CRISPR/Cas9 mediated knockout of cyclooxygenase-2 gene inhibits invasiveness in A2058 melanoma cells

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 tu-mor microenvironment, tumor growth, apoptosis resistance, angiogenesis, and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the effect of functional knockout 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 con-trols, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor devel-opment in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic ac-tivity of cells under normoxia and experimental hypoxia as demonstrated by using the radio-tracers [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.