ARTICLE ABSTRACTUlcerative colitis (UC) increases the risk of colorectal cancer (CRC), but the mechanisms involved in colitis-to-cancer transition (CCT) are not well understood. CCT may involve a inflammation-dysplasia-carcinoma progression sequence compared with the better characterized adenoma-carcinoma progression sequence associated with sporadic CRC. One common thread may be activating mutations in components of the Wnt/β-catenin signaling pathway, which occur commonly as early events in sporadic CRC. To examine this hypothesis, we evaluated possible associations between Wnt/β-catenin signaling and CCT based on the cancer stem cell (CSC) model. Wnt/β-catenin immunostaining indicated that UC patients have a level of Wnt-pathway-active cells that is intermediate between normal colon and CRC. These UC cells exhibiting activation of the Wnt pathway constituted a major subpopulation (52% + 7.21) of the colonic epithelial cells positive for aldehyde dehydrogenase (ALDH), a putative marker of precursor colon CSC (pCCSC). We further fractionated this subpopulation of pCCSC using a Wnt pathway reporter assay. Over successive passages, pCCSCs with the highest Wnt activity exhibited higher clonogenic and tumorigenic potential than pCCSCs with the lowest Wnt activity, thereby establishing the key role of Wnt activity in driving CSC-like properties in these cells. Notably, 5/20 single cell injections of high-Wnt pCCSC resulted in tumor formation, suggesting a correlation with CCT. Attenuation of Wnt/β-catenin in high-Wnt pCCSC by shRNA-mediated downregulation or pharmacological inhibition significantly reduced tumor growth rates. Overall, the results of our study indicates (i) that early activation of Wnt/β-catenin signaling is critical for CCT and (ii) that high levels of Wnt/β-catenin signaling can further demarcate high-ALDH tumor-initiating cells in the nondysplastic epithelium of UC patients. As such, our findings offer plausible diagnostic markers and therapeutic target in the Wnt signaling pathway for early intervention in CCT. Cancer Res; 72(19); 5091–100. ©2012 AACR.