extracellular domain. *Journal of Neuroimmunology*, 107, 42-49.

*Nature Reviews in Neuroscience*, 6, 603-614.

and chromosomal localizations. *Genomics,* 10, 558-568.

action. *Current Opinions in Neurobiology*, 11, 272-280.

*ceeding of the National Academy of Sciences, USA*, 89, 3060-3064.

cogene encodes a receptor for nerve growth factor. *Cell*, 65, 198-197.

*Genomics*, 7, 97-102.

80 Neuroblastoma

319, 743-748.

*lar Biology*, 9, 24-33.

*netics*, 5, 102-104.

NTRK1 gene. *Oncogene*, 13, 2463-2466.

er-assisted microscopy. *Genomics*, 26, 390-393.


copoulos, G. D. (1991). trkB encodes a functional receptor for brain-derived neurotro‐ phic factor and neurotrophin-3 but not nerve growth factor. *Cell*, 65, 885-893.

[95] Yano, H. & Chao, M. V. (2000). Neurotrophin receptor structure and function. *Phar‐*

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

83

[96] Wartiovaara, K.; Paavola, P.; Suvanto, P.; Paulin, L.; Saarma, M.; Peltonen, L. & Sario‐ la, H. (1997). Exclusion of the p75 neurotrophin receptor gene as a candidate gene for

[97] Von Shack, D.; Casademunt, E.; Schweigreiter, R.; Meyer, M.; Bibel, M. & Dechant, G. (2001). Complete ablation of the neurotrophin receptor p75NTR causes defects both

[98] Nykjaer, A.; Lee, R.; Teng, K. K.; Jansen, P.; Madsen, P.; Nielsen, M. S.; Jacobsen, C.; Kliemannel, M.; Schwarz, E.; Willnow, T. E.; Hempstead, B. & Petersen, C. M. (2004). Sortilin is essential for proNGF-induced neuronal cell death. *Nature,* 427, 843-847.

[99] Zampieri, N. & Chao, M. V. (2004). Structural biology. The p75 NGF receptor ex‐

[100] Harel, L.; Costa, B. & Fainzilber, M. (2010). On the death Trk. *Developmental Neurobi‐*

[101] Teng, H. K.; Teng, K. K.; Lee, R.; Wright, S.; Tevar, S.; Almeida, R. D.; Kermani, P.; Torkin, R.; Chen, Z. Y.; Lee, F. S.; Kraemer, R. T.; Nykjaer, A. & Hempstead, B. L. (2005). ProBDNF induces neuronal apoptosis via activation of a receptor complex of

[102] Yano, H.; Torkin, R.; Martin, L. A.; Chao, M. V. & Teng, K. K. (2009). Proneurotro‐ phin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing.

[103] Petersen, C. M.; Nielsen, M. S.; Nykjaer, A.; Jacobsen, L.; Tommerup, N.; Rasmussen, H. H.; Roigarard, H.; Gliemann, J.; Madsen, P. & Moestrup, S. K. (1997). Molecular identification of a novel candidate sorting receptor purified from human brain by re‐ ceptor-associated protein affinity chromatography. *Journal of Biological Chemistry*, 272,

[104] Vincent, J-P.; Mazella, J. & Kitabgi, P. (1999). Neurotensin and neurotensin receptors.

[106] Schneider, R. & Schweiger, M. (1991). A novel modular mosaic of cell adhesion mor‐ ifs in the extracellular domains of the neurogenic trk and trkB tyrosine kinase recep‐

[107] Holden, P. H.; Asopa, V.; Robertson, A. G.; Clarke, A. R.; Tyler, S.; Bennett, G. S.; Brain, S. D.; Wilcock, G. K.; Allen, S. J.; Smith, S. K. & Dawbarn, D. (1997). Immuno‐ globulin-like domains define the nerve growth factor binding site of the trkA recep‐

[105] Schweigreiter, R. (2006). The dual nature of neurotrophins. *Bioessays,* 28, 583-594.

p75NTR and Sortilin. *Journal of Neuroscience*, 25, 5455-5463.

Journal of Neuroscience, 29, 14790-14802.

*Trends in Pharmacological Sciences*, 20, 302-309.

tors. *Oncogene*, 6, 1807-1811.

tor. *Nature Biotechnology*, 15, 668-672.

in the nervous and vascular system. *Nature Neuroscience*, 4, 977-978.

*maceutica acta Helvetiae*, 74, 253-260.

posed. *Science,* 304, 833-834.

*ology,* 70, 298-303.

3599-3605.

Meckel syndrome. *Clinical Dysmorphology*, 6, 213-217.


[95] Yano, H. & Chao, M. V. (2000). Neurotrophin receptor structure and function. *Phar‐ maceutica acta Helvetiae*, 74, 253-260.

copoulos, G. D. (1991). trkB encodes a functional receptor for brain-derived neurotro‐

phic factor and neurotrophin-3 but not nerve growth factor. *Cell*, 65, 885-893.

nism. *Biochemical and Biophysical Research Communications*, 290, 1054-1065.

[83] Soilov, P.; Castren, E. & Stamm, S. (2002). Analysis of the human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mecha‐

[84] Eide, F. F.; Vining, E. R.; Eide, B. L.; Zang, K.; Wang, X. Y. & Reichardt, L. F. (1996). Naturally occurring truncated TrkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signalling. *Journal of Neuroscience*, 16, 3123-3129.

[85] Luberg, K.; Wong, J.; Weickert, C. S. & Timmusk, T. (2010). Human TrkB gene: novel alternative transcripts, protein isoforms and expression pattern in the prefrontal cere‐ bral cortex during postnatal development. *Journal of Neurochemistry*, 113, 952- 964.

[86] Fenner, B. M. (2012). Truncated TrkB: Beyond a dominant negative receptor. *Cytokine*

[87] Middlemas, D. S.; Kihl, B. K.; Zhou, J. F et al., (1999). Brain-derived neurotrophic fac‐ tor promotes survival and hemoprotection of human neuroblastoma cells. *Journal of*

[88] Lamballe, F.; Klein, R. & Barbacid, M. (1991). trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neutotrophin-3. *Cell*, 66, 967-979.

[89] Lamballe, F.; Tapley, P. & Barbacid, M. (1993). trkC encodes multiple neurotrophin-3 receptors with distinct biological properties and biological activities. *EMBO Journal,*

[90] Tsoulfas, P.; Soppet, D.; Escandon, E.; Tessarollo, L.; Mendoza-Ramirez, J. L.; Rosen‐ thal, A.; Nikolics, K. & Parada, L. F. (1993). The rat TrkC locus encodes multiple neu‐ rogenic receptors that exhibit differential response to neurotrophin-3 in PC-12 cells.

[91] Valenzuela, D. M.; Maisonpierre, P. C.; Glass, D. J.; Rojas, E.; Nunez, L.; Kong, Y.; Stitt, Ip, N. Y. & Yancopoulos, G. D. (1993). Alternative forms of rat TrkC with differ‐

[92] Menn, B.; Timsit, S.; Calothy, G. & Lamballe, F. (1998). Differential expression of trkC catalytic and noncatalytic isoforms suggests that they act independently or in associ‐

[93] Tsoulfas, P.; Stephens, R. M.; Kaplan, D. R. & Parada, L. F. (1996). trkC isoforms with insert in the kinase domain show impaired signalling responses. *Journal of Biological*

[94] Liepenish, E.; Llag, L. L.; Otting, G. & Ibanez, C. F. (1997). NMR structure of the death domain of the p75 neurotrophin receptor. *EMBO Journal*, 16, 4999-5005.

*& Growth factor Reviews,* 23, 15-24.

*Biological Chemistry,* 274, 16451-16460.

ent functional capabilities. *Neuron*, 10, 963-974.

ation. *Journal of Comparative Neurology*, 410, 47-64.

12, 3083-3094.

82 Neuroblastoma

*Neuron*, 10, 975-990.

*Chemistry*, 271, 5691-5697.


[108] Perez, P.; Coll, P. M.; Hempstead, B. L.; Martin-Zanca, D. & Chao, M. V. (1995). NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobu‐ lin-like domains. *Molecular and Cellular Neuroscience*, 6, 97-105.

[120] Mischel, P. S.; Smith, S. G.; Vining, E. R.; Valletta, J. S.; Mobley, W. C. & Reichardt, L. F. (2001). The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

85

[121] Murray, S. S.; Perez, P.; Lee, R.; Hempstead, B. L. & Chao, M. V. (2004). A novel p75 neurotrophin receptor-related protein, NRH2, regulates nerve growth factor binding

[122] Pratcha, G. & Ibanez, C. F. (2002). Lipid rafts and the control of neurotrophic factor signaling in the nervous system: variations on a theme. *Current Opinions in Neurobiol‐*

[123] Marsh, H. N.; Dubreuil, C. I.; Quevedo, C.; Lee, A.; Majdan, M.; Walsh, G. S.; Haus‐ dorff, S.; Said, F. A.; Zoueva, O.; Kozlowski, M.; Siminovitch, K.; Neel, B. G.; Miller, F. D. & Kaplan, D. R. (2003). SHP-1 negatively regulates neuronal survival by func‐

[124] Watson, F. L.; Porcionatto, M. A.; Bhattacharyya, A.; Stiles, C. & Segal, R. C. (1999). TrkA glycosylation regulates localisation and activity. *Journal of Neurobiology*, 39,

[125] Ostman, A. & Bohmer, F. D. (2001). Regulation of receptor tyrosine kinase signalling

[126] Sastry, S. K. & Elferink, L. A. (2011). Checks and balances: interplay of RTKs and PTPs in cancer progression. *Biochemical Pharmacology* http://dx.doi.org/10.1016/j.bcp.

[127] Kaplan, D. R. & Stephens, R. M. (1994). Neurotrophin signal transduction by the trk

[128] Green, L. A. & Kaplan, R. M. (1995). Early events in neurotrophin signaling via Trk

[129] Hallberg, B.; Ashcroft, M.; Loeb, D. M. & Kaplan, R. M. (1998). Nerve factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoi‐

[130] Meakin, S. O.; MacDonald, J. I. S.; Gryz, E. A.; Kubu, C. J. & Verdi, J. M. (1999). The signalling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkA. A model discriminating between proliferation and differentiation.

[131] Cunningham, M. E.; Stephens, R. M.; Kaplan, D. R. & Greene, L. A. (1997). Autophos‐ phorylation of activation loop tyrosines regulates signaling by the Trk nerve growth

tioning as a TrkA phosphatase. *Journal of Cell Biology*, 163, 999-1010.

by protein tyrosine phosphatases. *Trends in Cell Biology*, 11, 258-266.

and p75 receptors. *Current Opinions in Neurobiology*, 5, 579-587.

factor receptor. *Journal of Biological Chemistry*, 272, 10957-10967.

receptor. *Journal of Neurobiology*, 25, 1404-1417.

nositol 3-OH kinase. *Oncogene,* 17, 691-697.

*Journal of Biological Chemistry,* 274, 9861-9870.

signaling through TrkA. *Journal of Biological Chemistry*, 276, 11292-11301.

to the TrkA receptor. *Journal of Neuroscience*, 24, 2742-2749.

*ogy*, 12, 542-549.

323-336.

2011.06.016.


[120] Mischel, P. S.; Smith, S. G.; Vining, E. R.; Valletta, J. S.; Mobley, W. C. & Reichardt, L. F. (2001). The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA. *Journal of Biological Chemistry*, 276, 11292-11301.

[108] Perez, P.; Coll, P. M.; Hempstead, B. L.; Martin-Zanca, D. & Chao, M. V. (1995). NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobu‐

[109] Urfer, R.; Tsoulfas, P.; O'Connell, L. & Presta, L. G. (1997). Specificity determinants in neurotrophin-3 and design of nerve growth factor-based trkC agonists by changing central beta strand bundle residues to their neurotrophin-3 analogs. *Biochemistry,* 36,

[110] Arevalo, J. C.; Conde, B.; Hemstead, B. I.; Chao, M. V.; Martin-Zanca, D. & Perez, P. (2000). TrkA immunoglobulin-like ligand binding domains inhibit spontaneous acti‐

[111] Peng, X.; Green, L. A.; Kaplan, D. R. & Stephens, R. M. (1995). Deletion of a con‐ served juxtamembrane sequence in Trk abolishes NGF-promoted neuritogenesis.

