Melanoma and the Nervous System – Novel Pathways Mediated by Neurotrophins and Their Receptors

Human melanoma is the deadliest form of skin cancer in the world, and its incidence is rapidly increasing every year 3-7% on average of the last decades, pointing to melanoma as one of the biggest health problem worldwide (Perlis & Herlyn, 2004). Recent statistics show that melanoma represents 1-3% of all malignant tumors, with Australia and New Zealand having the highest incidence (40 new cases/100 000 inhabitants per year), followed by North European countries and the USA; on the other hand, Japan and central Africa share the lowest incidence. Melanoma is one of the most aggressive tumors. Up to one-fifth of patients progress to metastatic (stage IV) disease, with a median survival of 6 months and a 5-year survival rate of less than 5% (Balch et al., 2001). Melanoma is resistant to conventional therapy such as chemotherapy, radiotherapy and immuno-therapy, as only 5-20% of the patients show a positive response to these treatments. To date, the only FDA-approved chemotherapy for melanoma is the alkylating agent dacarbazine (DTIC), which gives clinical responses in 5–10% of patients and cures in about 1%. The only other approved agents for disseminated melanoma are interleukin (IL)-2 and interferon (IFN)-a, which also have low response rates. During the radial phase, melanoma cells spread horizontally, while during the vertical phase they are able to invade the dermis and become able to generate metastasis (Garbe et al., 2011). Usually, the two phases represent the step-process that leads to aggressive melanomas: once melanoma has spread beyond its original location, it is usually highly resistant to therapies. There are four major types of melanoma: superficial melanoma, the most common type, flat and irregular in shape and colour, nodular melanoma, lentigo maligna melanoma, usually occuring in the elderly, and finally, acral lentiginous melanoma, the least common form of melanoma, which develops at high incidence in African Americans. Melanomas may develop in or near a previously existing precursor lesion or in healthy skin, and they can appear in the mouth, eye, or retina at the back of the eye, vagina, esophagus, anus, urinary tract, and small intestine. Age, sunburns during childhood, close relatives with a history of melanoma, presence of displastyc moles, weak immune-system, long-term exposure to sunlight and exposure to carcinogens are the most common risk factors for melanoma development. Melanoma originates from mutated melanocytes, which share with neurons a common neuroectodermal origin. Among the molecules that act as

neurotrophic factors, neurotrophins (NTs) and their receptors constitute an important network.

Neurotrophins
NTs are a family of structurally and functionally related proteins, initially identified as promoters for neuronal survival.During a search for survival factors, nerve growth factor (NGF) was initially identified (Levi-Montalcini, 1987).Secondly, brain-derived neurotrophic factor (BDNF) was characterized as a survival factor for several neuronal populations not responsive to NGF (Barde et al., 1982).These two proteins revealed conserved features of the sequences, leading to isolation of clones encoding additional members of this family.NT expressed in mammals are four: NGF, BDNF, neurotrophin-3 (NT-3) and NT-4.NTs play a critical role in developmental neurobiology; they are crucial in cellular interactions, in regulating synapse formation and plasticity (Huang et al., 2003), in controlling cell survival and differentiation (Segal, 2003).NTs effects are mediated by two classes of cell-surface receptors, a family of tyrosine kinase receptors called Trks (TrkA, TrkB and TrkC) and the p75 neurotrophin receptor (p75NTR).Trk receptors bind NTs with higher affinity and specificity.In particular, TrkA binds NGF and NT-3, TrkB binds BDNF, NT-3, NT-4/5.TrkC only binds NT-3.On the other hand, p75NTR binds all NTs with low affinity and specificity.length Trk proteins, all three trk genes encode other protein isoforms by alternative splicing in the extracellular and intracellular domains, which include insertions in the extracellular domain (Barker et al., 1993;Tacconelli et al., 2005) and deletions or added residues in the intracytoplasmic domain (Reichardt, 2006).Several different mutation types in the trkA gene have been identified in congenital insensitivity to pain with anhydrosis (CIPA) patients (Indo, 2001).The phenotypes associated with CIPA are believed to result in large part from loss of NGF-dependent neurons, including nociceptive sensory and sympathetic neurons, during embryogenesis.A dominant mutation in TrkB (Y722C) that impairs TrkB kinase signalling has recently