Most solid tumors contain a subfraction of cells with stem/progenitor cell features. and WNT-signaling 2 three cell lines showed decreased expression of stem cell markers decreased aldehyde dehydrogenase activity attenuated WNT-signaling and lost the capacity to form colonospheres and 3) two cell lines displayed prominent expression of ABC transporters with a heterogeneous response for stem cell markers. While WNT-signaling could be attenuated in the HT-29/5-FU cells by the WNT-signaling inhibitors ICG-001 and PKF-118 this was not accompanied by any selective growth inhibitory effect suggesting that this cytotoxic activity of these compounds is not directly linked to WNT-signaling inhibition. We conclude that classical WNT-signaling inhibitors have toxic off-target activities that need to be addressed for clinical development. gene amplifications epithelial to mesenchymal transition (EMT) as well as a transformation from NSCLC into small cell lung cancer . Exposure of colorectal cancer (CRC) cells to oxaliplatin was associated with up-regulation of VEGF ligands and receptors  while chronic exposure to irinotecan was accompanied by activation of EGFR- and SRC-signaling in CRC models . Most if not all solid tumors contain a subpopulation that displays molecular and functional similarities to stem cells. Stem cells are naturally chemoresistant due to high expression of certain ATP-binding cassette transporters proficient DNA repair a slow cell cycle and activation of various signaling pathways including WNT . Therefore one potential mechanism of acquired resistance to chemotherapeutic stress would be selection of cells with increased “stemness”. The WNT-signaling F3 pathway plays an important role in the colon. Starting at the base of the crypt unit WNT-signaling is usually highest Altrenogest in the stem cell compartment and decreases as the cell moves upwards through the proliferative areas and into the differentiative compartment . The central role of WNT signaling is usually further emphasized by a recent molecular study of human colon and rectal cancers indicating that at least 90% of patient tumors display a deregulated WNT-signaling pathway . These findings support a role for WNT/beta-catenin-signaling inhibitors as potential novel brokers for treatment of CRC. Assuming that acquired chemoresistance is accompanied by increased “stemness” and upregulation of WNT-signaling such inhibitors might show preferential activity toward tumors with acquired drug resistance. To establish the influence of chronic chemotherapeutic stress on “stemness” we carried out a comprehensive molecular and functional analysis of six independently selected CRC cell lines with acquired resistance to three chemotherapeutic brokers with different mechanisms of action (5-fluorouracil oxaliplatin and irinotecan) derived from two parental cell lines with distinct molecular profiles HT-29 (chromosome instable CIN) and HCT-116 (microsatellite instable MSI). We here report that chronic chemotherapeutic stress drives the evolution of “stemness” in CRC cells in a complex manner which is relevant for the elaboration of future therapeutic strategies. In addition our results reveal that classical WNT-signaling inhibitors have toxic off-target activities that need to be addressed for their clinical development. RESULTS Chronic chemotherapeutic stress is accompanied by altered CD44 splicing CD44 is usually a broadly distributed multifunctional glycoprotein adhesion molecule that is widely used as a stem cell marker . Through alternative splicing cells can produce a large family of CD44 protein isoforms which are involved in a variety of biological processes. Normal epithelial cells in the colon express Altrenogest the standard form of CD44 (CD44s) whereas adenomas carcinomas and CRC metastasis may also express CD44 variants (CD44v) containing additional exons that are coding for insertions in the membrane-proximal extracellular region . Western blot analysis revealed Altrenogest that the parental HCT-116 cells predominantly expressed CD44v isoforms in contrast to the three drug-resistant HCT-116 variants that almost exclusively expressed Altrenogest CD44s (Figure ?(Figure1A1A upper panel). Parental HT-29 cells expressed exclusively CD44v isoforms while the oxaliplatin- and SN38-resistant variants showed increased.