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Proc Natl Acad Sci U S A. May 14, 2002; 99(10): 7021–7026.
PMCID: PMC124521
Medical Sciences

Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype

Abstract

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1α and HIF-2α are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2α in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1α and HIF-2α proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

Solid tumors often have areas in which circulation is compromised because of structurally disorganized blood vessels and tumor cells that grow faster than the developing tumor capillary network (1). The poor circulation results in selection of tumor cells that can grow or survive under conditions of hypoxia, poor nutrient supply, low pH, and high intratumor pressure (2). This microenvironment has prognostic implications, because cells in hypoxic areas are less vulnerable to ionizing radiation and cytotoxic drugs, and tumors with substantial hypoxia metastasize more efficiently (3, 4). In an adaptive response to hypoxic conditions, cells alter their gene expression program, primarily by action of the hypoxia-inducible factors (HIFs) 1α and HIF-2α (the latter also designated EPAS-1) (58). Hypoxia stabilizes these two transcription factors against degradation (912) and thereby induces expression of several target genes involved in maintaining homeostasis, for instance vascular endothelial growth factor (VEGF) and erythropoietin (5, 13, 14). HIF-1α and HIF-2α are essential for embryonic survival and proper vascularization (7, 1416). HIF-2α is also necessary for the developing sympathetic nervous system (SNS) and shows a transient embryonic expression pattern confined to sympathetic ganglia and paraganglia (7). HIF-2α-deficient mice have been reported to lack sympathetic tyrosine hydroxylase (TH) expression and die of catecholamine shortage (7). The SNS is neural crest-derived and is composed of two major cell types, neurons and neuroendocrine (chromaffin) cells (17), the latter forming the adrenal medulla and the paraganglia. In the developing embryo and fetus, paraganglia are the main source of catecholamines that regulate heart rate and blood pressure (18).

Neuroblastoma (NB) is a childhood malignancy originating from the developing SNS (1921). The tumor cells vary regarding differentiation stage, with immature cells forming more aggressive tumors (22). Most NBs exhibit characteristics of immature sympathetic neuroblasts, often with remaining neural crest traits (19, 21, 23). A phenotypically distinct subset of NBs contains a mixture of neuroblastic and neuroendocrine cell types that are organized in lobular structures with a central necrotic zone (19). A spontaneous neuronal-to-neuroendocrine lineage shift occurs toward the necrotic zone (20). Therefore, we recently hypothesized that low oxygen levels and compromised nutrient supply may cause spontaneous neuroendocrine differentiation (24). Here, we examined whether oxygen deprivation affects the differentiation status of cultured human NB cells. Unexpectedly, we found that hypoxic cells down-regulated SNS marker genes, including the lineage-specific transcription factors HASH-1 and dHAND. The cells further up-regulated genes expressed during early neural crest development, hence the hypoxic cells seemed to adopt an immature, neural crest-like phenotype.

Materials and Methods

Cell Culture.

Cells were maintained as monolayers in standard media with 10% FCS at 37°C in 5% CO2 and 95% air. Hypoxic conditions were created by flushing 5% CO2 and 95% N2 through a humidified chamber at 37°C, until an atmosphere containing 1% or 5% O2 was achieved, and measured with a MiniOX1 oxygen meter (Mine Safety Appliances Company, Pittsburgh). Anoxia (0% O2, 5–10% CO2 and 90–95% N2) was established in an anaerobic workstation (Electrotek, Keighleg, U.K.). Culture media were preequilibrated at the indicated oxygen levels.

Immunoblot Analysis.

Cells were lysed in 1% Nonidet P-40, 10% glycerol, 20 mM Tris[center dot]HCl (pH 8.0), 137 mM NaCl, and Complete protease inhibitor mixture (Roche Molecular Biochemicals). Western blotting was performed with 60 μg of total protein per lane. The following primary Abs were used: HIF-2α, affinity-purified rabbit antiserum directed against amino acids 138–152 of human HIF-2α (custom-made by Innovagen, Lund, Sweden); HIF-1α, mAb (Novus Biologicals, Littleton, CO); TH, mAb (Roche Molecular Biochemicals); chromogranin, rabbit antiserum (Euro-Diagnostica, Malmö, Sweden); and Notch-1, goat polyclonal IgG (Santa Cruz Biotechnology). Super Signal substrate (Pierce) was used for chemiluminescence detection of the secondary Abs.

Northern Blot Hybridizations and Semiquantitative Reverse Transcription (RT)-PCR.

