Neuroblastoma as an experimental model for neuronal differentiation and hypoxia-induced tumor cell dedifferentiation

Seminars in Cancer Biology 17 (2007) 248–256

Review

Neuroblastoma as an experimental model for neuronal differentiation and hypoxia-induced tumor cell dedifferentiation Anders Edsj¨o, Linda Holmquist, Sven P˚ahlman ∗ Department of Laboratory Medicine, Molecular Medicine, Lund University, University Hospital MAS, Entrance 78, SE-205 02 Malm¨o, Sweden

Abstract Neuroblastoma is a childhood tumor derived from precursor or immature cells of the sympathetic nervous system. Neuroblastomas show a tremendous clinical heterogeneity, encompassing truly benign as well as extremely aggressive forms. In vivo as well as in vitro data have shown that the degree of sympathetic neuronal tumor cell differentiation influences patient outcome. Unraveling mechanisms governing neuroblastoma cell differentiation is therefore a central issue in the neuroblastoma research field. In this communication, we discuss some of the in vitro models frequently used to study human neuroblastoma cell differentiation. We also review recent data demonstrating that oxygen shortage, hypoxia, shifts neuroblastoma cells toward an immature, stem cell-like phenotype and discuss the potential clinical impact of hypoxia on neuroblastoma behavior. © 2006 Elsevier Ltd. All rights reserved. Keywords: Neuroblastoma; Hypoxia; Neuronal differentiation; Sympathetic nervous system

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Neuroblastoma and its clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma—a sympathetic nervous system-derived tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma in vitro differentiation models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Neuroblastoma cell differentiation induced by retinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Phorbolesters as differentiating agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Growth factors and neuroblastoma differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Neurotrophins and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Neurotrophins and neurotrophin receptors in the developing SNS and in neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Neurotrophin-induced differentiation of cultured neuroblastoma cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma cell differentiation status in response to oxygen shortage, hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The hypoxic response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Neuroblastoma and hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Hypoxia-induced dedifferentiation of neuroblastoma cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Neuroblastoma and its clinical features Neuroblastoma is a childhood tumor, which can present at birth and throughout early childhood with only occasional cases ∗

Corresponding author. Tel.: +46 4033 7403; fax: +46 4033 7322. E-mail address: [email protected] (S. P˚ahlman).

1044-579X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2006.04.005

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diagnosed after 10 years of age. Neuroblastomas are clinically classified into five stages, ranging from the localized stage 1 tumors to stage 4 cases with extensive tumor dissemination [1]. Children with stage 1 and 2 tumors are basically treated with surgery alone, whereas children with stage 4 tumors receive advanced radiation and chemotherapy. Some tumors fall within the intriguing stage 4s category. This stage is characterized by localized primary tumors and dissemination limited to liver, skin

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and/or bone marrow. The tumors occur in infants, and can regress spontaneously, hence stage 4s and stage 4 patients are handled separately. Despite their sometimes extensively disseminated disease, the overall survival rate of stage 4s patients is approximately 80%, contrasting the close to 20% survival rate of stage 4 patients. Neuroblastoma cells are typically undifferentiated, round and small with scant cytoplasm. Many tumors, though, contain differentiated cells with larger nuclei and cytoplasms and they are classified as ganglioneuroblastoma. Some tumors exclusively contain ganglion-like cells, and these benign tumors are termed ganglioneuromas. This classification system early revealed that high differentiation stage correlates to favorable prognosis, which later was confirmed by differentiation marker studies [2]. Some 80 years ago, Cushing and Wolbach described a patient with differentiated, benign ganglioneuroma cells in a lymph node. As ganglioneuromas are non-metastasizing, Cushing and Wolbach assumed that these lymph node-located tumor cells were derived from a neuroblastoma metastasis, thus suggesting, for the first time, that neuroblastomas might spontaneously differentiate, a capacity later attributed to stage 4s tumors. Although it took almost 50 years before the concept of tumor cell differentiation became generally acknowledged, the report by Cushing and Wolbach provided clinical relevance and importance of the early in vitro observations of tumor cell maturation of cultured neuroblastoma and hematopoietic tumor cells. Through the pioneering work of June Biedler, Robert Seeger and many others, neuroblastoma cell lines were established already in the 1970s. The natural history and tumor biology of neuroblastomas could thus be studied as a model for tumor cell maturation processes in general, and human sympathetic differentiation in particular. However, not all established neuroblastoma cell lines appear to have the capacity to differentiate. One reason for this might be that most (if not all) existing human neuroblastoma cell lines are derived from immature and aggressive high stage tumors.