been described in a patient with severe hyperphagic obesity and severe impairments in nociception, learning and memory (Yeo et al., 2004).The biological effects induced by NTs strongly depend on the pattern of NT receptor/coreceptors expression in target cells as well as onset of intracellular adaptor molecules that link NT signaling to different biochemical pathways.The propensity of NTs to produce diametrically opposing effects on cell survival has led to propose a "yin and yang" model of neurotrophin action, where the binary actions of NTs depend on both the form of the neurotrophin (pro-versus mature) and the class of receptor that is activated (Lu et al., 2005).Central to the proposed "yin-yang" model of NTs function is the observation that a mature NT binds preferentially to Trk receptors to enhance cell survival, whereas an unprocessed proNT binds to p75NTR to induce cell death.NTs interaction with Trks require receptor dimerization, autophosphorylation, and the subsequent binding of adaptor molecules that couple Trk receptors to different intracellular signal transduction pathways (Reichardt, 2006).NTs interact with trks receptors at the membrane-proximal immunoglobulin-like domain.The three-dimensional structures of this domain in each of the Trk receptors have been solved (Ultsch et al., 1999).Expression of a specific Trk receptor confers responsiveness to the NT to which it binds.On the other hand the isoform of TrkA including an insert is also activated by NT-3 in addition to NGF (Clary & Reichardt, 1994), while the similar isoform of TrkB is activated by NT-3 and NT-4 in addition to BDNF (Strohmaier, 1996).36 novel isoforms of TrkB proteins with unique properties have been described recently.This suggests high complexity in the synthesis, regulation and function of this important NTs receptor, emphasizing the need for further study of these novel TrkB variants (Luberg et al., 2010).TrkC has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis.It was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand.(Tauszig-Delamasure et al., 2007).Differential splicing of trkC mRNA also results in expression of a TrkC isoform with an amino acid insert within the tyrosine kinase domain.This insert appears to modify the substrate specificity of this tyrosine kinase, inhibiting activation of several substrates and interfering with its ability to promote neuronal differentiation (Guiton et al. 1995).p75NTR was the first NT receptor to be identified as a low-affinity receptor for NGF, but it was subsequently shown to bind each of the neurotrophins with a similar affinity (Rodriguez-Tébar et al., 1990;Frade & Barde, 1998).p75NTR belongs to the tumor necrosis factor receptor superfamily.It has an extra-cellular domain containing four cysteine-rich motifs, a single transmembrane domain and a cytoplasmic domain that includes a 'death' domain, similar to those present in other members of this family (He & Garcia, 2004).This receptor does not contain a catalytic motif, however it interacts with proteins that transmit crucial signals for regulating neuronal survival and differentiation as well as synaptic plasticity.The three-dimensional structure of the extracellular domain of p75NTR in association with an NGF dimer indicates that each of the four cysteine-rich repeats participates in binding to NGF (He & Garcia, 2004).The binding of NGF to p75NTR may result in dissociation of p75NTR multimers and is compatible with the possibility that Trk and p75NTR monomers simultaneously bind the same NT dimer.A gene related to p75NTR, named NRH-2, has recently been identified.The product of this gene lacks the extracellular cysteine-rich repeats present in p75NTR and fails to bind NGF, but it is able to interact and influence the ligand-binding properties of TrkA (Murray et al., 2004).When p75NTR is expressed alone, mature NTs are capable of inducing apoptosis or promote survival depending on the intracellular adaptor molecules present in target cells by interacting with a mounting number of downstream molecules (Wang et al., 2000).However, signaling through p75NTR may also promote cell survival acting through the NF-κB pathway (Roux & Barker, 2002).The coexpression of Trks and p75NTR increases high affinity NT binding, enhances Trk ability to discriminate a preferred ligand from the other NT, and promotes NT survival effects (Teng & Hempstead, 2004).On the other hand, the proform of NT, proNGF, binds p75NTR in association with its co-receptor sortilin, but not Trk (Nykjaer et al., 2004).More specifically, sortilin, a member of the vps-10 protein family, binds the ''pro'' region of NGF, whereas p75NTR binds mature NGF.The p75NTR-sortilin complex couples with proNGF to induce apoptosis (Kaplan & Miller, 2004).

