Abstract

Background Dysbiosis and accumulation of reactive oxygen species (ROS) are hallmarks of ulcerative colitis (UC) and have been associated with tumor progression. We have previously shown that UC patient biopsies and colorectal adenomas overexpress toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs). A potential mechanism of TLR4-mediated tumorigenesis is by providing cancer stem cells with redox signals that they efficiently use to outgrow their non-transformed counterparts. We test the hypothesis that epithelial signaling via TLR4 induces the production of ROS through the NADPH oxidases Nox1 and Duox2. We also explore the main redox-sensitive cascades that participate in TLR4-driven tumorigenesis. Methods Colonoids from wild-type (WT), TLR4-KO, and Nox1-KO mice were stimulated with LPS and tested for H2O2 production in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium. IECs isolated from villin-TLR4 mice (which have constitutive activation of epithelial TLR4) and their WT littermates were analyzed for H2O2 production (Amplex Red) and expression of the NADPH oxidases Nox1 and Duox2 (qPCR). Villin-TLR4 and their littermates underwent a model of CAC by azoxymethane injection followed by 2 cycles of 3% dextran sulfate sodium (1.5% in villin-TLR4 mice). Cancer stem cells (aldehyde dehydrogenase positive) were sorted for RNA sequencing. Results LPS incubation enhanced the expression of Nox1 and Duox2 in WT but not TLR4-KO colonoids, demonstrating that these oxidative enzymes are regulated by TLR4. Consistently, LPS stimulation elicited H2O2 release by WT organoids in a NADPH oxidase-dependent manner. Nox1-KO colonoids showed attenuated production of H2O2 upon LPS stimulation, confirming the participation of this NADPH oxidase in TLR4-mediated production of ROS. Constitutive activation of TLR4 induced the production of H2O2 in IECs, which was accompanied by a marked upregulation of Nox1, Duox2, and DuoxA2. RNAseq done on cancer stem cells shows that cancer stem cells of villin-TLR4 tumors have increased expression of NADPH oxidases, as well as enrichment of redox-sensitive pathways, including PI3K, myc, Rac1, and NF-κB. All of these pathways have been implicated in the promotion of proliferative and antiapoptotic signals. Conclusions TLR4 controls the expression and function of oxidative enzymes in IECs, enhancing redox signals in cancer stem cells upon bacterial challenge. In the context of inflammation-associated dysbiosis, we speculate that overexpression of TLR4 provides additional activation of proliferative and antiapoptotic pathways, accelerating tumor development and growth. The results of our studies highlight how we might target critical signaling pathways in inflammatory-driven cancer stem cells.