DUOX2 is finely tuned by synergism between cytokines IL-6, IL-22, IL-17A, and TNFalpha in colon cancer

Gastrointestinal inflammation is associated with an increased risk for colorectal cancer (CRC) and is associated with elevated expression of NADPH oxidase (NOX) isoforms, NOX1 and DUOX2, which catalyze the synthesis of superoxide anion radical (O 2 ·- ) and hydrogen peroxide (H 2 O 2 ), respectively. To explore the mechanisms that regulate DUOX2, we investigated the effects of a combination of IL-6 and IL-17A on DUOX2 expression in CRC patient-derived cells (PDC) (T-280R, F725, F1126) vs. normal colon cell lines (CCD-112, CCD-841, CCD-18). DUOX2 mRNA levels were generally higher across CRC PDCs relative to the normal colon cell lines. Treatment of PDCs with IL-6 plus IL-17A for 4-6 days resulted in significant upregulation of DUOX2 mRNA and protein. This agrees with data demonstrating elevated DUOX2 levels in CRC tumors relative to corresponding non-malignant tissues. Treatment of HT-29 and Ls513 colon cancer cell lines with IL-6 plus IL-17A for 8-15 days yielded greater-than-additive increases in DUOX2 mRNA, protein, and DUOX2-dependent H 2 O 2 production as measured by Amplex Red assays. RNA-Seq analysis of cytokine-treated HT-29 cells indicated that STAT3 and NFkB signaling pathways mediated the IL-6/IL-17A-induced DUOX2 expression. We also investigated the regulation of DUOX2 using a triple-cytokine cocktail of IL-6, IL-17A, and TNFalpha. The inclusion of TNFalpha increased the expression of DUOX2 50-fold more than treatment with IL-6 plus IL-17A in HT29 cells, and even more in LS513 cells. There were also substantial increases in DUOX2 activity. In addition, the triple-cytokine treatment shortened the time course of mRNA and protein expression from several days to 24-48 h. When IL-22 was substituted for IL-6 in the triple-cytokine treatment, the fold-increase was even greater. The STAT3 pathway was activated by IL-6 or IL-22, and the NFkB pathway was activated by IL-17A and TNFalpha in these cell lines. Silencing of STAT3 or RELA by siRNA nearly abolished DUOX2 expression. The triple-cytokine treatments could also induce cell death within 48-72 h, as well as DNA damage as evidenced by phosphorylation of gamma-H2AX. In summary, DUOX2 expression and DUOX2-dependent H 2 O 2 production in HT-29 and LS513 cells and PDCs could be finely tuned by synergism amongst several pro-inflammatory cytokines known to be overexpressed in CRC.