NTs and skin microenviroment
NTs also operate in a number of non-neuronal tissues, including skin where a complex NT network exists with various cells that are either the target or the source of NTs, thus playing autocrine and paracrine functions (Botcharev et al., 2006).Moreover, given the common neuroectodermal origin of the skin with the nervous system, different studies have been carried out on the role of NTs and their receptors in the cutaneous system.First, NGF, which is synthesized in the epidermis, is retrogradelly transported to the ganglia to stimulate the release of neuropeptides in the skin, thus favoring cutaneous neurogenic inflammation (Davis et al., 1997).Moreover, null mutations of genes in the NTs and their receptors lead to loss/reduction of specific neurons in sensory ganglia; conversely, cutaneous overexpression of NTs results in skin hyperinnervation and increase in the number of sensory neurons innervating the skin (Montaño et al., 2010).Not only NGF is neurotrophic at the skin level, but it possesses a number of biological effects also in cutaneous cells.Normal human keratinocytes synthesize and release NGF that can act as a growth factor for these cells (Pincelli et al., 1994).The most common type of skin cells is the keratinocyte.These cells synthesize and secrete all NTs (Di Marco et al., 1991), NGF being secreted at highest levels as compared to the other NT.In human keratinocytes, NT-3 and NGF upregulate each other's secretion.UVB irradiation downregulate NGF, while UVA augment NT-3 release (Marconi et al., 1999;Stefanato et al., 2003).Human keratinocytes release NGF in increasing amounts while proliferating, whereas secretion ends in more differentiated cells (Pincelli et al., 1994).NGF induces human keratinocyte proliferation (Di Marco et al., 1993) and it can either stimulate or inhibit murine epidermal and hair follicle keratinocyte proliferation in situ (Paus et.al., 1994).Human keratinocytes express the high affinity receptors TrkA and TrkC, but not the functional form of the TrkB (Marconi et al., 2004).Endogenous NGF autocrinally sustains keratinocyte proliferation.NTs together with their receptors TrkA and TrkB can stimulate proliferation of mouse keratinocyte in ex-vivo cultured skin explants (Paus et al., 1994;Botchkarev et al., 1999).Moreover, NTs modulate susceptibility to www.intechopen.comMelanoma and the Nervous System -Novel Pathways Mediated by Neurotrophins and Their Receptors 267 apoptosis in the epidermis.Autocrine NGF protects human keratinocytes from UVBinduced apoptosis, while UVB downregulates both NGF and TrkA expression in these cells (Marconi et al., 1999).Because in normal human keratinocytes TrkB lacks functional isoform, NT may exert different functions by binding p75NTR alone.In this context, p75NTR acts as a proapoptotic receptor.This is exemplified by BDNF and NT4, which induce a higher rate of apoptosis in normal human keratinocytes overexpressing p75NTR, as compared to mocktransfected cells.On the other hand, p75NTRsiRNA-transfected keratinocytes fail to undergo cell death after administration of NT4 (Truzzi et al., 2010).Interestingly, stem keratinocytes (KSC) expresses most of the NGF produced by human keratinocytes (Marconi et al., 2004), and inhibition of TrkA reduces the proliferation of this keratinocyte subpopulation (Marconi et al., 2004).This suggests that the presence of a NGF-TrkA autocrine loop, which acts both as mitogenic and as survival factor, contributing to the maintenance of the so-called "stemness" in keratinocytes.Conversely, p75NTR is mostly expressed in the differentiated transit amplifying (TA) cells, and it is barely detected in KSC.TA cells have been shown to be more susceptible than KSC to apoptosis (Tiberio et al., 2002).Therefore, the espression of p75NTR predominantly in TA cells seems to be consistent with the pro-apoptotic role of this receptor in human keratinocytes (Truzzi et al., 2011).Another important epidermal cell is the melanocyte that localizes in the dermo-epidermal junction and in the hair matrix.NTs are important for melanocyte migration, viability and differentiation together with other paracrine signalling molecules (Pincelli et al., 1997).Normal human melanocytes are a target of the NTs skin network, because they express all the NTs receptors both in vitro and in vivo (Marconi et al., 2006).Normal human melanocytes also express p75NTR, which is upregulated by different stimuli, like UV irradiation (Peacocke et al., 1988).NTs influence melanocytes in a paracrine fashion.NGF is implicated in melanocyte