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2. Neuroblastoma—a sympathetic nervous system-derived tumor Neuroblastomas are derived from precursor or immature cells of the sympathetic nervous system (SNS), and primary tumors can be found at any location of SNS structures. A common sympathoadrenal precursor is thought to give rise to three distinct sympathetic neuronal/neuroendocrine lineages (Fig. 1), the neuronal/ganglionic, the small intensely fluorescent (SIF) and the chromaffin lineages [3]. The sympathetic ganglia proper, i.e. the sympathetic chain and truncus ganglia are composed of ganglion cells and during development also of SIF cells. Phenotypically, SIF cells share characteristics with both neuroblasts and chromaffin cells [3]. Chromaffin cells form the adrenal glands and the paraganglia, the latter being primarily fetal and early post-natal structures, which in humans appear to loose their function as major catecholamine producers 2–3 years after birth, when this role is taken over by the adrenal gland. The chromaffin cells are the major sympathetic cell type of the adrenal gland, but nests of sympathetic neuroblasts are frequently seen in developing human fetal adrenal glands [4]. Extensive characterization of marker genes expressed in human SNS structures during embryonal and fetal development and corresponding marker gene expression analyses of human neuroblastomas, reveals that neuroblastomas have immature neuronal characteristics suggesting that they are derived from precursors or immature cells of the sympathetic ganglionic lineage [4]. Thus, also adrenal neuroblastomas share these features, and Hoehner et al. [4] postulated that adrenal neuroblastomas are derived from neuroblasts present in immature adrenal glands. A small subset of neuroblastomas have regions of tumor cells that show a spontaneous neuronal to neuroendocrine lineage shift [5]. The lineage shift occurs in cell layers surrounding necrotic foci and might thus be hypoxia-driven, but so far this has not been convincingly demonstrated in experimental model systems (see hypoxia section below). To summarize, neuroblastomas are

Fig. 1. Sympathetic nervous system (SNS) cell lineages and their derived tumor forms.

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derived from immature sympathetic cells of the ganglionic lineage and they are arrested at various stages of differentiation (Fig. 1). In a subset of tumors, cells adjacent to necrotic zones show a spontaneous neuronal to neuroendocrine lineage shift. Marker gene expression studies indicate that these cells do not adopt an adrenal or paraganglionic chromaffin phenotype. They might become SIF-like (Fig. 1), but lack of discriminating and specific markers for SIF cells means that this issue has not been further addressed. 3. Neuroblastoma in vitro differentiation models In such a rare malignancy as neuroblastoma, access to fresh tumor material is limited and in vitro studies based on primary tumor explants are difficult to perform. Differentiation studies have instead utilized in vitro models in which neuroblastoma cell lines have been induced to differentiate in the presence of various agents and growth factors (Fig. 2A). The first and conceptually most important demonstration of in vitro differentiation of a human neuroblastoma cell line was published 25 years ago, when human SH-SY5Y neuroblastoma cells (a subclone of the SK-N-SH cell line) were shown to differentiate morphologically and biochemically in response to bioactive phorbolesters [6]. The induction of processes (neurites) in these cells was paralleled by an increased accumulation of norepinephrine and neuron specific enolase (NSE), differentiation markers also employed as diagnostic markers for neuroblastoma. Since the first report, a number of neuroblastoma differentiation protocols have been published employing among others retinoids and growth factors such as nerve growth factor (NGF) (Fig. 2B). These observations fostered a hope that patients with advanced neuroblastoma might successfully be treated by inducing their tumor cells to differentiate [6]. In retrospect, it turned out that naturally occurring and synthetic retinoids so far have had the greatest clinical impact and usage as differentiation inducers, although the mechanism(s) of action of retinoids in neuroblastoma patients with residual disease is not fully known [7].