[112] Monshipouri, M.; Jiang, H. & Lazarovici, P. (2000). NGF stimulation of erk phosphor‐ ylation is impaired by a point mutation in the transmembrane domain of the trkA re‐

[113] Kaplan, D. R.; Martin-Zanca, D. & Prada, L. F. (1991). Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. *Nature*,

[114] Wiesman, C.; Muller, Y. A. & de Vos, A. M. (2000). Ligand binding sites in Ig-like do‐ mains of receptor tyrosine kinases. *Journal of Molecular Medicine*, 78, 247-260.

[115] Arevalo, J. C.; Conde, B.; Hemstead, B. I.; Chao, M. V.; Martin-Zanca, D. & Perez, P. (2001). A novel mutation within the extracellular domain of TrkA causes constitutive

[116] Benedetti, M.; Levi, A. & Chao, M. V. (1993). Differential expression of nerve growth factors leads to altered binding affinity and neurotrophin responsiveness. *Proceeding*

[117] Mahadeo, D.; Kaplan, L.; Chao, M. V. & Hempstead, B. L. (1994). High affinity nerve growth factor binding displays a faster rate of association than p140trk binding im‐ plications for multi-subunit polypeptide receptors. *Journal of Biological Chemistry*, 269,

[118] Esposito, D.; Patel, P.; Stephens, R. M.; Perez, P.; Chao, M. V.; Kaplan, D. R. & Hemp‐ stead, B. L. (2001). The cytoplasmic and transmembrane domains of the p75 and trkA receptors regulate high affinity binding to nerve growth factor. *Journal of Biological*

[119] Bibel, H.; Hoppe, E. & Barde, Y-A. (1999). Biochemical and functional interactions be‐ tween the neurotrophin receptors trk and p75NTR. *EMBO Journal*, 18, 616-622.

lin-like domains. *Molecular and Cellular Neuroscience*, 6, 97-105.

vation of the receptor. *Molecular and Cellular Biology*, 20, 5908-5916.

ceptor. *Journal of Molecular Neuroscience*, 14, 69-76.

receptor activation. *Oncogene*, 20, 1229-1234.

*of the National Academy of Science. USA*, 90, 7859-7863.

4775-4781.

84 Neuroblastoma

*Neuron*, 15, 395-406.

350, 358-360.

6884-6891.

*Chemistry*, 276, 32687-32695.


[132] Obermeier, A.; Haltre, H.; Weismuller, K. H.; Jung, G.; Schlessinger, J. & Ullrich, A. (1993). Tyrosine 785 is a major determinant of Trk-substrate interaction. *EMBO Jour‐ nal*, 12, 933-941.

[145] Postigo, A.; Calella, A. M.; Fritzsch, B.; Knipper, M.; Katz, D.; Eilers, A.; Schimmang, T.; Lewin, G. R.; Klein, K. & Minichiello, L. (2002). Distinct requirements for TrkB and TrkC signalling in target innervation by sensory neurons. *Genes and Development*,

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

87

[146] Kong, H.; Boulter, J.; Weber, J. L.;lai, C. & Chao, M. V. (2001). An evolutionaily con‐ served transmembrane protein that is a novel downstream target of neurotrophin

[147] Arevalo, J. C.; Yano, H.; Teng, K. K. & Chao, M. V. (2004). A unique pathway for sus‐ tained neurotrophin signalling through an akyrin-rich membrane-soanning protein.

[148] Ginty, D. D.; Bonni, A. & Greeberg, M. E. (1994). Nerve growth factor activates a Rasdependent protein kinase that stimulates c-fos transcription via phosphorylation of

[149] Xing, J.; Ginty, D. D. & Greenberg, M. E. (1998). Coupling of the RAS-MAPK path‐ way to gene activation by RSK2, a growth factor-regulated CREB kinase. *Science*, 273,

[150] Deak, M.; Clifton, A. D.; Lucocq, L. M. & Alessi, D. R. (1998). Mitogen- and stressactivated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38,

[151] Atwal, J. K.; Massie, B.; Miller, F. D. & Kaplan, D. R. (2000). The TrkB-Shc site signals neuronal survival and local axon growth via MEK and PI3-kinase. *Neuron,* 27,

[152] Ecinas, M.; Iglesias, M.; Llecha, N. & Comella, J. X. (1999). Extracellular-regulated kinases and phosphoinositol 3-kinase are involved in brain-derived growth factormediated survival and neuritogenesis of the neuroblastoma cell line SH-SY5Y. *Jour‐*

[153] Florez, A. I.; Mallon, B. S.; Matsui, T.; Ogawa, W.; Rosenzweig, A.; Okamoto, T. & Macklin, W. B. (2000). Akt-mediated survival of oligodendrocytes induced by neuro‐

[154] Wooten, M. W.; Vandenplas, M. L.; Seibenhener, M. L.; Geetha, T. & Diaz-Meco, M. T. (2001). Nerve growth factor stimulates tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway. *Molecular and Cellular Biolo‐*

[155] Corbit, K. C.; Foster, D. A. & Rosner, M. R. (1999). Protein kinase Cdelta mediates neurogenic but not mitogenic activation of mitogen-activated protein kinase in neu‐

[156] Simpson, A. M.; Iyer, R.; Mangino, J. L.; Minturn, J. E.; Zhao, H.; Kolla, V. & Brodeur, G. M. (2012). TrkA3 isoform expression upregulates stem cell markers and correlates

and may mediate activation of CREB. *EMBO Journal,* 17, 4426-4441.

and aphrin receptors. *Journal of Neuroscience*, 21, 176-185.

16, 633-645.

*EMBO Journal*, 23, 2358-2368.

*nal of Neurochemistry*, 73, 1409-1421.

regulins. *Journal of Neuroscience,* 20, 7622-7630.

ronal cells. *Molecular and Cellular Biology*, 19, 4209-4218.

CREB. *Cell*, 77, 713-725.

959-963.

265-277.

*gy*, 21, 8414-8427.


[145] Postigo, A.; Calella, A. M.; Fritzsch, B.; Knipper, M.; Katz, D.; Eilers, A.; Schimmang, T.; Lewin, G. R.; Klein, K. & Minichiello, L. (2002). Distinct requirements for TrkB and TrkC signalling in target innervation by sensory neurons. *Genes and Development*, 16, 633-645.

[132] Obermeier, A.; Haltre, H.; Weismuller, K. H.; Jung, G.; Schlessinger, J. & Ullrich, A. (1993). Tyrosine 785 is a major determinant of Trk-substrate interaction. *EMBO Jour‐*

[133] Obermeier, A.; Bradshaw, R. A.; Seedorf, K.; Choidas, A.; Schlessinger, J. & Ullrich, A. (1994). Neuronal differentiation signals are controlled by nerve growth factor re‐

[134] Segal, R. A. & Greenberg, M. E. (1996). Intracellular signalling pathways activated by

[135] Yao, R. & Cooper, G. M. (1995). Requirement for phosphoinositol-3 kinase in the pre‐

[136] MacDonald, J. I.; Gryz, E. A.; Kubu, C. J.; Verdi, J. M. & Meakin, S. O. (2000). Direct binding of the signalling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA. *Journal of Biological Chemistry*,

[137] Hagag, N.; Halegoua, S. & Viola, M. (1986). Inhibition of growth factor induced dif‐ ferentiation of PC12 cells by microinjection of antibody to ras p21. *Nature*, 319,

[138] Hampstead, B. L.; Martin-Zanca, D.; Kaplan, D. R.; Parada, L. F. & Chao, M. V. (1991). High affinity NGF binding requires coexpression of the trk proto-oncogene

[139] Majdan, M.; Walsh, G. S.; Aloyz, R. & Miller, F. D. (2001). TrkA mediates develop‐ mental sympathetic neuron survival by silencing an ongoing p75NTR-mediated

[140] Zaccaro, M. C.; Ivanisevic, L.; Perez, P.; Meakin, S. O. & Saragovi, H. U. (2002). P75 coreceptors regulate ligand dependent and ligand independent Trk receptor activa‐ tion, in part by altering trk docking subdomains. *Journal of Biological Chemistry*, 276,

[141] Nykjaer, N.; Willnow, T. E.; & Munk-Peterson, C. (2005). P75NTR-liver or let die.

[142] York, R. D.; Yao, H.; Dillon, T.; Ellig, C. L.; Eckert, S. P.; McCleskey, E. W. & Stork, P. J. (1998). Rap1 mediates sustained MAP kinase activation induced by nerve growth

[143] Wu, C.; Lai, C. F. & Mobley, W. C. (2001). Nerve growth factor activates persistent

[144] Minichiello, L.; Casagrande, F.; Tatche, R. S.; Stucky, C. L.; Postigo, A.; Lewin, G. R.; Davies, A. M. & Klein, R. (1998). Point mutation in trkB causes loss of NT-4-depend‐ ent neurons without major effect on diverse BDNF responses. *Neuron*, 21, 335-345.

Rap1 signaling in endosomes. *Journal of Neuroscience*, 21, 5406-5416.

and low affinity NGF receptor. *Nature*, 350, 678-683.

death signal. *Journal of Cell Biology*, 155, 1275-1285.

*Current Opinions in Neurobiology,* 15, 40-57.

factor. *Nature*, 392, 622-626.

ceptor/Trk binding sites for Shc and PLCγ. *EMBO Journal*, 13, 1585-1590.

neurotrophic factors. *Annual Reviews in Neuroscience*, 19, 463-489.

vention of apoptosis by nerve growth factor. *Science,* 267, 2003-2005.

*nal*, 12, 933-941.

86 Neuroblastoma

275, 18225-18233.

680-682.

31023-31029.


with worse outcome in neuroblastomas (NBs). *Proceedings of the Advances in Neuro‐ blastoma Research (2012)* (Meeting Abstract POT055), p 164.

[168] Paul, C. E.; Vereker, E.; Dickson, K. M. & Barker, P. A. (2004). A pro-apoptotic frag‐ ment of the p75 neurotrophin receptor is expressed in p75NTR exon IV null mice.

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

89

[169] Gentry, J. J.; Barker, P. A. & Carter, B. D. (2004). The p75 neurotrophin receptor: mul‐ tiple interactors and numerous functions. *Progress in Brain Research,* 146, 25-39.

[170] Miller, F. D. & Kaplan, D. R. (2001). Neurotrophin signalling pathways regulating

[171] Bhakar, A. L.; Howell, J. L.; Paul, C. E.; Salehi, A. H.; Becker, E. B.; Said, F.; Bonni, A. & Barker, P. A. (2003). Apoptosis induced by p75NTR overexpression requires jun kinase-dependent phosphorylation of Bad. *Journal of Neuroscience,* 23, 11373-11381.

[172] Okuno, S.; Saito, A.; Hayashi, T. & Chan, P.H. (2004). The c-Jun N-terminal protein kinase signaling pathway mediates bax activation and subsequent neuronal apopto‐ sis through interaction with bim after transient focal cerebral ischemia. *Journal of*

[173] Barker, P. A. (2004). P75NTR is positively promiscuous: novel partners and new in‐

[174] Becker, E. B. E.; Howell, J.; Kodama, Y.; Barker, P. A. & Bonni, A. (2004). Characteri‐ sation of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal apopto‐

[175] Casademunt, E.; Carter, B. D.; Benzel, I.; Frade, J. M.; Dechant, G. & Barde, Y-A. (1999). The zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR) and participates in programmed cell death. *EMBO Journal*, 18, 6050-6061.

[176] Salehi, A. H.; Roux, P. P.; Kubu, C. J.; Zeindler, C.; Bhakar, A.; Tannis, L. L.; Verdi, J. M. & Barker, P. A. (2000). RAGE, a novel MAGE protein, interacts with the p75 neu‐ rotrophin receptor and facilitates nerve growth factor-dependent apoptosis. *Neuron*,

[177] Park, J. A.; Lee, J. Y.; Sato, T. A. & Koh, J. Y. (2000). Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture

[178] Chittka, A.; Arevalo, J. C.; Rodriguez-Guzman, M.; Perez, P.; Chao, M. V. & Sendt‐ ner, M. (2004). The p75NTR-interacting protein SC1 inhibits cell cycle progression by

[179] Arevalo, J. C. & Wu, S. H. (2006). Neurotrophin signalling: many exciting surprises.

