The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2

Abstract

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.

Figure 1
figure1

MSMB and EZH2 expression in prostatic epithelial cells and trimethylation of the MSMB gene on H3K27. (a) The relative amounts of EZH2 (left panel) and MSMB transcripts (right panel) in the indicated prostate cell lines were determined by quantitative RT–PCR with intron-spanning primers. Human prostate PC-3 cells (adenocarcinoma), LNCaP cells (carcinoma) and DU 145 cells (carcinoma) were cultured as monolayers in 50% Dulbecco's modified Eagle's medium (DMEM) and 50% Ham's F12, RPMI1640 and DMEM, respectively, supplemented with 10% fetal calf serum. PZ-HPV-7 cells, an immortalized cell line derived from normal human prostate cells, were cultured in keratinocyte-serum free medium supplemented with 5 ng/ml human recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract. Total RNA was isolated using the Genelute Mammalian Total RNA Miniprep kit (Sigma, St. Louis, MO, USA). A total of 1–5 μg RNA was reverse-transcribed with oligo dT primer (Sigma) and the M-MulV reverse transcriptase (Fermentas GMBH, St. Leon-Rot, Germany). cDNA (1.5%) was analysed by real-time PCR in triplicate using a Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Paisly, UK) in a Rotorgene detection system (Corbett Research, Cambridge, UK) and normalized to the housekeeping gene hypoxanthine-guanine-phosphoribosyl-transferase (HPRT). All used primer sequences are available on request. The data represent the means±s.e. of at least three independent experiments. (b) Schematic representation of the MSMB gene on scale. The four exons are indicated by the black boxes. The black lines below the MSMB locus represent the fragments (1–5) amplified by ChIP analysis. (c) ChIPs on PC-3 (left panel) and LNCaP (right panel) cells were performed with 10 μg of control antibodies (rabbit anti-mouse IgGs, Dakocytomation, Gostrup, Denmark) or 5 μg of antibodies against H3K27me3 (polyclonal anti-H3K27, Upstate, Dundee, UK). ChIP reactions were performed according to the protocol of Upstate, Dundee, UK. The DNA was recovered with the Genelute PCR clean-up kit (Sigma) and analysed by real-time PCR. Numbers 1–5 refer to the MSMB fragments that were amplified (see panel b). The data represent the means±s.e. of at least three independent experiments in duplicate and indicate the fold enrichment as compared to the negative control (IgGs). The enrichment of DNA was calculated using the formula: fold enrichment=2(CtIgG–CtAb), where Ct is the threshold cycle, IgG is the normal rabbit IgG and Ab is the specific antibody.

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).

Figure 2
figure2

EZH2 is recruited to the MSMB gene and causes trimethylation of H3K27. (a) ChIP assays were performed in PC-3 cells using rabbit antibodies against a synthetic peptide of human EZH2 (Viré et al., 2006), trimethylated H3K27 (Upstate), trimethylated H3K9 (Upstate) and control IgGs. ChIP results were revealed by EtBr staining of agarose gels containing PCR-amplified ChIP DNA. (b) Immunoblotting (upper panel) and quantitative RT–PCR analysis (lower panel) of PC-3 cell lysates, obtained after transfection with either a control siRNA (Ctr), that is, a scrambled version of a siRNA duplex for the housekeeping gene PPP1R8 (GGAACUCGAACCUCCACGAACAAUU, Invitrogen) or an EZH2 siRNA (KD; AAGACUCUGAAUGCAGUUGCU, Dharmacon, Chicago, IL, USA). The PC-3 cells were plated at 1.2 × 106 cells in a 10 cm plate. At 24 h after plating, the cells were transfected with 300 nM of siRNA duplex using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, the cells were harvested. SIPP1 served as a loading control for the immunoblotting and ACTIN was used as control for normalization in the quantitative RT–PCR. (c and d) ChIP assays were performed on chromatin obtained from PC-3 cells transfected with either control (Ctr) or EZH2 siRNAs, using control antibodies (rabbit IgGs), antibodies against EZH2 (c) or antibodies against H3K27me3 (d). The immunoprecipitated DNA was analysed by quantitative PCR using primers specific for the MSMB gene (Figure 1b). The enrichment on MSMB is expressed as a %±s.e. of the control value (n=3–4). (e) PC-3 cells were transfected with control (A lamin A/C siRNA duplex (AACUGGACUUCCAGAAGAACA, Dharmacon) or EZH2 siRNAs for 48 h. The steady-state transcript levels of MSMB, EZH2 and the housekeeping gene PPP1R8 were determined by quantitative RT–PCR analysis with intron-spanning primers specific for the indicated genes and were expressed relative to the transcript level in the control condition. ACTIN was used as a control for normalization. (f) PZ-HPV7 cells were transiently transfected with an expression vector encoding either Gal4-tag alone or its fusion with EZH2 using lipofectamine plus (Invitrogen), according to manufacturer's instructions. The analysis was carried out as described in (e), with the housekeeping gene HPRT as negative control.