Fig. 2. In vitro differentiation of neuroblastoma cells. (A) Summary of discussed differentiation protocols. (B) Neuroblastoma cells are generally non-responsive to neurotrophin stimulation. However, if TRKA expression is enhanced by mitogenic blockers or by transfection of exogenous TRKA (dashed arrow), some neuroblastoma cell lines, exemplified by the SH-SY5Y cells, have the capacity to differentiate into cells with neuron-like properties in response to NGF stimulation.

3.1. Neuroblastoma cell differentiation induced by retinoids The effects of retinoids are mediated by two classes of nonsteroid nuclear hormone receptors, the retinoic acid (RAR ␣, ␤, ␥) and the retinoic X (RXR ␣, ␤, ␥) receptors [8]. The naturally occurring all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid, and the synthetic 13-cis-retinoic acid are examples of retinoids studied in neuroblastoma. It was early established that ATRA stimulation of neuroblastoma cells results in growth inhibition, decreased anchorage-independent growth, and neuronal differentiation as indicated by morphology, increased NSE activity, a slight accumulation of norepinephrine, and upregulated expression of GAP43 coding for growth associated protein 43, a protein important in axonal growth [9,10]. Later, downregulation of the proto-oncogenes MYCN, MYB, HRAS was shown to precede the morphological differentiation, with changes in the expression of a set of other proto-oncogenes to follow [11,12]. Even though ATRA-induced differentiation appears to be neuronal,

the outcome differs depending on the neuroblastoma cell line studied. While some cell lines develop a sympathetic noradrenergic phenotype, a cholinergic switch has been suggested in other cell lines (Fig. 2A) [13–15]. As will be discussed in the neurotrophin section, ATRA treatment also results in the upregulation of the neurotrophin receptors TRKB and RET resulting in brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF) responsiveness (Fig. 2A). As a therapeutic tool, ATRA has proven effective in inducing differentiation of certain hematological malignancies, most notably acute promyelocytic leukemia [16]. In neuroblastoma, 13-cis-retinoic acid, administered as a high dose pulse treatment, is beneficial for patients with minimum residual disease [7]. The fact that 13-cis-retinoic acid has higher clinical effectiveness than ATRA is probably due to more favorable pharmacokinetic properties. In this clinical setting, the induction of

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neurotrophin receptor expression and neurotrophin responsiveness by retinoids might be important mechanisms contributing to the improved outcome of neuroblastoma patients. In vitro as well as clinical data indicate that synthetic retinoids such as Fenretinide might prove valuable also in cells resistant to retinoic acid. The effect seems independent of retinoid receptors and appears not to involve differentiation but induction of apoptosis as well as necrosis [7]. 3.2. Phorbolesters as differentiating agents Biologically active phorbolesters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) have drastic effects on cell growth and differentiation. In some neuroblastoma cell lines, the SHSY5Y cells being the best described, nanomolar concentrations of TPA promote differentiation. TPA-differentiated cells are characterized by outgrowth of varicosity-containing neurites terminated by growth cones and by induced expression of a number of neuronal sympathetic differentiation markers, e.g. tyrosine hydroxylase (TH), neuropeptide tyrosine (NPY), GAP43, NSE. TPA-treated cells also downregulate MYC and become growth retarded [6,17]. In addition, the phorbolester treated cells become functionally differentiated as they accumulate norepinephrine in dense core granules and build up an action potential, which is depolarized by acetylcholine resulting in release of stored neurotransmitters [18]. The biological effects of phorbolesters are primarily mediated by protein kinase C (PKC) isoforms and downregulation of PKCs by high phorbolester concentrations results in less differentiated neuroblastoma cells, suggesting that the differentiation process is PKC dependent [19]. The issue appears to be more complicated than first anticipated, as it was demonstrated that neurite outgrowth is dependent on the regulatory, and not the catalytic domains of novel PKCs [20]. Overall, the phenotype of TPA-treated SHSY5Y cells very much resembles that of sympathetic neurons and according to our view, is still one of the best model systems for mechanistic studies of human sympathetic neuronal differentiation. 3.3. Growth factors and neuroblastoma differentiation At an early stage during SNS development, neuronal survival is independent of the classical neurotrophins, NGF, BDNF and neurotrophins (NTFs) 3 and 5, but several other growth factors important for survival, proliferation, and differentiation have been identified. These factors include basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF1), GDNF and ciliary neurotrophic factor (CNTF) [21]. Consistent with their importance during SNS development, combinations of growth factors such as bFGF and IGF1 have also proved efficient in vitro inducers of neuroblastoma differentiation [22]. Neuroblastoma cell lines generally lack functional neurotrophin receptors of the tropomyosin receptor kinase (TRK) gene family and do not differentiate when stimulated with NGF, BDNF, NTF3 or NTF5. However, pre-treatment with ATRA or growth inhibiting agents will induce TRK expression and neurotrophin sensitivity as will introduction of exogenous TRKs (Fig. 2B), which will be

