Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones.

Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, (R,Z)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and (E)-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the Z isomer of DAG-lactones showed higher potency than the E isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.

Author Correction: EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

We wish to correct two mutations in Supplementary Table 4 of this Letter. The NCI-H460 cell line was annotated as being mutant for TP53. NCI-H460 has been verified to be TP53 wild type by several sources1. The NCI-H2009 cell line was annotated as being mutant for PIK3CA. As annotated by COSMIC (ref. 24 of the original Letter) and CCLE (ref. 25 of the original Letter), the NCI-H2009 cell line has a mutation in PIK3C3, rather than PIK3CA. The cell line is wild type for PIK3CA. The Supplementary Information of this Amendment contains the corrected Supplementary Table 4. These errors do not affect our conclusions. The original Letter has not been corrected.

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme.

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a North-methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2'-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.

Enzyme-Driven Chemo-and Radiation-Therapy with 12 Pyrimidine Nucleoside Analogs Not Yet in the Clinic.

Enzymatic activity from tumor and adjacent normal tissue of 200 patients involving deoxycytidine kinase (dCK), uridine/cytidine kinase (U/CK), cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD) was quantified. Patients with brain (17), colon (24), and breast (30) tumors, 53, 67, and 73%, respectively, had an elevated T/N value (Specific Activity of tumor/ Specific Activity of normal tissue) involving dCK and dCMPD suggesting chemotherapy with 5-fluorodeoxycytidine (5-FdC) alone or in combination with thymidine plus deoxytetrahydrouridine, or with the radiosensitizer, 5-chlorodeoxycytidine (5-CldC) plus tetrahydrouridine (H4U). Among patients with colon (19) and pancreatic tumors (40), 53 and 68 %, respectively, displayed T/N values >4 for CD suggesting chemotherapy with 5-FdC, 4-N-methylamino-5-FdC, 5-trifluoromethyldeoxycytidine and radiosensitization with 5- CldC, 4-N-methylamino-5-CldC, 5-iododeoxycytidine and 5-bromodeoxycytidine. The percent of patients with tumors with a T/N value >4 for U/CK in lung (72), colon (23) and breast (28) was 47, 61 and 68, respectively, suggesting zebularine (plus thymidine) treatment for tumors involving gene silencing. Evidence is presented that the 4-N-alkylamino-dC substituted nucleosides and those with large 5-substitutions are activated only via CD to thymidine kinase (TK) using end-points of cytotoxicity and/or radiosensitization: H4U, the inhibitor of CD is an antagonist, cells with low CD or no TK are resistant to the analogs, the end points are indifferent to the dCK status of cells, they are poor substrates for dCK and good substrates for CD, whereas 5-FdC and 5-CldC are good substrates for both enzymes. The analogs present opportunities for Collateral Sensitivity for 5-azacytidine and gemcitabine resistant tumors.

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells. RESULTS: Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells. CONCLUSIONS: Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.

Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells.

Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase. dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK. dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed. List of abbreviations: ddC, 2', 3'-didioxycytosine, Zalcitabine; 3TC, ß-L-(-)-2',3'-dideoxy-3'-thiacytidine, Lamivudine; CdA, 2-cloro-2'-deoxyadenosine, Cladribine; AraA, 9-ß-D-arabinofuranosyladenine; hCNT 1-3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1-4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Diacylglycerol lactones targeting the structural features that distinguish the atypical C1 domains of protein kinase C ζ and ι from typical C1 domains.

To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.

Inhibition of histone methyltransferase EZH2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16.

Leukemia stem cells (LSC) are resistant to conventional chemotherapy and persistent LSC after chemotherapy are supposed to be a major cause of relapse. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified. Epigenetic regulators are associated with many cellular processes including maintenance of stem cells. Of note are polycomb group proteins, because they potentially control stemness, and can be pharmacologically targeted by a selective inhibitor (DZNep). Therefore, we investigated the therapeutic potential of EZH2 inhibition in mixed lineage leukemia (MLL) fusion leukemia. Intriguingly, EZH2 inhibition by DZNep or shRNA not only suppressed MLL fusion leukemia proliferation but also reduced leukemia initiating cells (LIC) frequency. Expression analysis suggested that p16 upregulation was responsible for LICs reduction. Knockdown of p16 canceled the survival advantage of mice treated with DZNep. Chromatin immunoprecipitation assays demonstrated that EZH2 was highly enriched around the transcription-start-site of p16, together with H3K27 methylation marks in MLL/ENL and Hoxa9/Meis1 transduced cells but not in E2A/HLF transduced cells. Although high expression of Hoxa9 in MLL fusion leukemia is supposed to be responsible for the recruitment of EZH2, our data also suggest that there may be some other mechanisms independent of Hoxa9 activation to suppress p16 expression, because expression levels of Hoxa9 and p16 were not inversely related between MLL/ENL and Hoxa9/Meis1 transduced cells. In summary, our findings show that EZH2 is a potential therapeutic target of MLL fusion leukemia stem cells.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

