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. 2021 Jan 5;12(1):46.
doi: 10.1038/s41419-020-03326-8.

Loss of EHF facilitates the development of treatment-induced neuroendocrine prostate cancer

Zhi Long  1 , Liang Deng  1 , Chao Li  1 , Qiangrong He  1 , Yao He  2 , Xiheng Hu  2 , Yi Cai  2 , Yu Gan  3
Affiliations

Loss of EHF facilitates the development of treatment-induced neuroendocrine prostate cancer

Zhi Long et al. Cell Death Dis. .

Abstract

The rising of a highly aggressive subtype of castration-resistant prostate cancer (CRPC) named treatment-induced neuroendocrine prostate cancer (t-NEPC) after androgen deprivation therapy (ADT) is well known for its features of the neuroendocrine differentiation (NED) and androgen receptor (AR) independence. However, t-NEPC is still largely unknown. Here, we found that EHF is notably depressed in t-NEPC tumors, patient-derived xenografts, transgenic mice, and cell models. Results from cell lines uncovered that ADT represses EHF expression, which is required for the ADT-induced NED. Mechanism dissection revealed that ADT decreases the EHF transcription via relieving the AR binding to different androgen-responsive elements, which then promotes the expression and enzymatic activity of enhancer of zeste homolog 2 (EZH2), consequently catalyzing tri-methylation lysine 27 of histone H3 for transcriptional repression of its downstream genes to promote the NED. Furthermore, preclinical studies from cell and mice models proved that recovery of EHF expression or using EZH2 inhibitor can attenuate aggressive properties of CRPC cells, hinder the progression of t-NEPC, and promote the response of CPRC cells to enzalutamide. Together, we elucidate that the ADT/AR/EHF/EZH2 signaling is required for the ADT-enhanced NED and plays a critical role in the progression of t-NEPC.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. EHF expression is downregulated in t-NEPC.
A EHF mRNA expression of tumor tissues derived from Michigan 2012, SU2C/PCF 2015, and Aggarwal et al. clinical prostate cancer cohorts was plotted. B EHF expression in tumor or prostate tissues from t-NEPC, CRPC-Ad, AdPC, and BPH patients was measured by immunohistochemistry. Scale bar 100 μm. C EHF mRNA expression from the GEMM models was plotted. D EHF, AR, SYP, and SOX2 mRNA expressions during the progression of AdPC (LTL-331) to t-NEPC (LTL-331R) by castration surgery to the host mice were plotted. E AR, EHF, ENO2, and CHGA protein expressions in LNCaP, C4-2, 22Rv1, DU145, PC-3, and NCI-H660 cell lines were measured by immunoblotting. F ENO2 and CHGA levels in LNCaP cells with/without EHF knockdown were measured by qPCR and immunoblotting. G ENO2 and CHGA levels in PC-3 cells with/without EHF overexpression were measured by qPCR and immunoblotting. H GSEA revealed the enrichment of the “EHF low expression-specific genes” signature into the transcriptome of t-NEPC tumors, t-NEPC PDXs, and NEPC DuNE cell line in comparison with their prostate adenocarcinoma counterparts. All qPCR and immunoblotting assays were repeated in triplicate. The two-tailed Student’s t test or one-way ANOVA, followed by Dunnett’s t test was used to compare results between two groups with ** denoting p < 0.01. Bar graphs show means ± SD. AdPC localized prostate adenocarcinoma tumors, mCRPC metastatic castration-resistant prostate cancer, Ad adenocarcinoma, t-NEPC treatment-induced neuroendocrine prostate cancer, t-SCNC-e treatment-induced small cell neuroendocrine cancer-enriched, PCa prostate cancer, BPH benign prostate hyperplasia, TCGA The Cancer Genome Atlas, HE hematein–eosin, SKO/WT wild-type and single Pten loss, TKO/DKO Pten plus Trp53 double loss and Pten, Trp53 plus Rb1 loss, NE neuroendocrine, PDXs patient-derived xenografts, n.s. not significant.
Fig. 2
Fig. 2. ADT-induced EHF depression is important for NE differentiation.
A Bar graph showing EHF expression (left) and heatmap of AR-associated genes including EHF, t-NEPC markers, and an androgen-depressed gene in long-term androgen-deprived LNCaP cells (GSE8702). B, C Effects of androgen deprivation (by culturing in CSS medium) and AR ligand R1881 on EHF expression in LNCaP cells were measured by qPCR and immunoblotting. P values for each sample were obtained based on nontreated controls (*) and cells cultured in CSS medium for 5 days (#) or for 7 days (γ) for EHF and PSA, respectively. D, E Effects of ENZ on EHF expression in C4-2 cells were determined by qPCR and immunoblotting. P values for each sample were obtained based on nontreated controls (*) for EHF and PSA. F EHF, ENO2, and CHGA protein levels in LNCaP cells with/without EHF overexpression responsive to androgen deprivation (by culturing in CSS medium for 48 h) were measured by immunoblotting. G EHF, ENO2, and CHGA protein levels in C4-2 cells with/without EHF overexpression responsive to ENZ treatment for 48 h were measured by immunoblotting. All qPCR and immunoblotting assays were repeated in triplicate. The two-tailed Student’s t test or one-way ANOVA, followed by Dunnett’s t test was used to compare results between two groups with * denoting p < 0.05, **/##/γγ denoting p < 0.01, and ***/###/γγγ denoting p < 0.001. Bar graphs show means ± SD. AAG AR-associated genes, M markers, ADG androgen-depressed gene, AD androgen deprivation, CTL control, CSS charcoal-stripped serum, ENZ enzalutamide, n.s. not significant.
