EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m6A autoregulation pathway
Yang Yi, Joshua Fry, Chaehyun Yum, Rui Wang, Siqi Wu, Sharath Narayan, Qi Liu, Xingxing Zhang, Htoo Zarni Oo, Ning Xie, Yanqiang Li, Xinlei Gao, Xufen Yu, Xiaoping Hu, Qiaqia Li, Kemal Keseroglu, Ertuğrul M. Özbudak, Sarki A. Abdulkadir, Kaifu Chen, Jian Jin, Jonathan C. Zhao, Xuesen Dong, Daniel Arango, Rendong Yang, Qi Cao
Yang Yi, Joshua Fry, Chaehyun Yum, Rui Wang, Siqi Wu, Sharath Narayan, Qi Liu, Xingxing Zhang, Htoo Zarni Oo, Ning Xie, Yanqiang Li, Xinlei Gao, Xufen Yu, Xiaoping Hu, Qiaqia Li, Kemal Keseroglu, Ertuğrul M. Özbudak, Sarki A. Abdulkadir, Kaifu Chen, Jian Jin, Jonathan C. Zhao, Xuesen Dong, Daniel Arango, Rendong Yang, Qi Cao
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Research Article Cell biology Oncology

EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m6A autoregulation pathway

Abstract

N6-methyladenosine (m6A), the most predominant RNA modification in humans, participates in various fundamental and pathological bioprocesses. Dynamic manipulation of m6A deposition in the transcriptome is critical for cancer progression, though how this regulation is achieved remains understudied. Here, we report that, in prostate cancer (PCa), Polycomb group (PcG) protein Enhancer of Zeste Homolog 2 (EZH2) exerts an additional function in m6A regulation via its enzymatic activity. Mechanistically, EZH2 methylates and stabilizes FOXA1 proteins from degradation, which, in turn, facilitates the transcription of m6A reader YTHDF1. Through activating an m6A autoregulation pathway, YTHDF1 enhances the translation of METTL14 and WTAP, 2 critical components of the m6A methyltransferase complex (MTC), and thereby upregulates the global m6A level in PCa cells. We further demonstrate that inhibiting the catalytic activity of EZH2 suppresses the translation process globally through targeting the YTHDF1-m6A axis. By disrupting both the expression and interaction of key m6A MTC subunits, combinational treatment of EZH2 degrader MS8815 and m6A inhibitor STM2457 mitigates prostate tumor growth synergistically. Together, our study decodes a previously hidden interrelationship between EZH2 and mRNA modification, which may be leveraged to advance the EZH2-targeting curative strategies in cancer.

Authors

Yang Yi, Joshua Fry, Chaehyun Yum, Rui Wang, Siqi Wu, Sharath Narayan, Qi Liu, Xingxing Zhang, Htoo Zarni Oo, Ning Xie, Yanqiang Li, Xinlei Gao, Xufen Yu, Xiaoping Hu, Qiaqia Li, Kemal Keseroglu, Ertuğrul M. Özbudak, Sarki A. Abdulkadir, Kaifu Chen, Jian Jin, Jonathan C. Zhao, Xuesen Dong, Daniel Arango, Rendong Yang, Qi Cao

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Figure 3

EZH2 activates YTHDF1 transcription through FOXA1 to regulate m6A globally.

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EZH2 activates YTHDF1 transcription through FOXA1 to regulate m6A global...
(A) Violin plot showing the mRNA level of YTHDF1 in normal (n = 52), primary PCa (n = 497), and mCRPC (n = 101) specimens using data from TCGA and SU2C. P values were calculated by 2-tailed Wilcoxon rank-sum test and adjusted for multiple comparisons using Bonferroni correction. mCRPC: metastatic castration-resistant prostate cancer. (B) Scatter plot showing the relationship between EZH2 and YTHDF1 mRNA expressions using data from TCGA-PRAD and SU2C, with Spearman correlation coefficient (R) and P value as indicated. TPM, transcript per million. (C) Representative IHC images of YTHDF1 expression in BPH and high Gleason score PCa tissues as indicated. Scale bar: 100 μm. BPH, benign prostatic hyperplasia. (D) Graph showing the YTHDF1 protein levels based on the TMA results from BPH (n = 20), Gleason 6–7 PCa (n = 83) and Gleason 8–10 PCa (n = 44) tissues. (E) The association between YTHDF1 expression and PSA recurrence-free survival time of patients with PCa was analyzed by Kaplan-Meier analysis using the patient dataset corresponding to the TMA slide we used. Patients were divided into low (H-score 0–100) and high (H-score ≥ 150) YTHDF1 groups based on the median expression value, which coincided with a natural separation in the frequency distribution. P value was calculated by Log-rank (Mantel-Cox) test. PSA, prostate-specific antigen. (F) Representative IHC staining of PCa TMA slides using the indicated antibodies. Scale bar: 100 μm. (G) Scatter plot showing the correlation between protein levels of EZH2 and YTHDF1, as revealed by the PCa TMA IHC data. The R and P value were calculated as indicated. (H) Bar chart showing aggregate rank scores for the top 10 transcription factors based on YTHDF1 correlation and Regulatory Potential (RP) Score. (I) Scatter plot showing the relationship between YTHDF1 Pearson correlation (x-axis) and Regulatory Potential Score (y-axis) for various transcription factors, with FOXA1 position highlighted in red. (J) ChIP-qPCR assay to monitor the enrichment of FOXA1 at YTHDF1 gene loci in 2 PCa cell lines. (K) Western blot to detect the change of YTHDF1 protein level upon FOXA1 knockdown in 2 PCa cell lines. (L) Western blot to detect the change of 3 m6A mediators upon forced expression of FOXA1, EZH2-WT, or EZH2-H689A in EZH2-deficient PCa cells. (M) The m6A ELISA was conducted to measure the m6A levels in each group of L. (N) Western blot to validate the reexpression of METTL14 and WTAP in EZH2-deficient PCa cells. (O) The m6A ELISA was conducted to measure the m6A levels in each group of N. One-way ANOVA followed by Dunnett’s multiple-comparison test was used for statistical analysis in D, M, and O. Two-tailed Student’s t test was used in J.