survival (Zhai et al., 1996), migration and dendricity (Yaar et al., 1991), and its synthesis and secretion are enhanced by UV irradiation (Tron et al., 1990).Moreover, NGF reduces apoptosis when melanocytes are irradiated with UV, through upregulation of the anti-apoptotic Bcl-2 protein in vivo (Stefanato et al., 2003).Thus, NTs are important for protection of UV-induced oxidative stress and apoptosis in melanocytes.Melanocytes produce all NTs, while when these factors are added to the cultures, they fail to stimulate cell proliferation (Marconi et al., 2006).When melanocytes are maintained in growth factordepleted medium, NGF and NT-3 promote melanocyte survival (Yaar et al., 1994).Both NT3 and NT4 secretion promotes the synthesis of tyrosinase and tyrosinase-related peptide (TRP)-1, critical enzyme of melanin biosynthesis (Marconi A et al., 2006).Human melanocytes express Trk receptors (Marconi et al., 2006;Yaar et al., 1994).Interestingly, phorbol 12-tetra decantate 13 acetate (TPA), a strong activator of protein kinase C, induces the expression of TrkA (Yaar et al., 1994) and decreases the expression of TrkC, suggesting that NGF and NT3 mediate different signals through their specific high affinity receptors.Melanocytes express also the NT low affinity receptor p75NTR, which expression is upregulated after TPA treatment (Yaar et al., 1994).NTs and their receptors are expressed both in dermal fibroblasts and in the more differentiated myofibroblasts.p75NTR and TrkB are expressed at higher levels in myofibroblasts than in fibroblasts, which in contrast express higher levels of TrkA.Dermal fibroblast and myofibroblasts secrete all NTs, modulating dermal fibroblast proliferation.Interestingly, NTs also promote fibroblast differentiation into myofibroblasts, by inducing α-SMA expression.This indicates that NTs could have a functional role in the fibromyofibroblast system (Palazzo et al., 2011).It has been shown that NGF induces fibroblast-like keratinocyte differentiation into myofibroblasts (Micera et al., 2001), their contraction in 3D collagen matrix (Micera et al., 2001) and the expression of MMP-9 (Metalloprotease-9) in keratoconjunctivitis-derived fibroblasts (Micera et al., 2007b), and different works show the applicative possibility of this NGF function (Landi et al., 2003;Aloe et al., 2008;Sun et al., 2010).All NTs promote fibroblast migration, while NGF and BDNF promote their contractile activity.Therefore, NGF and BDNF, produced by dermal and epidermal cells, could be key regulators of the biomechanical properties in the dermis.In the skin network, NT and their receptors are also important for other epidermal cells.Skin mast cells express functional TrkA, produce NGF (Marshall et al., 1999), and express p75NTR (Fischer et al., 2008).Merkel cells are sensory cells of neural crest origin.During development, neither NT-3 nor TrkC and p75NTR are expressed by Merkel cells in the murine whisker.However, NT-3 is essential for mice postnatal survival (Szeder et al., 2003).Moreover, both TrkB and p75NTR were shown to be important for Merkel cell development (Perez-Pinera et al., 2008;Kinkelin et al., 1999).

NTs and the origin of melanoma
Melanocytes, the cells that originate melanomas, are melanin-producing cells responsible for skin, hair and eye pigmentation.The principal role of melanocytes is to protect human skin from UVB radiation-induced stress.To understand melanoma origin, it is of paramount importance to elucidate the characteristics of the cells that originate the tumor.Indeed, melanocytes have intrinsic properties such as migratory capacity and self-renewal, which are shared with melanoma cells, and that could be partially responsible for the high susceptibility of melanoma to metastasize and recur in patients.Human melanocytes derive from a group of embryonic cells that form, during embryogenesis, the neural crest (NC).Neural crest cells (NCC) are multipotent, giving rise to different cell lineages depending on their anatomic location, and are therefore referred to as NC stem cells (NCSC).Melanocytes appear to derive from both cranial and trunk neural crest, while cells derived from the vagal, sacral, mid/hindbrain regions of the crest originate neurons cartilage, bones, smooth muscle, peripheral and enteric neurons and glia (Anderson et al., 2000).These multipotent cells give rise to a bipotent NCSC that generate both glia and melanoblasts (Dupin et al., 2000).Although the mechanism of lineage restriction towards melanoblasts specification has been deeply studied, it is only partially understood.However, it