Cancer Letters

Cancer Letters

Volume 253, Issue 1, 8 August 2007, Pages 74-88
Cancer Letters

Genome-wide expression analysis using microarray identified complex signaling pathways modulated by hypoxia in nasopharyngeal carcinoma

https://doi.org/10.1016/j.canlet.2007.01.012Get rights and content

Abstract

Previously, we showed that hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase IX (CA IX), and vascular endothelial growth factor (VEGF) were frequently coexpressed in tumor biopsies from patients of nasopharyngeal carcinoma (NPC) and were associated with poor outcome after radiotherapy. Here, we further studied hypoxic induction of HIF-1α, HIF-2α, CA IX, and VEGF in NPC cell lines, investigated hypoxia-modulated gene expression in NPC cell lines by Affymetrix GeneChip Array expression profiling, and identified pathways influenced by hypoxia and novel genes not previously recognized as hypoxia-inducible. Differentially regulated genes under hypoxia were identified genome widely and selected genes validated by RT-PCR. We found that hypoxia induced HIF-1α, CA IX and VEGF expression but not HIF-2α in NPC cells. Microarray expression analysis showed that 222 genes were commonly up-regulated and 137 genes down-regulated in hypoxic-treated CNE-2 and HONE-1 cells. Hypoxia induced broad changes of both up- and down-regulated gene expressions involved in diverse biological processes in NPC cells. Elucidation of the coordinated functions modulated by hypoxia could lead to a better understanding of the clinical significance of the hypoxic tumor phenotype.

Introduction

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck cancer in Southern China and South East Asia. It is endemic in Hong Kong, with an incidence rate of 20.9 per 100,000 men in 2002 and is the sixth leading cause of cancer death in man [1]. This tumor is highly sensitive to radiation and chemotherapy. The current treatment strategy in non-metastatic NPC is a course of radical radiotherapy in early stage disease, and concurrent chemotherapy and radiotherapy in locally advanced stage [2]. Despite an aggressive approach combining both chemotherapy and radiotherapy, about 25–30% of patients still fail with local recurrence and/or distant metastases. The prognosis for patients with recurrence or metastases is poor [3].

Tumor hypoxia is associated with resistance to radiotherapy and chemotherapy, as well as a more malignant tumor phenotype with increased invasiveness, metastases, and poorer survival [4], [5], [6]. The transcriptional complex of hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors [7]. HIF-1 plays a pivotal role in essential adaptive responses to hypoxia and its expression increases exponentially with decreased level of cellular oxygen. Genes that are up-regulated by micro-environmental hypoxia through activation of HIF include erythropoietin, glucose transporters, glycolytic enzymes, and angiogenic factors. Among them, both carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF) are up-regulated by hypoxia via HIF-1-dependent pathways [8], [9], [10].

Over the last ten years, clinical studies have shown that hypoxia is an independent prognostic indicator of poor patient survival in different tumor types [5], [11]. Because this observation also holds true for surgically treated patients [6], it suggests that there are fundamental biological differences between hypoxic and non-hypoxic tumor cells. A major mechanism by which hypoxia confers its effect is by differential regulation of gene expression. In our previous clinical study in NPC, we found that the markers HIF-1α, CA IX and VEGF were frequently over expressed in about 60% of patients’ tumor samples. We found that tumors with a hypoxic phenotype, as defined by the coexpression of HIF-1α and CA IX, were associated with poor survival. Furthermore, tumors with both hypoxic and angiogenic phenotype (defined by the overexpression of VEGF) had the worst outcome after radiotherapy [12]. In the current study, we further investigated the expression of these hypoxic markers at both the mRNA and protein levels in NPC cell lines. We also investigated the global differential gene expression in normoxic and hypoxic-treated NPC cells by Affymetrix GeneChip Array expression analysis. We demonstrated that hypoxia induced coordinated up- and down-regulation of a broad range of genes involved in distinct biological pathways. We investigated genes not previously reported that may be of special relevance in NPC, since there is known variation in the global transcriptional response to hypoxia among different cell types [13]. Recognition of the pattern of the differential gene expressions in the hypoxic cells will lead to a better understanding of the hypoxic tumor phenotype in the clinical settings.

Section snippets

Culture of tumor cell lines and hypoxia treatment

Four nasopharyngeal carcinoma (NPC) cell lines (CNE-2, C666-1, HONE-1 and HK1) were employed. The CNE-2 cell line [14] and HONE-1 cell line [15] were established from poorly differentiated nasopharyngeal carcinomas. C666-1 was established from undifferentiated nasopharyngeal carcinoma and consistently carried the Epstein-Barr virus in long-term cultures [16]. The HK1 cell line was established from a well-differentiated squamous nasopharyngeal carcinoma [17]. CNE-2 and HONE-1 cells were cultured

Expression of cellular HIF-1α, HIF-2α, CA IX proteins in NPC cells

The effects of hypoxia on the protein expression of some known hypoxia markers including HIF-1α, HIF-2α, and CA IX were examined in four NPC cell lines. After exposure to hypoxic conditions for 16 h, cellular proteins were subjected to Western blotting analysis. As shown in Fig. 1, HIF-1α protein levels were significantly increased after hypoxia treatment in all cell lines examined. It was also observed that the HIF-1α protein from hypoxic NPC cells has lower electromobility than that from

Discussion

In this work, we have shown that the protein level of HIF-1α was induced but in contrast, hypoxia did not induce significant change in the protein level of HIF-2α in the four NPC cell lines. Hypoxia incubation also significantly increased protein level of CA IX and VEGF in 3 of 4 NPC cell lines. Our findings in NPC are consistent with the known hypoxia induction of HIF-1α, CA IX and VEGF protein in other tumor types reported in the literature [8], [9], [10]. The lack of hypoxic induction of

Acknowledgements

This study was supported by a grant from the Research Grants Council of Hong Kong Special Administrative Region (CUHK 4093/02M). We thank Dr Verena Jendrossek (Department of Radiation Oncology, University of Tuebingen, Germany) for providing the original dataset from reference [30] in the comparative analysis.

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    These authors contributed equally to this work.

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