Human neuroblastoma cells exposed to hypoxia: induction of genes associated with growth, survival, and aggressive behavior

https://doi.org/10.1016/j.yexcr.2004.01.013Get rights and content

Abstract

We have recently found that cells derived from human neuroblastoma, a sympathetic nervous system (SNS) tumor, dedifferentiate and acquire a neural crest-like phenotype when exposed to hypoxia. In the present study, global analysis of gene expression and quantitative PCR of relevant genes showed that hypoxia provokes a general adaptive response in neuroblastoma cells and confirm loss of the neuronal phenotype and gain of stem-cell characteristics. Of the approximately 17,000 genes and ESTs analyzed, 199 were consistently upregulated and 36 were downregulated more than 2-fold by hypoxia. As anticipated, several genes involved in glucose and iron metabolism and neovascularization were upregulated, the latter group we here show to include the gene encoding chromogranin C and its cleavage product, secretoneurin, a vascular smooth muscle cell mitogen. We also observed upregulation of genes implicated in cell survival and growth, such as vascular endothelial growth factor (VEGF), neuropilin 1, adrenomedullin, and IGF-2. Several metallothioneins, which are linked to tumor drug resistance, were upregulated, whereas the expression of MDR1 decreased. In hypoxic neuroblastoma cells, proneuronal lineage specifying transcription factors, and their dimerization partner E2-2, were downregulated, whereas their inhibitors Id2 and HES-1 were induced, providing a molecular mechanism for the hypoxia-provoked dedifferentiation of neuroblastoma cells.

Introduction

To be able to grow larger than a few millimeters in diameter, solid tumors need to induce neovascularization to ensure sufficient supply of oxygen and nutrients. However, tumor-induced vessels are often malformed, resulting in inadequate blood circulation and accompanying hypoxia [1], [2]. Hypoxic tumor cells are more resistant to therapy since they have a lower proliferation rate, are poorly exposed to cytotoxic drugs delivered via the blood, and are less sensitive to radiation therapy [1], [3], [4]. Furthermore, compared to tumors with a low degree of oxygen deficiency, those that exhibit extensive hypoxia are more prone to metastasize and to accumulate mutations [3], [4], [5]. In addition to these unfavorable features, we recently showed that hypoxic neuroblastoma cells attain a less mature phenotype [6], which is a characteristic that is associated with an adverse clinical stage in patients [7], [8], [9]. This phenomenon is not restricted to neuroblastoma cells, since hypoxia also induces dedifferentiation and expression of stem-cell features in breast cancer, both in tumor specimens and in cultured cells [10].

Neuroblastoma is a childhood malignancy that is derived from the developing sympathetic nervous system (SNS) and is often diagnosed during early infancy. The tumor cells retain many characteristics of the immature cells that establish the sympathetic nervous system [11], [12], [13]. Neuroblastomas exhibit significant variation in stage of differentiation, and immature lesions are generally considered to be more aggressive [7], [8], [9]. In neuroblastoma cells, exposure to hypoxia decreases the expression of several neuronal and neuroendocrine marker genes, including chromogranin A (CHGA), chromogranin B (CHGB), NPY, dHAND, and HASH-1, and concomitantly, expression of genes associated with early neural crest development such as Id2, Notch-1, HES-1, and c-kit are upregulated [6].

Adaptation of tumor cells to growth under hypoxic conditions has been attributed primarily to the accumulation of hypoxia inducible factors (HIFs) HIF-1α and HIF-2α [14], [15], [16]. These proteins form transcriptionally active complexes with the ARNT proteins, and induce expression of many genes associated with the ability of cells to survive under hypoxic conditions and induce neovascularization, such as glucose transporter 3 (GLUT3) and vascular endothelial growth factor (VEGF) [14], [15], [16], [17], [18], [19], [20]. The level of oxygenation in different tissues and organs also appears to have a substantial impact on embryonic development and low oxygen pressure has been shown to affect differentiation processes in several types of cells. For instance, rat primary neural crest cells can be induced to adopt a neuronal cell fate in vitro in 5% oxygen, although this was not possible to achieve in 21% oxygen [21], whereas hypoxia block adipocyte differentiation [22]. In the present experiments, our objective was to extend the phenotypic analysis of long-term-treated hypoxic neuroblastoma cells by using high-density cDNA microarrays in a large-scale gene expression analysis. The microarray results were employed to guide further gene expression analysis in seven different neuroblastoma cell lines exposed to normoxic and hypoxic conditions.

Section snippets

Cell maintenance and exposure to hypoxia

The SK-N-BE(2) and SH-SY5Y cells were maintained in Minimum Essential Medium with added Earle's salts and l-gluthamine, and the SK-N-F1, IMR-32, LA-N-2, LA-N-5, and SK-N-RA cells were kept in RPMI 1640 medium. The media were supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml; Invitrogen), and the cells were maintained at 37°C in 5% CO2 and 95% air. For the hypoxia experiments, the human neuroblastoma cells were sparsely seeded to avoid confluency (0.8 to

Changes in gene expression in hypoxic neuroblastoma cells

We used microarray hybridization technique to explore the gene expression profile of SK-N-BE(2) neuroblastoma cells grown for 72 h in 1% oxygen (hypoxia) because we had previously found that such conditions lead to HIF-1α and HIF-2α protein stabilization [6]. When analyzing the microarray hybridizations, we normalized the results by assuming that the total gene expression is essentially the same in hypoxic and normoxic cells, and this assumption was supported by data showing that several

Discussion

Global gene expression analysis showed consistent induction of about 200 genes illustrating that hypoxia has a broad, but restricted, impact on the neuroblastoma cell phenotype, which is in accordance with the many cell functions already known to be affected by hypoxia. We also observed hypoxia-provoked repression of many genes, several of which are associated with a neuronal/neuroendocrine phenotype, which supports the conclusion that shortage of oxygen leads to a less differentiated tumor

Acknowledgements

We thank Ms. Siv Beckman and Ms. Elise Nilsson for skillful technical assistance. This work was supported by grants from the Swedish Cancer Society, the Children's Cancer Foundation of Sweden, HKH Kronprinsessan Lovisas Förening för Barnasjukvård, Hans von Kantzows Stiftelse, American Cancer Society, and research funds administered by Malmö and Lund University Hospitals.

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