Abstract 5065: Cancer derived Escherichia coli induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner
Authors: Uribe Gabriela , Rourke Russel , Villeger Romain , Golovko George , Khanipov Kamil , Liu Zhiqing , Pimenova Maria , Fofanov Yuriy , Zhou Jia , Brasier Allen R. , Pinchuk Irina V. 
Published: August 2018

Abstract
Introduction: Chronic inflammatory responses within the colonic environment are critical to colorectal cancer (CRC) tumor development. While the exact causes of CRC development are unknown, recent data suggest that dysbiosis in the colonic microbiome results in the overgrowth of bacteria from Enterebacteriaceae, Fusobacteriaceae and Bacteroidaceae taxonomic families, contributing to CRC development. Among bacteria of the Enterebacteriaceae family, adherent invasive Esherichia coli has been shown to promote tumor cell growth via production of colibactin. However, the contribution of E.coli to the tumor promoting inflammatory microenvironment (TME) during sporadic CRC development remains unknown. Cancer associated fibroblasts (CAFs) are a major component of TME and contribute to tumor-promoting inflammatory responses via NF-κB dependent production of IL-6. Herein we investigate the effect of E. coli on CAFs. We hypothesize that stimulation of CAFs by cancer derived E.coli is a key processes in supporting tumor-promoting inflammation in CRC. Methods: Bacterial DNA was extracted from tumor and adjacent normal tissue of 28 CRC patients. High throughput sequencing was done using metabarcoding of 16S rDNA for bacteria and analyzed using CLC Genomics Workbench 8.0.5 Microbial Genomics Module, SILVA v119 database for 16S. E.coli PS092717 strain, isolated from CRC tumor site, was used in co-culture experiments. Real time RT-PCR and multiplex cytokine array was used to analyze fibroblasts gene expression and secretion, respectively. Results: We observed a reduction in microbial diversity in tumor vs adjacent normal tissue, with an increased prevalence of Fusobacteria, and Enterobactereacea, but not Bacteroidacea. We then tested the effect of E.coli PS092717 on colonic primary normal fibroblasts and CAFs. Exposure of N-CMFs to E.coli for 24 h only moderately increased IL-6, while CAF responded with a greater increase in IL-6 production. Analysis of the signaling mechanism demonstrated that this process was NF-κB dependent, and was abrogated in the presence of NF-kB specific inhibitor, triptolide (20 ng/mL). Bromodomain containing protein 4 (Brd4) is an epigenetic regulator that can recruit canonical NF-κB transcription factor RelA. Therefore, we analyzed its involvement in the above processes. NF-κB mediated increase in IL-6 expression and secretion induced by E.coli PS092717 was strongly reduced in CAFs than N-CMFs in the presence of BRD4 specific inhibitor (1 µM ZL0590). A similar observation was made when a major components of the E.coli cell wall, lipopolysaccharide (LPS), was used. Conclusion Taken together our data suggests that, E.coli derived from cancer tissue and its cell wall component LPS can contribute to the tumor promoting inflammation via stimulation of IL-6 production by CAFs and this process is BRD4/NF-κB-dependent.
Citation Format: Gabriela Uribe, Russel Rourke, Romain Villeger, George Golovko, Kamil Khanipov, Zhiqing Liu, Maria Pimenova, Yuriy Fofanov, Jia Zhou, Allen R. Brasier, Irina V. Pinchuk. Cancer derived Escherichia coli induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5065.