Abstract
Cyclic hypoxia in tumors originates from heterogeneities in RBC flux and influences not only tumor cells but also endothelial cells lining tumor blood vessels. Whether pO(2) fluctuations, particularly transient reoxygenation periods, alter the well-known hypoxia-inducible factor (HIF)-dependent gene program is largely unknown. We compared the transcriptomic profiles of endothelial and tumor cells exposed to cyclic hypoxia versus continuous hypoxia to uncover a possible differential effect on angiogenesis and metastases. Microarray analyses identified early genes that were selectively induced by cyclic hypoxia in endothelial cells. Among them, we focused on PTGS2 because the observed increase in mRNA expression led to a significant increase in the expression and activity of cyclooxygenase-2 (COX-2; the protein product of PTGS2). HIF-1alpha was shown to be stabilized by cyclic hypoxia (despite reoxygenation periods) and to favor COX-2 induction as validated by the use of echinomycin and HIF-1alpha targeting small interfering RNA. Using a specific COX-2 inhibitor and a dedicated COX-2 targeting small interfering RNA, we documented that COX-2 accounted for the higher endothelial cell survival and angiogenic potential conferred by cyclic hypoxia. Cyclic hypoxia also led to a preferential COX-2 induction in tumor cells and, contrary to continuous hypoxia, fostered a higher metastatic take of prechallenged tumor cells. Our study documents that PTGS2/COX-2 is part of a cyclic hypoxia gene signature and largely accounts for the unique phenotype of endothelial and tumor cells exposed to fluctuations in pO(2), thereby offering new perspectives for the clustering of tumors expressing COX-2 together with other cyclic hypoxia-responsive genes.
Highlights
This study identifies the prostaglandin synthase 2 (PTGS2) gene as part of a transcriptomic signature induced by cyclic hypoxia and its protein product, cyclooxygenase-2 (COX-2), as a key mediator of both angiogenesis and metastases formation promoted by oxygen fluctuations
Using a pharmacologic inhibitor of cyclooxygenase-2 (COX-2; the protein encoded by PTGS2) and a dedicated COX-2 targeting small interfering RNA strategy, we identified the functional roles of this enzyme in driving endothelial cell survival and angiogenesis as well as the metastatic take of tumor cells preexposed to cyclic hypoxia
We found that the expression of 231 genes was modified by a factor >2 in response to cyclic hypoxia versus normoxia
Summary
Human umbilical vein endothelial cells were routinely cultured in a specific endothelial cell growth medium (Lonza). Cells were serum starved; endothelial cells were incubated in endothelial basal medium (Lonza) and tumor cells were incubated in serum-free DMEM. To reach and control hypoxic conditions, cells were transferred in a modular incubator chamber (BillupsRothenberg) and flushed for 10 min with a gas mixture of 5% CO2-95% N2 gas until the chamber atmosphere reaches a pO2 value of 0.5% (as determined by oximetry). The cyclic hypoxia protocol consisted of three periods of 1 h hypoxia interrupted by 30 min reoxygenation, whereas 3 h of uninterrupted exposure to hypoxia were used for the continuous hypoxia protocol. Cells were collected either at the end of hypoxia (the last period of hypoxia in the cycling protocol) or after increasing times of reoxygenation (Fig. 1A).
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