Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigenesis.

Division of Gastroenterology, Department of Internal Medicine, Oklahoma City, OK, USA.
Oncogene (Impact Factor: 8.56). 06/2013; DOI: 10.1038/onc.2013.210
Source: PubMed

ABSTRACT DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-β (TGFβ), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8 h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.Oncogene advance online publication, 10 June 2013; doi:10.1038/onc.2013.210.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8) CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although β-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected Apc(Min/+) mice paralleled changes recorded in BLT1(-/-)Apc(Min/+), AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.Oncogene advance online publication, 8 December 2014; doi:10.1038/onc.2014.386.
    Oncogene 12/2014; DOI:10.1038/onc.2014.386 · 8.56 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Colorectal cancer (CRC) may develop slowly over years from precursor lesions, and thus, screening combined with early diagnosis is the key to disease prevention. Recent studies have elucidated specific traits in the gut microbiome associated with CRC and suggested that the microbiome may be useful in screening for CRC purposes but failed to provide protocols that can be applied in a practical situation. A recent study by Zackular and colleagues presented in this issue of Cancer Prevention Research provides an important way forward here in showing that specific analysis of multiple aspects of the microbiome composition in toto provides reliable detection of both precancerous and cancerous lesions. This important achievement when combined with other non-invasive techniques promises to provide highly effective tools for early CRC diagnosis and its prevention.
    Cancer Prevention Research 09/2014; 7(11). DOI:10.1158/1940-6207.CAPR-14-0273 · 5.27 Impact Factor


Available from
Jun 6, 2014