seems that signals from surrounding cells control this mechanism, both during cell homing and in their final environment.The bipotent progenitor is able to generate a melanocyte-restricted cell, which is referred to as melanoblast.Melanoblasts are unpigmented cells that migrate from the neural crest site along the dorsolateral pathway, throughout the developing epidermis.They colonize both interfollicular epidermis, localizing at the basement membrane, and developing hair follicles.In interfollicular epidermis, melanoblasts differentiate into mature melanocytes, upon stimulation from neighbor keratinocytes.In the hair follicle, melanoblast can then further segregate into a committed melanocyte or an adult melanocyte stem cell (Nishikawa & Osawa, 2007).The committed melanocyte resides in the hair matrix of the follicle and differentiates into a melanin-producing cell, thus being responsible for hair pigmentation.As opposite, melanocyte stem cells, which reside in the bulge area of hair follicles, are amelanotic and conserve characteristics of stem cells such as slow-cell cycle/quiescence and self-renewal capacity (Nishimura et al., 2002).The presence of these cells ensures the pigmentation of hair follicles in subsequent hair cycles.In humans, melanocyte stem cells have not been isolated yet (Sabatino et al., 2009).However, in patients affected by vitiligo, a progressive skin disease with defective pigmentation, the repigmentation process starts from perifollicular areas, thus implying that, during this process, undifferentiated melanocytes in interfollicular epidermis may derive from melanocyte stem cells of the hair follicle bulge area (Falabella & Barona 2009).Consistently, a migration process of bulge melanoblasts towards interfollicular epidermis has been previously observed (Cui et al., 1991).Overall, these observations may suggest that melanoblasts in the hair follicles function as a reservoir population both for the bulb hair follicle and for interfollicular epidermis.Upon UVB radiations and exogenous stress stimuli, melanocytes can acquire genetic mutations.Although apoptosis usually eliminates highly damaged mutated cells, some of these mutations can confer resistance to apoptosis and proliferative advantage to the cells, thus leading to accumulation of mutations and to the formation of melanocytic lesions.The old carcinogenesis model, namely "the stochastic model", suggests that virtually any cell could be the target of mutations and subsequent transformation.As opposite, different works suggest that only some cells retain the susceptibility to acquire mutations and generate cancer, and that these cells can either be differentiated or have stem cells characteristics.Whether melanoma arises from melanocyte stem cells or a differentiated melanocyte is still matter of debate (Hoek & Godin, 2010).It is possible that differentiated melanocytes undergo a process of de-differentiation, thus acquiring stem cell characteristics.Melanocyte stem cells in the hair follicle generate an amplifying progeny, which is differentiated, still retaining self-renewal capacity.After exiting stem cell niche, transit amplifying melanocytes colonize adjacent vacant spaces and repopulate them, thus functioning as stem cells (Nishimura et al., 2002).In quails, melanocytes removed from their niche and clonally cultured, can generate multipotent cells retaining self-renewal capacity, thus confirming that de-differentiation of melanocytes is indeed possible (Real et al., 2006).It has been proposed that melanoma arises in a stepwise process, starting from nevus and dysplastic nevus stages to in situ melanoma and finally, to metastatic melanoma.Because nevi seems to originate from mutations in a precursor cell, which then activates proliferative pathways, while suppressing apoptosis, it would be more likely that melanoma arises from a mutated cell with stem characteristics.However, this model does not mimic precisely melanoma biology, as melanoma can arise in absence of nevus precursor, and displastyc nevi do not necessarily evolve into melanomas (Beona et al., 2003).In many other systems, stem cells are considered the best candidate for accumulation of mutations, given their long persistence in the tissue and retaining the highest proliferative potential.Although the cell at the origin of melanoma is still unidentified, recent works suggest that melanocyte stem cells located in the hair bulge may not be the right candidate.Indeed, primary melanoma localizes at the junction between dermis and epidermis, suggesting that stem melanocytes