Abstract
2049
Introduction: Hypoxia inducible factor (HIF)-1α has been implicated in the progression of esophageal squamous cell carcinoma (ESCC). However, the genes regulated by hypoxia in esophageal tumors remain elusive. Methods: Gene array experiments were performed using an Affymetrix gene chip (U133+v2.0) upon RNAs representing paired primary ESCC and adjacent normal mucosa (n=5) as well as immortalized human esophageal cells (EPC2-hTERT) exposed to hypoxia or normoxia. Cells were retrovirally transduced with wild-type (WT) or dominant-negative (DN) HIF-1α, whose effects were documented by transfection assays with a luciferase reporter construct fused to an erythropoietin HIF responsive cis-element. Gene expression was determined by real-time RT-PCR, Western blotting, and Immunohistochemistry (IHC). Prostaglandin E synthase (PTGES) was also stably transduced into transformed EPC2-hTERT cell derivative (Genes & Development 2007;21:2788-803). Prostaglandin E2 (PGE2) level was quantitated by enzyme-linked immunosorbent assays (ELISA). Results: Principal component analysis of the gene array data showed a consistent relationship in gene expression patterns between tumor and normal mucosa (56.1% variation). Hypoxic exposure of EPC2-hTERT cells resulted in an induction of 2,225 probe sets (≥ 1.5x), including 37 of 52 (71%) known HIF target genes such as carbonic anhydrase 9 (9.6x), hexokinase 2 (6.8x), and cyclooxygenase (COX)-2 (16.6x). Comparison of the gene array data further extracted 113 probe sets, representing 69 known genes as well as 25 others with unknown functions, as commonly increased (≥ 2.5x) in both primary tumors and hypoxia treated cells. Amongst the robustly upregulated genes (>5x) were COX-2 and PTGES, and their downstream molecules, including interleukin (IL)-8 and chemokine ligand (CXCL)-1. IHC localized overexpression of COX-2 and PTGES in tumor cells while the latter was also detected in stromal inflammatory cells. In culture, COX-2 and PTGES were induced by hypoxia or cobalt chloride, an inhibitor of HIF prolylhydroxylase, and augmented by WT-HIF-1α and suppressed by DN-HIF-1α, implying a role of HIF-1α in their transcriptional activation. Moreover, hypoxia stimulated PGE2 production in a HIF-1α dependent manner while ectopically expressed PTGES further enhanced the PGE2 level. Interestingly, IL-1β was also highly expressed in tumors but only modestly in hypoxia exposed cells. Induction of PGE2, IL-8 and CXCL-1 by recombinant IL-1β was greater in PTGES overexpressing cells. However, IL-1β did not affect the expression of PTGES per se, implying multiple levels of regulation in PGE2 production. Conclusions: Genes involved in prostaglandin E biosynthesis and its downstream molecules are important in the esophageal tumor hypoxic microenvironment as revealed by our experiments. PTGES appears to be a novel HIF-1α regulated gene and may play a role in angiogenesis.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA