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

In a study published in the journal Molecular Diversity, Kader Sahin of Bahçeşehir University and Bandırma Onyedi Eylül University, Ajda Coker of Marmara University, and Serdar Durdağı of Bahçeşehir University describe a structure-based virtual screening campaign that combed through FDA-approved drugs in search of molecules capable of disrupting the interaction between β-catenin and T-cell factor 4, or Tcf4. This protein–protein interaction is the final, decisive step in Wnt signaling: when β-catenin accumulates in the cell nucleus and binds Tcf4, the complex switches on a battery of genes that drive tumor growth, survival, metastasis, and drug resistance. Block that binding, the logic goes, and the entire oncogenic program collapses.

The catch has always been the shape of the target. The β-catenin/Tcf4 interface sprawls across roughly 4,800 square angstroms, a vast and shallow surface with few of the deep, well-defined pockets that medicinal chemists traditionally exploit. For years, interfaces like this one were written off as undruggable. Yet structural biology has gradually revealed that even sprawling protein–protein interfaces concentrate their binding energy in a handful of so-called hot spots, short stretches of residues that contribute disproportionately to the affinity between the two partners. If a small molecule can be steered into those hot spots, the reasoning goes, it might achieve with a compact drug what would otherwise require a much larger molecule.

That is precisely the strategy the researchers pursued. Rather than screening synthetic chemical libraries filled with untested compounds, they turned to drug repurposing, focusing on medicines that have already passed safety scrutiny and are in clinical use. Repurposing offers a practical shortcut: known pharmacology, established safety profiles, and in many cases existing manufacturing and distribution infrastructure. The team first mapped the critical hot spot residues of β-catenin that engage Tcf4, then used molecular docking to position each candidate drug within the interface, asking which approved molecules could physically occupy and destabilize the contact points that hold the complex together.

The computational pipeline did not stop at docking. To separate plausible binders from artifacts, the researchers subjected the top candidates to physics-based molecular dynamics simulations, watching in atomic detail how each ligand behaved over time when bound to β-catenin. They then calculated binding free energies using the MM/GBSA method, a hybrid approach that combines molecular mechanics energies with implicit solvent models to estimate how strongly a ligand clings to its target. This layered filtering, docking followed by simulation followed by energetic scoring, is designed to weed out compounds that look good in a single static snapshot but fall apart under the thermal motion of a real cellular environment.

Three candidates emerged from the screen with stable binding to the critical hot spot residues of β-catenin, specifically asparagine 430, lysine 435, histidine 470, arginine 474, and lysine 508. They were Leucovorin, also known as folinic acid, a vitamin derivative routinely used to enhance chemotherapy; Carbenicillin, a penicillin-class antibiotic; and Ceforanide, a second-generation cephalosporin. All three are approved, well-characterized medicines, which means that if any of them proves effective against Wnt-driven cancers, the path from bench to bedside could be considerably shorter than for a brand-new chemical entity.

Computational predictions, however convincing, mean little until they are tested in living cells. The team moved their three hits into the laboratory, using HT-29 colorectal cancer cells, a widely studied cell line in which Wnt signaling is aberrantly active. The cells were treated with each compound and assessed for viability and for their ability to form colonies, a measure of long-term proliferative capacity that often correlates with tumorigenic potential. The results were striking: Carbenicillin and Ceforanide reduced cell viability and impaired colony formation, exhibiting superior anti-proliferative activity compared with the other candidates, including Leucovorin.

The convergence between the computational and experimental arms of the study is what gives the findings their weight. The two antibiotics that performed best in cell assays were the same molecules whose simulated binding poses anchored them most securely to the β-catenin hot spots. This consistency suggests that the anti-proliferative effects observed in HT-29 cells may indeed arise from disruption of the β-catenin/Tcf4 interaction, although the researchers are careful to frame the work as a proof of concept rather than a finished therapy. Further studies will be needed to confirm the mechanism directly, to characterize selectivity for cancer cells over healthy tissue, and to establish whether the compounds can achieve therapeutic concentrations in tumors.

Even so, the implications are considerable. Wnt/β-catenin signaling is implicated not only in colorectal cancer but in a broad family of malignancies, including hepatocellular carcinoma and other Wnt-driven tumors, and it also maintains cancer stem cell populations that fuel relapse and metastasis. A validated small-molecule inhibitor of the β-catenin/Tcf4 interaction would be a genuinely new class of anticancer agent, one that attacks the transcriptional engine of the tumor rather than a single downstream symptom. Prior efforts, including peptide mimetics and dedicated small molecules developed in academic labs, have struggled to reach the clinic, which makes the repurposing angle particularly attractive: these are drugs whose toxicity, metabolism, and drug interactions are already documented.

The study also offers a template for tackling other supposedly undruggable targets. By combining hot spot analysis, docking against a library of approved drugs, rigorous molecular dynamics validation, and energetic scoring, and only then moving to cell-based experiments, the researchers demonstrated a workflow that is both computationally disciplined and experimentally grounded. As the authors conclude, their findings provide compelling evidence that targeting the β-catenin/Tcf4 interaction with small molecules is feasible, and that Carbenicillin and Ceforanide may serve as promising repurposed candidates for therapeutic intervention in Wnt-driven malignancies. For patients with colorectal cancer, whose treatment options too often narrow as the disease progresses, the idea that answers might be hiding in plain sight among existing medicines is a hopeful one, and this study shows exactly how such hidden answers can be systematically found.

Subject of Research: Virtual drug repurposing to discover β-catenin/Tcf4 interaction inhibitors for colorectal cancer treatment

Article Title: Hot spot-driven discovery of β-catenin/Tcf4 interaction inhibitors via virtual drug repurposing study and experimental validation in colorectal cancer cells

Article References: Sahin, K., Coker, A., & Durdağı, S. (2026). Hot spot-driven discovery of β-catenin/Tcf4 interaction inhibitors via virtual drug repurposing study and experimental validation in colorectal cancer cells. Molecular Diversity. https://doi.org/10.1007/s11030-026-11720-1

Image Credits: AI Generated

DOI: 10.1007/s11030-026-11720-1

Keywords: β-catenin, Tcf4, Wnt signaling, colorectal cancer, drug repurposing, virtual screening, molecular docking, molecular dynamics, MM/GBSA, Carbenicillin, Ceforanide, protein-protein interaction inhibitors