EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent

Abstract

Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Fig. 1. Overexpression of EZH2-stimulated genes in clinical CRPC samples

(A) Immunoblot of nuclear extracts from LNCaP and abl cells treated without (-) or with (+) 5α-dihydrotestosterone (DHT) to control for effects of cell growth on protein expression. EED isoforms are numbered (22). (B) Growth of cells transduced with lentiviral shRNAs targeting scrambled control (shCtrl) or EZH2 (shEZH2#1 and #4). (C) Tumor growth curve of castrated male scid mice injected with CWR22Rv1 cells with or without EZH2 silencing. (D) Clustering of the union of differentially expressed genes in LNCaP and abl after transfection with siRNAs against control (siCtrl) or EZH2 (siEZH2). (E and F) Heat map of expression levels (E) and box plots of Pearson correlation coefficients (PCC) (F) of EZH2-repressed (top) and -stimulated (bottom) genes with EZH2 level in the Varambally cohort. Nor., normal tissues; PCA, primary tumors. *, EZH2 level.

Fig. 2. EZH2 binding without H3K27me3 is associated with gene activation in CRPC

(A) Heat maps of EZH2 and H3K27me3 ChIP-seq signal ±1 kb around the EZH2 peak summit in LNCaP and abl. The color scale indicates average signal using a 10 bp window. The numbered index of EZH2 peaks is shown to the left. (B) Heat maps of EZH2, H3K27me3, H3K4me2, H3K4me3, and PolII ChIP-seq signal ±1 kb around EZH2 solo or ensemble peak summit in abl. (C) Percentages of differentially expressed genes upon EZH2 depletion in LNCaP or abl cells containing EZH2 solo or ensemble peaks within 20 kb around transcription start sites (TSS). (D) Kaplan Meier plots of EZH2 directly activated genes in the Yu cohort. The number of genes is indicated at the top of each plot. n, numbers of patients.

Fig. 3. Requirement for the methyltransferase activity and interaction with AR for the EZH2 transactivation function

(A) Immunoblot of nuclear extracts from LNCaP and abl cells after gel filtration fractionation. Molecular mass standards are indicated. (B) Box plots of minimum to maximum RT-PCR values for EZH2-activated genes in abl after replacement with wild-type or mutant EZH2 as indicated. (C and D) Growth of abl (C) and LNCaP (D) in hormone-depleted medium, following replacement with wild-type or mutant EZH2 as indicated. (E) Co-immunoprecipitation of EZH2 and AR in LNCaP and abl without (-) or with (+) DHT.

Fig. 4. Critical role of EZH2 phosphorylation at S21 for its functional switch

(A) Immunoprecipitation in LNCaP and abl cells using IgG or EZH2 antibodies, followed by immunoblotting with indicated antibodies. (B) Box plots of ChIP-qPCR values for EZH2 recruitment to selected ensemble or solo sites using phosphorylation-specific antibodies. (C) Growth of abl cells in androgen-depleted medium after replacement with wild-type or mutant EZH2 as indicated. (D) Immunoblot of nuclear extracts from abl cells after gel filtration fractionation. Molecular mass standards are indicated. (E) Analysis of EZH2, S21 phosphorylated EZH2 [pEZH2(S21)] and H3K27me3 protein levels by quantitative immunohistochemistry in neoadjuvant prostate tumors (PCA) and CRPC.