Abstract
The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.
Highlights
COX-2 contributes to tumor progression [7,8], for instance, upregulation of the vascular endothelial growth factor (VEGF) inducing apoptosis resistance and angiogenesis [9]
CRISPR/Cas9 technology was utilized to generate a stable knockout of COX-2 expression in human melanoma A2058 cells and to evaluate its results in vitro and in vivo
To prove that the generated COX-2 knockout (COX-2KO) was stably incorporated into the cells and alteration had an impact on in vivo behavior of the resulting tumor, human melanoma A2058 and A2058-COX-2KO cells were subcutaneously implanted into
Summary
Cyclooxygenase-2 (COX-2; PTGS2 (prostaglandin-endoperoxide synthase-2) is an inducible enzyme that converts arachidonic acid to prostaglandins. The upregulation of COX-2 by a variety of factors, including cytokines and growth factors, and the resulting increase in prostaglandin E2 (PGE2) levels may promote chronic inflammatory conditions, initiating tumor development [1,2,3]. COX-2 contributes to tumor progression [7,8], for instance, upregulation of the vascular endothelial growth factor (VEGF) inducing apoptosis resistance and angiogenesis [9]. Modulation of COX-2 might offer this, as the enzyme frequently is expressed in malignant melanoma and correlates significantly with poor survival in patients [11,12,13]. Different COX-2 pathway and transcription factors are important for their role in inflammation and melanoma cancer progression
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