should migrate from hair follicles to the epidermis before generating melanomas.Moreover, while epidemiological studies demonstrate a correlation between melanoma formation and UV-radiation-induced sunburn in childhood, hair follicles are only marginally reached by UV, because of their deep localization in the tissue.On the other hand, transgenic mice overexpressing NRAS and β-catenin develop melanocytic lesion in the follicle bulge, thus indicating that melanocyte stem cells in the hair bulge are potentially able to mutate and generate melanocytic lesions (Delmas et al., 2007).Up to now, the exact localization of the melanoma precursor and the differentiation state of this cell are still unclear.Although NCSC contribution to melanoma formation is still controversial, NCSC migration during embryogenesis and tumor formation are tightly connected.Indeed, extravascular migration process, which guides cells towards their final sites, is shared between melanoma cells and NCSC (Lugassy & Barnhill, 2007).In addition, NCSC behavior is influenced by NTs and their receptors, which in turn are part of melanoma microenvironment.Epidermalrestricted NCSC express all NTs, while NGF and NT-3 support their proliferation as well as survival of daughter cells, in that NGF and NT-3 depletion results in loss of 70% and 60% neurons, respectively (Dasari et al., 2008;Zhang et al., 1997).Moreover, NTs can either support proliferation or induce apoptosis in NCC, depending on their lineage/differentiation stage (Langtimm-Sedlak et al., 1996).Consistently, different expression of trk receptors may identify distinct subpopulations of early NCC.While Trk receptors are not expressed at early stages of embryonic development, p75NTR is the first NT receptor to be expressed in NCC (Rifkin et al., 2000).NCSC have been previously isolated from mouse trunk neural tubes by using a monoclonal antibody against p75NTR.p75NTR-positive cells derived from mouse neural tubes are multipotent and have self renewal capacity, generating multipotent progeny.These cells can differentiate into smooth muscle cells, neurons and glial cells, melanocytes, cartilage and bone.They can also generate clonal subpopulations of cells able to generate only glia and neurons, thus suggesting their ability to generate also committed progenitors (Stemple & Anderson, 1992).p75NTR, also knwon as CD271 (Rogers et al., 2008), was successfully used to isolate multipotent stem cells also from rat sciatic nerves (Morrison et al., 1999).These cells generate neurons and glial cells when injected into chick embryos, and retain self-renewal capacity.Multipotent NCSC can be isolated from hairy skin in humans and mice (Sieber-Blum & Grim, 2004;Sieber-Blum et al., 2004).Consistently, p75NTR-positive cells, isolated from human hair follicle dermal papillae, retain multipotency and contribute to the renewal of neural and non-neural cells, including melanocytes (Yu et al., 2006).Recently, multipotent cells with NCSC characteristics have been extracted by sphere formation from the dermis of human foreskins lacking hair follicles.These cells, not only express stem cells markers such as Oct-4 and nestin, but display also multipotency and the ability to differentiate into multiple lineages, including melanocytes.Moreover, they express p75NTR, thus suggesting that p75NTR can be considered a good marker for NCSC (Li et al., 2010;Paratore et al., 2001).Melanoma development is considered a stepwise process in which mature melanocytes in the epidermis progressively acquire genetic mutations in oncogenes or tumor-suppressor genes that lead from benign to dysplastic nevi, progressing toward radial growth phase to vertical growth phase, ultimately followed by metastatic melanoma.Melanomas arise within the epidermis and then invade the basement membrane to eventually disseminate to multiple organs.During progression, melanoma moves from the epidermal to the dermal microenvironment by virtue of various mechanisms that involve different cell types, such as keratinocytes, fibroblasts and a number of cytokines and growth factors.While in human epidermis, a functional symbiosis exists between keratinocytes and melanocytes, melanoma cells are refractory to the regulation by keratinocytes and become independent, by autocrinally producing their growth factors (Crowson et al., 2007).The NTs network involves most skin cell types and is responsible for various activities.(Botchkarev et al., 2006).In particular, melanocytes, which share with neurons a common neuroectodermal origin, express all NTs and their receptors.

