Neuroblastoma Integrins

In the body, cells are surrounded and supported by an intricate network of glycoproteins and proteoglycans that make up a complex extracellular matrix, or ECM. Many constituents, such as collagen, laminin, and fibronectin, are locally produced within the tissues, where they act as physical scaffolds, growth factor depots, and points of anchorage [1]. The local rigidity and composition of the matrix also provide environmental cues that govern cell behavior.


Introduction
In the body, cells are surrounded and supported by an intricate network of glycoproteins and proteoglycans that make up a complex extracellular matrix, or ECM. Many constituents, such as collagen, laminin, and fibronectin, are locally produced within the tissues, where they act as physical scaffolds, growth factor depots, and points of anchorage [1]. The local rigidity and composition of the matrix also provide environmental cues that govern cell behavior.
The ECM surrounding cells can be considered in two broad classes. On one hand, there exists a 'physiologic' ECM, present in all tissues, that aids in structuring and maintaining homeostasis. Typical ECM components include several collagens and laminins, as well as proteoglycans. On the other hand, there is a provisional ECM that is deposited during wounding, hemostasis and tissue remodeling. This ECM is typically deposited, digested and replaced in a very dynamic manner, and contains proteins such as fibronectin, fibrin, vitronectin and even residual fragments of collagen and laminin. This type of ECM promotes tissue remodeling as well as cellular survival, proliferation and invasion. In both types of ECM, however, the diversity in the type and quantity of each individual ECM component present determines the physical properties of these tissues. In so doing, this modulates the mechanical forces sensed by cells that bind to the ECM, and provides yet another layer of information relayed to cells. This 'mechanosensation' requires integrins, receptors that can transmit extracellular forces to the actin cytoskeleton. [3]. There are 18 different α subunits and 8 β subunits, but there are a limited number of possible combinations that can form from these subunits. To date, at least 24 unique integrin complexes have been identified, each with its own binding specificity for different subsets of ligands ( Figure 1). Cells will generally express only a limited number of integrins, perhaps 10 of these combinations. The particular repertoire of integrins expressed by a given cell varies, but is typically closely tied to a cell's particular extracellular microenvironment. Differences in integrin binding to a ligand can be subtle. For example, approximately one third of human integrins bind to an arginine-glycine-aspartic acid (RGD) sequence of amino acid residues, but this can be profoundly conformation specific, and thus not all 'RGD-binding' integrins are capable of binding all RGD sequences with appreciable affinity.

Integrin structure
Each integrin is composed of a large extracellular region of 600-1000 amino acids, as well as a single transmembrane domain. The extracellular regions can be broadly thought of in terms of a head and stalk (leg/thigh) region; the head is the critical site for ligand binding and divalent cation binding, as well as heterodimerization between the α and β subunits. Most integrins also have a small (~30-50 amino acid) cytosolic domain, with the singular exception being integrin β4, which has a large cytosolic domain that interacts with intermediate filaments [4]. Integrins are cysteine-rich proteins, and have extensive crosslinking within domains that stabilize domain structure. Thus, integrins appear at different sizes when analyzed on reducing and non-reducing gels, and detection of integrins by some antibodies may require either condition, depending upon the linearity or conformation dependence of an epitope.
The integrin extracellular domains are required for and sufficient to bind to ECM or to 'receptor-ligands' present on the surface of adjacent cells. However, the binding of integrins to their ligands is controlled by their conformation, which is influenced by the stalk and cytosolic regions of the molecule. Inactive integrins adapt a 'folded back' conformation at a region halfway up the stalk (at the 'genou,' or knee, between the thigh and leg). Active integrins are extended molecules with stalks separated, and intermediates between these states tend to have intermediate affinities for ligands. Integrin-ligand binding requires the presence of divalent cations, with a typical preference for manganese, magnesium and calcium, although the relative preference for optimal affinity varies among the different heterodimers. These divalent cations, and Mn +2 in particular, directly influence integrin conformation, stabilizing them in the extended and high affinity conformation ( Figure 2).
With the exception of circulating hematopoietic cells, which tend to maintain their integrins in an inactive conformation, most cells that have been examined express both active and inactive integrins. Active integrins tend to form higher order clusters on the cell surface, which promotes their localization to sites of ligation. There, the integrins are further stabilized by interaction with ligand. The accumulation of integrins in these sites creates a 'Velcro-like' effect, with groups of integrins (rather than individual molecules) collaborating to strengthen anchorage and to induce downstream signaling points of extracellular matrix contact. This clustering effect is called integrin 'avidity' regulation, which is distinct from affinity. This permits the stable interaction with the ECM required for sustained cellular anchorage and signaling via the assembly of a 'focal adhesion complex' that accumulates proximal to the membrane.
The focal adhesion complex that forms is multifunctional, and is capable of signaling directly, scaffolding additional or alternative signals, and engaging the actin/myosin system. Thus, despite the absence of intrinsic kinase or proteolytic activity, integrins transform mechanical and chemical cues from the extracellular environment into intracellular signals that profoundly impact cell behavior and function.
The focal adhesion complex contains a complicated array of non-receptor kinases and adaptor proteins that mediate downstream signaling events. As will be discussed in more detail below, integrin effectors in the focal adhesion include diverse signaling elements such as: focal adhesion kinase (FAK), src kinase, cytoskeletal elements including talin, paxillin and vinculin, phosphoinositide 3 kinase, and small GTPases of the Ras and Rho families and their effectors [5,6]. Importantly, as part of the clustering process, integrins tend to undergo lateral associations with other cell surface receptors such as the receptor tyrosine kinases, EGFR and VEGFR, which are important for other global cellular signaling events. This type of signaling, in which the integrin ectodomain is ligated and transforms information from the extracellular environment into cues for cytosolic signaling events has been termed "outside-in signaling." However, in some cases, signals from inside the cell result in changes in integrin conformation. These are typically associated with cytosolic proteins binding to the cytosolic domains of the integrins. This type of regulation of integrin conformation is called "inside-out signaling." Both types of signaling are important for understanding the role of integrins in normal tissues and in disease pathology. The extracellular domain comprises a head region and a stalk region, which includes the "thigh" and "leg" areas of the integrin. Ligand binding occurs at the head region and requires the presence of divalent cations such as manganese, magnesium, and calcium. Integrins on the cell surface can exist in a range of conformations that affect their affinity for ligand. In the low affinity conformation, the extracellular domain is folded back at the knee (between the thigh and leg areas) and the intracellular tails are clasped together. In the high affinity conformation the extracellular stalk is straight, the subunits are slightly separated and the tails shift apart as well. Conformations between these low and high affinity states confer intermediate affinity for ligand. Changes in conformation can be regulated by intracellular signaling events such as the binding of cytosolic proteins to integrin tails leading to integrin clustering, focal adhesion formation and further interaction of cytoskeletal proteins.