Total RNA was prepared with Trizol reagent (Life Technologies, Rockville, MD), and 15 μg of total RNA per lane was analyzed with probes as described (23, 25, 26). The inhibitor of DNA binding 2 (Id2) probe covered nucleotides 111–515. Expression of c-kit was analyzed by RT-PCR with β2-microglobulin as an internal loading control as described (27). Total RNA was treated with DNase I (Appligene, Strasbourg, France), followed by cDNA synthesis and PCR amplification according to standard procedures (denaturation 30 s at 95°C; annealing 30 s at 55°C; extension 1 min at 72°C). The PCR products were analyzed by electrophoresis, stained with SYBR Green I (Roche Diagnostics), and evaluated with LAS-1000 CCD imaging equipment (Fuji).

Experimental Tumors, Immunohistochemistry, and in Situ Hybridization.

SK-N-BE(2) cells were injected s.c. into female athymic mice of the NMRI strain (nu/nu) as described (28). All procedures were approved by the regional ethical committee for animal research (Dnr.M101-99). Tumors were recovered, fixed in diethyl pyrocarbonate-treated 4% buffered formaldehyde, and embedded in paraffin. Immunohistochemistry and in situ hybridization were done as described (19, 20, 23).

Results

HIF-2α Expression in Embryonal Mouse Paraganglia.

Of the two published HIF-2α gene targeting studies, only one reports an effect on the SNS (7, 16). Therefore, we examined abdominal sections of embryonic day (E) 14.5 embryonic mice for HIF-2α expression in the SNS. In situ hybridization showed distinct HIF-2α expression in paraganglia cells of the organ of Zuckerkandl, identified morphologically and by high TH levels (Fig. (Fig.1).1). At this developmental stage, the adjacent sympathetic ganglion, which weakly expressed TH (Fig. (Fig.1)1) and GAP-43 (not shown), did not express HIF-2α in accordance with published data (7).

Figure 1
Expression of TH and HIF-2α in mouse embryonal (E14.5) paraganglion (PG) cells of the organ of Zuckerkandl. TH expression is strong in PG cells and weak in the neuroblasts of the adjacent sympathetic ganglion (SG) (Upper). HIF-2α is expressed ...

HIF-1α and HIF-2α Are Accumulated in Hypoxic NB Cells.

We subsequently studied the impact of 4 h of exposure to normoxic (21% O2) and hypoxic (5%, 1%, and 0% O2) conditions on stabilization of HIF-1α and HIF-2α protein in cultured human NB cells with HeLa cells as a positive control (29). Considering the extensive homology between HIF-1α and HIF-2α, anti-HIF Ab specificities were verified with HIF proteins translated in vitro (Fig. (Fig.22A). Hypoxic stress resulted in accumulation of HIF-1α in all five analyzed NB cell lines, whereas HIF-2α was detectable in three cell lines (Fig. (Fig.22A). Neither HIF-1α nor HIF-2α was detected in cells grown under normoxic conditions or at 5% oxygen, which approximates tissue levels (30). There was no correlation between the extent of hypoxia-induced accumulation of HIF-1α and HIF-2α, as exemplified by comparison of IMR-32 and SH-SY5Y cells (Fig. (Fig.22A). At 1% oxygen, all tested cells survived for at least 72 h with no apparent effect on cell morphology or increase in cell death (not shown). Lower oxygen levels (approaching 0%) had no effect on cell morphology after 4 h, but most of the cells died within 72 h. Therefore, subsequent studies were performed at 1% oxygen.

Figure 2
Hypoxia-induced gene expression in oxygen-deprived NB cells. (A) Accumulation of HIF-1α and HIF-2α in NB cells in response to oxygen deprivation. Human NB cells were exposed to the indicated oxygen levels for 4 h and subjected to Western ...

Expression of TH and Other Known Hypoxia-Inducible Genes.

Most NB tumors and cell lines produce catecholamines, hence they also express TH. One function of the SNS is to produce and excrete noradrenaline and adrenaline into the blood stream to regulate heart rate and blood pressure, for example in response to low oxygenation, and it is well established that low oxygen levels can induce TH expression (31). In six of seven NB cell lines, TH expression was induced after growth in 1% oxygen for 72 h (Fig. (Fig.22B). Both normoxic and hypoxic TH levels varied considerably between the cell lines, probably reflecting that these tumor cells are arrested at different stages of maturation or that expression levels are low because of a mixed cholinergic/noradrenergic phenotype, as exemplified by the LA-N-2 cells. The sympathetic catecholaminergic lineage specificity of hypoxia-dependent TH accumulation was demonstrated by the lack of TH expression in HeLa and SK-N-MC neuroepithelioma cells, irrespective of oxygen levels (Fig. (Fig.22B).

