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Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived ApcMin/+ mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of β-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies. Cancer Prev Res; 7(1); 105–13. ©2013 AACR.

Background: Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been thoroughly investigated. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas in the proximal colon. These cancers form via a non-canonical mechanism without aberrations in Wnt signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the vast majority of human colon cancers and often occurs concurrently with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine using novel murine models. Methods: B6 females carrying a transgene encoding for a chimeric protein with the iSH2 domain of the p85 subunit of PI3K fused to the N-terminus of p110 (PIK3ca*) were crossed to B6 males carrying the Min allele of Apc (ApcMin/+). The resulting B6 PIK3ca* ApcMin/+ mice were then crossed to mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3ca* ApcMin/+ progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon, as well as having lost one allele of Apc. An additional novel model system was developed to induce mutations in the cells of the mid to distal colon using a minimally invasive technique to instil adenovirus expressing the Cre recombinase. These tumors could then be monitored over time with colonoscopy with biopsies taken for histological and molecular analyses. Results: FC PIK3ca* ApcMin/+ mice develop multiple small intestinal and colon tumors, including invasive adenocarcinomas with lymphatic and mesenteric metastases. Tumor multiplicity and size were increased. Moreover, the tumors appeared less differentiated and were more invasive as compared to those in controls. Advanced adenomas and adenocarcinomas were easily detectible with a novel near-infrared phospholipid ether analog, CLR1502, that selectively accumulates in tumors. In the proximal colon, mucinous adenocarcinomas that were initiated by the dominant active PI3K rarely exhibited nuclear β-catenin and when observed, this localization was limited to select areas. This indicates that in these tumors loss of APC in these cells was a late event and can be a source of clonal outgrowth. The tumors that form in this model are microsatellite stable. Interestingly this synergy appears to be independent of PI3K mediated regulation of GSK-3β and β-catenin signaling. Conclusion: Expression of a dominant active PI3K synergizes with loss of APC activity resulting in dramatic changes in tumor multiplicity, size, morphology, and invasiveness. Citation Format: Dustin Deming, Alyssa Leystra, Laura Nettekoven, Chelsie Kohns, Linda Clipson, Dawn Albrecht, Jeff Bacher, Mary Kay Washington, Christopher Pazoles, Jamey Weichert, Richard Halberg. PIK3CA and APC mutations are synergistic in the development of intestinal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2013-2738

Background Evidence is steadily accumulating that tumors can have a polyclonal origin. In fact, a recent review finds that there is evidence for polyclonal origin in at least 24 different human tumors types. Insights into clonality of intestinal tumors have been achieved through the generation of aggregation chimeras from early-stage mouse embryos and statistical analysis combining information about the pattern of chimerism with tumor phenotype. However, this experimental approach is incredibly laborious and very expensive. Methods To overcome these significant limitations, we developed a mosaic mouse model that expresses fluorescent proteins (FPs) in a variegated pattern throughout the distal small bowel and colon and have used this model to investigate the clonal origin of intestinal tumors. Briefly, our model is based on the mosaic expression of Cre recombinase in the intestinal epithelium and it can be generated by simple crosses, eliminating the time and cost associated with generating aggregation chimeras. In addition, FPs allow us to use advanced in and ex vivo imaging techniques such as dual-photon excitation microscopy and fluorescent endoscopy to address fundamental questions. Experimental Design Mosaic mice carrying the Min allele of Apc are predisposed to developing tumors throughout the intestinal tract. Many of the tumors (41%) from the distal small bowel and colon contained both green and red neoplastic cells confirming that mosaic mice can be used to detect polyclonal architecture. Dual-photon excitation microscopy was performed on whole tumors that had been optically cleared revealing the sub-surface structure of the epithelium and tumors to a depth of ∼350 μM. This detailed analysis revealed that some tumors can be derived from as many as 4 progenitors. Mosaic mice were also treated with azoxymethane (AOM) and AOM in the presence the inflammatory agent dextran sodium sulphate (DSS). The length of exposure to DSS was varied across groups to determine the effects of chronic versus acute inflammation. Results demonstrated that the effect of chronic inflammation is subtle, whereas acute inflammation has a significant effect on clonal architecture of the tumors. One possible explanation for these results is that chronic inflammation supplants the need for interactions among different progenitors to establish a tumor, whereas acute inflammation enhances clonal interactions. Conclusion Together these data demonstrate that mosaic mice are useful for studying the clonal origin of tumors. We also find evidence that polyclonal architecture is important in multiple biological contexts within the mammalian intestine. Because FP expression is ubiquitous the same approach can be used to study clonal origin of tumors in any tissue in which mosaic Cre expression can be induced. Having a better understand of the complexity of tumor origin could profoundly change our strategies for the prevention and treatment of cancers. Citation Format: Christopher D. Zahm, Joseph Szulczewski, Terrah J. Paul Olson, Laura Nettekoven, Alyssa Leystra, Dawn M. Albrecht, Linda Clipson, Kevin W. Eliceiri, Micheal A. Newton, RIchard B. Halberg. The clonal architecture of tumors in the mammalian intestines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2013-2717

Background: Mutations in PIK3CA are a common feature of human cancers and multiple new PI3K inhibitors are in clinical development. These mutations are present in 20-30% of colorectal cancers in humans; however, the effects of such mutations on the intestinal mucosa have not been previously studied in animal models. Here we demonstrate that the expression of a dominant active form of the PI3K protein in the mouse intestine results not only in hyperplasia but also advanced neoplasia. Methods: Mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC) were crossed to mice carrying a transgene encoding a chimeric protein with the iSH2 domain of the p85 subunit fused to the N-terminus of p110 (PIK3ca*). Progeny carrying both transgenes (FC PIK3ca*) express this constitutively active form of p110 in epithelial cells of the distal small bowel and colon. Results: Invasive mucinous adenocarcinomas develop in the proximal colon by only 50 days of age. These tumors are very aggressive, spreading into the mesentery and adjacent organs, and their histological characteristics are strikingly similar to those of invasive colon cancers in humans. Neoplastic cells exhibit a high rate of cell proliferation as seen by increased Ki67 immunohistochemical staining and phosphorylation of AKT, pS6, and p4E-BP1 by western blot demonstrating downstream signaling of PI3K. Interestingly, these cells did not exhibit perturbations in ERK and WNT signaling. Consistent with the lack of aberrant WNT signaling, no benign polypoid intermediaries were identified. Together, the results indicate that tumors in this model form via a non-canonical mechanism. A cohort of FC PIK3ca* mice were imaged by FDG dual hybrid PET/CT colonography and mice with similar sized tumors were divided into control and experimental arms. The experimental arm was treated with rapamycin (6mg/kg/day) for 14 days. Both groups were re-imaged 14 days later. The full results of the rapamycin treatment will be presented. Conclusion: This unique model of advanced colonic invasive adenocarcinomas has the potential to further our understanding of human disease and facilitate the development of therapeutics, especially inhibitors targeting downstream of PI3K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1341. doi:1538-7445.AM2012-1341