Integrins in early development
The ability of cells to interact with their extracellular environment is crucial for most developmental processes. Consequently, it is perhaps not surprising that integrins, as mediators of the interplay between cells, the ECM and the microenvironment, have critical roles in early development. The early physiological relevance is evident in defects observed in murine genetic models lacking proper integrin function or expression. Overall, the loss of the β1, α5, and α4 subunits leads to an embryonic lethal phenotype. The loss of the αv or α3 subunits permits initial and subsequent development, but results in perinatal lethality. Other integrin subunits do not appear to be essential during development.

Integrins in nervous system development
The development of the nervous system is dependent on integrin function, in part, because it involves extensive migration of neuronal precursors which is mediated by integrins. During the process of neurulation, the neural crest forms in the region of the neural plate border. Upon formation of the neural tube, neural crest cells undergo an 'epithelial-to-mesenchymal-like transition' which permits them to move along migratory tracks. These tracks lead cells to a variety of destinations where they differentiate and help to form several different tissue types. During development, collagens, laminins, fibronectin and vitronectin are expressed along these migratory pathways [28]. Disruption of integrin-ligand binding inhibits neural crest cell migration and results in impaired function in the peripheral nervous system. Following the initial gross exodus of neurons from the neural crest, integrins also play other key roles in the development of the peripheral nervous system, including the establishment of Schwann cell polarity [29], neurite outgrowth [30,31] and myelination [32].
In addition to the requirement for integrins to support migration, integrins are also important for arresting migration at the proper time and place. In the central nervous system, for example, the presence of the α6 and β1 subunits appears to serves as stop signals for neuronal cells when they reach a laminin rich region. This is critical for cortical plate formation. In the absence of these integrins, neuronal precursors migrating outward to the outermost layer of the cortical plate overshoot their destination and disrupt the cortical plate structure [33,34].

