Activation of MMPs by EZH2 Figure 4. Repression of TIMP3 expression by histone methylation (3meH3K27) in prostate cancer cells. (A) Western blot analysis of endogenous levels of EZH2, TIMP3 and 3meH3K37 in the prostate cancer cells (PC3 cell) after treatment with DZNep (5 mM) or DMSO (control) for 2 days. Actin was used as an internal control. (B) qRT–PCR analysis of expression of TIMP3 in PC3 cells treated with DZNep (5 mM) or DMSO (control). (C) Invasion assay of PC3 cells treated with DZNep (5 mM) or DMSO (control). doi:10.1371/journal.pone.0030393.g004 expression of those genes. However, these results were not clearly observed in DU145 cells, perhaps due to different genetic backgrounds between the two cell lines [51]. While the transcrip- tional regulation of MMP genes by EZH2, which was out of scope of this study, is currently under investigation, our results support the possibility that overexpression of EZH2 in metastatic prostate cancer cells may elevate overall MMP activity both by decreasing the levels of TIMPs (TIMP2 and TIMP3) and by increasing the levels of some MMPs, such as MMP-9. Promoter DNA methylation is the most common epigenetic modification associated with gene silencing in cancer. EZH2 appears to recruit DNMTs to its target genes, resulting in methylation of adjacent CpG islands and subsequent gene silencing [37]. In this model, EZH2-mediated H3K27 methylation may be a prerequisite for promoter DNA methylation. Our results support this idea, as we have observed that TIMP2 and TIMP3, like other EZH2 target genes such as MSMB [31], RUNX3 [52], and SLIT2 [39], are silenced through histone (H3K27) methyla- tion followed by DNA methylation (Figures 5 and 7). However, promoter DNA methylation is not always required for repression of the EZH2 target genes. For example, EZH2-mediated silencing of the E-cadherin gene occurs through H3K27 methylation but does not require promoter DNA methylation [38]. In addition, H3K27 trimethylation is not associated with promoter DNA methylation for silencing of a substantial number of EZH2 target genes in prostate cancer [41]. Therefore, further studies are required to distinguish these mechanisms. In order for cancer cells to metastasize, they must break free from the normal physical barriers, to migration and invasion, such as ECM and cell-cell adhesion. Moreover, degradation of the ECM releases signaling molecules associated with the ECM, such as growth factors and cytokines, that significantly influence metastasis [53]. Thus, the ECM degradation by MMPs is of pivotal importance for not only eliminating the barriers but also making the signaling molecules accessible to cancer cells. As demonstrated in this study, EZH2, which acts as a potential oncogene in various malignancies, plays an active role in the upregulation of MMP activity and promote ECM degradation and subsequent invasion of prostate cancer cells, providing a novel insight into the role of EZH2 in prostate cancer metastasis. Materials and Methods Cell culture All cell lines used in this study were obtained from the American Type Culture Collection (ATCC; Rockville, MD). PC3, DU145 and LNCaP cells were cultured in RPMI 1640 with 10% FBS and penicillin/streptomycin at 37uC in a humidified atmosphere of 5% CO2. The RWPE-1 cells were cultured in keratinocyte serum-free medium (K-SFM) containing 50 mg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor as described previously [43]. The cells were fed with fresh growth medium every 3 days, and at confluence, they were subcultured at a 1:4 ratio using trypsin- EDTA (0.05%). Lentiviral infection Lentiviral transduction was used for EZH2 knockdown and EZH2 overexpression in prostate cancer cells. Lentiviral expression vectors for GFP (green fluorescent protein; as a control), wild-type EZH2-GFP, and mutant EZH2 (H689A)-GFP were constructed by PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30393