Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

Abstract

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

Keywords: Androgen receptor; DNA methylation; EZH2; epigenetics; histone methylation; neuroendocrine; prostate cancer; small-cell prostate carcinoma; xenograft.

Figure 1.

A. Unsupervised hierarchical clustering (Ward linkage, Euclidean distance) using all the MCAM M values for the 16,621 SmaI sites after LOWESS normalization. Black boxes group single patient xenograft sublines, whereas the asterisks indicates a xenograft subline that does not cluster with sublines from the same patient donor. B. Frequency of methylated SmaI fragments, normalized by the fraction of the gene, across all samples according to their relationship to CGIs and to the promoter region (±1 kb of TSS) of known Reference Sequence genes. C. Unsupervised hierarchical clustering (Ward linkage, Euclidean distance) using all β-values from the Illumina 450K array of six xenografts and their corresponding patient donor. D. Frequency of methylated probes, normalized by the fraction of the gene, total and by compartment, for primary patient samples and xenografts evaluated on Illumina 450K arrays.

Figure 2.

A. Unsupervised hierarchical clustering using MCAM probes with at least two methylated samples (resulting in 3,031 probes) segregated the tumors into three groups. The black boxes indicate AR⁻ xenografts. B. Differentially methylated sequences identified by MCAM using the Marker Selection tool (GENE-E software) validated by pyrosequencing the differentially methylated sequences identified by MCAM. C. The methylation levels of the validated genes in the patient DNA samples. Statistical significance based on Student t-test is indicated by asterisk marks. (*) represents P< 0.05 and (**) represents P < 0 .005.

Figure 3.

A. Map showing the sequence location of the pyrosequencing and ChIP q-PCR primers. B. MCAM M-values at the AR promoter in patient derived xenografts (AR-negative, dark gray; AR-positive, light gray). C. Frequency of AR promoter-associated methylation in prostate cancer patient derived xenografts and cell lines (AR-negative black, AR-positive gray) using bisulfite pyrosequencing.

Figure 4.

AR promoter histone modification using ChIP-PCR for repressive marks (H3K27me3 and H3K9me2) in gray and active histone marks (H3K4me3 and H3K9ac) in black were assayed using primers covering two regions of the AR exon 1 as shown in Fig. 3A. Shown here is the average for both regions. HBB and ACTB were used as repressive and active mark controls respectively. The y-axis indicates fold enrichment compared to total histone H3 using the CT method.

Figure 5.

A. Global levels of AR, EZH2 and H3K27me3 by protein gel blot analysis in AR⁺ cell line C42B and xenograft 180 and AR- SCPC cell line NCI H660 and xenografts (144-13, 146-10, 155-2). B. Western blot analysis of the AR, EZH2, and H3K27me3 protein following 72 hDZNep treatment in 144-13 and NCIH660 cell lines. C. Percent of viable cells after 72 h of DZNep treatment as compared to vehicle.