Integrin expression in the dorsal root ganglion and in neuroblasts
Neuroblastoma is a tumor that is considered to arise from ganglion or pre-ganglion cells. To begin to understand the pathological roles of integrins in this disease, it is helpful to be familiar with the normal expression patterns of these receptors in neural crest cells and how that expression changes over time. Neural crest cells express subsets of integrins that allow them to adhere to the fibrillar proteins that line their migratory pathways. Truncal neural crest cells, which give rise to dorsal root ganglia, sympathetic ganglia, and the adrenal medulla express receptors for vitronectin (αvβ1, αvβ3, and αvβ5: [35]), laminin (α1β1, α3β1,: [36] [37]), and fibronectin and associated molecules (α4β1, α5β1, α8β1, αvβ1 and β8 integrin: [37], [38].
Antibody blockade of any one type of these integrins is unable to completely abolish cell migration, consistent with a multi-receptor and complex ligand system. However, in studies on avian truncal neural crest cells, the α3β1, α4β1, and αv integrins appear to be the most crucial to maintain migration [38]. In particular, inhibition of the interaction between α4β1 and its ligands via blocking antibodies or ligand-mimicking peptides, leads to a marked reduction in neural crest cell migration [37].
As neural crest cells reach their target tissues and differentiate, their integrin expression changes. For example, neural crest cells do not express detectable levels of α6β1 until they differentiate into a peripheral nervous system cell type such as a Schwann cell precursor [39]. Conversely, neural crest cells express α1β1 but Schwann cell precursors do not [40,41]. This induction of expression of one class of integrins while another is eliminated is not well understood, however, and further study will be required to elucidate additional neuroblastspecific integrin expression and function.

Integrins in vascular system development
Similarly, the formation of the vascular system relies heavily on integrin function. During vasculogenesis, or de novo formation of blood vessels, and angiogenesis, the growth of new vessels from pre-existing vasculature, integrins play essential roles in endothelial cell migration, adhesion to basement membranes and cell survival. Endothelial cells are known to express a large number of β1 integrin heterodimers including the α1 through α6 subunits as well as integrins α6β4, αvβ5, and αvβ3. The expression of different subsets of these integrins is dependent on the activation state of the endothelial cells. For example, integrins αvβ3 and α4β1 are primarily expressed on activated or angiogenic endothelial cells [42]. Knockout of integrin αv leads to perinatal lethality due to vessel malformation [15] and studies on the αvβ3 heterodimer show that it is essential for the survival of angiogenic endothelial cells [43]. In addition, knock-out of integrin α4 in mice is embryonic lethal by day 14.5 due to placental and cardiac defects [11], likely due to a lack of binding to the α4 ligand, vascular cell adhesion molecule-1 (VCAM-1) which is present on endothelial and smooth muscle cells.
The formation of the vasculature, and angiogenesis in particular, is of interest to scientists who study neuroblastoma, which is typically a highly angiogenic disease. Although a focus has been placed on the roles of integrins in development of the neuronal and vascular systems, the ability of integrins to regulate such a large array of cellular functions renders them essential for most, if not all, developmental processes. Their roles may be directly associated with their adhesion and motility-related functions, or with the ability of integrins to indirectly enhance the efficiency of other signaling pathways [44].

Tumors exploit integrins for local invasion
As cells are transformed from a normal to malignant state, their integrin expression is modulated to support pathologic behaviors. In primary tumors, integrin signaling can impact cell growth, differentiation, and vascular infiltration and continues to be important as the cancer progresses through the stages of metastasis ( Figure 3). The initial steps of the metastatic process involve the degradation and remodeling of extracellular matrix adjacent to primary tumor cells, facilitating cancer cell migration into recruited blood vessels. This process is termed local invasion. Usually, for local invasion to begin, cells from the primary tumor shift from an epithelial or non-motile to a more mesenchymal phenotype. In addition, cells frequently create a pathway for themselves by inducing degradation of the matrix via enzymes such as matrix metalloproteases [45]. Integrins can regulate MMP expression and/or activity. For example, integrin α2β1 is a positive regulator of MMP-1 expression [46,47].

Integrins, tumor metastasis, and tissue tropism
For many types of cancer, metastasizing cells spread to a specific subset of secondary locations for establishment of metastatic nodules. This phenomenon, termed tissue tropism, has historically been explained by two major theories. The "seed and soil" hypothesis proposed by Stephen Paget in 1889 followed his observation of tissue-specific patterns of tumor metastasis in 735 breast cancer patients. Paget noted that the pattern of organs bearing metastases was not random, and suggested that certain tumor types preferentially metastasized to compatible environments [48]. He proposed that 'seeds' of tumors required compatible 'soil' to take root and grow. An alternative theory, by Ewing, suggests that tissue tropism is simply due to mechanical forces and circulatory patterns [49], and that tissue tropism results from this. These are not absolutely exclusive theories, and it is reasonable that blood flow patterns are important for the initial distribution of circulating tumor cells, while the propensity to invade, grow and survive may be dependent on the presence of the appropriate integrin ligands as well as other pro-survival factors.
Though there have been no studies specifically linking integrins to site-specific metastasis in neuroblastoma, integrins have been shown to play a role in tissue tropism. The primary sites of neuroblastoma metastasis are bone marrow, bone, lymph node and liver. In general, certain integrins have been linked to metastasis to these sites. For instance, integrin α4β1 can promote homing to the bone [50] and has been shown to enhance bone metastasis in melanoma [51]. This effect may be due to expression of VCAM-1 on bone marrow stromal cells. Integrin α4β1 may also promote lymphatic metastasis by enhancing binding to VCAM-1 present on lymphatic endothelial cells [52]. Integrin α2β1 is associated with enhanced liver metastasis. This is potentially due to its binding to collagen type IV expressed in liver sinusoids [53].

