N-Myc-mediated epigenetic reprogramming drives lineage plasticity in advanced prostate cancer

Abstract

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration resistant prostate cancers become androgen receptor (AR) signaling-independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome and interactome using in vivo, in vitro and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR-co-factors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc-induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for EZH2 inhibition to reverse the N-Myc-induced suppression of epithelial lineage genes. Altogether, our data provide insights on how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage-specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

Keywords: Epigenetics; Genetics; Oncology; Prostate cancer.

Figure 1. Clinical NEPC is associated with neural lineage.

(A) Top: Enrichment plots of the Neural Stem Cell Markers and Lee Neural Crest Stem Cell Up gene sets between indicated groups. Bottom: Targeted GSEA in the 5 NEPC samples with the highest (N-Myc^hi) or lowest (N-Myc^lo) level of MYCN expression versus PCa (n = 66) patient samples, NEPC N-Myc^hi versus NEPC N-Myc^lo, and on the 5 CRPC with the highest level of MYCN expression versus the 5 lowest. *FDR q value < 0.05, **FDR q value < 0.01, ***FDR q value < 0.001. (B) Kaplan-Meier plots of CRPC (n = 57) patients, NEPC (n = 24) patients, or CRPC plus NEPC (n = 81) patients, stratified into 2 categories according to the median value of MYCN mRNA expression. Survival analysis was performed using the Kaplan-Meier estimator (log-rank test). qNSC, quiescent NSC.

Figure 2. AR signaling alters the N-Myc transcriptome in vivo.

(A) Photomicrographs of H&E staining, vimentin (VIM), NCAM1, AR, cytokeratin 8 (KRT8), and 5 (KRT5) immunohistochemistry, and MYCN RNA in situ hybridization (RNAish) on primary prostate tumor region enriched with sarcomatoid differentiation (left) or liver metastatic lesion (right) from Pb-Cre^+/– Pten^fl/fl LSL-MYCN^+/+ mice 6 months after castration. Scale bars: 50 μm. (B) Photomicrographs of H&E staining or immunohistochemical staining for epithelial markers (AR and KRT8), a mesenchymal marker (VIM), or neural/ganglionic marker (S100) on 4-μm serial sections from mouse C1 (Supplemental Figure 2). Dotted lines indicate conventional adenocarcinoma adjacent to neural/ganglionic cells. Arrows indicate VIM⁺S100⁺ tumor cells that have invaded local vasculature. Scale bars: 50 μm. (C) Top: N-Myc signatures defined from 22Rv1-N-Myc xenografts versus 22Rv1 control (CTL) xenografts (–1 < log₂[fold change] < 1, adj. P value < 0.05, n = 3 biological replicates per condition). Bottom: GSEA analysis results comparing N-Myc castrated tumors versus the other 3 groups of tumors. FC, fold change.

Figure 3. N-Myc expression leads to neural lineage gene expression and reduced androgen response.

(A) Experimental schematic with LNCaP-N-Myc or CTL cells and corresponding time points for RNA-seq or ChIP-seq analyses (arrows) in the presence (+A, green) or absence (–A, red) of androgen. (B) Enrichment plots for the Androgen Response and the Neural Stem Cell Differentiation Pathways and Lineage-specific Markers gene sets from indicated conditions. (C) Gene expression (fragments per kilobase of transcript per million mapped reads, FPKM) of AR target genes (left) and neural lineage–associated genes (right) measured by RNA-seq in the indicated cells and conditions, on day 4 (D4, n = 3 biological replicates) and day 42 (D42, n = 2 biological replicates) of androgen withdrawal. *P < 0.05, **P < 0.01, Sidak-Bonferroni–adjusted 2-tailed t test. NS, not significant. (D) Targeted GSEA of RNA-seq data from LNCaP-N-Myc versus CTL cells, without androgen, at D4 or D42 as indicated. *FDR q value < 0.05, **FDR q value < 0.01, ***FDR q value < 0.001. (E) Number of genes differentially expressed (adj. P value < 0.05) in the indicated conditions. ASC, adult stem cell; qNSC, quiescent NSC.

Figure 4. The N-Myc cistrome is distinct from C-Myc and is altered by androgen signaling.

(A) Left: Distributions and heatmaps of N-Myc ChIP-seq data generated from cells on day 4 (D4). Right: Proportion of N-Myc–bound sites at the indicated genomic annotation. (B) Overlap of Myc family member binding in prostate cancer cells (LNCaP, top) or N-Myc in prostate cancer cells versus published N-Myc in neuroblastoma (NB) cells (LNCaP/BE2C, bottom). (C) Examples of ChIP-seq tracks for indicated genes. (D) GSEA performed on the uniquely bound genes as identified in B. (E) Representation of N-Myc binding sites determined by ChIP-seq in LNCaP-N-Myc cells with and without androgen (left) or in 22Rv1 xenografts grown in castrated or intact recipients (right), and their distribution throughout the genome (n = 2 biological replicates per condition). com., common; enr., enriched; uni., unique.

