RB1-deficient prostate tumor growth and metastasis are vulnerable to ferroptosis induction via the E2F/ACSL4 axis
Mu-En Wang, … , Jiaoti Huang, Ming Chen
Mu-En Wang, … , Jiaoti Huang, Ming Chen
Published March 16, 2023
Citation Information: J Clin Invest. 2023;133(10):e166647. https://doi.org/10.1172/JCI166647.
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Research Article Oncology

RB1-deficient prostate tumor growth and metastasis are vulnerable to ferroptosis induction via the E2F/ACSL4 axis

Abstract

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RB1-deficient cancers remain elusive. Here, we showed that RB1 loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid–containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss–induced sensitization to ferroptosis. Importantly, using cell line–derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a promising approach for the treatment of RB1-deficient malignancies.

Authors

Mu-En Wang, Jiaqi Chen, Yi Lu, Alyssa R. Bawcom, Jinjin Wu, Jianhong Ou, John M. Asara, Andrew J. Armstrong, Qianben Wang, Lei Li, Yuzhuo Wang, Jiaoti Huang, Ming Chen

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Figure 2

ACSL4 is a downstream target of the RB/E2F pathway.

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ACSL4 is a downstream target of the RB/E2F pathway. (A) E2F1 ChIP-Seq p...
(A) E2F1 ChIP-Seq peaks on the ACSL4 gene in different cell lines. Twelve putative E2F1 binding sites are indicated at the top of the ChIP-Seq peak. Data were derived from E2F1 ChIP-Seq data sets generated from prostate cancer cell lines (GSE36614 and GSE67809) and HeLa (GSM935484) and MCF7 (GSM935477) cell lines. Note that ChIP-Seq traces show fold enrichment over input for E2F1 binding to ACSL4 in all cell lines. (B) Schematic map of E2F1 binding sites on the 3 kb human ACSL4 promoter and its full-length and 4 progressive deletion mutant luciferase constructs. (C) Luciferase reporter assay of full-length ACSL4 promoter in the absence or presence of E2F1, E2F2, or E2F3. (D) Immunoblot analysis of cell lysates from RB stable–knockdown PC3 cells transfected with EV, E2F1, E2F2, or E2F3 cDNA for 48 hours. (E) Luciferase reporter assay of ACSL4 3 kb full-length promoter and its progressive deletion mutants in the absence or presence of E2F1. (F) ChIP-qPCR of E2F1 binding to the linker region (negative control), cluster I, and cluster II on the promoter of ACSL4 and CDK1 loci (positive control) in DU145 cells. (G) ChIP-qPCR of E2F1 binding to cluster I and II on the promoter of ACSL4 in control or RB stable–knockdown LNCaP cells. (H) ChIP-qPCR of E2F3 binding to the linker region (negative control), cluster I, and cluster II on the promoter of ACSL4 and CDK1 loci (positive control) in DU145 cells. (I and J) Immunoblot analysis of cell lysates from control or RB stable–knockdown LNCaP (I) or PC3 (J) cells. (K) Immunoblot analysis of cell lysates from PC3 cells transfected with EV or RB1 cDNA for 48 hours. (L) Immunoblot analysis of cell lysates from PC3 cells with RB stable–knockdown or combined with E2F1 knockdown. (M–P) Analysis of correlation between the mRNA levels of ACSL4 and RB1 copy number alterations (M and N) or the mRNA levels of E2F3 (O) or E2F1 (P) in the Beltran et al. (40) or Kumar et al. (41) data set. In C and E, 1-way ANOVA with Tukey’s multiple-comparison test was used to determine significance. In F, G, and H, unpaired 2-tailed t test was used to determine significance. In M and N, Kruskal-Wallis with Dunn’s multiple-comparison test was used to determine significance. In O and P, Spearman’s correlation coefficient was used to examine the correlation (r, correlation coefficient). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bar graphs in C, E–H, are mean ± SD from n = 3~4 biological replicates.