[180] Yamashito, H.; Avraham, S.; Jiang, S.; Dikic, I. & Avram, H. (1999). The Csk homolo‐ gous kinase associates with TrkA receptors and is involved in neurite outgrowth of

or transient ischemia in the rat. *Journal of Neuroscience*, 20, 9069-9103.

transcriptional repression of cyclin E. *Journal of Cell Biology*, 164, 985-996.

PC12 cells. *Journal of Biological Chemistry*, 274, 15059-15065.

neuronal apoptosis. *Cellular and Molecular Life Sciences,* 58, 7879-7887.

*Journal of Neuroscience,* 24, 1917-1923.

*Neuroscience,* 24, 7879-7887.

sights. *Neuron,* 42, 529-533.

*Cell Mol Life Sci* 63, 1523-1537.

27. 279-288.

sis. *Journal of Neuroscience,* 24, 8762-8770.


[168] Paul, C. E.; Vereker, E.; Dickson, K. M. & Barker, P. A. (2004). A pro-apoptotic frag‐ ment of the p75 neurotrophin receptor is expressed in p75NTR exon IV null mice. *Journal of Neuroscience,* 24, 1917-1923.

with worse outcome in neuroblastomas (NBs). *Proceedings of the Advances in Neuro‐*

[157] Moises, T.; Dreieir, A.; Flohr, S.; Esser, M.; Brauers, E.; Reiss, K.; Merken, D.; Weis, J. & Kruttgen, A. (2007). Tracking TrkA's trafficking: NGF receptor trafficking controls

[158] Howe, C. L. & Mobley, W. C. (2004). Signaling endosome hypothesis: a cellular mechanism for long distance communication. *Journal of Neurobiology*, 58, 207-216.

[159] Valdez, G.; Akmentin, W.; Philippidou, P.; Kuruvilla, R.; Ginty, D. D. & Halegoua, S. (2005). Pincher-mediated macroendocytosis underlies retrograde signalling by neu‐

[160] Rajagopal, R.; Chen, Z. Y.; Lee, F. S. & Chao, M. V. (2004). Transactivation of Trk neu‐ rotrophin receptors by G-protein-coupled receptor ligands occurs on intracellular

[161] Shi, G. X.; Jin, L. & Andres, D. A. (2010). Src-dependent TrkA transactivation is re‐ quired for pituitary adenylate cyclase- activating polypeptide 38-mediated Rit activa‐

[162] He, X. L. & Garcia, K. C. (2004). Structure of nerve growth factor complexed with the

[163] Huang, E. J. & Reichardt, L. F. (2003). Trk receptors: roles in neuronal signal trans‐

[164] Kuruvilla, R.; Zweifel, L. S.; Glebova, N. O.; Lonze, B. E.; Valdez, G.; Ye, H. & Ginty, D. D. (2004). A neurotrophin signaling cascade coordinates sympathetic neuron de‐ velopment through differential control of TrkA trafficking and retrograde signaling.

[165] Epa, W. R.; Markovska, K. & Barrett, G. L. (2004). The p75 neurotrophin receptor en‐ hances TrkA signaling by binding to Shc and augmenting its phosphorylation. *Jour‐*

[166] Hannila, S. S.; Lawreance, G. M.; Ross, G. M. & Kawaja, M. D. (2004). TrkA and mito‐ gen-activated protein kinase phosphorylation are enhanced in sympathetic neurons lacking functional p75 neurotrophin receptor expression. *European Journal of Neuro‐*

[167] Lad, S. P.; Peterson, D. A.; Bradshaw, R. A. & Neet, K. E. (2003). Individual and com‐ bined effects of TrkA and p75NTR nerve growth factor receptors. A role for high af‐

finity receptor sites. *Journal of Biological Chemistry,* 278, 24808-24817.

tion and neuronal differentiation. *Molecular Biology of the Cell*, 21, 1597-1608.

*blastoma Research (2012)* (Meeting Abstract POT055), p 164.

NGF receptor signalling. *Molecular Neurobiology,* 35, 151-159.

rotrophin receptors. *Journal of Neuroscience*, 25, 5236-5247.

membranes. *Journal of Neuroscience*, 24, 6650-6658.

shared neurotrophin receptor p75. *Science,* 304, 870-875.

duction. *Annual Reviews in Biochemistry,* 72, 609-642.

2004

88 Neuroblastoma

*Cell* 118, 243-255.

*science,* 19, 2903-2908.

*nal of Neurochemistry,* 89, 344-353.


[181] Frade, J. M. (2005). Nuclear translocation of the p75 neurotrophin receptor cytoplas‐ mic domain in response to neurotrophin binding. *Journal of Neuroscience*, 25, 1407-1411.

[193] Bernd, P. (2008). The role of neurotrophins during early development. *Gene Expres‐*

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

91

[194] Kahane, N. & Kalcheim, C. (1994). Expression of trkC receptor mRNA during devel‐ opment of the avian nervous system. *Journal of Neurobiology*, 25, 571-584.

[195] Chalazonitis, A.; Pham, T. D.; Rothman, T. P.; DiStefano, P. S.; Bothwell, M.; Blair-Flynn, J.; Tassarollo, L. & Gershon, M. D. (2001). Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons. *Journal of neuro‐*

[196] Ernfors, P.; Kucera, J.; Lee, K. F.; Loring, J. & Jaenisch, R. (1995). Studies on the phys‐ iological role of brain-derived neurotrophic factor and neurotrophin-3 in knockout

[197] Farinas, I.; Jones, K. R.; Backus, C.; Wang, X. Y. & Reichardt, L. F. (1994). Severe sen‐ sory and sympathetic deficits in mice lacking neurotrophin-3. *Nature*, 369, 658-661.

[198] Yao, L.; Zhang, D. & Bernd, P. (1994). The onset of neurotrophin and trk mRNA ex‐ pression in early embryonic tissue of the quail. *Developmental Biology*, 165, 727-730.

[199] Zhang, D.; Yao, L. & Bernd, P. (1994). Expression of trk and neurotrophin mRNA in dorsal root and sympathetic ganglia of the quail during development. *Journal of Neu‐*

[200] Henion, P. D.; Garner, A. S.; Large, T. H. & Weston, J. A. (1995). trkC-mediated NT-3 signaling is required for the early development of a subpopulation of neurogenic

[201] Kalcheim, C.; Carmeli, C. & Rosenthal, A. (1992). Neurotrophin-3 is a mitogen for cultured neural crest cells. *Proceeding of the National Acadeny of Science. USA*, 89,

[202] Maisonpierre, P. C.; Belluscio, L.; Friedman, B.; Alderson, R. F.; Wiegand, S. J.; Furth, M. E.; Lindsay, R. M. & Yancopoulos, G. D. (1990). NT-3, BDNF, and NGF in the de‐ veloping rat nervous system: parallel as well as reciprocal petterns of expression.

[203] Ernfors, P.; Merlio, J. P. & Persson, H. (1992). Cells expressing mRNA for neurotro‐ phins and their receptors during embryonic rat development. *European Journal of*

[204] Verdi, J. M.; Groves, A. K.; Farinas, I.; Jones, K.; Marchionni, M. A.; Reichardt, L. F. & Anderson, D. J. (1996). A reciprocal cell-cell interaction mediated by NT-3 and neure‐ gulins controls the early survival and development of sympathetic neuroblasts. *Neu‐*

[205] DiCicco-Bloem, E.; Friedman, W. J. & Black, I. B. (1993). NT-3 stimulates sympathetic neuroblast proliferation by promoting precursor survival. *Neuron,* 11, 1101-1111.

mice. *International Journal of Developmental Biology*, 39, 799-807.

neural crest cells. *Developmental Biology*,172, 602-613.

*sion*, 14, 241-250.

*science*, 21, 5620-5636.

*robiology*, 25, 1517-1532.

1661-1665.

*Neuron,* 5, 501-509.

*ron*, 16, 515-527.

*Neuroscience*, 4, 1140-1158.


[193] Bernd, P. (2008). The role of neurotrophins during early development. *Gene Expres‐ sion*, 14, 241-250.

[181] Frade, J. M. (2005). Nuclear translocation of the p75 neurotrophin receptor cytoplas‐ mic domain in response to neurotrophin binding. *Journal of Neuroscience*, 25,

[182] Datta, S. R.; Dudek, H.; Tao, X.; Masters, S.; Fu, H.; Gotoh, Y. & Greenberg, M. E. (1997). Akt phosphorylation of BAD couples survival signals to the cell-intrinsic

[183] Orike, M.; Middleton, G.; Borthwick, E.; Buchman, V. Cowen, T. & Davies, A. M. (2001). Role of PI-3 kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-dependent adult sympathetic neurons. *Journal of Cell Biology*,

[184] Brunet, A.; Bonni, A.; Zigmond, A. J.; Lin, M. Z.; Juo, P.; Hu, L. S.; Anderson, M. J.; Arden, K. C.; Blenis, J. & Greenberg, M. E. (1999). Akt promotes cell survaival by phosphorylating and inhibiting a Forkhead transcription factor. *Cell,* 96, 857-868.

[185] Wyttenbach, A. & Tolkovsky, A. M. (2006). The BH3-only protein Puma is both nec‐ essary and sufficient for neuronal apoptosis induced by DNA damage in sympathet‐

[186] Putcha, G. V.; Moulder, K. L.; Golden, J. P.; Bouillet, P.; Adams, J. A.; Strasser, A. & Johnson, E. M. (2001). Induction of BIM, a proapoptotic BH3-only BCL-2 family

[187] Gilley, J.; Coffer, P. J. & Ham, J. (2003). FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. *Journal of Cell*

[188] Whitfield, J.; Neame, S. J.; Parquet, L.; Bernard, O. & Ham, J. (2001). Dominant-nega‐ tive c-Jun promotes neuronal survival by reducing BIM expression and inhibiting

[189] Du, K. & Mintiminy, M. (1998). CREB is a regulatory target for the protein kinase

[190] Riccio, A.; Ahn, S.; Davenport, C. M.; Blendy, J. A. & Ginty, D. D. (1999). Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic

[191] Tauszig-Delamasure, S.; Yu, L-Y.; Cabrera, J. R.; Bouzas-Rodriguez, J.; Mermet-Bouv‐ ier, C.; Giux, C.; Bordeux, M-C.; Arumae, U. & Mehlen, P. (2007). The TrkC receptor induces apoptosis when the dependence receptor meets the neurotrophin paradigm.

[192] Nikoletopoulou, V.; Lickert, H.; Frade, J. M.; Rencurel, C.; Giallonardo, P.; Zhang, L.; Bibel, M. & Barde, Y-A. (2010). Neurotrophin receptors TrkA and TrkC cause neuro‐

*Proceedings of the National Acadamy of Sciences, USA,* 104, 13361-13366.

ic neurons. *Journal of Neurochemistry*, 96, 1213-11226.

member, is critical for neuronal apoptosis. *Neuron*, 29, 615-628.

mitochondrial cytochrome c release. *Neuron*, 29, 629-643.

Akt/PKB. *Journal of Biological Chemistry*, 273, 32377-32379.

nal death whereas TrkB does not. *Nature,* 467, 59-64.

1407-1411.

90 Neuroblastoma

154, 995-1005.

*Biology*, 162, 613-622.

neurons. *Science*, 286, 2358-2361.

death machinary. *Cell,* 91, 231-241.


[206] Levi-Montalcini, R. (1987). The nerve growth factor: thirty-five years later. *EMBO Journal*, 6, 1145-1154.

[220] Scarisbrick, I. A.; Jones, E. G. & Isackson, P. J. (1993). Coexpression of mRNAs for NGF, BDNF, and NT-3 in the cardiovascular system of pre- and postnatal rats. *Jour‐*

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

93

[221] Hamberger, V. & Levi-Montalcini, R. (1949). Proliferation, differentiation and degen‐ eration in the spinal ganglia of the chick embry under normal and experimental con‐

[222] Saltis, J. & Rush, R. A. (1995). Effects of nerve growth factor on sympathetic neuron development in normal and limbless chick embryos. *International Journal of Develop‐*

[223] Lee, K. F.; Davies, A. M. & Jaenisch, R. (1994). P75-deficient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF.

