PSP94, for prostatic secretory protein of 94 amino acids, is secreted by the prostate gland and functions as a suppressor of tumor growth and metastasis. The expression of PSP94 is lost in advanced, hormone-refractory prostate cancer and this correlates with an increased expression of the Polycomb protein EZH2 (enhancer of zeste homolog 2), which represses transcription via trimethylation of histone H3 on Lys27 (H3K27). We show here that these events are causally related and that the MSMB gene, which encodes PSP94, is trimethylated on H3K27 in androgen-refractory, but not in androgen-sensitive prostate cancer cells. Chromatin immunoprecipitation experiments confirmed an association of EZH2 with the MSMB gene. The RNAi-mediated knockdown of EZH2 resulted in a loss of H3K27 trimethylation and an increased expression of the MSMB gene. Conversely, the overexpression of EZH2 was associated with a decreased expression of the MSMB gene. We also demonstrate that MSMB is additionally repressed in androgen-refractory prostate cancer cells by the hypoacetylation of histone H3K9 and the hypermethylation of a CpG island in the promoter region. Our data disclose a hitherto unexplored link between the putative oncogene EZH2 and the tumor suppressor PSP94, and show that MSMB is silenced by EZH2 in advanced prostate cancer cells.
Introduction, results and discussion
The tumor suppressor PSP94, also known as β-microseminoprotein or prostatic inhibin, is a small (10.7 kDa), non-glycosylated and cysteine-rich protein that is abundantly secreted by the prostate gland and is found in both seminal fluid and blood (Garde et al., 1999; Shukeir et al., 2003; Annahi et al., 2005; Lamy et al., 2006). It is not known how the expression of the PSP94-encoding MSMB gene is regulated. However, it is well established that the expression of PSP94 progressively decreases during the development of prostate cancer from an early, low-invasive, androgen-dependent state to a late, highly invasive, androgen-refractory state (LaTulippe et al., 2002; Vanaja et al., 2003; Stanbrough et al., 2006). The gradual loss of PSP94 is likely to contribute to the development of prostate cancer because PSP94 impedes prostate cancer growth and metastasis (Garde et al., 1999; Shukeir et al., 2003, 2004). The molecular basis for the tumor-suppressor function of PSP94 is complex as this protein has been found to promote tumor cell apoptosis (Garde et al., 1999), to inhibit the secretion of a matrix metalloproteinase that is implicated in tumor metastasis (Annahi et al., 2005), and to decrease tumor-associated, vascular endothelial growth factor (VEGF)-mediated vascularization (Lamy et al., 2006). Interestingly, the anti-tumor effects of PSP94 can be recapitulated with a synthetic peptide comprising an N-terminal fragment of PSP94 and this peptide is currently clinically tested for the treatment of metastatic prostate cancer (Shukeir et al., 2004; Annahi et al., 2005; Lamy et al., 2006).
The Polycomb group proteins (PcG) constitute a cellular memory system that maintains the heritable repression of genes (Ringrose and Paro, 2004; Martin and Zhang, 2005). The Polycomb targets include genes that function in cell proliferation, differentiation and tumorigenesis. Two distinct polycomb repressive complexes (PRC) have been identified. PRC2 is involved in the initiation of silencing and consists in mammals of a core of the proteins EZH2, EED, SUZ12 and RbAP46/48. When complexed to EED and SUZ12, EZH2 functions as a methyltransferase for Lys27 of histone H3 (H3K27). Trimethylated H3K27 serves as a docking site for the PRC1 complex, which maintains gene silencing by various mechanisms, including the inhibition of SWI/SNF-chromatin remodelling complexes and the transcription-initiation machinery.
The PcG protein EZH2 has the hallmarks of an oncogene (Varambally et al., 2002; Bracken et al., 2003). For example, EZH2 is essential for cancer cell proliferation and is overexpressed, often as a result of EZH2 gene amplification, in a wide range of cancers, including hormone-refractory, metastatic prostate cancer (Bracken et al., 2003; Saramäki et al., 2006). One mechanism by which EZH2 could promote tumorigenesis is by the repression of tumor suppressor genes. DNA microarray data revealed that the increased expression of EZH2 in metastatic prostate cancer is correlated with a loss of the tumor suppressor PSP94 (LaTulippe et al., 2002; Stanbrough et al., 2006). We have used quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) to examine whether these changes can also be detected in prostate-derived cell lines. The EZH2 transcript was indeed 4–14-fold overexpressed in the androgen-refractory PC-3 and DU 145 cell lines, as compared to the EZH2 expression level in the androgen-sensitive PZ-HPV-7 and LNCaP cells (Figure 1a). Furthermore, the PSP94 transcript was readily detected in the PZ-HPV-7 and LNCaP cells, but was at least three orders of magnitude less abundant in the PC-3 and DU 145 cells.
Having confirmed an inverse relationship between the transcript levels of EZH2 and MSMB, we subsequently examined whether these changes are causally related and whether the MSMB gene is a target for H3K27 trimethylation. Using a chromatin immunoprecipitation procedure (ChIP) with anti-H3K27me3 antibodies, we found that the MSMB gene in PC-3 cells was heavily trimethylated on H3K27 in nucleosomes that were associated with the promoter region (primer set 2) and with flanking sequences (primer sets 1, 3 and 4) (Figure 1b and c). In contrast, nucleosomes from a fragment of intron 3 of the MSMB gene (primer set 5) were much less trimethylated on H3K27. In these experiments MYT1 (myelin-transcription-factor 1), a well-established Polycomb target gene (Kirmizis et al., 2004), served as a positive control and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) as a negative control. Importantly, whereas the MSMB gene was heavily trimethylated on H3K27 in PC-3 cells, which hardly express PSP94, this gene was only mildly trimethylated on H3K27 in LNCaP cells, which express a lot of PSP94 (Figure 1a and c).
As EZH2 is the major histone methyltransferase known to trimethylate H3K27 in vivo, the above data suggested that the MSMB gene is a target for repression by EZH2. Consistent with this notion, ChIP experiments with anti-EZH2 antibodies showed an association of EZH2 with all the analysed regions of the MSMB gene (Figure 2a). Binding of EZH2 to the MSMB gene was as robust as its binding to the MYT1 gene, a well-known EZH2 target gene. Interestingly, neither MSMB nor MYT1 was abundantly trimethylated on H3K9. As H3K9 trimethylation also correlates with transcriptional repression but is EZH2-independent, these data attest to the specificity of the detected EZH2-H3K27me3 association. Little or no enrichment of GAPDH DNA was observed in the ChIP experiments with the different antibodies (Figure 2a).
To obtain more direct evidence for a role of EZH2 in the transcriptional repression of MSMB, we have subsequently examined the effect of the RNAi-mediated knockdown of EZH2 on the H3K27 trimethylation of the MSMB gene in PC-3 cells. As expected, less EZH2 was associated with the MSMB gene (Figure 2c) following the knockdown of EZH2 (Figure 2b). Within the time frame of the experiment (48 h), the loss was only evident in intron 1 (primer sets 3 and 4) and was not detected in upstream (primer sets 1 and 2) or downstream (primer set 5) sequences. This is reminiscent of the local loss of the association of PRC2 component SUZ12 with the MYT1 gene following the knockdown of SUZ12 (Cao and Zhang, 2004). Importantly, the loss of the targeting of EZH2 to intron 1 was associated with a loss of H3K27 trimethylation in this region (Figure 2d).
We have also investigated whether a change in the level of EZH2 affects the expression of MSMB. In Figure 2e, it is shown that the knockdown of the EZH2 transcript in PC-3 cells by about 70% was associated with a threefold increase in the expression of MSMB. The transcript level of a control housekeeping gene, PPP1R8, was not affected. As the knockdown of EZH2 only affected the targeting of EZH2 to intron 1 (Figure 2c), this suggests that intron 1 harbors important regulatory elements of MSMB expression. Conversely, the overexpression of EZH2, fused to a Gal4-tag, in PZ-HPV-7 cells resulted in a 50% drop of the MSMB transcript level but was without effect on the transcript level of the housekeeping gene HPRT (Figure 2f). Collectively, the above data demonstrate that MSMB is a canonical EZH2 target gene and that repression of MSMB is associated with trimethylation of H3K27.
Polycomb target genes are often additionally silenced through histone deacetylation and DNA methylation of CpG islands (van der Vlag and Otte, 1999; Viré et al., 2006). This is explained by the ability of PcG proteins to bind histone deacetylases and to recruit DNA methyltransferases. We used trichostatin A (TSA), a cell permeable inhibitor of histone deacetylases, to examine whether the MSMB gene is also controlled by histone (de)acetylation. The addition of TSA (50 ng/ml) to PC-3 cells for 9 h indeed resulted in a sixfold increase of the MSMB transcript level (Figure 3a). As TSA did not affect the expression level of EZH2, these data strongly indicate that the MSMB gene is additionally silenced by histone deacetylation. To examine this hypothesis in more detail, we performed ChIP experiments with antibodies against acetylated Lys 9 of Histone H3 (H3K9ac). Three of the four examined regions of the MSMB gene were hypoacetylated in PC-3 cells, as compared to their acetylation status in LNCaPs (Figure 3b). This fits nicely with the decreased expression of the MSMB gene in PC-3 cells (Figure 1a) and is further evidence for a role of deacetylation in the repression of this gene in PC-3 cells.