Indirect roles for integrins in metastasis
Since the metastatic cascade involves several steps, including local tumor invasion, intravasation, survival in the lymphatics/blood stream, extravasation, invasion into the new tissue parenchyma and growth and establishment of metastatic nodules, there are many opportunities for integrins to facilitate this process. The role of integrins in local invasion is clear. Once cells gain entry into the vasculature, integrins are important for cell-cell and cell-platelet adhesion leading to increased formation of cell emboli [54] and subsequent lodging in capillary beds. Integrins are also important for the endothelial transmigration that follows. At the site of distant metastasis, the microenvironment and composition of the extracellular matrix may be different from that of the native tissue of the invading tumor cells. Here, the balance of ligated and unligated integrins impacts cell behavior and survival, as discussed in Section 4.
The shedding of gangliosides also impacts neuroblastoma metastasis. Gangliosides are glycosphingolipids with one or more sialic acids linked to them. In circulation, gangliosides are associated with lipoproteins. There are several different types of gangliosides that are classified based on the number of associated sialic acids. Some of these gangliosides, such as G M3 , are normally present in circulation. Conversely, elevated levels of circulating G D2 , a disialoganglioside, have been found in neuroblastoma patients and its concentration is inversely related to progression-free survival. Shedding of gangliosides enhances integrin α2β1-dependent platelet activation, leading to platelet aggregation, and increased adhesion to vascular basement membranes [55]. These events can enhance tumor cell embolization impacting the occurrence of cells lodging in capillary beds and invading into surrounding tissue.
Finally, it is worth noting that at any phase of tumor progression, cancer cells must evade the immune system. Some T-cell lysis mechanisms are dependent on integrin expression. For instance, binding of T-cell integrin LFA-1 (αLβ2) to its ligands ICAM-1 on tumor cells is important in CD3-mediated T-cell lysis [56,57]. Of note, ICAM expression on neuroblastoma cells is associated with increased susceptibility to lymphokine-activated killer (LAK) cell lysis following interferon gamma treatment [58].

Trends in integrin expression with neuroblastoma stage and grade
Since integrins impact cell differentiation and invasion, there has been an interest in linking the expression of subsets of integrins with a particular tumor stage, or more appropriately, with tumor 'risk.' Key risk predictors to date have been established by the Children's Oncology Group, and include status of the MYCN gene, the pathology of the tumor according to guidelines established by Shimada [59], and in some cases the relative ploidy of the tumor. Since integrins are associated with neuronal cell developmental stages and activities, it is reasonable that integrin expression could offer insights into tumor activities.
In pioneering studies, using 45 clinical samples, Favrot et al. showed that the α2 and α6 subunits were associated with low grade, well-differentiated neuroblastoma samples. The finding is consistent with observations of normal 'neural crest cell to neuronal' differentiation. The β1 subunit was expressed on all samples while the α5 subunit was not expressed on any samples examined. Samples expressing the α4, αv, β3, and β4 subunits revealed no N-Myc amplification, and were associated with a good prognosis. In addition, expression of α4 and β4 subunits was found selectively on Schwannian stromal cells [60].
Conversely, more recent studies have found that many neuroblastoma cell lines express integrin α4 and that α4 expression is associated with increased tumor stage (stages 3 and 4) in clinical samples [61]. At least on cell lines, integrin α5β1 also appears to be expressed [45] and integrin αvβ3 has been described to be present on some malignant neuroblastomas [62]. In addition, by flow cytometry, our lab consistently observes low levels of integrin αvβ5 on established neuroblastoma cell lines, although whether this is a tissue culture adaptation or reflects actual expression in situ remains unclear. Indeed, neuroblasts exhibit significant plasticity, and although integrins may be associated with specific stages of neuroblastoma, or specific developmental states where transformation of the neuroblast initially occurred, an alternative hypothesis is that neuroblastoma may retain the capacity to alter their relative integrin expression, and that this type of plasticity may itself be a malignancy factor. Neuroblastomas fall into three common morphological/adhesive categories when grown in vitro: S (Substrate adherent), N (Neuroblastic), and I (Intermediate) types [63,64]. These different types are sometimes ascribed to a particular cell line, though in many cases a cell line may contain cells of all three types. Studies using tissue culture cell lines have shown that, relative to S-type, N-type neuroblastomas exhibit decreased expression of β1 integrin and greater expression of αvβ3, and are more migratory in vitro. However, the expression of αvβ3 on these cells is still relatively low, at least when one compares with tissues well known to express αvβ3, such as angiogenic endothelium or melanoma. N-type cells also form more colonies in soft agar and are more tumorigenic when implanted in mice than S-type, which are rarely able to form xenograft tumors [65]. S-type cells express fibronectin; it is therefore not surprising that they represent the group of neuroblastoma that express α5β1integrin, the fibronectin receptor [66].

