Notch ligand DLL1 shields ovarian cancer cells from ferroptosis, fueling cisplatin resistance

Ovarian cancer remains one of the deadliest gynecologic malignancies, and its most stubborn obstacle is cisplatin resistance: the first-line chemotherapy works initially, but tumors frequently adapt and recur. A new open-access study in Cancer & Metabolism points to an unexpected contributor to that adaptation, the Notch ligand DLL1, which the authors report is elevated in ovarian tumors and tied to poorer patient survival. According to the paper, DLL1 helps cancer cells resist ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, and that protective state blunts cisplatin’s killing power.

The research team, led by Dan Ye and Gaoting Huang with senior author Jian Shen, combined public transcriptome datasets, clinical samples from three ovarian cancer patients, and several ovarian cancer cell lines. In the GEO dataset GSE6008, DLL1 messenger RNA was significantly higher in tumor tissue than in normal ovarian tissue (p = 0.00075). In GSE148003, DLL1 was also elevated in cisplatin-resistant ovarian cancer samples compared with normal tissue (p = 0.026). A neoadjuvant chemotherapy cohort, GSE201600, showed a slight rise in DLL1 expression in tumor tissue after carboplatin and paclitaxel treatment, which the authors interpret as a hint that the ligand participates in chemosensitivity regulation.

Survival analysis based on The Cancer Genome Atlas data strengthened the clinical association. Patients whose tumors had high DLL1 expression had significantly worse overall survival than those with low expression (p = 0.0165; hazard ratio 1.779, 95% confidence interval 1.093 to 2.89). Disease-free survival showed a similar pattern (p = 0.0258; hazard ratio 1.710, 95% CI 1.052 to 2.77). At the protein level, western blotting detected strong DLL1 expression in the ovarian cancer cell lines A2780 and SK-OV-3, with SK-OV-3 reaching 1.8-fold the level seen in the normal ovarian epithelial line IOSE80 (p < 0.001). In paired clinical samples from three patients, tumor tissue showed higher DLL1 protein than adjacent normal tissue, and immunohistochemical quantification confirmed greater DLL1 positivity in tumor regions.

To probe function, the researchers knocked down DLL1 with short hairpin RNA in SK-OV-3 and A2780 cells. The knockdown slowed proliferation measured by CFDA-SE staining and cut colony formation, reducing clone numbers by 103 in SK-OV-3 cells and 99 in A2780 cells (both p < 0.001). Transwell assays showed migration fold change falling to 0.43 in SK-OV-3 cells and 0.25 in A2780 cells after DLL1 knockdown (p < 0.001). Western blots revealed shifts in epithelial-mesenchymal transition markers: in SK-OV-3 cells, the epithelial marker E-cadherin dropped 21% (p < 0.01), while mesenchymal markers N-cadherin and Vimentin fell 64% and 53% respectively (p < 0.001). Annexin V/PI staining showed cell death rising to 13% in SK-OV-3 cells and 14% in A2780 cells after knockdown (p < 0.001).

The nature of that cell death was the study’s central clue. When DLL1 was knocked down, CCK-8 viability assays showed that the ferroptosis inhibitor ferrostatin-1 and the necroptosis inhibitor necrostatin-1 partially rescued cell survival, whereas the caspase inhibitor z-VAD-FMK offered little protection, pointing to ferroptosis as the dominant death pathway. A Pearson correlation across the data found a significant but weak negative association between DLL1 expression and ferroptosis indicators (r = -0.10, p = 0.04), which the authors acknowledge is small in magnitude yet statistically meaningful. Direct biochemical measurements filled in the picture: DLL1 knockdown raised malondialdehyde, a lipid peroxidation end product, to 3.4-fold control levels, depleted glutathione by roughly 87%, and increased ferrous iron 2.2-fold, a trio consistent with lipid peroxidation, antioxidant exhaustion, and iron overload.

Mechanistically, the paper links DLL1 to the Nrf2/GPX4 antioxidant axis through Notch signaling. Western blotting showed that DLL1 knockdown reduced GPX4 to 50% of control and Nrf2 to 29% in SK-OV-3 cells (p < 0.001 and p < 0.01), while adding exogenous DLL1 protein restored them to 77% and 71% of baseline. The knockdown also lowered xCT, the cystine importer that feeds glutathione synthesis, and raised ACSL4, an enzyme that promotes lipid peroxidation susceptibility. ChIP-qPCR experiments indicated that Notch1 binds directly to the Nrf2 promoter, and a dual-luciferase reporter assay showed that Notch activation increases Nrf2 promoter activity, supporting Nrf2 as a transcriptional target of the pathway. Lipid ROS measured by C11-BODIPY staining rose sharply after DLL1 knockdown and fell again when exogenous DLL1 was supplied, dropping by 88% in SK-OV-3 cells and 56% in A2780 cells relative to knockdown levels.