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discussed below. ATRA treatment of neuroblastoma cells also induces the expression of the proto-oncogene RET, which codes for a tyrosine kinase growth factor receptor specifically binding GDNF [23]. GDNF is required for proper development of enteric and parasympathetic neuroblasts [23], and ATRA-treated neuroblastoma cells differentiate in response to GDNF (Fig. 2A) [24]. Taken together, data show that many neuroblastoma cell lines have the capacity to respond to physiological differentiation stimuli, although they uniformly seem to have lost this capacity with regard to stimulation with NGF or with other neurotrophins unless the neuroblastoma cells are triggered in some way. 3.3.1. Neurotrophins and their receptors Corroborated by data with blocking antibodies towards individual neurotrophins, targeted deletions of genes encoding neurotrophins (NGF, BDNF, NTF3, NTF5) and neurotrophin receptors (TRKA, TRKB, TRKC, p75(NTR)) have established essential roles for neurotrophins in the survival and differentiation of neuronal subpopulations, including those of the SNS [25]. The neurotrophins bind to two types of cell surface receptors, the tyrosine kinase (TRK) receptors and the neurotrophin receptor p75(NTR), which can complex with each other resulting in the formation of high-affinity neurotrophin receptors. The preferred ligand–receptor interactions are NGF/TRKA, BDNF and NTF5/TRKB and NTF3/TRKC, but NTF3 can under some circumstances activate TRKA and TRKB [26,27]. All three neurotrophin receptors have spliced or truncated variants [26]. In some cases the splice variants have an altered ligand specificity [28,29], in others they appear to be simply non-functioning [30]. Recently a constitutively active TRKA splice variant, lacking the NGF-binding domain, with oncogenic potential was described [31]. This variant is expressed in sympathetic precursor cells and in some neuroblastomas. TRKB and TRKC receptor proteins come in two classes, with and without the cytoplasmic tyrosine kinase region. A specific role in neural crest cell differentiation has been suggested for TRKCTK− variants [32], while TRKBTK− isoforms seems to be dominant negative variants [33]. The transmembrane glycoprotein p75(NTR) is the founding member of the tumor necrosis factor (TNF) receptor superfamily of receptors and binds all neurotrophins [27]. It is structurally unrelated to the TRK proteins and its downstream signaling pathways are less well described than that of the Trk receptors. 3.3.2. Neurotrophins and neurotrophin receptors in the developing SNS and in neuroblastoma As already mentioned, the developing SNS becomes dependent on neurotrophins comparatively late, i.e. after the formation of SNS ganglia and paraganglia. During human development only trace amounts of TRKA protein can be immunohistochemically detected in sympathetic ganglia at fetal weeks 8–9, whereas adrenal neuroblasts, paraganglia, and adrenal chromaffin cells are negative at this developmental stage [34]. Around fetal weeks 13–15, TRKA is clearly expressed in major SNS structures, and TRKC expression can also be detected. TRKB expression is found in some high stage neuroblastomas, but, interestingly,

