The investigation was designed around a possible link between Wnt/β-catenin signaling, stem-like cellular characteristics and chemoresistance. The canonical pathway promotes cancer-stem-cell renewal, proliferation and differentiation, while its activation has also been associated with epithelial–mesenchymal transition and metastatic dissemination in earlier colorectal cancer research cited by the authors. Wnt/β-catenin activity may arise through mutations affecting β-catenin or proteins in its destruction complex, as well as through overexpression of Wnt ligands, Frizzled receptors or Dkk-1. Because the pathway has been associated with both tumor progression and therapeutic failure, the researchers examined whether it was active in their established model of long-term 5-FU resistance.

The experimental system consisted of parental HCT-116 colorectal cancer cells and HCT-116 5FUR cells derived through long-term exposure to 5-FU. Previous work with this model had shown that the resistant cells acquired epithelial–mesenchymal transition characteristics together with enhanced migration and invasion. The current study did not determine whether stemness and epithelial–mesenchymal transition are controlled by the same signaling mechanism. Its narrower objective was to measure Wnt/β-catenin activity in the resistant cells, assess selected stemness markers and test whether pharmacological pathway inhibition could make the resistant population more sensitive to 5-FU.

The researchers first used bioinformatic analysis to examine the relationship between two gene signatures in 275 colon cancer samples available through the GEPIA2 platform from The Cancer Genome Atlas. The 5-FU-resistance signature included genes associated with resistance, DNA-damage repair and 5-FU metabolism, while the cancer-stem-cell signature included CD44, PROM1, EPCAM, KLF4, MYC, STAT3, SALL4 and ALDH1A1. Pearson analysis found a positive correlation between the signatures, with R = 0.41 and p < 0.001. This is an association in patient-derived expression data rather than evidence that either signature causes treatment failure, but it provided the clinical-sample context for the subsequent cell experiments.

Laboratory assays showed that the thawed HCT-116 5FUR cells retained their resistant phenotype after recovery from frozen storage. In MTT viability measurements performed 48 hours after exposure to increasing concentrations of 5-FU, the resistant cells tolerated substantially more drug than the parental line. Reverse-transcription quantitative PCR also detected differences in stemness-associated transcripts. ALDH1A1 expression was significantly higher in the resistant cells, whereas POU5F1 showed an upward trend that did not meet the study’s threshold for statistical significance, with p = 0.08. ALDH1A1 was therefore treated as evidence of a stem-like characteristic in this model, not as proof that the entire resistant population consisted of cancer stem cells.

Several measurements indicated stronger canonical Wnt/β-catenin signaling in the 5-FU-resistant cells. Western blotting detected higher levels of both total β-catenin and active, dephosphorylated β-catenin in HCT-116 5FUR cells than in parental cells. Total glycogen synthase kinase 3 beta, or GSK-3β, was lower in the resistant population, while the measured phosphorylated GSK-3β at serine 9 did not differ between the groups. Immunofluorescence imaging likewise showed greater total and active β-catenin staining in the cytoplasm and nuclei of the resistant cells. A TCF/LEF luciferase reporter showed greater β-catenin-dependent transcriptional activity in HCT-116 5FUR cells, and cyclin D1 protein levels were also higher. Taken together, these findings associated the acquisition of 5-FU resistance in this cell line with increased pathway activity.

To test whether that activity contributed to resistance, the investigators used LF3, a 4-thiourea benzenesulfonamide derivative that inhibits the interaction between β-catenin and TCF4. Its negatively charged sulfonamide group binds to Lys435 on β-catenin and competitively prevents the interaction with Asp16 in TCF4, blocking downstream pathway activation. The researchers selected this downstream strategy because colorectal cancer mutations in APC, RNF43 or AXIN2 can generate truncated proteins and constitutive pathway activation, which may limit approaches aimed only at upstream components. In the resistant cells, 60 µM LF3 reduced relative TCF/LEF luciferase activity by approximately sixfold after 24 hours and also reduced cyclin D1 protein levels, confirming both functional pathway inhibition and a downstream molecular effect.

LF3 at the concentration used for pathway blockade did not by itself eliminate the resistant cells. After 48 hours, the maximum tested concentration of 60 µM reduced viability by approximately 30% in both parental and resistant cultures. The LF3 half-maximal inhibitory concentration was 103.5 µM in HCT-116 cells and 135.2 µM in HCT-116 5FUR cells. Combination experiments showed a different effect. The 5-FU IC50 in the resistant cells was 4.8 µM, compared with 0.035 µM in the parental line, documenting the large shift associated with long-term resistance. Adding LF3 lowered the 5-FU IC50 in HCT-116 5FUR cells to 0.138 µM and further reduced viability in the combination condition.

The authors interpret these measurements as evidence that Wnt/β-catenin activity regulates cellular events involved in chemoresistance and that blocking the pathway can impair the resistant phenotype in this model. The stemness and epithelial–mesenchymal transition findings came from separate analyses, so the study did not establish that the two phenotypes are interconnected. The authors propose that cells in intermediate epithelial–mesenchymal states may acquire stem-like characteristics when canonical Wnt signaling is active, but that mechanism was not directly tested here. They also identify long-term 5-FU-resistant cellular models as experimental systems for examining resistance, epithelial–mesenchymal transition and stemness within the same framework.

The scope of the evidence limits how the findings can be interpreted. All experimental results came from the HCT-116 5FUR cell model, while the patient-data analysis established only a correlation between gene signatures. The study did not assess LF3 safety, pharmacokinetics, effects on normal tissues or clinical outcomes, and it did not test combination treatment in animals, organoids or people. The measured result is therefore confined to cultured colorectal cancer cells: LF3 inhibited Wnt/β-catenin transcriptional activity, lowered cyclin D1 and made long-term 5-FU-resistant cells more sensitive to 5-FU in viability assays. The work identifies Wnt/β-catenin inhibition as a candidate combination approach for further preclinical evaluation, but the efficacy and clinical applicability of that approach remain unestablished.

Subject of Research: Wnt/β-catenin inhibition and 5-fluorouracil resistance in a colorectal cancer cell model.

Article Title: Pharmacological inhibition of the canonical Wnt/β-catenin signaling pathway sensitizes 5-fluorouracil-resistant colorectal cancer cells

Article References: Ramos, D. A. A., de Pina, D. F. T., Sousa-Squiavinato, A. C. M., de Araújo, W. M., Rocha, M. R., & Morgado-Diaz, J. A. (2026). Pharmacological inhibition of the canonical Wnt/β-catenin signaling pathway sensitizes 5-fluorouracil-resistant colorectal cancer cells. Molecular Biology Reports, 53(1), Article 1522. https://doi.org/10.1007/s11033-026-12711-z

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12711-z

Keywords: colorectal cancer, 5-fluorouracil resistance, Wnt signaling, β-catenin, LF3 inhibitor, cancer stem cells, HCT-116 cells, chemotherapy sensitization, TCF4, preclinical research, Pharmacological, inhibition