[224] Birren, S.; Lo, L. & Anderson, D. J. (1993). Sympathetic neuroblasts undergo a devel‐

[225] Straub, J. A.; Saulnier Sholler, G. L. & Nishi, R. (2007). Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brian-derived neuro‐

[226] Elkabes, S.; Dreyfus, C. F.; Schaar, D. G. & Black, I. B. (1994). Embryonic sensory de‐ velopment: local expression of neurotrophin-3 and target expression of nerve growth

[227] Pizzuti, A.; Borsani, G.; Falini, A.; Sidoli, A.; Baralle, F. E.; Scarlato, G. & Silani, V. (1990). Detection of beta-nerve growth factor mRNA in the human fetal brain. *Brain*

[228] Quartu, M.; Geic, M. & Del Flacco, M. (1997). Neurotrophin-like immunoreactivity in

[229] Schober, A.; Wolf, N.; Huber, K.; Hertel, R.; Krieglstein, K.; Minichiello, L.; Kahane, N.; Widenfalk, J.; Kalcheim, C.; Olsen, L.; Klein, R.; Lewin, G. R. & Unsicker, K. (1998). TrkB and neurotrophin-4 are important for development and maintenance of sympathetic preganglion neurons innervating the adrenal medulla. *Journal of Neuro‐*

[230] Schober, A.; Minichiello, L.; Keller, M.; Huber, K.; Layer, P. G.; Roig-Lopez, J. L.; Gar‐ cia-Arraras, J. E.; Klein, R. & Unsicker, K. (1997). Reduced acetylcholinesterase (AChE) activity in adrenal medulla and loss of sympathetic preganglionic neurons in TrkA-deficient, but not TrkB-deficient, mice*. Journal of Neuroscience,* 17, 891-903. [231] Snider, W. D. (1994). Functions of the neurotrophins during nervous system develop‐

[232] Bode, K.; Hofmann, H. D.; Muller, T. H.; Otten, U.; Schmidt, R. & Unsicker, K. (1986). Effects of pre- and postnatal administration of antibodies to nerve growth factor on

opmental switch in trophic dependence. *Development* ,119, 597-610.

trophic factor in vitro. *BMC Developmental Biology,* 7, 1-13.

the human trigeminal ganglion. *Neuroreports*, 8, 3611-3617.

ment: what the knockouts are teaching us. *Cell*, 77, 627-638.

factor. *Journal of Comparative Neurology*, 341, 204-213.

*nal of Neuroscience*, 13, 875-893.

*mental Neuroscience*, 13, 577-584.

*Development*, 120, 1027-1033.

*Research*, 518, 337-341.

*science*, 18, 7272-7284.

ditions. *Journal of Experimental Zoology,* 111, 457-501.


[220] Scarisbrick, I. A.; Jones, E. G. & Isackson, P. J. (1993). Coexpression of mRNAs for NGF, BDNF, and NT-3 in the cardiovascular system of pre- and postnatal rats. *Jour‐ nal of Neuroscience*, 13, 875-893.

[206] Levi-Montalcini, R. (1987). The nerve growth factor: thirty-five years later. *EMBO*

[207] Verdi, J. M. & Anderson, D. J. (1994). Neurotrophins regulate sequential changes in neurotrophin receptor expression by sympathetic neuroblasts. *Neuron,* 13, 1359-1372.

[208] Wetmore, C. & Olson, L. (1995). Neuronal and non neuronal expression of neurotro‐ phins and their receptors in sensory and sympathetic ganglia suggest new intercellu‐

[209] Rosenthal, A.; Goeddel, D. V.; Nguyen, T.; Lewis, M.; Shih, A.; Laramee, G. R.; Nikol‐ ics, K. & Winslow, J. W. (1990). Primary structure and biological activity of a novel

[210] Reichardt , L. F. (2006). Neurotrophin-regulated signalling pathways. *Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences*, 361, 1545-1564.

[211] ElShamy, W. M.; Linnarsson, S.; Lee, K. F.; Jaenisch, R. & Enfors, P. (1996a). Prenatal and postnatal requirements of NT-3 for sympathetic neuroblast survival and inner‐

[212] ElShamy, W. M. & Enfors, P. (1996b). A local action of neurotrophin-3 prevents death

[213] Schecterson, L. C. & Bothwell, M. (1992). Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons. *Neuron*, 9, 449-463.

[214] Zhou, X-F. & Rush, R. A. (1996). Functional roles for neurotrophin-3 in the develop‐ ing and mature sympathetic nervous system. *Molecular Neurobiology,* 13, 185-197. [215] Francis, N.; Farinas, I.; Brennan, C.; Rivas-Plata, K.; Backus, C.; Reichardt, L. & Landis, S. (1999). NT-3, like NGF, is required for survival of sympathetic neurons,

[216] Davies, A. M. (1994). The role of neurotrophins in the developing nervous system.

[217] Ockel, M.; von Schack, D.; Schropel, A.; Dechant, G.; Lewin, G. R. & Barde, Y-A. (1996). Roles of neurotrophin-3 during early development of the peripheral nervous system. *Philosophical Transactions of the Royal Society: Biological Science*, 351, 383-387.

[218] Rush, R. A.; Chie, E.; Liu, D.; Tafreshi, A.; Zettler, C. & Zhou, X. F. (1997). Neurotro‐ phic factors are required by mature sympathetic neurons for survival, transmission and connectivity. *Clinical and Experimental Pharmacology and Physiology*, 24, 549-555.

[219] Schecterson, L. C. & Bothwell, M. (2009). Neurotrophin receptors: old friends with

of proliferating sensory neuron precusor cells. *Neuron*, 16, 963-972.

but not their precursors. *Developmental Biology*, 210, 411-427.

new partners. *Developmental Neurobiology,* 70, 332-338.

*Journal of Neurobiology*, 25, 1134-1148.

lar trophic interactions. *Journal of Comparative Neurology*, 353, 143-159.

human neurotrophic factor. *Neuron*, 4, 767-773.

vation of specific targets. *. Development*, 122, 491-500.

*Journal*, 6, 1145-1154.

92 Neuroblastoma


the morphological and biochemical behaviour of the rat adrenal medulla: a reinvesti‐ gation. *Brain Research*, 392, 139-150.

[246] Causing, C. G.; Gloster, A.; Aloyz, R.; Bamj, S. X.; Chang, E.; Fawcett, J.; Kuchel, G. & Miller, F. D. (1997). Synaptic innervation density is regulated by neuron-derived

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

95

[247] Timmusk, T.; Belluardo, N.; Metsis, M. & Persson, H. (1993). Widespread and devel‐ opmentally regulated expression of neurotrophin-4 mRNA in rat brain and peripher‐

[248] Kondo, Y.; Saruta, J.; To, M.; Shiiki, N.; Sato, C. & Tsukinoki, K. (2010). Expression and role of the BDNF receptor-TrkB in rat adrenal gland under acute immobilisation

[249] Morrison, S. J.; White, P. M.; Zock, C.; & Anderson, D. J. (1999). Prospective identifi‐ cation, isolation by flow cytometry, and in vivo self-renewal of multipotent mamma‐

[250] Boiko, A, D.; Razorenova, O. V.; van de Rijn, M.; Swetter, S. M.; Johnson, D. L.; Ly, D. P.; Butler, P. D.; Yang, G. P.; Joshua, B.; Kaplan, M. J.; Longaker, M. T. & Weissman, I. L. (2010). Human melanoma initiating cells express neural crest nerve growth factor

[251] Baker, D. L.; Molenaar, W. M.; Trojanowski, J. Q.; Evans, A. E.; Ross, A. H.; Rorke, L. B.; Packer, R. J.; Lee, V. M-Y. & Pleasure, D. (1991). Nerve growth factor receptor ex‐ pression in peripheral and central neuroectodermal tumors, other pediatric brain tu‐ mors and during development of the adrenal gland. *American Journal of Patholology*,

[252] Garin-Chesa, P.; Rettig, W.; Thomson, T. M.; Old, L. J. & Melamed, M. R. (1988). Im‐ munohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. *Journal of Histochemistry and Cytochemistry,* 36, 383-389.

[253] Barrett, G. L.; Georgiou, A.; Ried, K.; Bartlett, P. F. & Leung, D. (1998). Rescue of dor‐ sal root sensory neurons by nerve growth factor and neurotrophin-3, but not brainderived neurotrophic factor or neurotrophin-4, is dependent on the level of the p75

[254] Bamji, S. X.; Majdan, M.; Pozniak, C. D.; Belliveau, D. J.; Aloyz, R.; Kohn, J.; Causing, C. G. & Miller, F. D. (1998). The p75 neurotrophin receptor mediates neuronal apop‐ tosis and is essential for naturally occurring sympathetic neuron death. *Journal of Cell*

[255] Ibanez, C. F. & Simi, A. (2012). P75 neurotrophin receptor signalling in nervous sys‐ tem injury and degeneration: paradox and opportunity. *Trends in Neuroscience,* 35,

[256] Lorentz, C. U.; Woodward, W. R.; Tharp, K. & Habecker, B. A. (2011). Altered norepi‐ nephrine content and ventricular function in p75NTR-/- mice after myocardial infarc‐

al tissues. *European Journal of Neuroscience*, 5, 605-613.

stress. *Acta Histochemica Cytochemica,* 43, 139-147.

neurotrophin receptor. *Neuroscience*, 85, 1321-1328.

tion. *Autonomic Neuroscience,* 164, 13-19.

lian neural crest stem cells. *Cell,* 96, 737-749.

receptor CD-271. *Nature*, 466, 133-137.

139, 115-122.

*Biology*, 140, 911-923.

431-440

BDNF. *Neuron*, 257-267.


[246] Causing, C. G.; Gloster, A.; Aloyz, R.; Bamj, S. X.; Chang, E.; Fawcett, J.; Kuchel, G. & Miller, F. D. (1997). Synaptic innervation density is regulated by neuron-derived BDNF. *Neuron*, 257-267.

the morphological and biochemical behaviour of the rat adrenal medulla: a reinvesti‐

[233] Shibayama, E. & Koizima, H. (1996). Cellular localisation of the Trk neurotrophin re‐ ceptor family in human non-neural tissues. *American Journal of Pathology,* 148,

[234] Lillien, L. E. & Claude, P. (1985). Nerve growth factor is a mitogen for cultured chro‐

[235] Aloe L.; Alleva, E.; Bohm, A. & Levi-Montalcino, R. (1986). Aggressive behaviour in‐ duces release of nerve growth factor from mouse salivary glands. *Proceedings of the*

[236] Otten, U.; Schwab, M.; Gangnon, C. & Thoenen, H. (1977). Selective induction of ty‐ rosine hydroxylase and beta-hydroxylase by nerve growth factor: comparison be‐ tween adrenal medulla and sympathetic ganglia of adult and newborn rats. *Brain*

[237] Yamamoto, M. & Iseki, S. (2003). Co-expression of NGF and its high-affinity receptor trkA in the rat Carotic body chief cells. *Acta Histochem Cytochem* 36, 377-383.

[238] Suter-Crazzolara, C.; Lachmund, A.; Arab, S. F. & Unsicker, K. (1996). Expression of neurotrophins and their receptors in the developing and adult rat adrenal gland. *Mo‐*

[239] Unsicker, K. & Kriegelstein, K. (1996). Growth factors in chromaffin cells. *Progress in*

[240] Unsicker, K.; Huber, K.; Schutz, G. & Kalcheim, C. (2005). The chromaffin cell and its

[241] Doupe, A. J.; Landis, S. C. & Patterson, P. H. (1985). Environmental factors in the de‐ velopment of neural crest derivatives: glucocorticoids, growth factors, and chromaf‐

[242] Wyatt, S. & Davies, A. M. (1995). Regulation of nerve growth factor receptor gene ex‐ pression in sympathetic neurons during development. *Journal of Cell Biology,* 130,

[243] Jungbluth, S.; Koentges, G. & Lumsden, A. (1997). Coordination of early neural tube

[244] Pinon, L. G.; Minichiello, L.; Klein, R. & Davies, A. M. (1996). Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sen‐

[245] Dixon, J. E. & McKinnon, D. (1994). Expression of trk gene family of neurotrophin re‐ ceptors in prevertebral sympathetic ganglia. *Brain Research Developmental Brain Re‐*

sory neuron dependence on Trk signalling. *Development*, 122, 3255-3261.

gation. *Brain Research*, 392, 139-150.

maffin cells. *Nature*, 317, 632-634.

