Blocking ZDHHC2 Reawakens Ferroptosis and Overcomes Enzalutamide Resistance in Prostate Cancer
A new ZDHHC2 inhibitor, TTZ1, restores ACSL4 and triggers ferroptosis, re-sensitizing resistant prostate tumors to enzalutamide in cell and mouse models.
Summary
Enzalutamide is a standard drug for advanced prostate cancer, but tumors often stop responding. Researchers compared tumors from five responders and five non-responders and found that resistant tumors have more of an enzyme called ZDHHC2, which attaches fatty acid tags to proteins. This tagging removes a protective brake on a protein called ACSL4, which normally helps fats in the cell membrane oxidize and kill the cell through ferroptosis, an iron-dependent form of cell death. With less ACSL4, cancer cells avoid ferroptosis and survive treatment. A synthesized ZDHHC2 blocker, TTZ1, restored ACSL4, brought back ferroptosis, and made resistant tumors respond to enzalutamide again in cell lines and patient-derived mouse tumors, without obvious toxicity. The work is preclinical, but it points to a new drug target.
Detailed Summary
Enzalutamide is a next-generation androgen receptor signaling inhibitor used in castration-resistant prostate cancer (CRPC), but resistance is a major clinical problem. Lipid metabolism helps shape prostate cancer progression, and ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, is a potential therapeutic vulnerability. Earlier work linked palmitoylation of GPX4 and SLC7A11 to suppression of ferroptosis. This study investigated whether palmitoylation also regulates lipid peroxide production.
The team performed proteomic, lipidomic, and palmitoyl-proteomic profiling on prostate tumor specimens from five enzalutamide-sensitive and five enzalutamide-resistant patients. Sphingomyelin metabolism differed between groups, but blocking sphingolipid synthesis with myriocin only slightly improved enzalutamide sensitivity. Palmitic acid was significantly higher in resistant patient tumors and increased in resistant C4-2 and 22Rv1 cell lines; adding it reduced drug sensitivity. Palmitoylation patterns differed markedly, with affected proteins enriched in pathways including ferroptosis. The general palmitoylation inhibitor 2-BP increased enzalutamide sensitivity in vitro and in resistant patient-derived xenografts.
Among the ZDHHC enzymes examined, ZDHHC2 showed the largest increase in resistant cells. Knockdown partly reversed resistance, and overexpression promoted it, whereas an enzymatically inactive C129A mutant did not. In ZDHHC2-knockout mice with prostate tumors induced by intraprostatic plasmid injection, knockout limited tumor growth and enhanced enzalutamide sensitivity. The reported animal experiment was small (n=3 per group).
The authors report that a FOXA1/CXXC5/TET2 complex transcriptionally upregulates ZDHHC2. ZDHHC2 then palmitoylates the deubiquitinase USP19, impairing its interaction with ACSL4. This disrupts USP19-mediated stabilization of ACSL4 and promotes ACSL4 degradation through the ubiquitin–proteasome pathway. Reduced ACSL4 consequently suppresses lipid peroxidation and ferroptosis, contributing to enzalutamide resistance.
The synthesized ZDHHC2 inhibitor TTZ1 restored ACSL4 protein, reactivated ferroptosis, and reversed enzalutamide resistance in CRPC cell lines and patient-derived xenograft models. TTZ1 no longer affected enzalutamide sensitivity when ZDHHC2 was knocked out, supporting target dependence for that response. In mice, TTZ1 plus enzalutamide improved treatment effects in resistant patient-derived xenografts without reported changes in body weight or the measured liver- and kidney-function indicators. These limited measurements do not establish comprehensive or human safety.
The work is preclinical. Limitations include the small patient comparison (5 versus 5) and small knockout-mouse groups. No human TTZ1 treatment results are presented in the supplied material, which also does not establish comprehensive inhibitor selectivity, pharmacokinetics, or long-term safety.
Key Findings
- Palmitic acid was significantly higher in tumors from 5 enzalutamide-resistant patients than in 5 sensitive patients, and increased in resistant C4-2 and 22Rv1 cell lines
- ZDHHC2 showed the largest increase among ZDHHC enzymes in resistant cells, and its knockdown partly reversed resistance
- Catalytically dead ZDHHC2 (C129A) failed to confer resistance, showing the effect depends on palmitoylation activity
- ZDHHC2 knockout in a spontaneous prostate cancer mouse model (n=3 per group) limited tumor growth and enhanced enzalutamide response (p<0.05 to p<0.01)
- A FOXA1/CXXC5/TET2 complex transcriptionally upregulates ZDHHC2
- ZDHHC2-mediated palmitoylation of USP19 impairs its interaction with ACSL4, promoting ACSL4 degradation and suppressing lipid peroxidation and ferroptosis
- TTZ1 restored ACSL4 and ferroptosis and enhanced enzalutamide responses in resistant PDX models; it no longer affected enzalutamide sensitivity after ZDHHC2 knockout. Combined treatment caused no reported changes in mouse body weight or measured liver- and kidney-function indicators
Methodology
The study combined proteomics, lipidomics, and palmitoyl-proteomics on tumor specimens from 5 enzalutamide-sensitive and 5 resistant patients with experiments in C4-2 and 22Rv1 cells, including shRNA knockdown and overexpression of wild-type or catalytically inactive ZDHHC2. In vivo work included cell-derived and patient-derived xenografts and a CRISPR/Cas-generated ZDHHC2-knockout mouse model. In the latter, tumors were induced by intraprostatic plasmid injection at 6 weeks of age, followed by 2 weeks of enzalutamide or vehicle treatment; Figure 1 reports enzalutamide at 10 mg/kg and n=3 per group. Figure 1 reports mean ± SD and significance thresholds of p<0.05, p<0.01, or p<0.001 for specified panels. Specific statistical tests were not detailed in the supplied text.
Study Limitations
Patient profiling cohorts were small (5 versus 5), and the knockout-mouse experiment reported n=3 per group. The supplied experiments relied on cell lines and mouse tumor models, with no human TTZ1 treatment results presented. Mouse safety observations were limited to reported body-weight, liver-function, and kidney-function indicators; they do not establish comprehensive or long-term safety. Comprehensive selectivity and pharmacokinetic data are not provided in the supplied excerpt. Conflicts of interest were not stated in the text provided.
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