Epidermal Growth Factor-activated Aryl Hydrocarbon Receptor Nuclear Translocator/HIF-1β Signal Pathway Up-regulates Cyclooxygenase-2 Gene Expression Associated with Squamous Cell Carcinoma

Abstract

Hypoxia-inducible factor (HIF) accumulates when tumors grow under hypoxic conditions. The genesis of tumors, however, usually involves normoxic conditions. In this study, we were interested in examining the potential role of aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1␤ in tumor growth under normoxic conditions, specifically when cells are treated with epidermal growth factor (EGF), which is known to affect the gene expression of tumor growth-related protein COX-2 (cyclooxygenase-2). The results showed that EGF receptor inhibitor, AG1478, abolished EGF-induced nuclear accumulation of ARNT as well as the expression of COX-2. ARNT small interfering RNA inhibited the promoter activity, mRNA level, and protein expression of COX-2 in cells treated with EGF. In contrast, CoCl 2 -induced HIF-1␣ exhibited no effect on COX-2 expression. EGF also stimulated the formation of the ARNT⅐c-Jun complex as well as the complex binding to the COX-2 promoter. ARNT small interfering RNAs blocked EGF-activated cell migration. Moreover, COX-2 and ARNT were cohorts present distinctively in clinical specimens of human cervical squamous cell carcinoma and were almost nondetectable in adjacent normal or noncancerous cervical tissues. Our results revealed that ARNT plays an important role in EGF-regulated COX-2 gene expression and may thus be related to either a cause or a consequence of tumorigenesis in cervical cancer.The aryl hydrocarbon receptor nuclear translocator (ARNT) 3 (also known as HIF-1␤ (hypoxia-inducible factor-1␤)) is a member of the basic helix-loop-helix Per/aryl hydrocarbon receptor (AhR)/ARNT/Sim (bHLH-PAS) family of transcription factors and a general partner factor for bHLH-PAS proteins such as the AhR, HIF-1␣ (hypoxia-inducible factor-1␣), and single-minded (SIM) proteins (1). Although the in vivo importance of ARNT homodimers remains unclear, each of the heterodimeric ARNT-containing complexes plays a critical function in pathways used to respond to environmental conditions. As in exposure to xenobiotic compounds, the AhR/ ARNT heterodimer recognizes and promotes transcription from xenobiotic response elements found upstream of 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive genes (1). ARNT also cooperates with HIF-1␣ to form a heterodimer and regulates several genes involved in tumorigenesis under hypoxia (2). In addition, ARNT is essential for normal embryonic development (3). Loss of ARNT results in reduced tumor growth, decreased angiogenesis, and increased response to radiotherapy (4, 5). Inactivation of ARNT suppresses the development of liver hemangioma, polycythemia, and HIF-induced gene expression. Unlike ARNT, however, HIF-1␣ is insufficient to suppress the development of the Von Hippel-Lindau-associated polychthemia (6). These results demonstrate that the development of Von Hippel-Lindau-associated vascular tumors in the liver depends on functional ARNT but not in an HIF-1␣/hypoxia-dependent manner. Prostaglandin endoperoxide synthase, also known as cyclooxygenase, plays a regulatory role in...

Expression of Aryl Hydrocarbon Receptor Nuclear Translocator Enhances Cisplatin Resistance by Upregulating MDR1 Expression in Cancer Cells

“…Under hypoxic conditions, HIF-1a/ARNT dimers activate the transcription of numerous target genes whose the promoters contain a binding motif termed the hypoxia-response element (Semenza, 2001). Our previous studies indicated that ARNT regulates the transcriptional activation of genes that do not contain ARNTbinding sites, such as COX-2, 12(S)-lipoxygenase, and p21 WAF1/CIP1 (Chang et al, 2009;Huang et al, 2010). Herein, we provided evidence to show that ARNT indeed regulates gene expression in a hypoxia response element-independent manner.…”

“…Transient transfection of cells with plasmids was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions but with slight modification. The luciferase activity in cell lysate was determined as described previously (Chang et al, 2009).…”

“…Quantitation of the change in ARNT and Sp1 binding to the MDR1 promoter element was achieved by DNA affinity precipitation assay according to the method reported previously (Chang et al, 2009). In brief, 59-biotinylated oligonucleotides corresponding to the sense (256 to 246 bp and 1258 to 1269 bp) and antisense strands of the MDR1 promoter element were annealed.…”

“…It serves as a dimerization partner for a number of other transcription factors, such as the AhR, HIF-1a, single-minded, and c-Jun proteins (Kewley et al, 2004;Chang et al, 2009). Previous studies suggested that ARNT is a new regulatory factor that interacts with c-Jun and bridges the binding of c-Jun with Sp1 to regulate epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2), 12(S)-lipoxygenase, and p21 WAF1/CIP1 gene expression under normoxic conditions, thus contributing to tumorigenesis (Chang et al, 2009;Huang et al, 2010). In addition, ARNT is essential for normal embryonic development (Kozak et al, 1997).…”

“…Among the ABC transporters, P-glycoprotein/MDR1 mediates resistance to a wide variety of anticancer agents, and the regulation of MDR1 expression might contribute to the development of appropriate agents to combat MDR and improve chemotherapeutic efficacies (Liu et al, 2007). Because tumorigenesis usually involves normoxic conditions, and given that ARNT is important for gene expression networks in cervical carcinoma and oral cavity squamous cell carcinoma (Gu et al, 2000;Chang et al, 2009;Huang et al, 2010), we propose that ARNT not only plays a role in regulating tumorigenesis but also contributes to drug resistance. Indeed, our data revealed that ARNT-regulated expression of MDR1 confers cisplatin resistance, which is associated with chemotherapeutic efficacy.…”