Sox9 induction, ectopic Paneth cells, and mitotic spindle axis defects in mouse colon adenomatous epithelium arising from conditional biallelic Apc inactivation.

We generated transgenic mice in which human CDX2 gene elements control expression of a tamoxifen-regulated Cre protein (CDX2P-CreER(T2)) to allow for inducible gene targeting in intestinal epithelium. After tamoxifen dosing of CDX2P-CreER(T2) mice, Cre activity was detected in the distal ileal, cecal, colonic, and rectal epithelium, with selected crypt base, transit amplifying, and surface cells all capable of activating Cre function. Four weeks after tamoxifen dosing of CDX2P-CreER(T2) mice carrying a Cre-activated fluorescent reporter, single crypts were uniformly fluorescence positive or negative, reflecting Cre activation in crypt stem cells. Biallelic inactivation of the Apc tumor suppressor gene via the CDX2P-CreER(T2) transgene in colon epithelium led to acute alterations in cell proliferation, apoptosis, and morphology, along with mitotic spindle misorientation, ß-catenin nuclear localization, and induction of the intestinal stem cell markers Lgr5 and Musashi-1 and the Sox9 transcription factor. Normal mouse colon epithelium lacks Paneth cells, a key small intestine niche cell type, and Paneth cell differentiation is dependent on Sox9 function. In Apc-deficient colon epithelium, ectopic Paneth-like cells were seen outside the crypt base, such as new crypt budding sites. Our data indicate Apc inactivation via CDX2P-CreER(T2) targeting in mouse colon epithelium is sufficient to induce adenomatous changes and the generation of Paneth-like cells from neoplastic progenitors, with potentially significant roles in colon adenoma development and progression.

Mutant KRAS promotes hyperplasia and alters differentiation in the colon epithelium but does not expand the presumptive stem cell pool.

BACKGROUND & AIMS: Adenomatous polyps are precursors to colorectal cancer (CRC), whereas hyperplastic polyps (HPPs) have low risk of progression to CRC. Mutations in KRAS are found in ∼40% of CRCs and large adenomas and a subset of HPPs. We investigated the reasons why HPPs with KRAS mutations lack malignant potential and compared the effects of Kras/KRAS activation with those of Apc/APC inactivation, which promotes adenoma formation. METHODS: We activated a KrasG12D mutant allele or inactivated Apc alleles in mouse colon epithelium and analyzed phenotypes and expression of selected genes and proteins. The mouse data were validated using samples of human HPPs and adenomas. Signaling pathways and factors contributing to Kras/KRAS-induced phenotypes were studied in intestinal epithelial cells. RESULTS: Activation of Kras led to hyperplasia and serrated crypt architecture akin to that observed in human HPPs. We also observed loss of Paneth cells and increases in goblet cell numbers. Abnormalities in Kras-mediated differentiation and proliferation required mitogen-activated protein kinase signaling and were linked to activation of the Hes1 transcription factor. Human HPPs also had activation of HES1. In contrast to Apc/APC inactivation, Kras/KRAS activation did not increase expression of crypt stem cell markers in colon epithelium or colony formation in vitro. Kras/KRAS activation was not associated with substantial induction of p16(INK4a) protein expression in mouse colon epithelium or human HPPs. CONCLUSIONS: Although Kras/KRAS mutation promotes serrated and hyperplastic morphologic features in colon epithelium, it is not able to initiate adenoma development, perhaps in part because activated Kras/KRAS signaling does not increase the number of presumptive stem cells in affected crypts.