NTs and melanoma
As for many other growth factors, dysregulation of NT signal transduction is found in a number of tumors inside and outside the nervous system where they accompany or contribute to malignant transformation (Kruttgen et al., 2006).Yet, the precise role of NTs and their receptors has to be clarified (Thiele et al., 2009;Papatsoris et al., 2009).On the other hand, NTs have been widely investigated in neuroblastoma (NB) and medulloblastoma, tumors derived from the neural crest.Trk-receptors have been identified as important prognostic factors, influencing the heterogeneous clinical behavior of NB (Nakagawara, 1993).TrkA and/or TrkC expression is present in favorable NBs, which tend to show a more differentiated neuronal phenotype and highly correlates with patient survival (Nakagawara, 2001;Brodeur, 2009;Yamashiro, 1996).The tendency of NBs to regress may be related to their dependence on an inadequate amount of NGF supplied from stromal cells and a differentiating rather than a proliferative response to this NT.Therefore, an autocrine or paracrine TrkB/BDNF pathway may contribute to an unfavorable outcome in primary NBs.The different clinical implications of the Trk receptors and their isoforms strongly suggest that the activation of full-length and truncated TrkA and TrkB may exert different influences on the biological behavior of NB cells (Barbacid, 1994).Like NB, medulloblastoma can be subdivided in many subtypes with different prognosis (Johnsen et al., 2009).TrkA or TrkC has been detected in such areas, in parallel with concentrations of apoptotic or differentiating cells (Ohta et al., 2006).TrkC has been correlated to positive prognosis in medulloblastoma (Segal et al., 1994) and to apoptosis in vitro in primary medulloblastoma (Kim et al.,1999).Nevertheless, the effect of p75NTR on the cellular response to NT is complex and may depend on the concentration of ligand, the ratio of receptors, the cell type in which it is expressed, and its stage of differentiation (Greene et al., 1995;Chao et al., 1995).Overexpression of p75NTR increases the number of high-and low-affinity NGF binding sites in TrkA-expressing PC12 cells (Hempstead et al., 1992).Moreover, p75NTR induces apoptosis in the presence of NGF (Bunone et al., 1997), but this apoptotic signaling is inhibited by the presence of TrkA receptors (Eggert et al., 2000).Recently, it has been demonstrated that the effects of necdin, a protein known to interact with NTs receptors, on the susceptibility of NB cells to oxidant stress depend on the p75NTR/TrkA ratio in the cell (Ingraham et al., 2011).Although melanoma is a tumor derived from the neural crest, and shares with cancers of the same origin embryogenic and oncogenic pathways as well as common transcription factors (Wang et al., 2008;Gershon et al., 2005), it has been given less attention, as far as the role of NT.Melanomas can be morphologically subdivided in several subtypes, such as epithelioid, pleomorphic spindle cell and desmoplastic melanoma.Iwamoto et al. found detectable p75NTR in 13 of 14 benign nevi, primarily in the spindled nevocytic structures within the dermis (Iwamoto et al., 2001).Moreover, p75NTR is weakly expressed in the epithelioid melanomas, while it is highly expressed in desmoplastic and spindle cell melanomas (Iwamoto et al., 1996).This was confirmed by others who showed p75NTR staining to be more diffuse and intense as compared with S100 (Kanik et al., 1996;Lazova et al., 2010).On the contrary, Huttenbach and co-workers detected p75NTR positive cells only in 33% of desmoplastic melanomas (Huttenbach et al., 2002).Marchetti et al, by using melanoma cell lines, observed that NGF/p75NTR signaling promotes the survival of melanoma cells (Marchetti et al., 2003).They also observed the presence of NGF and NT-3 in tumor adjacent tissues at the invasive front of melanoma brain metastases, which might indicate a paracrine activation of p75NTR and TrkC in melanoma cells by NGF and NT-3 produced by nearby glial cells.Besides promoting melanoma cell survival, NTs also induce the expression in melanoma cells of heparanase, an important enzyme for local invasion and metastasis, that cleaves heparan sulfate chains of proteoglycans, thus modifying the extracellular matrix of tumor cells (Walch et al., 1999).Moreover, Shonukan et al showed that NTs are chemotactic for melanoma cells, and that the actin-bundling protein fascin co-immunoprecipitates with p75NTR in an NGF-dependent manner (Shonukan et al., 2003).In addition, TrkA is expressed by primary and metastatic melanomas and is associated with poor clinical outcome (Flørenes et al., 2004).Recently, during a functional characterization of human cancer-derived TrkB mutations, one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line was found, even if it was functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo (Geiger et al., 2011).As melanoma could originate from melanocytes and melanocytes seem to benefit from the cutaneous neurotrophic network mainly as paracrine targets, once malignant transformation occurs, melanoma cells acquire self-renewal capabilities mediated also by autocrine NTs loops.Indeed, all NTs, particularly NT-3 and NT-4, have been detected in conditioned medium of different melanoma cell lines (Truzzi et al., 2008).Recent results show that melanoma cells proliferate through autocrine NTs stimulation.K252a significantly reduces melanoma cell proliferation by inhibiting Trks phosphorylation.Proliferation is significantly reduced when endogenous NTs are removed from the culture medium by soluble Trk/Fc receptors.NTs appeared to be important for melanoma cell migration in vitro, with special respect for metastatic cell lines.The migratory phenotype is necessarily dependent on the presence of both the high-and low-affinity NTs receptors.Cells treated with p75NTR small interfering RNA (p75NTRsiRNA) fail to respond to NTs stimulation.Similarly, the administration of K252a blocks melanoma cell migration, confirming that NTs stimulate melanoma cell migration and invasion with the cooperation of the low-and high-affinity receptors (Truzzi et al., 2008).

