Abstract 5193: Wnt signaling in colitis derived colon cancer initiating cells promotes the tumorigenicity in colitis to cancer transition

Abstract

Abstract Introduction: Wnt (Wingless) signaling in colon stem cells is involved in the development and maintenance of crypt architecture. In addition, activating mutations in the Wnt signaling pathway initiate and perpetuate colon cancer initiating cells (CCIC). Recently, we have isolated and propagated CCICs from colitis. Here, we hypothesize that Wnt signaling in the colitis derived CCICs potentiates colitis-associated tumor initiation. Methods: In order to test the above hypothesis, we have designed a dual fusion Wnt reporter construct (TTLG). The construct comprises of luminescent firefly luciferase and enhanced green fluorescent protein (eGFP), which are preceded by the TCF/LEF binding site. TTLG was transduced into colitis sphere isolates that were derived from Aldehyde dehydrogenase (ALDH) high cells. Immunohistochemistry validated the staining for activated β-Catenin in TTLG GFP high vs eGFP low colitis sphere isolates Tumorigenicity was evaluated by injecting eGFP high and eGFP low cells (1000 each) in the flanks of NOD-SCID mice. Secondary tumors were generated from 10% eGFP bright vs dimmest cells (1000, 100 and 10 each) obtained from eGFP high xenograft derived cells. In parallel, in vitro limiting dilution assays (LDA) were performed. Results: The eGFP high sphere cell population had nuclear localized Active Beta Catenin staining, indicating active Wnt signaling (95%). Conversely, little to no (<5%) Active Beta Catenin (ABC) staining was observed in the nucleus of eGFP low sphere cells. In vitro studies demonstrated that the clonogenic potential of eGFP high colitis sphere cells was significantly higher than that of eGFP low cells. This was further confirmed by in vivo studies, where the tumors generated from eGFP high cells grew more rapidly and larger when compared to eGFP low cells. The ability of the eGFP low cells to induce the tumor relates to the fact that eGFP low cells are a subset of the ALDH high population. Furthermore, secondary tumors that were generated from the 10% eGFP bright cells derived from eGFP high xenograft were initiated within few days after injection. At the time of harvest, the tumors formed from 10% eGFP dimmest cells were less than ¼ the size of the 10% eGFP bright cells derived tumor. The clonogenic potential of these 10% eGFP bright cells was about 50-100 fold higher than the 10% eGFP dimmest cells. Conclusions: The Wnt high ALDH high cells have higher clonogenic potential when compared to Wnt low ALDH high cells. Also, the tumors generated from Wnt high ALDH high colitis cells demonstrated decreased tumor latency and increased tumorigenicity compared to Wnt low ALDH high colitis cells. Collectively, our observations indicate that Wnt high CCICs from colitis promote colitis-associated tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2011-5193