recruiting DNA methyltransferases (DNMTs) to its target genes that catalyze de novo DNA methylation [37]. However, recent reports have also shown that H3K27 trimethylation by EZH2 is not always associated with promoter DNA methylation for the silencing of certain EZH2-target genes [38–41]. The functional role of EZH2 in prostate cancer progression has been identified by gene expression profiling of RNA from nontumorigenic human prostate epithelial cells overexpressing EZH2 [27]. However, the results were found not to alter significantly expression levels of many metastasis–associated genes that were identified by genetic profiling of human prostate cancer cells [42]. The nature of this discrepancy is unclear but might arise from differences in expression levels of EZH2 in the prostate cancer cells tested. In this study, we investigated the role of EZH2 in activation of MMPs to promote the invasion and metastasis of prostate cancer cells. To identify the metastasis-associated genes regulated by EZH2 in prostate cancer, mRNA expression in highly invasive prostate cancer cells in which EZH2 expression was knocked down was profiled using human metastasis PCR arrays. We found that high levels of EZH2 expression during cancer progression induce repression of TIMP genes (TIMP2 and TIMP3), leading to increased activity of MMP-9 and thus to increased invasive activity of prostate cancer cells. These results provide for the first time evidence that EZH2 plays an active role in shifting the MMPs/TIMPs balance towards MMPs and thereby promoting metastasis of prostate cancer cells. Results EZH2 knockdown significantly reduces the invasive and migratory activities of prostate cancer cells To investigate whether high levels of EZH2 expression is correlated with the invasive phenotype of prostate cancer cells, we first determined protein levels of EZH2 in human prostate cell lines (LNCaP, PC3, and DU145). Our Western blot analysis showed that EZH2 levels are significantly elevated in the cancer cell lines than in the benign human prostate epithelial cell line RWPE-1 (Figure 1A). We then examined the invasive activity of prostate cancer cells expressing different levels of EZH2 protein using Transwell Boyden chamber assay. To this end, EZH2 expression was knocked down using two different EZH2-specific shRNAs, sh1 or sh2 (Figure 1C, top). PC3 and DU145 cells were highly invasive, compared with the benign RWPE-1 cells (Figure 1B). However, EZH2 knockdown significantly decreased invasiveness of the prostate cancer cells (Figure 1C, middle); only 18,20% of the prostate cancer cells infected with lentivirus expressing EZH2 targeting shRNA penetrated the BME membrane in the chamber (sh1 and sh2), compared with those cells infected with the control (NT, nontargeting shRNA) lentivirus (Figure 1C, bottom). On the contrary, overexpression of EZH2 conferred an invasive phenotype on RWPE-1 cells (Figure 1D, WT); however, RWPE-1 cells overexpressing a mutant EZH2 protein (EZH2-H689A) with significantly reduced HMT activity [44] did not display invasive activity (Figure 1D, H689A). Because EZH2 plays an active role in prostate cancer invasion (Figures 1C and 1D), we further examined the migratory activity of DU145 cells using the wound-healing migration assay (Figure 1E, top). We found that DU145 cells fill ,80% and ,50% of the wounded areas before (NT) and after (sh1) knockdown of EZH2 expression, respectively, at 24 h after scratching (Figure 1E, bottom). These results strongly suggest that high levels of EZH2 expression promote invasion and migration of prostate cancer cells. Activation of MMPs by EZH2 Identification of the downstream target genes of EZH2 associated with metastasis of prostate cancer To identify metastasis-associated genes whose expressions are regulated by EZH2, we investigated gene expression changes in invasive prostate cancer cells after EZH2 knockdown. Expression of different mRNAs in prostate cancer cells was assessed by using the human tumor metastasis RT2 ProfilerTM PCR Array (PAHS- 028D) designed to represent 84 genes known to be involved in metastasis (Figures 2A and 2B). The results showed that expression of 12 and 32 genes is altered .2-fold in DU145 (Table S1) PC3 (Figures S1A and S1B and Table S2) cells, respectively, by knockdown of EZH2 expression. Among those genes, TIMP3 was found to be the gene that is most highly upregulated in both DU145 and PC3 cells (Figures 2B and S1B), leading to the hypothesis that TIMP3 may be a primary target for repression by EZH2. To test this hypothesis, we examined protein levels of TIMP3 and EZH2 by immunohistochemistry using anti-EZH2 and anti-TIMP3 antibodies (Figure 2C). As shown in the immunostaining images, TIMP3 protein levels were high in normal prostate tissues (Figures 2C-e and 2C-f), but barely detectable in prostate cancer tissues (Figures 2C-g and 2C-h). Similar results were obtained by RT-PCR analysis showing that TIMP3 mRNA levels are markedly decreased in PC3 and DU145 cells, compared with the RWPE-1 cells (Figure S1C). In contrast, EZH2 protein levels were very high in prostate cancer tissues (Figures 2C-c and 2C-d) compared with normal prostate tissues (Figures 2C-a and 2C-b). These observations suggest that expression of TIMP3 and EZH2 is inversely correlated, probably due to downregulation of TIMP3 expression by EZH2. Downregulation of TIMP3 expression by EZH2 in prostate cancer cell lines To investigate further the transcriptional repression of TIMP3 expression by EZH2, we quantitatively determined TIMP3 mRNA and protein levels in three prostate cancer cell lines (LNCaP, PC3, and DU145) with and without knockdown of EZH2 expression. TIMP3 mRNA levels were found to be ,10–13-fold higher in the cancer cells treated with EZH2-specific shRNA (sh1) than control cells untreated with the shRNA (Figure 3A), and as a result, TIMP3 protein levels were significantly increased by knockdown of EZH2 expression (Figure 3B). In contrast, overexpression of the functional EZH2 (WT), but not of the mutant EZH2 protein (H689A), was shown to decrease TIMP3 mRNA and protein levels in RWPE-1 cells (Figure 3C). Down-regulation of TIMP3 expression by EZH2 was also confirmed by immunostaining of TIMP3 in DU145 cells infected with EZH2-specific shRNA lentivirus (sh1) or control (nontargeting shRNA) lentivirus (NT) (Figure 3D). TIMP3 protein levels were low in DU145 cells (Figure 3D-c) but significantly elevated by knockdown of EZH2 expression (Figure 3D-h). Thus, TIMP3 may be a target of EZH2 for repression in prostate cancer cells. Epigenetic silencing of TIMP3 expression by EZH2 EZH2 is a component of PRC2 that catalyzes trimethylation of H3K27 (3meH3K27) [24,25]. To explore the underlying mech- anism of the epigenetic repression of TIPM3 by EZH2, we investigated effects of the histone methylation inhibitor 3-Deaza- naplanocin A (DZNep) on expression of TIMP3. DZNep treatment resulted in decreased levels of TIMP3 protein (Figure 4A), probably by causing derepression of TIMP3 expression (Figure 4B). Moreover, the invasive potential of PC3 cells was ,5-fold decreased by DZNep treatment (Figure 4C). To study further the molecular mechanism of the transcrip- tional repression of TIMP3 expression by EZH2, we performed PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30393