*lecular Brain Research,* 43, 351-355.

*Neurobiology,* 48, 307-324.

1435-1446.

*search*, 77,177-182.

*Research,* 133, 291-303.

*National Acadamy of Sciences USA,* 83, 6184-6187.

development. *Neurochemistry Research,* 30, 921-925.

fin cell plasticity. *Journal of Neuroscience*, 5, 2119-2142.

development by BDNF/trkB. *Development*, 124, 1877-1885.

1807-1818.

94 Neuroblastoma


[257] Nakagawara, A.; Arima, M.; Azar, C. G.; Scavards, N. J. & Brodeur, G. M. (1992). In‐ verse relationship between trk expression and N-myc amplification in human neuro‐ blastomas. *Cancer Research,* 52, 1364-1368.

[267] Tajiri, T.; Higashi, M.; Souzaki, R.; Tatsuta, K.; Kinoshita, Y. & Taguchi, T. (2007). Classification of neuroblastomas based on an analysis of the expression of genes re‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

97

[268] Weinreb, I.; Goldstein, D.; Irish, J. & Perez-Ordonez, B. (2009). Expression patterns of Trk-A, Trk-B, GRP78, and p75NRT in olfactory neuroblastoma. *Hum Pathol* 40,

[269] Light, J. E.; Koyama, H.; Minturn, J. E.; Ho, R.; Simpson, A. M.; Iyer, R.; Mangino, J. L.; Kolla, V.; London, W. B. & Brodeur, G. M. (2012). Clinical significance of NTRK

family gene expression in neuroblastomas. *Pediatric Blood Cancer,* 59, 226-232.

[270] Shimada, H.; Nakagawara, A.; Peters, J.; Wang, H.; Wakamatsu, P- K.; Lukens, J. N.; Matthay, K. K.; Siegel, S. E. & Seeger, R. C. (2004). TrkA expression in peripheral neu‐

[271] Cheng, A. J.; Cheng, N. C.; Ford, J.; Smith, J.; Murray, J. E.; Flemming, C.; Lastowska, M.; Jackson, M. S.; Hackett, C. S.; Weiss, W. A.; Marshall, G. M.; Kees, U. R.; Murray, D. N. & Haber, M. (2007). Cell lines from MycN transgenic murine tumours reflect molecular and biological characteristics of human neuroblastoma. *European Journal of*

[272] Cohn, S. L.; Look, T. A.; Joshi, V. V.; Holbrook, T.; Salwern, H.; Chagnovich, D.; Chesler, L.; Rowe, S. T.; Valentine, M. B.; Komuro, H.; Castleberry, R. P.; Bpwman, L. C.; Rao, P. V.; Seeger, R. C. & Brodeur, G. M. (1995). Lack of correlation of N-myc gene amplification with prognosis in localised neuroblastoma: a pediatric oncology

[273] Comstock, J. M.; Willmore-Payne, C.; Holden, J. A. & Coffin, C. M. (2009). Composite pheochromocytoma: a clinicopathologic and molecular comparison with ordinary pheochromocytoma and neuroblastoma. *American Journal of Clinical Phology*,132,

[274] Iraci, N.; Diolati, D.; Papa, A. et al., (2011). A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TrkA and p75NTR promoters and effects neuroblastoma ma‐

[275] Lau, D. T.; Hesson, L. B.; Norris, M. D.; Marshall, G. M.; Haber, M. & Ashton, L. J. (2012). Prognostic significance of promoter DNA methylation in patients with child‐

[276] De Preter, K.; Vandesompele, J.; Heimann, P.; Yigit, N.; Beckman, S.; Schramm, A.; Eggert, A.; Stallings, R. L.; Benoit, Y.; Renard, M.; De Paepe, A.; Laureys, G.; Pahl‐ man, S. & Speleman, F. (2006). Human fetal neuroblasts and neuroblastoma tran‐ scriptome analysis confirms neuroblast origin and highlights neuroblastoma

lignancy by inhibiting the cell response to NGF. *Cancer Research,* 71, 404-412.

hood neuroblastoma. *Clinical Cancer Research*, 18, 5690-5700.

candidate genes. *Genome Biology,* 7 (R84) 3-17.

lated to prognosis. *Journal of Pediatric Surgery,* 42, 2046-2049.

roblastic tumours. *Cancer,* 101, 1873-1881.

group study. *Cancer Research,* 55, 721-726.

1330-1335.

*Cancer*, 43, 1467-1475.

69-73.


[267] Tajiri, T.; Higashi, M.; Souzaki, R.; Tatsuta, K.; Kinoshita, Y. & Taguchi, T. (2007). Classification of neuroblastomas based on an analysis of the expression of genes re‐ lated to prognosis. *Journal of Pediatric Surgery,* 42, 2046-2049.

[257] Nakagawara, A.; Arima, M.; Azar, C. G.; Scavards, N. J. & Brodeur, G. M. (1992). In‐ verse relationship between trk expression and N-myc amplification in human neuro‐

[258] Nakagawara, A.; Arima-Nakagawara, M.; Scavarda, N. J.; Azar, C. G.; Cantor, A. B. & Brodeur, G. M. (1993). Association between high levels of expression of the trk gene and favourable outcome in human neuroblastoma. *New England Journal of Medi‐*

[259] Kogner, P.; Barbany, G.; Dominici, C.; Castello, C.; Raschella, G. & Persson, H. (1993). Coexpression of messenger RNA for TRK protooncogene and low affinity nerve growth factor receptor in neuroblastoma with favourable prognosis. *Cancer Research,*

[260] Cheung, N. –K. V.; Kushner, B. H.; LaQuaglia, M. P.; Kramer, K.; Ambros, P.; Am‐ bros, I.; Ladanyi, M.; Eddy, J.; Bonilla, M.-A. & Gerald, W. (1997). Survival from nonstage 4 neuroblastoma without cytotoxic therapy: an analysis of clinical and

[261] Combaret, V.; Gross, N.; Lasset, C.; Balmas, K.; Bouvier, R.; Frappaz, D.; Beretta-Brognara, C.; Philip, T.; Favrot, M. C. & Coll, J-L. (1997). Clinical relevance of trkA expression on neuroblastoma: camparison with Nmyc amplification and CD44 ex‐

[262] Matsunaga, T.; Shirasawa, H.; Enomoto, H.; Yoshida, H.; Iwai, J.; Tanabe, M.; Kawa‐ mura, K.; Etoh, T. & Ohnuma, N. (1998). Neuronal Src and Trk A protoncogene ex‐ pression in neuroblastomas and patient prognosis. *International Journal of Cancer*

[263] Matsunaga, T.; Shirasawa, H.; Hishiki, T.; Yoshida, H.; Kouchi, K.; Ohtsuka, Y.; Ka‐ wamura, K.; Etoh, T. & Ohnuma, N. (2000). Enhanced expression of N-myc messen‐ ger RNA in neuroblastoma found by mass screening. *Clinical Cancer Research*, 6,

[264] Suzuki, T.; Bogenmann, E.; Shimada, H.; Stram, D. & Seeger, R. C. (1993b) Lack of high-affinity nerve growth factor receptors in aggressive neuroblastomas. *Journal of*

[265] Terui, E.; Matsunaga, T. ; Yoshida, , H.; Kouchi, K.; Kuroda, H.; Hishiki, T.; Saito, T.; Yamada, S-I.; Shirasawa, H. & Ohnuma, N. (2005). Shc family expression in neuro‐ blastoma: high expression of Shc C is associated with a poor prognosis in advanced

[266] Warnat, P.; Oberthuer, A.; Fischer, M.; Westermann, F.; Eils, R. & Brors, B. (2007). Cross-study analysis of gene expression data for intermediate neuroblastoma identi‐

biological markers. *European Journal of Cancer*, 33, 2117-2120.

pression. *British Journal of Cancer* , 75, 1151-1155.

*(Predictive Oncology),* 79, 226-231.

*the National Cancer Institute,* 85, 377-384.

neuroblastoma. *Clinical Cancer Research,* 11, 3280-3287.

fies two biological subtypes. *BMC Cancer*, 7, 89-100.

blastomas. *Cancer Research,* 52, 1364-1368.

*cine,* 328, 847-854.

96 Neuroblastoma

53, 2044-2050.

3199-3204.


[277] Lucarelli, E.; Kaplan, D. & Thiele, C. J. (1995). Selective regulation of TrkA and TrkB receptors by retinoic acid and interferon-γ in human neuroblastoma cells. *Journal of Biological Chemistry*, 270, 24725-24731.

[289] Eggert, A.; Ikegaki, N.; Liu, X-G.; Chou, T. T.; Lee, M. V. & Brodeur, G. M. (2000). Molecular dissection of TrkA signal transduction pathways leading to differentiation

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

99

[290] Olsson, A-K. & Nanberg, E. (2001). A functional role for ERK in gene induction, but not in neurite outgrowth in differentiating neuroblastoma cells. *Experimental Cell Re‐*

[291] Eggert, A.; Grotzer, M. A.; Ikegaki, N.; Liu, X-G.; Evans, A. E. & Brodeur, G. M. (2000). Expression of neurotrophin receptor TrkA inhibits angiogenesis in neuroblas‐

[292] Eggert, A.; Grotzer, M. A.; Ikegaki, N.; Liu, X. G.; Evans, A. E. & Brodeur, G. M. (2002). Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells. *Cancer Research*,

[293] Schulte, J. H.; Kuhfittig-Kulle, S.; Klein-Hitpass, L.; Schramm, A.; Baird, D. S. F.; Pfeiffer, P. & Eggert, A. (2008). Expression of the TrkA and TrkB receptor tyrosine kinase alters the double-strand break (DSB) repair capacity of SY5Y neuroblastoma

[294] Matsumoto, K.; Wada, R. K. & Yamashiro, J. M. (1995). Expression of brain derived neurotrophic factor and p145TrkB affects survival, differentiation, and invasiveness

[295] Lavoie, J-F.; LeSauteur, L.; Kohn, J.; Wong, J.; Furtoss, O.; Thiele, C. J.; Miller, F. D. & Kaplan, D. R. (2005). TrkA induces apoptosis of neuroblastoma cells and does so via

a p53-dependent mechanism. *Journal of Biological Chemistry*, 280, 29199-29207.

signaling by the TrkA receptor tyrosine kinase. *Neuron*, 63, 585-591.

[296] Harel, L.; Costa, B.; Tcherpakov, M.; Zapatka, M.; Oberthuer, A.; Hansford, L. M.; Vojvodic, M.; Levy, Z.; Chen, Z-Y.; Lee, F. S.; Avigad, S.; Yaniv, I.; Shi, L.; Eils, R.; Fischer, M.; Brors, B.; Kaplan, D. R. & Fainzilber, M. (2009). CCM2 mediates death

[297] Jung, E. J. & Kim, D. R. (2008). Apoptotic death in TrkA-overexpressing cells: kinetic regulation of ERK phosphorylation and caspase-7 activation. *Molecular Cells*, 26,

[298] Kogner, P.; Barbany, G.; Bjork, O.; castello, C.; Donfrancesco, A.; Falkmer, U. G.; Hedborg, F.; Kouvidou, H.; Persson, H.; Raschella et al., (1994). Trk mRNA and low affinity nerve growth factor receptor mRNA expression and triploid DNA content in favourable neuroblastoma tumors. *Progress in Clinical and Biological Research,* 385,

[299] Schulte, J. H.; Pentek, F.; Hartmann, W.; Schramm, A.; Friedrichs, N.; Ora, I.; Koster, J.; Versteeg, R.; Kirfel, J.; Buettner, R. & Eggert, A. (2009). The low affinity neurotro‐ phin receptor, p75, is upregulated in ganglioneuroblastoma/ ganglioneuroma and re‐

of human neuroblastoma cells. *Cancer Research*, 55, 1798-1806.

in human neuroblastoma cells. *Oncogene*, 19, 2043-2051.

toma. *Medical and Pediatric Oncology,* 35, 569-572.