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.

Figure 3
figure3

The MSMB gene is regulated by histone (de)acetylation. (a) PC-3 cells were treated for 9 h with 50 ng/ml of the histone deacetylase inhibitor trichostatin A (TSA, Sigma). Subsequently, the steady-state levels of the MSMB and EZH2 transcripts were determined by quantitative RT–PCR analysis with intron-spanning primers specific for the indicated genes. The data are expressed relative to the transcript level in the control condition. HPRT was used as a control for normalization. (b) ChIPs on PC-3 (left panel) and LNCaP (right panel) cells were performed with 10 μg of control antibodies (rabbit anti-mouse IgGs, Dakocytomation) or 5 μg of antibodies against H3K9ac (polyclonal anti-H3K9ac, Upstate). The data represent the means±s.e. of three independent experiments in duplicate and indicate the fold enrichment as compared to the negative control with IgG.

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.

Figure 4
figure4

The MSMB gene is regulated by methylation of a CpG island in the promoter region. (a) PC-3 cells were treated for 48 h with 10 μ M of the DNA methyltransferase inhibitor 5′-azacytidine. Subsequently, the steady-state levels of the MSMB and EZH2 transcripts were determined by quantitative RT–PCR analysis with intron-spanning primers specific for the indicated genes. The data were expressed relative to the transcript level in the control condition. HPRT was used as a control for normalization. (b) A schematic representation of the MSMB gene on scale. The four exons are indicated by the black boxes and the two analysed CpG regions by black stars. TSS, transcriptional start site. Methylated CG dinucleotides are denoted underneath by closed circles and unmethylated CGs by open circles. Genomic DNA of PC-3 cells or LNCaP cells was purified with the GenElute Mammalian Genomic DNA Miniprep kit of Sigma. Two microgram was digested overnight with BglII. The DNA was denatured with 0.3 M NaOH at 42°C for 30 min. Sodium bisulfite (3.3 M) and hydroquinone (0.5 mM) were added to the DNA and the mixture was incubated overnight at 55°C. The DNA was purified with the PCR purification kit of Sigma, St Louis, Mo, USA. The DNA was desulfonated with 0.3 M NaCl for 15 min at 37°C and precipitated by adding NH4Ac and ethanol. The pellet was air-dried and dissolved in 10 mM Tris and 1 mM ethylene-diaminete-traacetic acid at pH 8. PCR was performed with Jumpstart Taq Polymerase (Sigma). The primers GTTTAGGTTGGAGTGTAGTGG (sense) and ATCCTAACTAACATAATAAAACCCC (antisense) were used to amplify the first CpG island and the primers AGTTTTTTTATTTAGGGGTGGATTTTA (sense) and CCAAACTAATCTCAAATACCTAACCTC (antisense) were used to amplify the second CpG island. The PCR products were subcloned in the pGem-T vector of Promega according to the manufacturer's protocol. At least 10 clones for each condition were sequenced. The plasmids were sequenced on a MegaBace sequencer. The percentages of methylated CpG dinucleotides are indicated in the bar diagrams.

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.

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Acknowledgements

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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Correspondence to A Van Eynde.

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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

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Keywords

  • EZH2
  • tumor suppressor
  • Polycomb group proteins
  • prostate cancer PSP94
  • epigenetics

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