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convincing TRKB expression is only detected in paraganglia from fetal week 21 and onwards, whereas sympathetic neuroblasts in fetal SNS ganglia and adrenal glands are TRKB negative [34]. In neuroblastoma, TRKA expression is a strong predictor of positive outcome and can together with MYCN copy number be used as a powerful prognostic tool [35,36]. While the expression pattern of TRKC is similar to that of TRKA [37,38], expression of full-length TRKB is predominantly seen in MYCN amplified tumors with poor prognosis [39]. The effect of p75(NTR) expression in neuroblastoma is not established, but, while ubiquitously expressed in primary tumors, it seems downregulated in tumors with MYCN amplification and high expression seems to be associated with favorable outcome [40,41,36]. It has not been possible to correlate neurotrophin expression with patient outcome. However, cell line and tumor tissue data suggest that neuroblastoma cells express no or low NTF3 and NGF, while BDNF and NTF5 seem to be expressed at high levels [39]. In addition to neurotrophins produced by neuroblastoma cells, surrounding cells might serve as a supply of neurotrophic support. Indeed, experimental data suggest that Schwann cells might regulate survival and differentiation of neuroblastoma cells [42]. 3.3.3. Neurotrophin-induced differentiation of cultured neuroblastoma cells Neuroblastoma cell lines transfected with either TRKA or TRKC can differentiate in response to treatment with their cognate ligand, and the phenotype induced is sympathetic neuronal with increased expression of e.g. TH, GAP43, NPY [43,44]. Blocked proliferation of SH-SY5Y cells also results in NGF responsiveness through induced expression of TRKA, again demonstrating that high-stage, TRK-negative cells retain a capacity to respond to TRK-mediated signals [43,45,46]. The recently described TRKAIII splice variant lacking the NGFbinding domain is constitutively active but does not seem to activate the Ras/Raf/MEK/MAPK pathway, which is activated during classical NGF-induced differentiation. Instead, it appears as if an aggressive neuroblastoma cell behavior is promoted via Akt activation [31]. The blocked differentiation and uncontrolled proliferation of neuroblastoma cells might in part be the result of an aberrant expression of this TRK variant, explaining the impaired neurotrophin-driven differentiation of high-stage neuroblastomas [35]. If TRKA- and TRKC-mediated signals can be expected to decrease the neuroblastoma aggressiveness, the effects of BDNF/TRKB signaling seem to be more in line with their association to neuroblastomas with poor outcome. Even though BDNF treatment of neuroblastoma cells transfected with TRKB cDNA appears to induce differentiation and inhibit growth [47], BDNF stimulation of neuroblastoma cell lines expressing TRKB as a result of RA treatment does not affect proliferation, but increases survival and invasiveness [48]. In addition to these effects of TRKA and TRKB signaling, recent data seem to suggest a difference also with respect to therapy resistance, invasiveness, angiogenesis, and possibly also genomic stability [49].

4. Neuroblastoma cell differentiation status in response to oxygen shortage, hypoxia 4.1. The hypoxic response Efficient mechanisms for cellular adaptation to hypoxia probably evolved as a necessary consequence of the development of oxygen dependent multi-organ organisms. These mechanisms seem to be universal and conserved during evolution and primarily involve the activation of hypoxia inducible factors, HIFs [50]. The adaptation to hypoxia occurs at a systemic as well as at a cellular level. Examples of systemic effects include induced vascularization, which is promoted by induction of angiogenic factors such as VEGF and IGF-II and increased blood pressure via induction of the catecholamine production. At the cellular level, hypoxia is associated with a switch to anaerobic metabolism, decreased protein synthesis and decreased DNA repair capacity. One important net effect of the cellular and systemic hypoxic responses is to maintain ATP pools by a combination of reduced energy consumption and increased energy production. From a tumor biological point of view, it is noteworthy that the hypoxic response through the activation of HIF transcription factors results in phenotypical changes much resembling those of neoplastic progression. In general, solid tumors are hypoxic, defined in the studies by H¨ockel and Vaupel as having an oxygen tension at which cells cannot keep ATP stores intact (corresponding approximately to 1% oxygen). These and other investigators showed that hypoxic tumors are more aggressive when compared to tumors that are better oxygenated [51]. The tumor biological and physiological rationales behind this observation are not fully understood, although several mechanisms leading to an aggressive behavior of hypoxic tumor cells have been unraveled. Hypoxic tumor cells have an increased metastatic capacity, impaired or suboptimal DNA repair mechanisms and enhanced cytotoxic resistance, and increased expression of the key HIF target gene VEGF, which leads to enhanced tumor vascularization. Finally, hypoxic tumor cells appear to dedifferentiate and acquire stem cell-like properties, as demonstrated in neuroblastoma, breast carcinoma and prostate carcinoma cells [52–54]. Given the impact by which hypoxia and HIF activation affect tumor cell aggressiveness, targeting of the hypoxic phenotype in general and HIF-1␣ in particular are currently evaluated novel treatment strategies against solid tumors [55]. 4.2. Neuroblastoma and hypoxia HIF-2␣ is selectively expressed in the developing SNS during discrete time periods [56]. This observation together with data suggesting that hypoxia might drive a neuronal/neuroendocrine lineage shift in some neuroblastomas [4,5], prompted an investigation of the impact of hypoxia on neuroblastoma cell differentiation status in controlled in vitro and in vivo systems [52]. Unexpectedly, it was found that a panel of neuroblastoma cell lines grown at hypoxic (1% oxygen) conditions reduced their expression of a number of neuronal and neuroendocrine marker genes such as neurofilament, NPY, GAP43, and chromogranin A and B [52,57]. Furthermore, SNS lineage specifying transcrip-