The third type of cells is the 'intermediate cells.'
Noted as potential 'cancer stem cells' as early as 1989 by Ross and colleagues, these cells look like an intermediate between the N and S types via diverse measures including phase contrast microscopy, intermediate filament expression, tyrosine hydroxylase activity, and norepinephrine uptake [64]. Consistent with being a tumor stem-like cell (or tumor initiating cell), I-type cells are by far the most tumorigenic in mice and in in vitro surrogate assays of tumor formation. Treatment with 13-cis retinoic acid or 5bromo-2'-deoxyuridine can differentiate I-type cells into N-type or S-type, respectively. Retinoic acid has significant effects on integrins, consistent with changes seen during neuronal differentiation, and can differentiate some neuroblastomas into a benign growth-arrested state [67]. Clinically, retinoic acid has also been demonstrated to improve event free and overall survival in a long-term follow-up on a large cohort of neuroblastoma patients [68]

Signaling by integrins
In addition to key roles in cell anchorage and migration, integrin-mediated ligation of the extracellular matrix results in the initiation of signaling events exerting both local and cellular effects. Thus, the extracellular matrix encodes information via the local milieu of cell surface or diffusible factors presented to the cell (Figure 4). Most of these signals have been studied in rigorously defined systems in vitro with cell lines, rather than primary in vivo investigation.

Integrin ligation promotes the activation of the nonreceptor tyrosine kinases FAK and Src
Signaling that follows the ligation of integrins by extracellular matrix components can be studied by introducing suspended neuroblastoma cells to a surface coated with an extracellular matrix component, such as fibronectin. This results in cell attachment and spreading. Concurrent with these events, phosphorylation is observed on cytosolic nonreceptor tyrosine kinases like FAK (tyrosine residue 397) and Src (tyrosine residue 418), which indicate activation of the tyrosine kinases. At least some of this activity is physically present in the integrin associated focal adhesion complex, and these kinases can be co-purified with integrins from this complex.
FAK and Src can associate with each other and with an array of cytosolic adaptor proteins and other effectors. For example, FAK can associate with the cytoskeletal adaptor protein talin, which also binds to integrins. The adhesion of NB7 neuroblastoma cells to fibronectin or collagen has been shown to promote co-association of these molecules together in a complex with the protease calpain. Calpain in turn cleaves talin in a cell-adhesion dependent manner, which faciliates more rapid turn over of the focal adhesion, and promotes neuroblastoma cell migration. The same cleavage is observed in other neuroblastoma cells, including NB5 and NB16, suggesting it may be a conserved pathway [69].
FAK also associates with Grb2 and SoS [70], key regulators of Ras-GTP mediated activation of the Raf/MEK/ERK pathway of MAP kinase signaling. This pathway helps to drive proliferation of the tumor cells, and may account for adhesion-based induction of cyclin E in neuroblastoma (and other) cells [71]. FAK is perhaps best known for its capacity to support and promote integrin-mediated cell migration on an ECM, and performs this function in neuroblastoma cells as well, although this appears to be integrin specific [61]. For example, integrin α5β1 activates FAK and uses this kinase for migration, while integrin α4β1 migration is dependent upon the non-receptor kinase Src. Both integrins can bind to a fibronectin substrate, thus the particular integrin ligated can have an impact on the cells' response. Other effects of specific integrin ligation have been reported in non-neuroblastoma cell lines, such as the FAK and α5β1-induced expression of the pro-survival gene Bcl-2 [72]. Thus, signals from FAK can play a role in regulating cell survival in an ECM and integrin-dependent manner. Signaling from integrins and focal adhesion-associated receptor tyrosine kinases (RTK) leads to downstream pro-survival signaling pathways such as the PI3K /AKT and Erk axes. By contrast, the presence of an incompatible ECM or of unligated or antagonized integrins promotes cell death via anoikis pathways, including integrin-mediated death. N-Myc exerts pleiotropic effects via transcription (or inhibition thereof) of many downstream genes, enhancing proliferation and survival, and attenuating the expression of integrins, and therefore decreasing anoikis signaling.