*search*, 265, 21-30.

62, 1802-1808.

12-17.

137-145.

cells. *DNA Repair*, 7, 1757-1764.


[289] Eggert, A.; Ikegaki, N.; Liu, X-G.; Chou, T. T.; Lee, M. V. & Brodeur, G. M. (2000). Molecular dissection of TrkA signal transduction pathways leading to differentiation in human neuroblastoma cells. *Oncogene*, 19, 2043-2051.

[277] Lucarelli, E.; Kaplan, D. & Thiele, C. J. (1995). Selective regulation of TrkA and TrkB receptors by retinoic acid and interferon-γ in human neuroblastoma cells. *Journal of*

[278] Chang, B. B.; Persengiev, S. P.; de Diego, J. G.; Sacristan, M. P.; Martin-Zanca, D. & Kilpatrick, D. L. (1998). Proximal promoter sequences mediate cell specific and ele‐ vated expression of favourable prognosis marker TrkA in human neuroblastoma

[279] Condello, S.; Caccamo, D.; Curro, M.; Ferlazzo, N.; Parisi, G. & Ientile R. (2008). Transglutaminase and NF-kB interplay during NGF-induced differentiation in neu‐

[280] Azar, C. G.; Scavarda, N. J.; Nakagawara, A. & Brodeur, G. M. (1994). Expression and function of the nerve growth gactor receptor (TRK-A) in human neuroblastoma cell

[281] Matsushima H & Bogenmann, E. (1993). Expression of TrkA cDNA in neuroblasto‐ mas mediates differentiation in vitro and in vivo. *Molecular and Cellular Biology,* 13,

[282] Hartman, D. S. & Hertel, C. (1994). Nerve growth factor-induced differentiation in neuroblastoma cells expressing trkA but lacking p75NTR. *Journal of Neurochemistry,*

[283] Poluha, W.; Poluha, D. K. & Ross, A. H. (1995). TrkA neurogenic receptor regulates

[284] Gryz, A. A. & Meakin, S. O. (2003). Acidic substitution of the activation loop tyro‐ sines in TrkA supports nerve growth factor-dependent, but not nerve growth factorindependent differentiation and cell cycle arrest in the human neuroblastoma cell

[285] Kim, C. J.; Matsuo, T.; Lee, K-H. & Thiele, C. J. (1999). Up-regulation of insulin-like growth factor-II expression is a feature of TrkA but not TrkB activation in SH-SY5Y

[286] Peterson, S. & Bogemann, E. (2004). The RET and TrkA pathways collaborate to regu‐

[287] Tsuruda, A.; Suzuki, S.; Maekawa, T. & Oka, S. (2004). Constitutively active Src facili‐ tates NGF-induced phosphorylation of TrkA and causes enhancement of MAPK sig‐

[288] Fagerstrom, S.; Pahlman, S. Gestblom, C. & Nanberg, E. (1996). Protein kinase C-ε is implicated in neurite outgrowth in differentiating neuroblastoma cells. *Cell Growth &*

lines. *Progress in Clinical and Biological Research*, 385, 169-175.

differentiation of neuroblastoma cells. *Oncogene*, 10, 185-189.

neuroblastoma cells. *American Journal of Pathology*, 155, 1661-1670.

late neuroblastoma differentiation. *Oncogene*, 23, 213-215.

nalling in SK-N-MC cells. *FEBS Letters*, 560, 215-220.

*Biological Chemistry*, 270, 24725-24731.

cells. *Journal of Biological Chemistry*, 273, 39-44.

roblastoma cells. *Brain Research*, 1207, 1-8.

line, SY5Y. *Oncogene*, 22, 8774-8785.

*Differentiation*, 7, 775-785.

7447-7456.

98 Neuroblastoma

63, 1261-1270.


duces tumorigenicity of neuroblastoma in vivo. *International Journal of Cancer,* 124, 2488-2494.

[309] Brodeur, G. M.; Nakagawara, A.; Yamashiro, D. J.; Ikegaki, N.; Liu, X. G.; Azar, C. G.; Lee, C. P. & Evans, A. E. (1997). Expression of TrkA, TrkB and TrkC in human neuro‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

101

[310] Brodeur, G. M; Minturn, J. E.; Ho, R.; Simpson, A. M.; Iyer, R.; Varela, C. R.; Light, J. E.; Kolla, V. & Evans A. E. (2009). Trk expression and inhibition in neuroblastomas.

[311] Nakagawara, A.; Azar, C. G.; Scavarda, N. J. & Brodeur, G. M. (1994). Expression and function of Trk-B and BDNF in human neuroblastomas. *Molecular and Cellular Biolo‐*

[312] Aoyama, M.; Sai, K.; Shishikura, T.; Kawamoto, T.; Miyachi, T.; Yokoi, T.; Togari, H.; Wada, Y.; Kato, T. & Nakagawara, A. (2001). Human neuroblastomas with unfavour‐ able biologies express high levels of brain-derived neurotrophic factor mRNA and a

[313] Baj, G. & Tongiorgi, E. (2008). BDNF splice variants from the second promoter cluster support cell survival of differentiated neuroblastoma upon cytotoxic stress. *Journal of*

[314] Bouzas-Rodrigues, J.; Cabrera, J. R.; Delloye-Bourgeois, C.; Ichim, G.; Delcros, J-G.; Raquin, M-A.; Rousseau, R.; Combaret, V.; Bénard, J.; Tauszig-Delamasure, S. & Mehlen, P. Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis. *Journal of Clinical Investigation,* 120, 850-858.

[315] Stemple, D. L. & Anderson, D. J. (1992). Isolation of a stem cell for neurons and glia

[316] Biagiotti, T.; D'Amico, M.; Marzi, I.; Di Gennaro, P.; Arcangeli, A.; Wanke, E. & Oli‐ votto, M. (2006). Cell renewing in neuroblastoma: electrophysiological and immuno‐ cytochemical characterization of stem cells and derivatives. *Stem Cells*, 24, 443-453.

[317] Marzi, I.; D'Amico, M. ; Biagiotti, T.; Giunti, S.; Carbone, M. V.; Fredducci, D.; Wanke, E. & Olivotto, M. (2007). Purging of the neuroblastoma stem cell component and tumor regression on exposure to hypoxia or cytotoxic treatment. *Cancer Research*,

[318] Islam, O.; Loo, T. X. & Heese, K. (2009). Brain-derived neurotrophic factor (BDNF) has proliferative effects on neural stem cells through the truncated Trk-B receptor, MAP kinase, Akt, and STAT-3 signaling pathways. *Current Neurovascular Reseach*, 6,

[319] Fanburg-Smith, J. C. & Miettinen, M. (2001). Low-affinity nerve growth factor recep‐ tor (p75) in Dermatofibrosarcoma Protuberans and other neural tumors: A study of

1,150 tumors and fetal and adult normal tissues. *Human Pathology,* 32, 976-983. [320] Perosio, P. M. & Brooks, J. J. (1988). Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors. *American Journal of Pathology,* 132, 152-160.

blastomas. *Journal of Neurooncology,* 31, 49-55.

variety of its variants. *Cancer Letters*, 164, 51-60.

from the mammalian neural crest. *Cell*, 71, 973-985.

*Clinical Cancer Research,* 15, 3244-3256.

*gy,* 14, 759-767.

*Cell Science*, 122, 36-43.

67, 2402-2407.

42-53.


[309] Brodeur, G. M.; Nakagawara, A.; Yamashiro, D. J.; Ikegaki, N.; Liu, X. G.; Azar, C. G.; Lee, C. P. & Evans, A. E. (1997). Expression of TrkA, TrkB and TrkC in human neuro‐ blastomas. *Journal of Neurooncology,* 31, 49-55.

duces tumorigenicity of neuroblastoma in vivo. *International Journal of Cancer,* 124,

[300] Bogemann, E. (1996). A metastatic neuroblastoma model in SCID mice. *International*

[301] Morandi, F.; Scaruffi, P.; Gallo, F.; Stigliani, S.; Moretti, S.; Bonassi, S.; Gambini, C.; Mazzocco, K.; Fardin, P.; Haupt, R.; Arcamone, G.; Pistoia, V.; Tonini, G. P. & Cor‐ rias, M. V. (2012). Bone marrow infiltrating human neuroblastoma cells express high

[302] Lipska, B. S.; Drozynska, E.; Scaruffi, P.; Tonini, G. P.; Izycka-Swieszewska, E.; Ziet‐ kiewicz, S.; Balcerska, A.; Perek, D.; Chybicka, A.; Biernat, W. & Limon, J. (2009). C. 1810C>T polymorphism of NTRK1 gene is associated with reduced survival in neu‐

[303] Cao, F.; Liu, X.; Zhang, L.; Wang, Y. & Zhang, N. (2010). Expression of TrkA splice isoforms in neuroblastoma and its clinical significance. *Chinese Journal of Clinical On‐*

[304] Minturn, J. E.; Evans, A. E.; Villablanca, J. G.; Yanik, G. A.; Park, J. R.; Shusterman, S.; Groshen, S.; Hellriegel, E. T.; Bensen-Kennedy, D.; Matthay, K. K.; Brodeur, G. M. & Maris, J. M. (2011). Phase I trial of laustaurtinib for children with refractory neuro‐ blastoma: a new approaches to neuroblastoma therapy consortium study. *Cancer Che‐*

[305] Schramm, A.; Vandesompele, J.; Schulte, J. H.; Dreesman, S.; Kaderali, L.; Brors, B.; Eils, R.; Speleman, F. & Eggert, A. (2007). Translating expression profiling into a clini‐ cally feasible test to predict neuroblastoma outcome. *Clinical Cancer Research,* 13,

[306] Guo, X.; Chen, Q-R.; Song, Y. K.; Wei, J. S. & Khan, J. (2011). Exon array analysis re‐ veals neuroblastoma tumors have distinct alternative splicing patterns according to

[307] Hishiki, T.; Saito, T.; Terui, K.; Sato, Y.; Takenouchi, A.; Yahata, E.; Ono, S.; Nakaga‐ wara, A.; Kamijo, T.; Nakamura, Y.; Matsunga, T. & Yoshida, H. (2010). Reevaluation of trkA expression as a biological marker of neuroblastoma by high-sensitivity ex‐ pression analysis – a study of 106 primary neuroblastomas treated in a single study.

[308] Farina, A. R.; Cappabianca, L.; Ruggeri, P.; Di Ianni, N.; Ragone, M.; Merolla, S.; Gu‐ lino, A. & Mackay, A. R. (2012). Alternative TrkA Splicing and neuroblastoma. In: *Neuroblastoma-Present and Future* (Ed. Hiroyuki Shimada) Intech, Rijeka Croatia,

stage and MYCN amplification status. *BMC Medical Genomics*, 4, 35-50.

levels of calprotectin and HLA-G- proteins. *PloS ONE*, 7, e2pp22

roblastoma patients. *BMC Cancer,* 9, 436-444.

*motherapy and Pharmacology,* 68, 1057-1065.

*Journal of Pediatric Surgery,* 45, 2293-2298.

2488-2494.

100 Neuroblastoma

*Journal of Cancer*, 67, 379-385.

*cology*, 37, 1282-1285.

1459-1465.

pp111-136.


[321] Kimura, N.; Nakamura, M.; Kimura, I. & Nagura, H. (1996). Tissue localisation of nerve growth factor receptors: TrkA and low-affinity nerve growth factor receptor in neuroblastoma, pheochromocytoma, and retinoblastoma. *Endocrine Pathology,* 7, 281-289.