Finally, we have found that 5′-azacytidine, an inhibitor of DNA methyltransferases, promotes the expression of the MSMB gene in PC-3 cells by about fivefold (Figure 4a), indicating that DNA methylation also contributes to the repression of MSMB. In further agreement with this notion, we found that the MSMB gene harbors two CpG islands (Figure 4b). DNA bisulfite sequencing revealed that these islands are indeed methylated, both in PC-3 and in LNCaP cells. Interestingly, the methylation of the CpG island in the promoter region was significantly more pronounced in PC-3 cells, as compared to its methylation in LNCaP cells, in agreement with the lesser expression of the MSMB gene in PC-3 cells. In addition, the methylation of this CpG island in PC-3 was decreased following the addition of 5′-azacytidine, which is additional evidence that MSMB is controlled by DNA methylation.
In summary, our data firmly establish MSMB as a novel EZH2 target gene. Consistent with this conclusion, Bracken et al. (2006) recently performed a genome-wide screening for PcG targets and included MSMB in the resulting list of candidate-PcG target genes. The identification of MSMB as an EZH2 target gene can explain why the expression of this tumor suppressor gene is lost in advanced stages of prostate cancer. We suggest that the increased expression of EZH2 in metastatic prostate cancer results in H3K27 trimethylation of the MSMB gene. This leads to the recruitment of the PRC1 complex and MSMB silencing. In addition, EZH2 binds to DNA methyltransferases and, indirectly, histone deacetylases and these enzymes also contribute to the maintenance of MSMB silencing. Our data indicate that specific inhibitors of EZH2 could be useful for the treatment of metastatic prostate cancer, at least in part because such inhibitors are expected to reverse the downregulation of the tumor suppressor PSP94.
Annahi B, Bozeghrane M, Currie JC, Hawkins R, Dulude H, Daigneault L et al. (2005). A PSP94-derived peptide PCK3145 inhibits MMP-9 secretion and triggers CD44 cell surface shedding: implications in tumor metastasis. Clin Exp Mestas 22: 429–439.
Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K . (2006). Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes and Dev 20: 1123–1136.
Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K . (2003). EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 20: 5323–5335.
Cao R, Zhang Y . (2004). SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 15: 57–67.
Garde SV, Basrur VS, Li L, Finkelman MA, Krishnan A, Wellham L et al. (1999). Prostate secretory protein (PSP94) suppresses the growth of androgen-independent prostate cancer cell line (PC-3) and xenografts by inducing apoptosis. Prostate 38: 118–125.
Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green R et al. (2004). Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev 18: 1592–1605.
Lamy S, Ruiz MT, Wisniewski J, Garde S, Rabbani SA, Panchal C et al. (2006). A prostate secretory protein 94-derived synthetic peptide PCK3145 inhibits VEGF signalling in endothelial cells: implication in tumor angiogenesis. Int J Cancer 118: 2350–2358.
LaTulippe E, Satagopan J, Smith A, Scher H, Scardino P, Reuter V et al. (2002). Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease. Cancer Res 62: 4499–4506.
Martin C, Zhang Y . (2005). The diverse functions of histone methylation. Nat Rev Mol Cell Biol 6: 838–849.
Ringrose L, Paro R . (2004). Epigenetic regulation of cellular memory by the polycomb and trithorax group proteins. Annu Rev Genet 38: 413–443.
Saramäki OR, Tammela TLJ, Martikainen PM, Vessella RL, Visakorpi T . (2006). The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer. Gen Chrom Cancer 45: 639–645.
Shukeir N, Arakelian A, Chen G, Garde S, Ruiz M, Panchal C et al. (2004). A synthetic 15-mer peptide (PCK3145) derived from prostate secretory protein can reduce tumor growth, experimental skeletal metastasis, and malignancy-associated hypercalcemia. Cancer Res 64: 5370–5377.
Shukeir N, Arakelian A, Kadhim S, Garde S, Rabbani SA . (2003). Prostate secretory protein PSP-94 decreases tumor growth and hypercalcemia of malignancy in a syngenic in vivo model of prostate cancer. Cancer Res 63: 2072–2078.
Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA, Penning TM . (2006). Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res 66: 2815–2825.
van der Vlag J, Otte AP . (1999). Transcriptional repression mediated by the human polycomb protein EED involves histone deacetylation. Nat Genet 23: 474–478.
Vanaja DK, Cheville JC, Iturria SJ, Young CYF . (2003). Transcriptional silencing of zinc finger protein 185 identified by expression profiling is associated with prostate cancer progression. Cancer Res 63: 3877–3882.
Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG et al. (2002). The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419: 624–629.
Viré E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C et al. (2006). The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439: 871–874.
This work was financially supported by the Fund for Scientific Research-Flanders (Grant G.0290.05), a Flemish Concerted Research Action and the Prime Minister's office (IAP/V-05). Fabienne Withof provided expert technical assistance.
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Cite this article
Beke, L., Nuytten, M., Van Eynde, A. et al. The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2. Oncogene 26, 4590–4595 (2007). https://doi.org/10.1038/sj.onc.1210248
- tumor suppressor
- Polycomb group proteins
- prostate cancer PSP94
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