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tion factors such as ASCL1 and HAND2 were down-regulated, whereas genes associated with a neural crest phenotype, ID1, ID2, NOTCH1, NOTCH3, HES1, HEY1 and KIT became upregulated [52,57–59]. The conclusion drawn from these data was that hypoxia pushes neuroblastoma cells toward an immature, stem cell-like phenotype ([52], reviewed in [60]), a conclusion supported by the upregulation of the oncogenic TRKAIII variant by hypoxia mimics [31]. The in vitro dedifferentiating effect of oxygen shortage was also observed in vivo, as the expression of differentiation marker genes were downregulated also in hypoxic regions of tumors of human neuroblastoma cells xenotransplanted into nude mice [52]. Based on similar experimental approaches, Hedborg et al. later claimed that hypoxia instead induces chromaffin differentiation in neuroblastoma cells [61]. The distinction between a sympathetic neuroendocrine, chromaffin phenotype on one hand and a sympathetic neuronal phenotype on the other hand is subtle and when these cell types are taken out of their normal developmental context, like in the tumor setting, the distinction between the two has to rely on distinct markers. While there are several reliable sympathetic neuronal markers (e.g. NPY, GAP-43), specific sympathetic extra-adrenal chromaffin/neuroendocrine markers are presently lacking and those in use today (e.g. neurofilaments and chromogranin A and B) are also expressed in sympathetic neuroblasts, albeit at lower levels [4]. Hedborg et al. analyzed a limited number of marker genes, and the only bonafide neuroendocrine marker analyzed, chromogranin A, was not upregulated by hypoxia [61] and the results presented are all in agreement with reported findings [62,52,57,63,64]. The important point, though, is that chromogranin A and B and neurofilaments are highly expressed in chromaffin cells and hence the hypoxia-induced downregulation of these proteins in all tested neuroblastoma cell lines is incompatible with a

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hypoxia-driven enhancement of a chromaffin phenotype. Extensive microarray analyses of hypoxic/normoxic neuroblastoma cells have strengthened this conclusion ([57] and unpublished data (Fredlund, Ovenberger and P˚ahlman)) by identifying novel hypoxia-regulated genes with suggested involvement in the dedifferentiation process. The fact that the in vivo neuronal to neuroendocrine lineage shift observed in a subset of neuroblastoma tumors cannot be recapitulated in in vitro models, suggest that established cell lines with their high-stage tumor origin are not good models for this subset of neuroblastomas with neuroendocrine differentiation capacity. 4.3. Hypoxia-induced dedifferentiation of neuroblastoma cells Severe hypoxia will cause cell death but it has been speculated that tumor stem cells are more resistant to oxygen shortage than the more mature bulk of tumor cells and that hypoxia even might promote stem cell survival. The rationale behind such presumptions is the observation that the bone marrow is largely hypoxic and that hematopoietic stem cells reside and survive in the bone marrow [65]. It is not known if also other types of stem cells have a similar resistance, but if that is the case, the observation that hypoxic (1% oxygen) neuroblastoma cells become more stem cell-like and loose their differentiated characteristics might be explained by a hypoxia-induced selection pressure in favor of putative stem cell pools in these neuroblastoma cultures (Fig. 3A). We find that explanation less likely, as the hypoxic neuroblastoma cells proliferate and limited, if any cell death occurs at this and even lower oxygen tensions. An alternative explanation would be that tumor stem cells indeed are selected and that hypoxia also promotes growth of this subset of cells (Fig. 3B). To experimentally address that possibility, neurob-