Mitochondria emerged as a visible casualty of DLL1 loss. MitoTracker-Red staining showed mitochondrial activity falling about 61% in DLL1-knockdown SK-OV-3 cells (p < 0.001), recovering to 77% of control after exogenous DLL1 supplementation. MitoTracker-Green imaging revealed fragmentation, blurred cristae, and loss of structural continuity after knockdown, with a marked shift from intact, interconnected mitochondria to fragmented forms. Importantly, ferrostatin-1 co-treatment restored xCT, GPX4, and Nrf2 expression, normalized MDA, glutathione, and iron levels, and partially rescued mitochondrial activity and morphology, which the authors take as confirmation that the damage caused by DLL1 loss runs through ferroptosis rather than an unrelated lethal process.

The chemoresistance link came from dose-response testing. CCK-8 assays showed that DLL1 knockdown lowered the cisplatin IC50 in SK-OV-3 cells from 2.0 to 1.5 micromolar and in A2780 cells from 2.5 to 0.9 micromolar (p < 0.05 for both), with the gap widening at higher cisplatin concentrations of 5 to 10 micromolar. Under 10 micromolar cisplatin, DLL1-deficient cells showed stronger lipid peroxidation, greater glutathione depletion, and a burst of ferrous iron that the authors describe as potentially amplifying toxic ROS through the Fenton reaction. The team then built cisplatin-resistant derivatives, SK-OV-3-R and A2780-R, by gradual cisplatin exposure, and found DLL1 upregulated in the resistant cells alongside elevated Nrf2, GPX4, and xCT and reduced ACSL4. Knocking down DLL1 in those resistant lines raised lipid ROS by 52% and 55% (p < 0.05), increased MDA and free iron, lowered glutathione, and reduced mitochondrial viability while increasing fragmentation.

Therapeutically, the study tested whether blocking GPX4 could defeat DLL1-driven protection. In DLL1-overexpressing SK-OV-3 and A2780 cells, cisplatin alone or the GPX4 inhibitor RSL3 alone each reduced GPX4 expression and cell viability, but the combination pushed viability below 52% (51% in SK-OV-3, 49% in A2780) and raised lipid ROS by 191% and 199% compared with single-agent treatment. Similar results appeared in the cisplatin-resistant cell models, where combined treatment increased lipid ROS, MDA, and iron while depleting glutathione. In nude mice bearing DLL1-overexpressing SK-OV-3 tumors, average tumor weight reached 1.44 grams versus 0.86 grams in controls (p < 0.01); cisplatin monotherapy reduced tumor weight, and adding RSL3 brought it down to 0.28 grams (p < 0.001 versus monotherapy), with Ki-67 staining showing the lowest proliferative index in the combination group.

The authors are candid about the study’s limits. The clinical cohort comprised only three patient sample pairs, the in vivo work lacked independent controls such as DLL1 knockdown alone or RSL3 alone, and the experiments used subcutaneous xenografts of cisplatin-sensitive cells rather than orthotopic models of resistant disease. How Notch signaling precisely activates Nrf2 also remains to be confirmed with methods such as ChIP-seq. The discussion also clarifies a subtle point: DLL1 overexpression creates a ferroptosis-resistant state that depends partly on GPX4, so RSL3 bypasses that shield rather than DLL1 overexpression itself sensitizing cells to ferroptosis. Within those boundaries, the work frames DLL1 as a candidate biomarker and a preclinical target whose blockade, paired with ferroptosis induction, may offer a route around cisplatin resistance in ovarian cancer.

Subject of Research: How the Notch ligand DLL1 promotes ferroptosis resistance and cisplatin resistance in ovarian cancer

Article Title: DLL1-mediated ferroptosis resistance via the Notch-Nrf2/GPX4 axis drives cisplatin resistance in ovarian cancer

Article References: Ye, D., Huang, G., Yan, X., Suntan, L., Shen, H., & Shen, J. (2026). DLL1-mediated ferroptosis resistance via the Notch-Nrf2/GPX4 axis drives cisplatin resistance in ovarian cancer. Cancer & Metabolism, 14(1), Article 28. https://doi.org/10.1186/s40170-026-00444-3

Image Credits: AI Generated

DOI: 10.1186/s40170-026-00444-3

Keywords: ovarian cancer, cisplatin resistance, ferroptosis, DLL1, Notch signaling, Nrf2, GPX4, lipid peroxidation, mitochondria, RSL3, preclinical study, drug resistance