We also analyzed VEGF expression as part of a more detailed characterization of the hypoxic NB phenotype. At 1% oxygen, all four tested cell lines showed pronounced up-regulation of VEGF within 4 h, and hypoxia-induced expression of this gene persisted 3 days later (Fig. (Fig.22C). Initially, VEGF expression was not detected in cells grown under normoxic conditions, except for weak expression in KCN-69n cells (Fig. (Fig.22C, 4-h time points), but after 72 h, VEGF was expressed in some of the cell lines, showing that VEGF can be transcribed in a nonhypoxic environment. We analyzed the expression of two additional hypoxia-induced genes: insulin-like growth factor 2 (IGF-2) (32) and the glycolytic enzyme and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (33). IGF-2 was not expressed in NB cells exposed to normoxia or hypoxia for 4 h but it was distinctly expressed in three of four cell lines after 3 days of hypoxia, and GAPDH expression was augmented in all NB cell lines grown in 1% oxygen, although with some variation in the expression kinetics (Fig. (Fig.22C).

Down-Regulation of SNS Marker Genes in Hypoxic NB Cells.

Although elevated expression levels of TH and IGF-2 are known indicators of a hypoxic phenotype, such a rise can also be the result of induced sympathetic neuroendocrine differentiation (19). Therefore, we examined the effects of oxygen deprivation on the differentiation status of NB cells by investigating the expression patterns of the neuroendocrine chromaffin marker protein, chromogranin, and the sympathetic neuronal peptide neurotransmitter gene, neuropeptide tyrosine (NPY) (19, 20). All five tested NB cell lines expressed chromogranin, and under hypoxic conditions the level decreased in four of these (Fig. (Fig.33A). In the fifth cell line, SH-SY5Y, hypoxia did not alter the chromogranin level. The same samples were analyzed for TH expression, confirming increased TH levels in the hypoxic cells. NPY mRNA was abundant in SK-N-BE(2), KCN-69n, and SH-SY5Y cells but absent in IMR-32 cells (Fig. (Fig.33B). Under hypoxic conditions, NPY was down-regulated within 4 h in SK-N-BE(2) and KCN-69n cells, and the differences between hypoxic and control samples were more pronounced after 3 days. In contrast, hypoxia did not down-regulate NPY in SH-SY5Y cells (Fig. (Fig.33B), which is noteworthy because these cells differ from the other cell lines in at least one important aspect: N-myc is not amplified in SH-SY5Y cells (25). We therefore tested two additional NB cell lines without N-myc amplification, SK-N-F1 and SK-N-RA. TH expression increased at 1% oxygen, whereas chromogranin and NPY levels decreased (Fig. (Fig.33 A and C), suggesting that the weak response to hypoxia in SH-SY5Y cells was not associated with the N-myc expression status.

Figure 3
Down-regulation of SNS marker genes and induced expression of neural crest genes in hypoxic NB cells. (A) Western blot analysis of chromogranin and TH in NB cells exposed to 21% or 1% oxygen for 72 h. (B and C) Northern blot analysis of ...

The chromogranin and NPY expression data suggested that, compared with cells grown at normoxic conditions, hypoxic NB cells acquire a less mature phenotype. Therefore, we investigated the expression patterns of HASH-1, dHAND, and N-myc, three transcription factor genes involved in early sympathetic lineage specification and development (23, 34). Hypoxia caused down-regulation of HASH-1 and dHAND, but SH-SY5Y cells were again an exception (Fig. (Fig.33 B and C). N-myc is expressed during neural crest cell migration and early phases of ganglionic neural crest differentiation (34), and human embryonal sympathetic ganglia express N-myc at least up to week 8.5 (S.P., unpublished observation). In hypoxic NB cells with N-myc amplification, N-myc expression was distinctly down-regulated after 72 h (Fig. (Fig.33B).

Hypoxia-Induced Expression of Neural Crest Genes.

Because our results suggested that hypoxia-treated NB cells lose their sympathetic ganglionic phenotype, we examined the effects of hypoxia on expression of neural crest genes. Id2, Notch-1, HES-1, and c-kit are involved in determination of neural crest cell fate (3538) and are expressed in sympathetic precursor cells at earlier developmental stages than, for instance, N-myc, HASH-1, and dHAND (21). We observed Id2 expression in all four tested cell lines, and this expression decreased with time in culture. Hypoxia led to an increased Id2 expression after both 4 and 72 h (Fig. (Fig.33B). Expression of c-kit was detected in only two cell lines at normoxia but occurred in all four tested NB cell lines after growth at low oxygen levels (Fig. (Fig.33E). Hypoxia also increased Notch-1 levels (Fig. (Fig.33D), whereas HES-1 protein was detected in three cell lines under normoxic conditions, and hypoxia increased the levels in two of these cell lines (not shown).

