Abstract Colorectal cancer (CRC) develops through a multistep process that is enhanced by chronic inflammation and includes complex interactions between the colonic epithelium and pro-inflammatory cytokines capable of stimulating cancer initiation and progression due, in part, to enhanced production of reactive oxygen species (ROS). Expression of colonic DUOX2, a H2O2 generator, and NOX1, a superoxide producer, is increased in patients with inflammatory bowel disease and adenomatous polyps. For these experiments, we investigated the effect of prolonged treatment with pro-inflammatory cytokines known to be associated with CRC (IL-4, IL-6, and IL-17A alone and in combination) on NADPH oxidase (NOX) expression in human colon cancer lines, and early passage, two-dimensional cultures of conditionally-reprogrammed patient-derived colon cancer cells (PDCs). HT-29 colon cancer cells were treated with IL-4, IL-6, IL-17A, IL-4+IL-17A, and IL-6+IL-17A at a concentration of 25ng/ml each for 4, 6, 8, 15, and 30 days. We measured the effect of these cytokines on expression of NOX family members NOX1, DUOX2, and DUOXA2 by quantitative RT-PCR, and by Western analysis. The production of ROS by NOX proteins was measured with chemiluminescence (superoxide) and Amplex Red assays (H2O2), respectively. DUOX2 protein, which is absent at baseline in HT-29 cells, was easily detectable after 4 days, and increased as a function of the duration of cytokine exposure and for cytokine combinations. In addition to HT-29 cells, similar results were obtained using Ls513 and DLD-1 colon cancer lines. Six PDC lines were also studied: T 280-R, F725, F1126, CK7962, CK7354 and 027-R1. Modest increases in NOX1 expression were observed for 2 lines (T 280-R, CK7962) following 6 days of treatment with IL-6+IL-17A or IL-4; however, NOX1 was highly expressed in 4 cell lines following exposure to the combination of IL-4+IL-17A. Moreover, DUOX2 mRNA and protein were highly expressed after IL-6+IL-17A treatment for 6 days in the T 280-R and F725 cell lines, and very highly expressed after IL-4+IL-17A treatment in all 6 PDCs. Enhanced NOX1 and/or DUOX2 expression in CRC cells was accompanied by concomitant enhancement of NOX-specific ROS. RNA-Seq analysis of cytokine-modulated NOX expression in HT-29 cells and a NOX1 shRNA inhibited subline of HT-29 cells, suggests that both NF-κB and Stat signaling pathways are involved in the control of DUOX2 expression in both established and early passage human CRC cells. These experiments suggest that multiple pro-inflammatory cytokines known to be present in the CRC microenvironment may contribute to the development of a hyperoxidant milieu through the up-regulation of the two NOX species, NOX1 and DUOX2, expressed in the colon. Citation Format: Agnes N. Juhasz, Mariam Konate Monnard, Guojian Jiang, YongZhong Wu, Abdalla Amr Abdelmaksoud, Jinqiu Chen, Xialong Luo, Noemi Kedei, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Iris M. Dahan, Krishnendu Roy, James H. Doroshow. Long-term treatment with IL-4, IL-6, and/or IL-17 increases the expression of the NADPH oxidase DUOX2 in human colon cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5240.