Integrin activation of the phosphoinositide 3' kinase signaling axis
Integrins stabilized and ligated to correct ECM promote signaling via class I phosphoinisotol-3 kinases (PI3K). PI3K's are a family of lipid bound kinases found at the cell membrane or intracellular endosomes, and can promote cell motility, intracellular trafficking and survival. Among the four class I PI3K's, neuroblastoma tend to express P110α and p110β, with the latter more likely to be associated with N-Myc expressing tumors. Nonetheless, P110γ and p110δ are also sometimes detected [73]. Activation of the PI3K signaling axis promotes malignancy in numerous cancer cell lines and models of human cancer [74]. PI3K signaling also enhances turnover of pro-mitochondrial apoptotic proteins like Bad and promotes downstream prosurvival pathways, such as AKT and mTOR [75]. PTEN, a suppressor of PI3K, is frequently lost in cancer, although studies in neuroblastoma have shown a lesser degrees of loss, in the range of ~5% for homozygous deletion [76]. Mutations of PI3K that enhance kinase activity have been reported in other cancers [77], yet they have been proven to be infrequent in neuroblastoma [78]. Thus, the activity of PI3K appears to frequently depend upon extrinsic regulatory factors, mediated by receptor tyrosine kinases (eg., IGFR-1, ALK) and integrins.
Given the lack of effective therapies for malignant neuroblastoma, it is perhaps not surprising that the PI3K pathway is being pursued for pharmacological intervention [79]. In neuroblastoma, inhibition of PI3K has been demonstrated to decrease migration and survival of tumor cells in vitro, and inhibit tumor growth in vivo [80,81]. The efficacy of pharmacological PI3K inhibition may be enhanced by combining a pro-drug with an RGDS peptide to target the agent to tumor sites [81]. The relative affinity for this linear peptide for integrin, however, is quite low, and it is improbable that enhanced efficacy is due to direct action on integrins, rather, it is likely due to improved pharmacokinetics associated with the targeting peptide.

Interplay between integrins and signature neuroblastoma signaling pathways
N-Myc is a transcription factor normally expressed during early lymphocyte development and in embryonic brain and kidney tissues [82], and is critical for survival of neural crest-derived neurons [83]. Amplification of greater than ten copies of the MYCN gene has long been recognized as a strong negative prognostic indicator of outcome in neuroblastoma [84]. N-Myc interacts with integrins in an antagonistic manner; while N-Myc seems to increase expression of FAK, it has also been shown to down-regulate the expression of integrins such as α3β1 and α1β1 [85][86][87][88]. Transcriptional analysis of the β3 and αv promoters have revealed negative transcriptional regulatory elements in their promoters by the closely related c-Myc [76], suggesting why αvβ3 is not highly expressed in neuroblastoma relative to other tumors. In fact, the loss of integrin expression may be important for survival in specific circumstances, particularly among tumors that retain intrinsic apoptotic capacity, as discussed below.
ALK is a tyrosine kinase that is expressed largely during development within the nervous system. ALK belongs to the 'insulin-like tyrosine kinase' family of receptors that is frequently upregulated or subject to oncogenic mutation in neuroblastoma [89]. Signaling by tyrosine kinases generally requires integrin ligation [5], activating downstream targets (such as FAK, Src, PI3K etc.). This suggests that there is an intrinsic requirement for ECM adhesion to permit a tumor to 'leverage' amplified ALK. However, mutant forms of ALK also exist, particularly a F1174 mutation that drives neuroblastoma malignancy cooperatively with MYCN. In this case, it is unclear whether integrin-mediated adhesion is actually required for cell proliferation, although it is likely to enhance signaling in keeping with the rationale described above. MYCN also leads to increased expression of a close ALK relative, insulin-like growth factor I receptor (IGF-IR). In this case, crosstalk between IGF-IR and integrins is also observed [90].

Integrins and cell survival signaling
Cells that lose anchorage for extended periods of time will typically undergo apoptosis. This phenomenon encompasses one aspect of anoikis (gr., homelessness), a phenomenon wherein a cell that finds itself in an inappropriate environment is signaled to undergo apoptosis. However, there is no 'central cell death pathway' associated with anoikis, and in fact many different pathways have been validated in the literature. This underscores the critical need for cell adhesion. One anoikis pathway is focused on the activation of caspase-9. Although many neuroblastomas lose expression of one copy of caspase-9 (as many are LOH1p21), this does not appear to impact the capacity of caspase-9 to activate [91]. Antagonism of b1 integrins on differentiated neuroblastoma, but not undifferentiated, promotes this apoptotic pathway [92].
Integrin-mediated death is an anoikis pathway in which the presence of unligated, or antagonized, integrins on the cell surface promote cell death via the activation of caspase-8. Neuroblastoma avoid this death pathway via several mechanisms. First, the amplification of MYCN can lead to an overall decrease in integrin expression, which lowers the capacity of the pathway to trigger. Secondly, stage III and IV neuroblastoma tend to methylate, delete, or disrupt the caspase-8 gene [93,94], preventing the triggering of the apoptotic pathway, and this results in a survival advantage in vitro and a metastasis advantage in vivo. Finally, neuroblastoma that are seeded as individual cells in an 'inappropriate' three dimensional matrix will tend to either die or, within only a couple days, find each other and form small cell clusters. These islands of cells promote their own survival and can persist, although they are sometimes surrounded by apoptotic bodies as errant progeny try to migrate away from the original cell mass.
Opposing the induction of death by unligated or antagonized integrins, it is worth noting that a cell that has a robust interaction with the ECM is more resistant to certain insults than others, and integrin ligation has been linked to chemo and radiation resistance. Mechanistically, this is likely to result from remodeling of the ECM, combined with transcriptional alterations of survival promoting genes such as Bcl-2 family members, IAPs and others. However, direct effects, such as maturation-inhibiting phosphorylation of procaspase-8, cannot be excluded from contributing to this effect [95,96].

Integrin αvβ3
Integrin αvβ3 is the most 'promiscuous' member of the integrin family, in that it binds a variety of different RGD conformations, and thus binds to ligands that include vitronectin, fibronectin, fibrinogen, von Willebrand factor and others. Gladson et al. found that αv was present in all tumors they examined regardless of stage. While αvβ1 and αvβ5 heterodimers were found in normal adrenal tissues and ganglioneuroblastomas which exhibit lower levels of dissemination, the αvβ3 integrin was found to be expressed in highly metastatic, undifferentiated neuroblastomas [62]. By contrast, we observe only very low levels of integrin αvβ3 on our neuroblastoma specimens relative to melanoma or cultured endothelial cells, which express robust levels of αvβ3. However, it remains possible that the techniques originally used by Gladson were simply very sensitive and detected this modest but important level of integrin expression. Indeed, αvβ3 is, in some systems, a stem cell marker, and this may reflect the advanced stage and poor prognosis of her positive cohort.
In addition, on a variety of tumor cells, αvβ3 expression has been demonstrated to promote tumor progression by its ability to bind to a wide array of different ligands, facilitating anchorage and invasion. Integrin αvβ3 also stimulates MMP activity, promotes the activation of receptor and non-receptor tyrosine kinases including src, and the release of growth factors such as TGF that promote tumor response. This vascularization provides the growing tumor with the nutrients it needs and brings tumor cells proximal to vessels, which may facilitate invasion and metastasis. As previously mentioned, αvβ3 is also expressed on angiogenic endothelial cells where it promotes cell survival and migration. One study showed that there is higher β3 expression on invasive and metastatic melanomas than on noninvasive melanomas [97], although the levels demonstrated in these cases appear to be logarithmically higher than those seen on neuroblastoma cell lines [98].

Integrin α4β1 and tumor spread
Integrin α4β1 is primarily known as a trafficking integrin, as it is present on most leukocytes. Binding to its ligand VCAM-1, present on activated endothelial cells, enhances the transendothelial migration of white blood cells into surrounding tissues. Cancer cells that express α4β1 acquire this same enhanced trafficking potential and show increased tumor cell arrest in circulation and increased extravasation and colony formation. α4β1 may also enhance invasion and metastasis through promotion of angiogenesis and lymphangiogenesis [99,100]. In [97], α4β1 expression was found on 40% of invasive and metastatic melanomas, although not on non-malignant melanocytes.
It is important to note that, though the expression of α4β1 can indeed promote extravasation, the overall role of integrin α4β1 in tumor progression and metastasis is highly controversial and is dependent on the level of expression and the phase of tumor progression. For example, high α4β1 expression in some primary tumors can enhance homotypic cell-cell adhesion [101], preventing cells from breaking away from the tumor and invading into surrounding tissues [102]. In addition, α4β1 expression can lead to a reduction in MMPs and impair the ability of the cells to degrade the matrix and create a pathway for invasion [103]. If cells do successfully metastasize to distant sites, α4β1 expression may promote or inhibit metastatic growth depending on the microenvironment.

Drugs that target integrins
The involvement of integrins in multiple stages of tumor progression makes them attractive therapeutic targets. Inhibition of integrin signaling can be achieved using several approaches including blocking ligand binding, preventing the formation of functional focal adhesion complexes and disrupting integrin association with the cytoskeleton. Because the structure of integrins has been extensively studied and because having an extracellular target eliminates the challenges of intracellular delivery, the most common approach has been to target the integrin ligand-binding site. This has been accomplished using blocking antibodies, cyclic and ligand-mimicking peptides, small molecule antagonists and disintegrins [104] ( Table 2).

