Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition

nature cell biology Article https://doi.org/10.1038/s41556-024-01464-1 Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition Received: 23 June 2023 A list of authors and their affiliations appears at the end of the paper Accepted: 20 June 2024 Published online: xx xx xxxx Check for updates Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial–mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFβ stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very longchain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1. The two major obstacles in combating cancer progression are metastasis and therapy resistance, underscoring the urgent need for innovative therapeutic strategies. Both processes are often triggered by a transient and partial activation of the EMT programme in cancer cells, which is exerted by EMT transcription factors (EMT-TFs), mainly of the Zeb, Snail and bHLH families1,2. These highly plastic, partially mesenchymal cancer cells turned out to be the most crucial and fatal; they combine high tumorigenic and metastatic capacity with high resistance to any kind of current therapy modalities3–5, which makes them the ‘ultimate’ target in many cancer types. However, targeting these elusive cancer cell populations (either with a transient or intrinsic mesenchymal phenotype) has remained a considerable challenge until now. e-mail: ; Nature Cell Biology A mesenchymal phenotype has been shown to predispose to a higher susceptibility to ferroptotic cell death6. Ferroptosis is an ancestral, highly conserved death pathway, depending on an iron and oxygen-radical-mediated peroxidation of phospholipids. Notably, such phospholipids must be composed of polyunsaturated fatty acids (PUFAs)7–9. In contrast to PUFAs, monounsaturated fatty acids (MUFAs) are resistant to peroxidation and excess MUFAs can even protect cells from ferroptosis and counteract PUFA biosynthesis10,11. Moreover, MUFAs can protect cells from death, as demonstrated for the MUFA-containing lipokine phosphatidylinositol (PI) (oleate (18:1/18:1))12. To prevent spontaneous ferroptosis, cells have efficient protection systems, leading to detoxification of lipid hydroperoxides Article (lipid reactive oxygen species (ROS)). Two well-known systems involve the enzymes GPX4 and FSP1, and their pharmacological inhibition can induce ferroptosis7,13. In contrast to apoptosis, the prevailing death pathway in differentiated cells (for example epithelial cells), ferroptosis can be executed predominantly in cells with a mesenchymal phenotype6, which is activated by the expression of EMT-TFs. Hereby, the expression of the EMT-TF Zeb1 has been associated with ferroptosis susceptibility, but the underlying molecular links are not known6. We and others have previously described that Zeb1 is a hallmark transcription factor of aggressive cancer types involved in all stages of fatal tumour progression, including therapy resistance and metastasis1,4,14,15. Therefore, the unexpected link of this metastasis-promoting EMT-TF with ferroptosis sensitivity offers a therapeutic window. The aim of our study was to elucidate molecular mechanisms of the described Zeb1/EMT-associated ferroptosis sensitivity as basis for future therapeutic strategies. Results Ferroptosis sensitivity of mesenchymal cancer cells relies on Zeb1 We investigated whether different ways of EMT activation all lead to increased ferroptosis susceptibility and if this relies on common key factors. First, we analysed cancer cell models with intrinsically different phenotypes. The KPC genetic mouse model gives rise to metastatic pancreatic cancers with highly variable phenotypes15,16. Ferroptosis sensitivity in various cell lines isolated from such tumours was closely linked to their intrinsic phenotype, with mesenchymal tumour cells showing the highest susceptibility (Fig. 1a). Moreover, GPX4 inhibition selects for an epithelial phenotype in KPCmix cell lines with a mixed, plastic phenotype (Supplementary Video 1). The same association is evident in established human cancer cell lines, for example from breast cancer, as exemplified for the lines MDA-MB-231 (mesenchymal) and MCF7 (epithelial) (Extended Data Fig. 1a). Analyses of published datasets confirmed a strong association of sensitivity to various ferroptosis-inducing compounds with high expression of the EMT-TF Zeb1 (Fig. 1b). CRISPR–Cas9 essentiality screens (CERES) identify Zeb1 as an important gene in MDA-MB-231 cells, one of the most ferroptosis sensitive cancer cell lines (Fig. 1b). Accordingly, depletion of Zeb1 reduced ferroptosis susceptibility (Fig. 1c and Extended Data Fig. 1b). Zeb1 dependency could be confirmed in vivo in a zebrafish model. Here tumour growth was strongly enhanced by ferrostatin-1 for MDA-MB-231 wild-type cells, indicating spontaneous ferroptosis, but not for Zeb1-depleted cells (shZeb1) (Fig. 1d). In human cancers a partial mesenchymal state is often transiently activated in plastic epithelial cancer cells by environmental factors, for example TGFβ, or in the course of therapy resistance. This clinically relevant type of a transient EMT activation by TGFβ also increased the sensitivity to ferroptosis in various epithelial cancer cell types Fig. 1 | Zeb1 is important for increased ferroptosis sensitivity of mesenchymal cancer cells. a, Representative immunofluorescence and immunoblots of KPC cells, classified according to an epithelial (KPCe), mixed (KPCmix) or mesenchymal (KPCm) phenotype. Relative viability and death rate treated with ML210 (46 h) ± 1 µM ferrostatin-1 (Fer-1). Data are mean ± s.e.m. from n = 3 independent experiments, two-way analysis of variance (ANOVA) (with multiple comparisons). Scale bar, 50 µm. b, High expression of Zeb1 correlates (...truncated)