[333] Chen, J. & Zhe, X. (2003). Cotransfection of TrkA and p75(NTR) in neuroblastoma cell line (IMR32) promotes differentiation and apoptosis of tumor cells. *Chinese Medi‐*

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

103

[334] Yan, C.; Liang, Y.; Nylander, K. D.; Wong, J.; Rudavsky, R. M.; Saragovi, H. U. & Schor, N. F. (2002). P75-Nerve growth factor as an antiapoptotic complex: Independ‐ ence versus cooperativity in protection from enediyne chemotherapeutic agents. *Mo‐*

[335] Holub, J. L.; Qui, Y. Y.; Chu, F. & Madonna, M. B. (2011). The role of nerve growth factor in caspase-dependent apoptosis in human BE(2)C neuroblastoma. *Journal of Pe‐*

[336] Bunone, G.; Mariotti, A.; Compagni, A.; Morandi, E. & Della Valle, G. (1997). Induc‐ tion of apoptosis by p75 neurotrophin receptor in human neuroblastoma cells. *Onco‐*

[337] Eggert, A.; Sieverts, H.; Ikegaki, X-G. & Brodeur, G. M. (2000). P75 mediated apopto‐ sis in neuroblastoma cells is inhibited by expression of TrkA. *Medical and Pediatric*

[338] Giraud, S.; Lautrette, B.; Bessette, B.; Decourt, C.; Mathonnet, M. & Jauberteau, M. O. (2005). Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a hu‐

[339] Bai, Y.; Qiang, Li.; Yang, J.; Zhou.; X.; Yin, X. & Zhao, D. (2008). P75NTR activation of NF-kB is involved in prP106- 126-induced apoptosis in mouse neuroblastoma cells.

[340] Rogers, M-L.; Beare, A.; Zola, H. & Rush, R. A. (2008). CD-271 (P75 neurotrophin re‐

[341] Kuwako, K-I.; Taniura, H. & Yoshikawa, K. (2004). Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin re‐

[342] Kenchappa, R. S.; Zampieri, N.; Chao, M. V.; Barker, P. A.; Teng, H. K.; Hempstead, B. L. & Carter, B. D. (2006). A ligand-dependent cleavage of P75 neurotrophin recep‐ tor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neu‐

[343] Christiansen, H.; Christiansen, N. M.; Wagner F et al., (1990). Neuroblastoma-inverse relationship between expression of N-Myc and NGF-R. *Oncogene,* 5, 437-440.

[344] Wang, C.; Liu, Z.; Woo, C-W.; Li, Z.; Wang, L.; Wei, J. S.; Marquez, V. E.; Bates, S. E.; Jin, Q.; Khan, J.; Ge, K. and Thiele, C. J. (2011). EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR. *Cancer Research*,

ceptor). *Journal of Biological Regulation & Homeostatic Agents,* 22, 1-6.

man neuroblastoma cell line. *Apoptosis*, 10, 1271-1283.

ceptor. *Journal of Biological Chemistry,* 279, 1703-1712.

*cal Journal,* 116, 906-912.

*lecular Pharmacology*, 61, 710-719.

*diatric Surgery*, 46, 1191-1196.

*gene*, 14,1463-1470.

*Oncology,* 35, 573-576.

*Neuroscience Research*, 62, 9-14.

rons. *Neuron,* 50, 219-232.

72, 315-324.


[333] Chen, J. & Zhe, X. (2003). Cotransfection of TrkA and p75(NTR) in neuroblastoma cell line (IMR32) promotes differentiation and apoptosis of tumor cells. *Chinese Medi‐ cal Journal,* 116, 906-912.

[321] Kimura, N.; Nakamura, M.; Kimura, I. & Nagura, H. (1996). Tissue localisation of nerve growth factor receptors: TrkA and low-affinity nerve growth factor receptor in neuroblastoma, pheochromocytoma, and retinoblastoma. *Endocrine Pathology,* 7,

[322] Ho, R.; Minturn, J. E.; Simpson, A. M.; Iyer, R.; Light, J. E.; Evans, A. E. & Brodeur, G. M. (2011). The effect of P75 on Trk receptors in neuroblastoma. *Cancer Letters,* 305,

[323] Zhao, S. P. (2003). Co-expression of TrkA and p75 neurotrophin receptor in extracra‐ nial olfactory neuroblastoma cells. *Humnan Yi Ke Da Xue Xue Bao*, 28, 50-52.

[324] Matsushima, H. & Bogenmann, E. (1994). NGF induces terminal differentiation in trkA expressing neuroblastoma cells in vitro and in vivo. *Progress in Clinical and Bio‐*

[325] Ehrehard, P.B.; Ganter, U.; Schmutz, B.; Bauer, J. & Otten, U. (1993). Expression of low affinity nerve growth factor receptor and TrkB messenger RNA in human SH-

[326] Roux, P. P.; Bhakar, A. L.; Kennedy, T. E. & Barker, P. A. (2001). The p75 neurotro‐ phin receptor activates Akt (protein kinase B) through a phosphoinositol 3-kinase-

[327] Levrerrier, Y.; Thomas, J.; Mathieu, A. L.; Low, W.; Blanquier, B. & Marvel, J. (1999). Role of PI-3kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or

[328] Gargano, N.; Levi, A. & Alema, S. (1997). Modulation of nerve growth factor internal‐ isation by direct interaction between p75 and trkA receptors. *Journal of Neuroscience*

[329] Wehrman, T.; He, X.; Raab, B.; Dukipatti, A.; Blau, H. & Garcia, K. C. (2007). Structur‐ al and mechanistic insights into nerve growth factor interactions with the TrkA and

[330] Makkerth, J. P.; Ceni, C.; Auld, D. S.; Vaillancourt, F.; Dorval, G. & Barker, P. A. (2005). P75 neurotrophin receptor reduces ligand-induced Trk receptor ubiquination and delays Trk receptor internalisation and degradation. *EMBO Reports*, 6, 936-941.

[331] Zhang, C.; Helmsing, S.; Zagrebelsky, M.; Schirrmann, T.; Marschall, A. L. J.; Schun‐ gel, M.; Korte, M.; Hust, M. & Dubel, S. (2012). Suppression of p75 neurotrophin re‐ ceptor surface expression with itraantibodies influences Bcl-X mRNA expression and

[332] Ito, H.; Nomoto, H. & Furukawa, S. (2003). Growth arrest of PC12 cells by nerve growth factor is dependent on the phosphoinositol 3-kinase pathway via p75 neuro‐

dependent pathway. *Journal of Biologcal Chemistry*, 276, 23097-23104.

281-289.

102 Neuroblastoma

76-85.

*logical Research,* 385, 177-183.

*Research*, 50, 1-12.

p75 receptors. *Neuron*, 53, 25-38.

SY5Y neuroblastoma cells. *FEBS letters,* 330, 287-292.

IGF in Baf-3 cells. *Cell Death & Differentiation*, 6, 290-296.

neurite outgrowth in PC12 cells. *PloS ONE*, 7, e30684.

trophin receptor. *Journal of Neuroscience*, 72, 211-217.


[345] Cortazzo, M. H.; Kassis, E. S.; Sproul, K. A. & Schor, N. F. (1996). Nerve growth fac‐ tor (NGF)-mediated protection of neural crest cells from antimitotic agent-induced apoptosis: the role of low-affinity NGF receptor. *Journal of Neuroscience*, 16, 3895-3899.

[356] Esposito, C. L.; D'Alessio, A.; de Franciscis, V. & Cerchia, L. (2008). A cross-talk be‐ tween TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differ‐

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

105

[357] Shirohira, H.; Kitaoka, A.; Enjoji, M.; Uno, T. & Nakashima, M. (2012). AM80 induces neuronal differentiation via increased tropomyosin-related kinase B expression in

[358] Middlemas, D. S.; Kihl, B. K.; Zhou, J. & Zhu, X. (1999). Brain-derived neurotrophic factor promotes survival and chemoprotection of human neuroblastoma cells. *Journal*

[359] Scala, S.; Wosikowski, K.; Giannakakou, P.; Valle, P.; Biedler, J.; Spengler, B. A.; Lu‐ carelli, E.; Bates, S. E. & Thiele, C. J. (1996). Brain-derived neurotrophic factor pro‐ tects neuroblastoma cells from vinblastine toxicity. *Cancer Research,* 56, 3737-3742. [360] Ho, R.; Eggert, A.; Hishiki, T.; Minturn, J. E.; Ikegaki, N.; Foster, P.; Camoratto, A. M.; Evans, A. E. & Brodeur, G. M. (2002). Resistance to chemotherapy mediated by TrkB

[361] Jaboin, J.; Kim, C. J.; Kaplan, D. R. & Thiele, C. J. (2002). Brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy-induced apoptosis via phosphatidylinositol 3'-kinase pathway. *Cancer Research*, 62, 6756-6763.

[362] Jaboin, J.; Hong, A.; Kim, C. J. & Thiele, C. J. (2003). Cisplatin-induced cytotoxicity is blocked by brain-derived neurotrophic factor activation of TrkB signal transduction

[363] Li, Z. & Thiele, C. J. (2007). Targeting Akt to increase the sensitivity of neuroblastoma to chemotherapy: lessons learned from the brain derived neurotrophic factor/TrkB signal transduction pathway. *Expert Opinions in Therapeutic Targets*, 11, 1611-1621. [364] Hecht, M.; Schulte, J. H.; Eggert, A.; Wilting, J. & Schweigerer, L. (2005). The neuro‐ trophin receptor TrkB cooperates with c-Met in enhancing neuroblastoma invasive‐

[365] Cimmino, F.; Schulte, J. H.; Zollo, M.; Koster, J.; Versteeg, R.; Iolascon, A.; Eggert, A. & Schramm, A. (2009). Galectin-1 is a major effector of Trk-B-mediated neuroblasto‐

[366] Nakamura, K.; Martin, K. C.; Jackson, J. K.; Beppu, K.; Woo, C-W. & Thiele, C. J. (2006). Brain-derived neurotrophic factor activation of TrkB induces vascular endo‐ thelial growth factor expression via hypoxia-inducible factor-1a in neuroblastoma

[367] Geiger, T. R. & Peeper, D. S. (2005). The neurotrophic receptor TrkB in anoikis and

[368] Haapsalo, A.; Saarelainen, T.; Moshynakov, M.; Arumae, U.; Kiema, T. R.; Saarma, M.; Wong, G. & Castrén, E. (1999). Expression of the naturally occurring truncated

human neuroblastoma SH-SY5Y cell line. *Biomedical Research*, 33, 291-297.

entiation. *PloS ONE*,3(2):e1643.doi:10.1371/journal.pone. 001643

*of Biological Chemistry*, 274, 16451-16460.

in neuroblastoma. *Cancer Research*, 62, 6462-6466.

path in neuroblastoma. *Cancer Letters*, 193, 109-114.

ness. *Carcinogenesis,* 26, 2105-2115.

cells. *Cancer Research*, 66, 4249-4255.

ma aggressiveness. *Oncogene*, 28, 2015-2023.

metastasis: a perspective. *Cancer Research*, 65, 7033-7036.


[356] Esposito, C. L.; D'Alessio, A.; de Franciscis, V. & Cerchia, L. (2008). A cross-talk be‐ tween TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differ‐ entiation. *PloS ONE*,3(2):e1643.doi:10.1371/journal.pone. 001643

[345] Cortazzo, M. H.; Kassis, E. S.; Sproul, K. A. & Schor, N. F. (1996). Nerve growth fac‐ tor (NGF)-mediated protection of neural crest cells from antimitotic agent-induced apoptosis: the role of low-affinity NGF receptor. *Journal of Neuroscience*, 16, 3895-3899.

[346] Panicker, J.; Li, Z.; McMahon, C.; Sizer, C.; Steadman, K.; Piekarz, R.; Bates, S. E. & Thiele, C. J. (2010). Romidepsin (FK228/depsipeptide) controls growth and induces

[347] Borrello, M. G.; Bongarzone, I.; Pierotti, M. A.; Luksch, R.; Gasparini, M.; Collini, P.; Pilotti, S.; Rizzetti, M. G.; Mondellini, P.; De Bernardi, B.; Di Martino, D.; Garaventa, A.; Brisigotti, M. & Tonini, G. P. (1993). Trk and ret proto-oncogene expression in hu‐ man neuroblastoma specimens: high frequency of trk expression in non-advanced

[348] Edsjo, A.; Lavinius, E.; Nilsson, H.; Hoehner, J. C.; Simonsson, P.; Culp, L. A.; Mar‐ tinsson, T.; Larsson, C.; Pahlman, S. (2003). Expression of trkB in human neuroblasto‐ ma in relation to MycN expression and retinoic acid treatment. *Laboratory*

[349] Fung, W.; Hasan, M. Y.; Loh, A. H.; Chua, J. H.; Yong, M. H.; Knight, L.; Hwang, W. S.; Chan, M. Y.; Seow, W. T.; Jacobsen, A. S. & Chui, C. H. (2011). Gene expression of Trk neurotrophin receptors in advanced stage neuroblastomas in Singapore - a pilot

[350] Zhang, J.; Zhend, Y.; Wang, Y. & Tong, H. (2010). The studies on the correlation for gene expression of tyrosine-kinase receptors and vascular endothelial growth factor in human neuroblastomas. *Journal of Pediatric Hematology and Oncology,* 32, 180-184.