Fig. 3. NTs and their receptors in melanoma biology
Although melanoma may develop in a stepwise manner from a pre-existing lesion, only 26% of melanomas evolve from nevi, while the majority of melanomas arise from normalappearing skin, suggesting that melanoma development may not follow the classical linear mode of progression (Zabierowsky & Herlyn, 2008).The constant recurrence of melanoma after any therapy is likely due to the survival of a subset of cancer cells that display a resistance to treatment.The "Cancer Stem Cell hypothesis" states that within tumor heterogeneity, only few cells are responsible for tumor initiation and maintenance, sharing with normal stem cells the ability to self renew and to persist in the tissue for years.Although in some tumors, such as leukemia, the CSC model is valid, in melanoma, cells able to initiate tumor formation are not a rare population (Quintana et al., 2010;Held et al., 2010;Roesch et al., 2010).Given the role of NTs and their receptors in melanoma biology, this complex network of growth factors is likely to be involved both in melanoma recurrence and metastasis in the patients.NCSC, that have been proposed as the cells responsible for melanoma origin (see above), have been previously isolated both from neural crest and from adult tissues by using p75NTR (Kruger et al., 2002).Similarly, Boiko and coworkers showed that p75NTR expression characterizes a population of melanoma cells with tumor initiating properties (Boiko et al., 2010).However, the number of cells capable of recapitulating the tumors in vivo may vary depending on the assay conditions (Quintana et al., 2008).The use of sensitive mouse models, such as the NOD/SCID IL2R_(null) mice, demonstrated that cell isolation through p75NTR does not enrich melanoma CSC, and melanoma cells are able to metastasize independently from their expression pattern, being able to reverse their phenotype through clonal expansion (Quintana et al., 2010;Civenni et al., 2011).Consistently, in another study CD34-neg/p75NTR-pos cells from mouse melanomas only rarely form tumors, while the p75NTR-negative counterpart frequently forms tumors, depending on CD34 expression (Held et al., 2010).This suggests that although some lines of evidence point at p75NTR as a good marker for the isolation of melanoma CSC, it may not be the right candidate.It is interesting to mention though that the frequency of p75NTRpositive cells in melanoma samples correlates with higher metastasis and worse patient prognosis (Civenni et al., 2011).Altogether, these results suggest that p75NTR-positive cells are not the CSC population in melanoma, but may retain characteristics of aggressiveness and susceptibility to metastasize.Further characterization of these cells and evaluation of the role of p75NTR in melanoma stem cells is still needed.

Conclusions
A significant progress in understanding NTs signaling in skin has been done during the last fiftheen years.Although more studies are needed to better elucidate the role of these molecules in cutaneous physiology and pathology, there is a large body of evidence indicating that NTs form a complex network with autocrine and paracrine functions.In this context, NTs and their receptors seem to play an important role in the origin, development and invasiveness of malignant melanoma.Given the premise and the common neuroectodermal origin of the skin and the nervous system, future studies will allow a better understanding of the cell of origin of melanoma and of the environment that favors the growth and metastasis of the tumor.Translating these data into clinically oriented research will possibly unravel novel strategies for the treatment of melanoma.In particular, blocking Trks will inhibit melanoma cell migration and survival, thus acting in a less toxic and more specific manner, as compared to chemotherapy.Interestingly, Lestaurtinib (CEP-701), a Trk-selective inhibitor, is effective and well tolerated in patients with refractory neuroblastoma (Minturn JE et al., 2011;Evans AE et al., 2001).In addition, inhibitors of Trk kinase activity, such as K-252a family members, have a significant anti-tumor activity in prostatic and pancreatic carcinomas, in preclinical studies.

Fig. 1 .
Fig. 1.NTs and their receptors Trks are tyrosine kinase receptors with similar structure.They are activated by ligandinduced formation of noncovalently associated receptor dimers.In addition to the three full-