Fig. 3. Alternative models to explain the loss of differentiated characteristics seen in hypoxic neuroblastoma cells. In (A) and (B), a stem cell population is favored by hypoxia, without (A) or with (B) net proliferation of that pool of cells. In (C), no selection occurs, and the bulk of tumor cells change towards an immature, stem cell-like phenotype. Available data suggest that a stem cell pool is not selected, thus favoring the alternative in (C).

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lastoma cells were cultured at 1% oxygen for varying lengths of time and during cycles of reoxygenation, and changes in the expression of differentiation markers were monitored [63]. To summarize the outcome of these studies, prolonged exposure to hypoxia (up to 12 days) did not enhance the dedifferentiated phenotype and did not cause severe cell death. Hypoxic cells reverted to their initial phenotype within 3 days when reoxygenated, and when such cultures were brought back to hypoxic conditions, the tumor cells again developed an immature phenotype. We conclude from these experiments that the hypoxic phenotype is reversible and that hypoxia does not promote a selection of immature stem cell-like cells, thus favoring the model presented in Fig. 3C. The molecular mechanisms underlying the hypoxia-driven dedifferentiation are most likely complex, but might recapitulate, in reverse, the molecular steps activated during the differentiation of sympathetic precursor cells into neuroblasts and eventually ganglion cells. Although this process is far from delineated in any detail, some essential genes have been identified. For instance, the tissue specific basic helix–loop–helix (bHLH) transcription factor genes ASCL1 and HAND2 are required for proper development of the SNS [21]. As both these genes are downregulated by hypoxia, the involvement of this differentiation pathway was investigated in some detail. These tissue-specific bHLH factors form active heterodimeric complexes with other bHLH family members, the so-called E-proteins (E12/E47, E22 and HEB). The E-proteins, in turn, can complex with a group of inhibitory proteins, ID1, ID2, ID3 and ID4, which are HLH domain containing factors lacking the basic DNA-binding domain [66]. When ID proteins are complexing with E-proteins, the transcriptionally active heterodimers between E-proteins and ASCL1 or HAND2 are disrupted and transcription of neuron-specific genes decreases. As reviewed in more detail elsewhere [59], hypoxic neuroblastoma cells downregulate ASCL1, HAND2, and E2-2, the preferential partner of HAND2 [62,52,57]. In addition, both ID1 and ID2 expression is upregulated, and at least the increase in ID2 expression is HIF-1␣ driven [58]. All these changes in the expression of these transcription factors will work in concert and promote a less differentiated phenotype. Furthermore, the dedifferentiation process is probably enhanced by the hypoxiainduced activation of the Notch pathway, which could explain the downregulation of ASCL1 (for reviews, see [59]). 5. Concluding remarks It has been known for decades that neuroblastoma cells have the capacity to differentiate, spontaneously in vivo, and in vitro when triggered by various agents and growth factors. Together with the recent observation that hypoxia pushes neuroblastoma cells toward an immature, stem cell-like phenotype, there is an emerging picture of neuroblastoma cells as being truly instable with regard to differentiation stage (Fig. 4 and the article by Ross and Spengler in this issue). It is apparent that the differentiation stage of cultured cells could shift depending on growth conditions, but more importantly, tumor physiological properties such as oxygenation, hypoglycemia, acidity, and stromal/growth factor influences, will contribute to the heterogeneity frequently

Fig. 4. Environmental effects on neuroblastoma (NB) cell differentiation. The differentiation stage at which neuroblastoma cells are arrested can alter depending on environmental factors such as hypoxia and differentiation stimulating agents. The relation between high/low differentiation stage and favorable/unfavorable clinical outcome is indicated.

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