In Vivo Gene Expression Patterns in Human NB Xenografts.

To study the effects of hypoxia on gene expression in solid tumors in vivo, we mimicked such conditions by growing SK-N-BE(2) cells as xenografts in nude mice (28). Viable cells surrounding necrotic areas in the tumors exhibited high VEGF expression, whereas most cells in other parts of the tumors were VEGF-negative (Fig. (Fig.44 BD and not shown). The VEGF-positive cells also expressed high levels of TH and IGF-2, but low levels of chromogranin (not shown). Furthermore, expression of the neuronal differentiation marker genes dHAND and GAP-43 was low in cells expressing VEGF, IGF-2, and TH, as compared with the expression in nonnecrotic, well vascularized areas of the tumor (Figs. (Figs.44 and and5).5). In summary, hypoxic SK-N-BE(2) cells seem to develop essentially the same immature phenotype, regardless of whether they are grown in experimental tumors or under hypoxic conditions in vitro. To test whether the hypoxia-induced phenotypical changes render SK-N-BE(2) cells other growth properties in vivo, these cells were cultured in vitro under normoxic or hypoxic conditions for 3 days before injection into nude mice. The hypoxia-treated cells tended to form palpable tumors earlier than control cells (Fig. (Fig.66A). In addition, the tumors generated from hypoxia-pretreated cells appeared to grow faster and become slightly larger in a shorter time than the corresponding tumors from control cells (Fig. (Fig.66 A and B).

Figure 4
Marker gene expression in human SK-N-BE(2) NB cells grown for 3 weeks as xenograft tumors in nude mice. (A) Hematoxylin/eosin (H&E)-stained section of a tumor, indicating areas analyzed in the experiments illustrated in this figure and ...
Figure 5
Neuronal marker gene expression in hypoxic and vascularized regions of an NB xenograft tumor. Analysis of tumor sections taken adjacent to those analyzed in Fig. Fig.4.4. In situ hybridization of GAP-43 (AD) and dHAND (EH) in ...
Figure 6
(A and B) Formation of tumors in nude mice after injection of SK-N-BE(2) cells precultured for 72 h at 1% or 21% O2. The animals were killed when the tumors reached a diameter of 15 mm. (A) Mean time until tumor take (open bars) and termination ...

Discussion

The observations that NB tumors display distinct differences in stages of cell maturation, and that there is a strong correlation between tumor cell differentiation stage and prognosis, have provided an important basis for the concept of tumor cell differentiation. We have found that spontaneous neuroendocrine differentiation can occur in tumor areas with poor oxygenation in a subset of NBs (20, 24). Given the apparent requirement of HIF-2α for proper SNS development and the expression of HIF-2α in SNS cells during embryogenesis (7), these observations prompted us to determine whether hypoxia influences the differentiation status of NB cells. We found that all hypoxic NB cell lines accumulated HIF-1α but not all accumulated HIF-2α, which may reflect that these cell lines are arrested at different stages of maturation, given the temporal SNS expression of HIF-2α during normal development. The hypoxic NB cells increased the expression of TH and other hypoxia-inducible genes, such as VEGF, IGF-2, and GAPDH. Concurrent with the induction of these genes, hypoxic NB cells down-regulated neuronal and neuroendocrine marker genes and up-regulated genes expressed during normal development of the neural crest (Fig. (Fig.66C). Furthermore, xenografted hypoxia-pretreated cells tended to form tumors earlier and grow slightly faster than grafted control cells. Thus, over a period of days, hypoxia induces complex changes in the gene expression pattern both in vitro and in vivo, which strongly suggest that dedifferentiation and acquisition of a neural crest-like phenotype in NB cells is an overall effect of growth at low oxygen levels. The initiating molecular event leading to dedifferentiation has not been explored, but the hypoxia-induced expression of Id2, Notch-1, and HES-1 will contribute to development of a nonneuronal phenotype. Id2 would act by sequestering E proteins, thereby preventing the proneuronal effect of HASH-1 and dHAND and Notch-1/HES-1 by inhibition of HASH-1 expression (36, 37). Thus, these changes in expression will act in concert and counteract a neuronal phenotype.

