The trans-differentiation of epithelial cells into mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT) occurs throughout embryonic development and is recapitulated during epithelial tissue injury and in carcinoma progression. This plasticity is enabled by underlying shifts in epigenetic regulation. Little is known however, regarding our understanding of how these pathways coordinately suppress the epithelial phenotype and induce a mesenchymal program that generates cells with properties of stem cells and whether the acquisition of EMT phenotype in genetically predisposed individuals could be an early event triggered by enteric pathogens. Citrobacter rodentium (CR) causes infectious colitis and shares a remarkable functional similarity to enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli in promoting attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. We have shown previously that functional cross-talks between Wnt/b-catenin, Notch and NF-kB pathways, regulate crypt hyperplasia and/or tumorigenesis in response to CR infection while inflammation and/or colitis is regulated by the MEK/ERK and NF-kB pathways. We have also discovered differential expression of stem cell markers Dclk1 and Lgr5 during progression (days 6-12) and regression (days 20-34) phases of crypt hyperplasia. CR- infected crypt epithelial cells and adenomas stain positive for vimentin, fibronectin and Dclk1 but negative for E-cadherin thereby exhibiting an EMT-like phenomenon. These events seem to be epigenetically regulated as isolated crypts exhibit elevated levels of EZH2 (Enhancer of Zeste Homolog-2) that transcriptionally represses E-cadherin and WIF1 (Wnt Inhibitory Factor 1) expression via histone methylation and deacetylation. Elevated levels of EZH2 and b-catenin with concomitant decrease in WIF1 expression in the adenomas of CR-infected ApcMin/+ mice also parallel changes recorded in BLT1-/-ApcMin/+ mice, a spontaneous model of colon cancer, in AOM/DSS model and in human adenocarcinomas. Finally, orally administered Tributyrin (TBT) blocks EZH2, upregulates WIF1 and inhibits crypt hyperplasia. Based on these findings, we hypothesize that CR infection- induced epigenetic signaling via EZH2 in stem and/or progenitor cells of genetically susceptible mice will promote EMT of colonic crypts and that TBT/butyrate will mitigate epigenetically-linked EMT and/or metastatic process by upregulating WIF1. We propose three specific aims to test this hypothesis.
In Aim 1, we will investigate whether alteration in EZH2/WIF1 axis within the stem cells in response to bacterial infection precedes or accompanies EMT, local invasion or metastasis.
Aim 2 will attempt to establish specificity of the epigenetic and EMT changes in the colon in response to CR infection and, Aim 3 will examine if TBT blocks stem cell-driven and epigenetically regulated process of EMT and/or tumor spread. The new found role for TBT as an inhibitor of EZH2 is expected to eventually pave the way for developing TBT as a therapy to eliminate cancer-initiating cells thereby helping to prevent tumor recurrence or distant metastasis.

Public Health Relevance

Microorganisms are causally implicated in slightly over 20% of all human cancers. Viruses, bacteria and parasites are among the most important pathogens linked to carcinogenesis. It is therefore imperative to focus on understanding the mechanistic basis of malignant transformation initiated by pathogens, an area that promises exciting prophylactic, diagnostic, and therapeutic applications. Utilizing an infection-induced model of hyperplasia and/or carcinogenesis, we intend to examine how enteric pathogens and dietary interventions modulate the process of epigenetically regulated and stem cell-driven Epithelial-Mesenchymal-Transition and metastasis which is expected to help us design novel inhibitors to prevent diseases with infectious etiology.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Chemo/Dietary Prevention Study Section (CDP)
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Ross, Sharon A
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University of Kansas
Schools of Medicine
Kansas City
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Ahmed, Ishfaq; Roy, Badal C; Raach, Rita-Marie T et al. (2018) Enteric infection coupled with chronic Notch pathway inhibition alters colonic mucus composition leading to dysbiosis, barrier disruption and colitis. PLoS One 13:e0206701
Iyer, Swathi V; Ranjan, Atul; Elias, Harold K et al. (2016) Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-?B and osteosarcoma progression. Nat Commun 7:13561
Ahmed, Ishfaq; Roy, Badal C; Subramaniam, Dharmalingam et al. (2016) An ornamental plant targets epigenetic signaling to block cancer stem cell-driven colon carcinogenesis. Carcinogenesis 37:385-96
Ahmed, Ishfaq; Roy, Badal C; Khan, Salman A et al. (2016) Microbiome, Metabolome and Inflammatory Bowel Disease. Microorganisms 4: