The research, led by Celal Cengiz of Batman Training and Research Hospital together with Canan Vejselova Sezer, Bahri Gür, Hatice Mehtap Kutlu, and colleagues at several Turkish universities, focused on the Yes-Associated Protein 1, or YAP-1, and its binding partner TEAD, the Transcriptional Enhancer Factor domain family of transcription factors. YAP-1 is the principal downstream effector of the Hippo signaling pathway, a network that normally governs organ size, tissue regeneration, and cell contact inhibition. When Hippo signaling is dampened, YAP-1 accumulates in the nucleus, pairs with TEAD proteins, and switches on a battery of genes that promote cell survival, proliferation, and epithelial-to-mesenchymal transition. In many cancers, including ovarian carcinoma, this YAP-1/TEAD axis behaves like an engine of malignancy, and pharmacologically breaking the pair apart has become a major goal of targeted drug discovery.

What makes the new work distinctive is the identity of the disruptor. Carmofur, known chemically as 1-hexylcarbamoyl-5-fluorouracil, has historically been appreciated as an antimetabolite that is converted in the body to 5-fluorouracil, interfering with nucleotide synthesis. But carmofur has a second, less celebrated identity: it is a potent inhibitor of acid ceramidase, the enzyme that breaks down the pro-apoptotic lipid ceramide. Previous studies by other groups have linked acid ceramidase inhibition to reduced YAP/TAZ signaling, and the Turkish team reasoned that carmofur might therefore suppress ovarian cancer by simultaneously raising ceramide levels and destabilizing the YAP-1/TEAD transcriptional complex.

To test that hypothesis, the investigators used the Ovcar-3 cell line, a well-characterized human ovarian adenocarcinoma model, and exposed the cells to progressively diluted carmofur solutions. Cell viability was measured with the MTT assay, and the results were striking. Carmofur reduced Ovcar-3 survival in both a concentration-dependent and time-dependent manner, with half-maximal inhibitory concentrations, or IC50 values, of 17.26 micromolar at 24 hours, 10.15 micromolar at 48 hours, and 9.22 micromolar at 72 hours. In other words, the longer the cells were exposed, the more sensitive they became, a pattern consistent with a drug that undermines a slowly turning transcriptional engine rather than simply poisoning one metabolic step.

Flow cytometry revealed the fate of the affected cells: at the 24-hour IC50 concentration, carmofur drove 26.65 percent of the Ovcar-3 population into apoptosis, the programmed cell death pathway that cancer cells typically resist. Confocal microscopy documented the morphological hallmarks that accompany this collapse, including the cellular shrinkage and structural disorganization characteristic of dying cells. Beyond killing cells outright, the team asked whether carmofur could blunt the metastatic behaviors that make ovarian cancer so dangerous. Scratch assays, which measure how quickly a wound in a cell monolayer closes as cells migrate into the gap, showed that carmofur significantly suppressed migratory capacity. Colony formation assays told a similar story, demonstrating that treated cells lost their ability to establish new proliferative outposts, a laboratory proxy for tumor seeding and relapse.

At the molecular level, western blotting provided the mechanistic centerpiece of the study. Carmofur treatment reduced the relative expression of YAP-1 in Ovcar-3 cells from 9.72 to 2.98, a drop of roughly seventy percent. Because YAP-1 requires TEAD to activate its oncogenic gene program, this depletion effectively starves the malignancy program of its key transcriptional driver. The authors report that this is the first study to provide combined in vitro and in silico evidence that carmofur induces apoptosis in ovarian cancer cells by disrupting the YAP-1/TEAD heterodimer, thereby inhibiting both proliferation and migration.

The computational half of the study added atomic-level detail to that conclusion. Using molecular modeling, the researchers examined how carmofur and ceramide interact with the YAP-1/TEAD interface across its three structural components: the beta-strand region, the alpha-helix, and the omega-loop. These interfaces are the contact points through which the two proteins grip each other, and prior structural work has shown that the alpha-helix of YAP’s TEAD-binding domain initiates complex formation. The modeling suggested that both carmofur and ceramide can wedge into and destabilize these contact zones, loosening the heterodimer’s grip. As a validation step, the team modeled verteporfin, a clinically approved drug and the best-known experimental YAP-TEAD disruptor, and confirmed that it exerts a comparable disruptive effect on the same interface, lending credibility to their docking approach and to the proposed mechanism for carmofur.

The broader significance of the work lies in the strategy it exemplifies: repurposing an established, clinically familiar molecule against a signaling axis that has proven difficult to drug. Directly inhibiting transcription factors such as YAP-1 has been notoriously challenging, which is why most efforts have targeted the protein-protein interaction with TEAD instead. Carmofur, with its dual capacity to inhibit acid ceramidase and destabilize YAP-1/TEAD, could offer a two-pronged attack, elevating the lipid signals that push cells toward death while simultaneously removing the transcriptional support that keeps them proliferating and migrating. Prior studies have already implicated acid ceramidase inhibition in sensitizing colon cancer cells to oxaliplatin, reducing glioblastoma migration, and curbing fibrosis through YAP/TAZ suppression, suggesting that the pathway explored here is part of a wider therapeutic logic.

Cautions remain, as they always do at this stage of translation. The findings come from a single cell line studied in vitro and supported by computational models, and Ovcar-3 cells, while representative of an aggressive ovarian adenocarcinoma subtype, cannot capture the full heterogeneity of patient tumors. Carmofur’s own pharmacology is complex, since its metabolite 5-fluorouracil carries cytotoxic effects of its own, and the drug’s stability in biological systems has been a known limitation, with prior work exploring lipid formulations to improve it. Clinical validation will require animal studies and, eventually, trials that test whether the YAP-1/TEAD disruption observed in dishes translates into tumor control in patients. Still, the study opens an intriguing possibility: that a drug sitting in the oncology pharmacopoeia for decades has been quietly harboring a mechanism that modern cancer biology has only now learned to recognize. For a disease as stubborn as ovarian cancer, that kind of second look can be the beginning of something genuinely new.

Subject of Research: Carmofur as a disruptor of the YAP-1/TEAD pathway in ovarian cancer cells

Article Title: Carmofur inhibits ovarian cancer cell proliferation, migration, and invasion via disrupting the YAP-1/TEAD pathway: in vitro and in silico approaches

Article References: Carmofur inhibits ovarian cancer cell proliferation, migration, and invasion via disrupting the YAP-1/TEAD pathway: in vitro and in silico approaches. (n.d.). https://doi.org/10.1007/s13205-026-05065-7

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05065-7

Keywords: carmofur, ovarian cancer, YAP-1, TEAD, Hippo pathway, acid ceramidase, apoptosis, cell migration, Ovcar-3, molecular docking, drug repurposing, ceramide