Fig. 3
Fig. 3. AR directs transcriptional activation of EHF in PCa.
A ChIP-Seq profiles in androgen-stimulated LNCaP cells (GSE55007) indicating AR enrichment on four genomic sites (peaks 1–4) located in the EHF’s first intron region. B Schema showing AR-binding motif obtained from the JASPAR database (top). Bottom panel showing two putative direct AR-binding sites selected from the DNA sequences represented by peaks 1–4. C ChIP-qPCR data showing recruitment of AR on the EHF’s first intron upon R1881 (10 nM) stimulation in LNCaP cells. D Same condition as in C, except H3K9Ac marks on the EHF’s first intron. E ChIP-qPCR data indicating enrichment of AR on the EHF’s first intron in R1881 (10 nM)-stimulated C4-2 cells with/without ENZ treatment (10 μM). PSA (KLK3) promoter was employed as a positive control for R1881 or ENZ treatment (C, D). F Transcriptional activation ability of ARE1 and ARE2 as indicated by increased luciferase reporter activity. Luminescence units were normalized by Renilla luciferase signal. G Transcriptional inhibitory function of ARE mutants (sequences are presented) was indicated by dual-luciferase reporter assay. Nucleotides in red are sequences of consensus ARE1, mutated ARE1, and deleted ARE1, respectively. The two-tailed Student’s t test or one-way ANOVA, followed by Dunnett’s t test was used to compare results between two groups. Experiments were performed with three biologically independent samples. Bar graphs show means ± SD. ARE androgen-responsive element, H3K9Ac H3 lysine 9 acetylation.
Fig. 4
Fig. 4. EZH2 is essential for NE phenotype induced by EHF loss.
A GSEA analysis indicated that EZH2 might function as a downstream effector molecule of EHF in prostate cancer. B EHF, EZH2, H3K27me3, and H3 protein expressions in LNCaP cells with/without EHF knockdown and PC-3 cells with/without EHF overexpression were measured by immunoblotting. C The mRNA level of three known EZH2-repressed targets (SLIT2, DABIP, and ADRB2) was measured by qPCR in LNCaP cells with/without EHF knockdown. D EHF, EZH2, ENO2, SOX2, H3K27me3, and H3 protein levels were measured by immunoblotting in CSS cultured LNCaP cells with/without GSK126 treatment (5 μM) or EZH2 knockdown. E Same tests as in D, except that measured in ENZ-treated (10 μM) C4-2 cells with/without GSK126 treatment or EZH2 knockdown. The two-tailed Student’s t test was used to compare results between two groups with * denoting p < 0.05 and ** denoting p < 0.01. All qPCR and immunoblotting assays were repeated in triplicate. Bar graphs show means ± SD. DN down, H3K27me3 tri-methylation lysine 27 of histone H3, H3 histone H3.
Fig. 5
Fig. 5. EHF knockdown promotes the aggressive properties of PCa cells.
A MTS assays measured cell proliferation of PC-3 cells with/without EHF overexpression and PC-3 cells with EHF and EZH2 overexpression. B Same cells as in (A) were used to perform colony formation assays. Representative images are shown. C Same cells as in (A) were used to perform cell invasion and migration assays in transwell chambers with/without coating with Matrigel. Representative images are shown. Scale bar 100 μm. D C4-2 cells with/without EHF knockdown were treated with/without ENZ (10 μM) and GSK126 (5 μM), and cell proliferation was determined by MTS assays. E Colony formation assays were performed in the same context as in D. Representative images are shown. F Cell invasion and migration assays were performed in the same context as in D. Representative images are shown. Scale bar 100 μm. One-way ANOVA, followed by Dunnett’s t test was used to compare results between two groups with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Experiments were performed with three biologically independent samples. Bar graphs show means ± SD. OD optical density.
Fig. 6
Fig. 6. EHF loss contributes to the progression of CRPC and NE differentiation in mice.
A Growth of tumor xenografts in male nude mice 25 days after subcutaneous inoculation with PC-3 cells with/without EHF overexpression. n = 4 mice per group. B Representative tumor images in (A). C EHF, EZH2, H3K27me3, and H3 protein expressions from the xenografts in (A) were measured by immunoblotting. D Ki-67, ENO2, and SOX2 expressions from the xenografts in (A) were measured by immunohistochemistry. Scale bar 100 μm. E Growth of tumor xenografts in male nude mice treated with/without ENZ (30 mg/kg, intraperitoneal injection, every other day) 25 days after subcutaneous inoculation with C4-2 cells with/without EHF knockdown. n = 4 mice per group. F Representative tumor images in (E). G Same proteins in (C) from the xenografts in (E) were measured by immunoblotting. (H) Same markers in (D) from the xenografts in (E) were measured by immunohistochemistry. Scale bar 100 μm. The two-tailed Student’s t test or one-way ANOVA, followed by Dunnett’s t test was used to compare results between two groups with *** denoting p < 0.001. Immunoblotting assays were repeated in triplicate. Bar graphs show means ± SD.
Fig. 7
Fig. 7. Proposed model of EHF loss-mediated therapeutic resistance and NE differentiation of prostate cancer.
AR androgen receptor, ADT androgen deprivation therapy, PRC2 polycomb repressive complex 2, t-NEPC treatment-induced neuroendocrine prostate cancer, AdPC prostate adenocarcinoma, NE neuroendocrine.
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