Integrin αv
The primary rationale for targeting integrin αvβ3 in cancer is to reduce primary tumor growth and metastasis via nutrient deprivation due to inhibition of tumor angiogenesis. Several αvβ3 antagonists have gone to clinical trials with the most notable being cilengitide. Cilengitide is a cyclic peptide containing the RGD integrin-binding motif. It inhibits both αvβ3 and αvβ5. Cilengitide produces both anti-angiogenic and anti-tumor effects through inhibition of VEGF stimulation and FAK-Src and Erk signaling, respectively [105]. In vitro, cilengitide reduces cell growth and survival and inhibits endothelial and tumor cell migration. In clinical trials, cilengitide has been evaluated as a single agent and in combination with radiation, DNAalkylating agents and gemcitibine. Importantly, cilengitide in combination with radiotherapy and temozolomide (a DNA-alkylating agent) has reached phase III trials in glioblastoma multiforme patients. Other small molecule antagonists are in development for noncancer indications.

Integrin α4
The integrin α4 subunit is predominantly expressed in lymphocytes and leukocytes and supports endothelial transmigration of these cells via binding to VCAM-1. Consequently, α4 is important for immune function and has been targeted in diseases such as multiple sclerosis (MS), Crohn's disease and asthma that are characterized by excessive inflammation or an improper immune response. Natalizumab, the only FDA approved α4 antagonist, is a humanized mouse monoclonal antibody that binds both α4 heterodimers. The use of natalizumab was successful in clinical trials in MS [106,107] and Crohn's disease [108] with the exception of rare cases of progressive multi-focal leukoencephalopathy (PML) caused by reactivation of latent JC virus associated with immunosuppression [109]. Unfortunately, this side effect was detrimental enough to lead to limitation of the use of natalizumab to patients who are unresponsive to other treatments. Other α4 antagonists under clinical evaluation include MLN-00002 (human α4β7 antibody), firategrast and IVL745 (small molecules: [104]). Though the rationale for the use of most α4 antagonists is to reduce excessive infiltration of immune cells, these therapies have the potential for use against cancer cells that exploit α4 for tumor cell extravasation. The success of targeting α4 in cancer will depend on the ability to minimize immunosuppression or to indirectly impair α4 function via downstream targets.

Integrin αIIbβ3
Integrin αIIbβ3 is also a frequently targeted integrin. This heterodimer is expressed selectively on platelets and megakaryocytes and is mostly known for its role in blood coagulation.
Antagonists of this receptor are primarily employed in diseases such as stroke, sickle cell anemia and acute coronary syndromes [104].

Summary and considerations
Integrins are a unique group of receptors that provide anchorage, mediate cell migration and invasion, and signal via cell survival and proliferation pathways. Aptly named, integrins integrate extracellular cues with intracellular signaling and serve to regulate many cellular processes that are mediated by other receptors, such as receptor tyrosine kinases. The importance of integrins in cancer development of the nervous system is well established; it seems inevitable therefore that they play a major role in neuroblastoma progression. In fact, integrin expression has been linked to malignancy in neuroblastoma, possibly due to alterations in invasiveness and the ability to evade cell death in foreign tissue environments. Aggressive disease may modulate integrin expression (i.e. N-Myc).
Targeting integrins has shown great clinical promise. By inhibiting ligand binding, many antagonists successfully disrupt cellular connections to the extracellular environment and prosurvival pathways that are necessary for tumor progression. As we continue to learn more about the downstream signaling activity of integrin receptors, we can also explore more therapeutic avenues against these targets, attacking the problem from both sides. However, the logical use of integrin antagonists in complex, multi-agent regimens is lacking. Given the synergy of integrins with signaling through receptor tyrosine kinases and in the induction of susceptibility to apoptosis, this is where one would suspect that these relatively non-toxic agents would have their greatest impact.
Though clinical studies of integrin-targeted drugs in neuroblastoma have not been performed, in vitro antagonism has been shown to decrease cell survival, migration and invasion. Despite these characteristics, integrin-targeted drugs are well tolerated. Given that current treatment for neuroblastoma still has a significant failure rate, the addition of new, low toxicity adjuncts to current treatment regimens seems a logical step forward. In the future, an increased understanding of the roles of specific integrins in neuroblastoma has the potential to provide better prognostic information regarding disease course, while targeting integrins, perhaps in combination with other targeted therapies as a cocktail addition to standard chemotherapy approaches, may lead to increased effectiveness in managing this disease.