Figure 5. N-Myc interacts with known AR cofactors to alter DNA binding.

(A) Motif analysis of unique N-Myc peaks with or without androgen obtained by ChIP-seq in LNCaP-N-Myc cells. Scores correspond to log₂(% target/% background). All motifs shown are enriched with a P value < 10^–5 and are listed with their best predicted match to a known protein family. *Percentage background of MEF2A motif was 0% and was subsequently adjusted to 0.001% to calculate a score. (B) Overlap between FOXA1 or HOXB13 ChIP-seq peaks in the LNCaP-N-Myc cells in the presence or absence of androgen on day 4. (C) Comparison of N-Myc binding with AR (GSE69045), FOXA1 (CTL cells: GSE69045) and HOXB13 binding with or without androgen, N-Myc in BE2C neuroblastoma cells (GSE80151), and C-Myc in LNCaP. Numbers represent the percentage of N-Myc peaks in each condition overlapping with the indicated cofactor. (D) Overlap of N-Myc peaks (enriched and unique in –A) with AR, HOXB13, or FOXA1 peaks in the indicated conditions. (E) Distribution of FOXA1 and HOXB13 binding at N-Myc–bound sites ± 4 kb in the indicated conditions. (F) Top: ChIP-seq tracks of genes cobound by N-Myc and FOXA1, independently of AR (in CTL cells), in the indicated conditions. Bottom: Effect of FOXA1 knockdown by siRNA (see Western blot inset) on N-Myc binding assessed by ChIP-qPCR. **P < 0.01 by Sidak-Bonferroni–adjusted 2-tailed t test. (G) Scatter plot of log₂(fold change of N-Myc–bound peptides versus IgG-bound peptides, identified by RIME) with (x axis) and without (y axis) androgen (n = 4 biological replicates per condition). Lines correspond to the regression line ± 1.7 Z. com., common; enr., enriched; uni., unique; FC, fold change; NB, neuroblastoma.

Figure 6. N-Myc promotes bivalency on neural lineage genes.

(A) H3K27me3 binding profiles within 8 kb centered at H3K4me3 peaks in LNCaP CTL and N-Myc cells, with and without androgen as specified. (B) Left: Number of H3K4me3, H3K27me3, or H3K4me3/H3K27me3 bivalent peaks in common (com.) or unique to the conditions on day 4 (D4) as indicated. Right: Top 5 gene sets from GSEA for uniquely bivalently marked genes in the absence of androgen from LNCaP-N-Myc cells (red) or CTL cells (black). (C) Left: Number of H3K4me3, H3K27me3, and bivalent peaks also bound by N-Myc with or without androgen. Right: H3K4me3, H3K27me3, and N-Myc binding profiles on bivalent peaks within 8 kb centered at H3K4me3 peaks, in LNCaP-N-Myc cells in the absence of androgen. (D) Examples of N-Myc and histone mark ChIP-seq tracks as indicated. (E) Enrichment plot of the bivalent genes identified in LNCaP-N-Myc –A cells on D4 measured in LNCaP-N-Myc –A cells versus LNCaP-CTL –A cells on D42. (F) Unsupervised clustering of the genes that were bivalent on D4 and differentially expressed (adj. P < 0.05) between LNCaP-N-Myc and LNCaP-CTL cells without androgen on D42.

Figure 7. N-Myc–induced bivalent genes are clinically relevant.

(A) Left: Unsupervised clustering of PCa (n = 66), CRPC (n = 73), and NEPC (n = 36) patient samples based on the expression level of the 966 bivalent and N-Myc–bound genes in LNCaP-N-Myc cells on day 4 (D4) without androgen. Right: NEPC score for each cluster group. Graph depicts the median value between the 25th and 75th percentiles, with whiskers indicating the range within 1.5 IQR, Student’s unpaired 2-tailed t test. *P < 1 × 10^–3, **P < 1 × 10^–5, ***P < 1 × 10^–10. (B) Targeted GSEA of bivalent-related gene sets in the 5 NEPC samples with the highest (N-Myc^hi) or lowest (N-Myc^lo) level of MYCN expression versus PCa (n = 66) patient samples, on the 5 CRPC with the highest level of MYCN expression versus the 5 lowest, and on PM154 cells treated with an EZH2 inhibitor versus vehicle. *FDR q value < 0.05, **FDR q value < 0.01, ***FDR q value < 0.001. (C) Heatmap of log₂(fold change) of genes in NEPC (n = 36) versus PCa (n = 66) or NEPC versus CRPC (n = 73) patient samples. Illustrated genes are bivalent and bound by N-Myc in LNCaP-N-Myc cells without androgen at D4. (D) Fold change expression of the indicated genes based on qRT-PCR data (n = 3 technical replicates) in PM154 following EZH2 knockdown (see Western blot inset). ***P < 0.001 by Sidak-Bonferroni–adjusted 2-tailed t test. FC, fold change.