BACKGROUND: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts. METHODS: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo. RESULTS: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo. CONCLUSIONS: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

EZH2 down-regulation exacerbates lipid accumulation and inflammation in in vitro and in vivo NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent, chronic liver diseases, worldwide. It is a multifactorial disease caused by complex interactions between genetic, epigenetic and environmental factors. Recently, several microRNAs, some of which epigenetically regulated, have been found to be up- and/or down-regulated during NAFLD development. However, in NAFLD, the essential role of the Polycomb Group protein Enhancer of Zeste Homolog 2 (EZH2), which controls the epigenetic silencing of specific genes and/or microRNAs by trimethylating Lys27 on histone H3, still remains unknown. In this study, we demonstrate that the nuclear expression/activity of the EZH2 protein is down-regulated both in livers from NAFLD rats and in the free fatty acid-treated HepG2. The drop in EZH2 is inversely correlated with: (i) lipid accumulation; (ii) the expression of pro-inflammatory markers including TNF-α and TGF-ß; and (iii) the expression of miR-200b and miR-155. Consistently, the pharmacological inhibition of EZH2 by 3-Deazaneplanocin A (DZNep) significantly reduces EZH2 expression/activity, while it increases lipid accumulation, inflammatory molecules and microRNAs. In conclusion, the results of this study suggest that the defective activity of EZH2 can enhance the NAFLD development by favouring steatosis and the de-repression of the inflammatory genes and that of specific microRNAs.

Lysine methylation promotes VEGFR-2 activation and angiogenesis.

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase, promotes tumor angiogenesis and ocular neovascularization. We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys(1041), a residue that is proximal to the activation loop of the kinase domain. Methylation of VEGFR-2 was independent of ligand binding and was not regulated by ligand stimulation. Methylation of Lys(1041) enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that methylation of Lys(1041) was required for VEGFR-2-mediated angiogenesis in zebrafish and tumor growth in mice. We propose that methylation of Lys(1041) promotes the activation of VEGFR-2 and that similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.

Effects of substitutions at the 4' and 2 positions on the bioactivity of 4'-ethynyl-2-fluoro-2'-deoxyadenosine.

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4'-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2'-exo/C-3'-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4'-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4'-ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.

PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer.

Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.

Enhancer of zeste homolog 2 silences microRNA-218 in human pancreatic ductal adenocarcinoma cells by inducing formation of heterochromatin.

BACKGROUND & AIMS: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is overexpressed by pancreatic ductal adenocarcinoma (PDAC) cells and increases their aggressiveness. We identified microRNAs (miRs) that are regulated by EZH2 and studied their functions in PDAC cells. METHODS: We performed miR profile analysis of PDAC cells incubated with EZH2 inhibitor 3-deazaneplanocin A, and pancreatic ductal epithelial cells that overexpressed EZH2. Expression levels of miRs and the targets of miRs were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We expressed different forms of EZH2 to analyze functional domains and used small interfering RNAs to reduce its level in PDAC cells. RESULTS: Expression of miR-218 was repressed by EZH2 in PDAC cells. Levels of miR-218 were significantly reduced in primary PDAC tumor samples compared with paired, adjacent nontumor tissue. Overexpression of miR-218 in SW1990 cells reduced their proliferation and tumor formation and metastasis in nude mice. Loss of miR-218 from SW1990 cells increased levels of UDP-glycosyltransferase 8 and miR-218 was found to bind to its 3'-UTR. Levels of UDP-glycosyltransferase protein and messenger RNA were associated with the metastatic potential of PDAC cell lines and progression of tumors in patients. EZH2 was found to silence miR-218 by binding to its promoter, promoting heterochromatin formation, and recruiting the DNAs methyltransferase 1, 3A, and 3B. CONCLUSIONS: EZH2 is up-regulated in PDAC samples from patients and silences miR-218. MicroRNA-218 prevents proliferation of PDAC cells in culture, and tumor growth and metastasis in nude mice. MicroRNA-218 reduces levels of UDP-glycosyltransferase, which is associated with the metastatic potential of PDAC tumors in mice and progression of human PDAC.