The tested NB cell lines showed no induced neuroendocrine or neuronal differentiation at 1% oxygen. Indeed, oxygen pressure can be important for SNS precursor cell development, and it was recently shown that growth of primary neural crest cells at 5% oxygen, which resembles physiological levels, promotes sympathoadrenal differentiation (30). At this oxygen level there was no accumulation of either HIF-1α or HIF-2α in the NB cells (Fig. (Fig.22A), suggesting that major hypoxia-driven changes in transcription were not activated under this condition. SH-SY5Y cells, which have a normal N-myc copy number, seemed resistant to hypoxia-induced decrease in neuronal/neuroendocrine marker gene expression. This lack of response could not be attributed to low N-myc expression as two other non-N-myc-amplified NB cell lines did down-regulate these marker genes. Our data could suggest that SH-SY5Y cells are phenotypically closer to differentiating NB cells found in tumors exhibiting a spontaneous neuronal-to-neuroendocrine lineage shift in hypoxic regions (20, 24). Such tumors are usually of low clinical stage, whereas virtually all NB cell lines are derived from high-stage tumors. Consequently, cell line studies may be inappropriate for elucidating the putative capacity of low-stage tumor cells to undergo neuroendocrine differentiation in response to hypoxia.

To our knowledge, no investigations have addressed the question of whether cells in hypoxic regions of aggressive solid tumors are generally less mature than oxygenated cells. However, it has been reported that hypoxia leads to up-regulation of telomerase activity (39), which is a characteristic sign of dividing immature progenitor cells. Moreover, the level of HIF-1α protein in breast carcinomas, presumably reflecting hypoxic conditions, is higher in poorly differentiated than in well differentiated lesions (40). Tumor aggressiveness and tumor cell differentiation stage are clearly correlated in NBs, because, when highly malignant (clinical stages 3 and 4), these tumors express low levels of neuronal differentiation marker genes (22). In light of the data presented here, it will be important to examine whether there is a reciprocal correlation between neuronal marker gene expression and expression of neural crest genes in high-stage tumors. Clinically, NB aggressiveness depends on the extent to which they metastasize (41). Accordingly, the metastatic phenotype of high-stage NBs might reflect that these cells have features that mimic the high migratory capacity of neural crest-derived progenitor cells. Therefore, we suggest that the hypoxia-induced shift toward a neural crest-like phenotype reported here results in more aggressive tumor cells with increased potential to metastasize (Fig. (Fig.6).6). This suggestion is in accordance with recent investigations demonstrating that cells in hypoxic tumors (other than NBs) show an increased tendency toward metastatic spread (3, 4). Interestingly, and consistent with our findings, a correlation has been found between congenital heart disease with cyanosis in infants and occurrence of NB (42).

Hypoxia-induced cell heterogeneity may also affect tumor cell selection mechanisms. The subpopulation of treatment-resistant cells in solid tumors includes hypoxic cells, which, in poorly vascularized and hypoxic regions, can elude treatment because they are inaccessible to cytostatic drugs, and irradiation-induced free-radical production is low because of low oxygen levels. Moreover, the mutation rate has been shown to increase in tumor cells in hypoxic regions (43). Many of the genes expressed in hypoxic NB cells determine stem cell features, such as self-renewal, survival, and migration, and these genes are also implicated in the growth and spread of aggressive cancers. If mutations or rearrangements occur in such genes and lead to constitutive active proteins, the affected cells may acquire a growth and/or survival advantage that improves their ability to withstand selection pressure.

Together, our results show that low oxygen tension in NB cells leads to dedifferentiation and an immature neural crest-like phenotype. We hypothesize that dedifferentiation is a general phenomenon in solid tumors, and that a low oxygen level, which is known to increase mutation frequency and promote metastatic spread, contributes to selection of immature, highly malignant tumor cells with stem cell characteristics. This phenomenon would define a novel mechanism by which hypoxia contributes to the malignant progression of solid tumors.

Acknowledgments

We thank Dr. Kristian Riesbeck for valuable help and Ms. Carolin Jönsson for skillful technical assistance. This work was supported by grants from the Swedish Cancer Society, the Children's Cancer Foundation of Sweden, the Swedish Foundation for Strategic Research, HKH Kronprinsessan Lovisas Förening för Barnasjukvård, Hans von Kantzows Stiftelse, and by the research funds of Malmö and Lund University Hospitals.

Abbreviations

HIF
hypoxia-inducible factor
NB
neuroblastoma
SNS
sympathetic nervous system

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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