[351] Pastor, R.; Bernal, J. & Rodriguez-Pena, A. (1994). Unliganded c-erbA/thyroid hor‐ mone receptor induces trkB expression in neuroblastoma cells. *Oncogene*, 1081-1089.

[352] Martens, L. K.; Kirschner, K. M.; Warnecke, C. & Scholz, H. (2007). Hypoxia-induci‐ ble factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor

[353] Hoehner, J. C.; Olsen, L.; Sandstedt, B.; Kaplan, D. R. & Pahlman, S. (1995). Associa‐ tion of neurotrophin receptor expression and differentiation in human neuroblasto‐

[354] Lucarelli, E.; Kaplan, D. & Thiele, C. J. (1997). Activation of trk-A but not trk-B signal transduction pathway inhibits growth of neuroblastoma cells. *European Journal of*

[355] Kaplan, D. R.; Matsumoto, K.; Lucarelli, E. & Thiele, C. J. (1993). Induction of TrkB by retinoic acid mediates biologic responsiveness to BDNF and differentiation of human

apoptosis in neuroblastoma tumor cells. *Cell Cycle*, 9, 1830-1838.

stages. *International Journal of Cancer*, 54, 540-545.

study. *Pediatric Hematology and Oncology,* 28, 571-578.

gene. *Journal of Biological Chemistry*, 282, 14379-14388.

ma. *American Journal of Pathology,* 147, 102-113.

neuroblastoma cells. *Neuron*, 11, 321-331.

*Cancer*, 33, 2068-2070.

*Investigation*, 83, 813-823.

104 Neuroblastoma


trkB neurotrophin receptor induces outgrowth of filopodia and processes in neuro‐ blastoma cells. *Oncogene,* 18, 1285-1296.

form of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells. *BMC*

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

http://dx.doi.org/10.5772/55536

107

[379] Thress, K.; Macintyre, T.; Wang, H.; Whitston, D.; Liu, Z. Y.; Hoffmann, E.; Wang, T.; Brown, J. L.; Webster, K.; Omer, C.; Zage, P. E.; Zeng, L. & Zweidler-McKay, P. A. (2009). Identification and preclinical characterisation of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway. *Molecular Cancer Therapeutics*,

[380] Zage, P. E.; Graham, T. C.; Zeng, L.; Fang, W.; Pien, C.; Thress, K.; Omer, C.; Brown, J. L. & Zweidler-McKay, P. A. (2011). The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor-mediated neuroblastoma cell proliferation and sig‐

[381] Iyer, R.; Varela, C. R.; Minturn, J. E.; Ho, R.; Simpson, A. M.; Light, J. E.; Evans, A. E.; Zhao, H.; Thress, K.; Brown, J. L. & Brodeur, G. M. (2012). AZ64 inhibits TrkB and enhances the efficacy of chemotherapy and local radiation in neuroblastoma xeno‐

[382] Evans, A. E.; Kisselbach, K. D.; Yamashiro, D. J.; Ikegaki, N.; Camoratto, A. M.; Dio‐ nne, C. A. & Brodeur, G. M. (1999). Antitumor activity of CEP-751 (KT-6587) on hu‐ man neuroblastoma and medulloblastoma xenografts. *Clinical Cancer Research*, 5,

[383] Evans, A. E.; Kisselbach, K. D.; Liu, X.; Eggert, A.; Ikegaki, N.; Camoratto, A. M.; Dio‐ nne, C. A. & Brodeur, G. M. (2001). Effect of CEP-751 (KT-6587) on neuroblastoma

[384] Iyer, R.; Evans, A. E.; Qi, X.; Ho, R.; Minturn, J. E.; Zhao, H.; Balamuth, N.; Maris, J. M. & Brodeur, G. M. (2010). Lestaurtinib enhances the antitumour efficacy of chemo‐ therapy in murine xenograft models of neuroblastoma. *Clinical Cancer Research*, 16,

[385] Minturn, J. E.; Evans, A. E.; Villablanca, J. G.; Yanik, G. A.; Park, J. R.; Shusterman, S.; Groshen, S.; Hellriegel, E. T.; Bensen-Kennedy, D.; Matthay, K. K.; Brodeur, G. M. & Maris, J. M. (2011). Phase I trial of laustaurtinib for children with refractory neuro‐ blastoma: a new approaches to neuroblastoma therapy consortium study. *Cancer Che‐*

[386] Norris, R. E.; Minturn, J. E.; Brodeur, G. M.; Maris, J. M. & Adamson, P. C. (2011). Preclinical evaluation of lesaurtinib (CEP-701) in combination with retinoids from

[387] Saulnier Sholler, G. L.; Brard, L.; Straub, J. A.; Dorf, L.; Illeyne, S.; Koto, K.; Kalkunte, S.; Bosenberg, M.; Ashikaga, T. & Nishi, R. (2009). Nifurtimox induces apoptosis of neuroblastoma cells in vitro and in vivo. *Journal of Pediatric Hematology and Oncology*,

neuroblastoma. *Cancer Chemotherapy and Pharmacology*, 68, 1469-1475.

xenografts expressing trkB. *Medical Pediatric Oncology*, 36, 181-184.

*motherapy and Pharmacology,* 68, 1057-1065.

grafts. *Cancer Chemotherapy and Pharmacology,* 10.1007/s00280-012-1879-x

nalling and is synergistic with topotecan. *Cancer*, 117, 1321-1329.

*Molecular Biology*, 11, 95.

8, 1818-1827.

3594-3602.

1478-1485.

31, 187-193.


form of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells. *BMC Molecular Biology*, 11, 95.

[379] Thress, K.; Macintyre, T.; Wang, H.; Whitston, D.; Liu, Z. Y.; Hoffmann, E.; Wang, T.; Brown, J. L.; Webster, K.; Omer, C.; Zage, P. E.; Zeng, L. & Zweidler-McKay, P. A. (2009). Identification and preclinical characterisation of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway. *Molecular Cancer Therapeutics*, 8, 1818-1827.

trkB neurotrophin receptor induces outgrowth of filopodia and processes in neuro‐

[369] Olivieri, G.; Otten, U.; Meier, F.; Baysang, G.; Dimitriades-Schmutz, B.; Muller-Spahn, F. & Savaskan, E. (2003). β-Amyloid modulates tyrosine kinase B receptor ex‐ pression in SHSY5Y neuroblastoma cells: influence of the antoxidant melatonin.

[370] Ryden, M.; Sehgal, R.; Dominici, C.; Schilling, F. H.; Ibanez, C. F. & Kogner, P. (1996). Expression of mRNA for the neurotrophin receptor TrkC in neuroblastomas with fa‐ vourable tumour stage and good prognosis. *British Journal of Cancer* ,74, 773-779. [371] Yamashiro, D. J.; Liu, X-G.; Lee, C. P.; Nakagawara, A.; Ikegaki, N.; McGregor, L. M.; Baylin, S. B. & Brodeur, G. M. (1997). Expression and function of Trk-C in favourable

[372] Svensson, T.; Ryden, M.; Schilling, F. H.; Dominici, C.; Sehgal, R.; Ibanez, C. F. & Kogner, P. (1997). Coexpression of mRNA for the full-length neurotrophin receptor Trk-C and trk-A in favourable neuroblastoma. *European Journal of Cancer,* 33,

[373] Menn, B.; Timsit, S.; Represa, A.; Mateos, S.; Calothy, G. & Lamballe, F. (2000). Spa‐ tiotemporal expression of noncatalytic TrkC NC2 isoform during early and late CNS neurogenesis: a comparative study with TrkC catalytic and p75NTR receptors. *Euro‐*

[374] Edsjo, A.; Hallberg, B.; Fagerstrom, S.; Larsson, C.; Axelson, H. & Pahlman, S. (2001). Differences in the early and late responses between neurotrophin-stimulated TrkA and TrkC transfected SH-SY5Y neuroblastoma cells. *Cell Growth & Differentiation*, 12,

[375] Bassili, M.; Birman, E.; Schor, N. F. & Saragovi, U. H. (2010). Differential roles of Trk and p75 neurotrophin receptors in tumorigenesis and chemoresistance ex vivo and in

[376] Nara, K.; Kasafuka, T.; Yoneda, A.; Oue, T.; Sangkhathat, S. & Fukuzawa, M. (2007). Silencing MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification. *Inter‐*

[377] Laneve, P.; Di Marcotullio, L.; Gioia, U.; Fiori, M. E.; Ferretti, E.; Gulino, A.; Bozzoni, I. & Caffarelli, E. (2007). The interplay between microRNAs and the neurotrophin re‐ ceptor tropomyosin-related kinase C controls proliferation of human neuroblastoma

[378] Guidi, M.; Muinos-Gimeno, M.; Kagerbauer, B.; Martl, E.; Estivilli, X. & Espinosa-Parrilla, Y. (2010). Overexpression of miR-128 specifically inhibits the truncated iso‐

cells. *Proceedings of the National Academy of Science. USA.* 104, 7957-7962.

human neuroblastomas. *European Journal of Cancer* 33, 2054-2057.

blastoma cells. *Oncogene,* 18, 1285-1296.

*pean Journal of Neuroscience,* 12, 3211-3223.

vivo. *Cancer Chemother Pharmacol* 65, 1047-1056.

*national Journal of Oncology,* 30, 1189-1196.

*Neuroscience,* 120, 659-665.

2058-2063.

106 Neuroblastoma

39-50.


[388] Yamaguchi, Y.; Tabata, K.; Asami, S.; Miyake, M. & Suzuki, T. (2007). A novel cyclo‐ phane compound, CPPy, facilitates NGF-induced TrkA signal transduction and in‐ duces cell differentiation in neuroblastoma. *Biological and Pharmacological Bullitin*, 30, 638-643.

**Chapter 5**

**Connexin36 is a Negative Regulator of Differentiation in**

Neuroblastoma is the most common extracranial solid tumor to present in children [1]. Neuroblastoma arises from bipotential sympathoadrenal progenitor cells of the trunk neural crest. During normal development, sympathoadrenal progenitors differentiate to form the sympathetic ganglia and adrenal medulla, however in neuroblastoma they form tumors at

Retinoic acid is now employed in multi-modal therapy to treat high-risk neuroblastoma and eradicate minimal residual disease [3]. All-trans retinoic acid (ATRA) diminishes MYCN oncogene expression, arrests proliferation, and induces differentiation of neuroblastoma cells

Gap junctional intercellular communication (GJIC) and connexins (Cx) have been implicated in carcinogenesis and differentiation. GJIC is often perturbed and connexins are typically downregulated or aberrantly localized in cancer cells, including IMR-32 neuroblastoma cells [6, 7]. Many connexins have been identified as tumor suppressors when overexpressed in cancer cells [6]. For example, overexpression of Cx43 resulted in growth suppression of communication deficient Neuro-2A murine neuroblastoma cells [8]. In addition, connexins have been shown to enhance the differentiation of cancer cell lines [8, 9]. For example, overexpression of Cx32 and Cx43 resulted in enhanced nerve growth factor induced neurite

Gap junctions are membrane channels that allow intercellular communication between adjacent cells [11]. GJIC involves the passage of ions, second messengers, and metabolites less than 1kDa in size between cells [11, 12]. Gap junctions and their constituent proteins, connex‐ ins, regulate cellular processes such as homeostasis, growth, and differentiation [6, 13]. Gap

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sidhu and Belliveau; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Human Neuroblastoma**

http://dx.doi.org/10.5772/55643

**1. Introduction**

these sites instead [1, 2].

outgrowth in PC12 cells [10].

[4, 5].

Mandeep Sidhu and Daniel J. Belliveau

Additional information is available at the end of the chapter

