The Role of EZH2 in the Regulation of the Activity of Matrix Metalloproteinases in Prostate Cancer Cells Yong Jae Shin, Jeong-Ho Kim* Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, Washington, D.C., United States of America Abstract Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells; however, this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells. Citation: Shin YJ, Kim J-H (2012) The Role of EZH2 in the Regulation of the Activity of Matrix Metalloproteinases in Prostate Cancer Cells. PLoS ONE 7(1): e30393. doi:10.1371/journal.pone.0030393 Editor: Dean G. Tang, The University of Texas M.D Anderson Cancer Center, United States of America Received November 2, 2011; Accepted December 20, 2011; Published January 17, 2012 Copyright: 2012 Shin, Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant GM087470 (J-HK) from the National Institute of General Medical Sciences, the National Institute of Health. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bcmjxk@gwu.edu Introduction Metastasis—the spread of cancer cells from a primary site to other parts of the body—is a common feature of malignant tumors. The process of cancer metastasis consists of multiple, sequential steps; cancer cells escape from the primary tumor, enter the bloodstream, travel to distant sites, and extravasate to form secondary tumor sites [1]. During metastasis, cancer cells invade and migrate through the normal molecular constraints, such as the extracellular matrix (ECM) [2]. The ECM, often referred to as the connective tissue, is an organized network of extracellular materials surrounding and supporting cells. The ECM is composed of a wide variety of polysaccharides and proteins, such as laminins, collagens, fibronectin, and proteoglycan, and plays an integral role in determining the shape, development, and biochemical function of cells [2]. The fabric of the ECM proteins makes up the basement membrane (BM) that underlies the basal surface of epithelial tissues and forms a physical barrier against tumor invasion. Cancer cells are capable of degrading the ECM barrier by using enzymes, resulting in dissolution of the BM. Most prominent among the enzymes are the matrix metalloproteinases (MMPs) [3]. MMPs are a large family of zinc-dependent endopeptidases and responsible for degradation of the ECM [4]. MMPs have long been known to be associated with physiological and pathological processes such as tissue remodeling, wound healing, angiogenesis, and cancer progression [5–8]. MMPs appear in latent proteins in the cytosol (pro-MMPs) and undergo proteolytic processing to yield the mature enzymes, which are, in turn, secreted and associated with the cell surface and the ECM [9,10]. When displayed at the cell surface, however, MMPs are inhibited by the endogenous tissue inhibitors of metalloproteinases (TIMPs), which directly bind to the catalytic domains of MMPs in a 1:1 stoichiometry [11]. Therefore, the balance between MMPs and TIMPs is critical for eventual ECM remodeling and degradation. The human genome encodes four TIMPs (TIMP1–TIMP4) that are functionally redundant and inhibit 23 human MMPs [12]. In many malignant tumors, expression of TIMPs is down-regulated, consistent with their role as MMP inhibitors [13,14]. Suppression of TIMP expression by antisense RNA confers oncogenicity on Swiss 3T3 cells [15,16]. Conversely, overexpression of TIMPs results in the inhibition of invasion and metastasis of cancer cells [17–23]. These observations indicate that repression of TIMP genes may be an important regulatory mechanism for cancer progression, but the underlying mechanism is still not fully understood. EZH2 (polycomb group protein enhancer of zeste homolog 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) [24,25] and is overexpressed in a variety of human cancers [26]. Early studies showed that high levels of EZH2 expression are associated with invasion and metastasis of malignant tumors such as breast and prostate cancers [27–31] and that EZH2 overexpression transforms the benign prostate cells RWPE-1 [32] and BPH1 [33] and the immortalized breast epithelial cells [28]. Recently, EZH2 has also been found to regulate signaling pathways associated with cellular metabolism such as the Ras GTPase-activating protein DAB2IP [34,35] and the adrenergic receptor-beta-2 ADRB2 [36], promoting cancer progression. EZH2 appears to mediate transcriptional silencing by either methylating lysine 27 in histone H3 (3meH3K27) [23,24] or PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30393