EZH2-mediated concordant repression of Wnt antagonists promotes ß-catenin-dependent hepatocarcinogenesis.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/ß-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear ß-catenin, T-cell factor-dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of ß-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/ß-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and ß-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/ß-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor.

Practical limitations of convalescent plasma collection: a case scenario in pandemic preparation for influenza A (H1N1) infection.

BACKGROUND: To ensure a good preparedness for pandemic influenza A (H1N1), a study was conducted to investigate clinical effectiveness of hyperimmune intravenous globulin (H-IVIG) prepared from convalescent plasma donated by recovered patients. This article reports on the outcome of the collection phase of the study. STUDY DESIGN AND METHODS: Starting on August 26, 2009, all confirmed patients aged between 18 and 55 years were invited for participation into the study and screen for plasma donation eligibility. Effective September 17, 2009, those who were unwilling to consider screening for plasma were asked to donate whole blood. Plasma collected or separated from whole blood had to demonstrate sufficient neutralization antibodies titers of 40 or more before being channeled for H-IVIG production. RESULTS: By October 31, 2009, a total of 9101 persons were successfully contacted. A total of 1309 screening and 619 whole blood donation appointments were made. In the former 786 (60.0%) attended screening but only 301 could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. For those who opted for whole blood donation, 379 (61.2%) had attended and donated. A total of 276 L of convalescent plasma with sufficient neutralization antibodies titers was collected for H-IVIG production. DISCUSSION: The study highlighted a number of practical limitations in convalescent plasma collection programs and plasmapheresis is always the preferred mode of collection. It provided valuable learning experience for the blood transfusion service in future planning when large-scale collection is required.