Myc enforces overexpression of EZH2 in early prostatic neoplasia via transcriptional and post-transcriptional mechanisms

Abstract

EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.

Figure 1. Elevated EZH2 Expression in the Lo-MYC Murine Model of Prostate Cancer

(A) Increased EZH2 protein expression in the ventral prostates of Lo-MYC mice (right panel), as compared to wild type controls (left panel). (B) Semi-quantitative image analysis of EZH2 protein expression in 9 wild type and 14 Lo-MYC mice. EZH2 expression was elevated in PIN, cribriform PIN/CIS, and invasive adenocarcinoma lesions, as compared to normal prostate. (C) EZH2 mRNA is elevated in PIN lesions from the ventral prostates of Lo-MYC (6 mice), as compared to ventral prostates from age-matched wildtype FVB mice (3 mice).

Figure 2. Correlation between MYC and EZH2 expression in primary prostate cancer specimens

(A) Western blot showing increased EZH2 protein expression in localized prostate cancer specimens, as compared to matched normal tissue. (B) Elevated EZH2 mRNA levels in localized prostate cancer specimens, as compared to matched normal tissue, determined by quantitative real-time PCR. (C) Positive correlation between the expression of MYC and EZH2 mRNA levels.

Figure 3. Myc binds to the EZH2 promoter region and activates EZH2 transcription in prostate cancer cell lines

(A) Reduced EZH2 protein (Left Panel) and mRNA (Right Panel) expression following siRNA-mediated MYC depletion. (B) Enrichment of Myc binding at the E-box-containing promoter region of EZH2 in 2 prostate cancer cell lines, as determined by ChIP. Immunoprecipitation was carried out with an anti-Myc antibody (black bars) and a control IgG antibody (white bars). (C) Decreased EZH2 promoter activity after MYC depletion (black bars), as compared to control cells (white bars) in human prostate cancer cell lines.

Figure 4. Myc Regulates miR-26a and miR-26b in Human and Murine Prostate Cancer Cells

(A) (Upper Panel) Induction of miR-26a, its primary forms miR-26a1 and miR-26a2, and the genes in which they are embedded, CTDSPL and CTDSP2, following siRNA-mediated Myc depletion. (Lower Panel) Induction of miR-26b, its primary form, and the gene in which it is embedded, CTDSP1. (B) Enriched Myc binding was observed at the promoter regions of CTDSPL, CTDSP2 and CTDSP1. Immunoprecipitation was carried out with an anti-Myc antibody (black bars) and a control IgG antibody (white bars). (C) Reduced miR-26a (Left Panel) and miR-26b (Right Panel) expression in the Lo-MYC mice, as compared to wildtype controls. (D) Induction of miR-26a and miR-26b after Myc depletion in MYC-CaP cells.

Figure 4. Myc Regulates miR-26a and miR-26b in Human and Murine Prostate Cancer Cells

(A) (Upper Panel) Induction of miR-26a, its primary forms miR-26a1 and miR-26a2, and the genes in which they are embedded, CTDSPL and CTDSP2, following siRNA-mediated Myc depletion. (Lower Panel) Induction of miR-26b, its primary form, and the gene in which it is embedded, CTDSP1. (B) Enriched Myc binding was observed at the promoter regions of CTDSPL, CTDSP2 and CTDSP1. Immunoprecipitation was carried out with an anti-Myc antibody (black bars) and a control IgG antibody (white bars). (C) Reduced miR-26a (Left Panel) and miR-26b (Right Panel) expression in the Lo-MYC mice, as compared to wildtype controls. (D) Induction of miR-26a and miR-26b after Myc depletion in MYC-CaP cells.

Figure 5. MiR-26a and miR-26b Regulate EZH2 Expression in Human and Murine Prostate Cancer Cells

(A,B) MiR-26a and miR-26b repress EZH2 protein (A) and mRNA (B) expression. (C) miR-26a and miR-26b bind to the 3′UTR of EZH2 in prostate cancer cells. (D) Reduced proliferation of prostate cancer cell lines after transfection with miR-26a (dotted lines) and miR-26b (dashed lines), as compared to control miR (solid lines).

Figure 5. MiR-26a and miR-26b Regulate EZH2 Expression in Human and Murine Prostate Cancer Cells

(A,B) MiR-26a and miR-26b repress EZH2 protein (A) and mRNA (B) expression. (C) miR-26a and miR-26b bind to the 3′UTR of EZH2 in prostate cancer cells. (D) Reduced proliferation of prostate cancer cell lines after transfection with miR-26a (dotted lines) and miR-26b (dashed lines), as compared to control miR (solid lines).

Figure 6. MiR-26a Expression in Primary Prostate Cancer Specimens

(A) Reduced miR-26a expression in primary prostate cancer cases, normalized to matched benign prostate tissue. Inverse correlations were observed between MYC and miR-26a/b (B), as well as miR-26a/b and EZH2 (C).

Figure 7. Model of Regulation of EZH2 by Myc by Two Distinct Mechanisms in Prostate Cancer

(A) In normal prostate luminal cells, Myc protein expression is low. Myc-induced EZH2 transcription occurs at a low level, resulting in basal expression levels of EZH2. Since MYC represses the transcription of CTDSPL, CTDSP2 and CTDSP1, when MYC levels are low, these genes, which harbor miR-26a and miR-26b, are actively transcribed. MiR-26a and miR-26b are incorporated into the RISC complex and bind specifically to their complementary site on the 3′ UTR of EZH2, destabilizing EZH2 mRNA and repressing its translation. As such, EZH2 is maintained at a low level by transcriptional and post-transcriptional regulatory mechanisms. (B) In prostate cancer, elevated Myc levels drive EZH2 overexpression. MYC binds to the E-box-containing promoter region of EZH2 and activates its transcription. Concurrently, MYC represses CTDSPL, CTDSP2 and CTDSP1, in which miR-26a and miR-26b are embedded. As such, miRNA-mediated post-transcriptional silencing of EZH2 mRNA is reduced. This results in elevated EZH2 protein expression.