Alfred S.L. Cheng

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.

NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

Cyclooxygenase-2 Expression in Helicobacter pylori-Associated Premalignant and Malignant Gastric Lesions

Expression of cyclooxygenase-2 (COX-2) in various stages of the Helicobacter pylori-associated gastric carcinogenesis pathway has not been elucidated. We investigated the distribution and intensity of COX-2 expression in premalignant and malignant gastric lesions, and monitored the changes after H. pylori eradication. Gastric biopsies from H. pylori-infected patients with chronic active gastritis, gastric atrophy, intestinal metaplasia (IM), gastric adenocarcinoma, and noninfected controls were studied. Expression of COX-2 was evaluated by immunohistochemistry and in situ hybridization. Endoscopic biopsies were repeated 1 year after successful eradication of H. pylori in a group of IM patients for comparing COX-2 expression and progression of IM. In all H. pylori-infected patients, COX-2 expression was predominantly found in the foveolar and glandular epithelium and, to a lesser extent, in the lamina propria. In the noninfected group, only 35% of cases demonstrated weak COX-2 expression. Intensity of COX-2 was not significantly different between the chronic active gastritis, gastric atrophy, IM, and gastric adenocarcinoma groups. In 17 patients with IM, COX-2 expressions in the epithelial cells and stromal cells were reduced 1 year after H. pylori eradication. However, the changes in COX-2 expression did not correlate with progression/regression of IM. Both premalignant and malignant gastric lesions demonstrate strong COX-2 expression. Successful eradication of H. pylori leads to down-regulation of COX-2 expression but failed to reverse IM at 1 year.

Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant.

The development of targeted therapies for antiestrogen-resistant breast cancer requires a detailed understanding of its molecular characteristics. To further elucidate the molecular events underlying acquired resistance to the antiestrogens tamoxifen and fulvestrant, we established drug-resistant sublines from a single colony of hormone-dependent breast cancer MCF7 cells. These model systems allowed us to examine the cellular and molecular changes induced by antiestrogens in the context of a uniform clonal background. Global changes in both basal and estrogen-induced gene expression profiles were determined in hormone-sensitive and hormonal-resistant sublines using Affymetrix Human Genome U133 Plus 2.0 Arrays. Changes in DNA methylation were assessed by differential methylation hybridization, a high-throughput promoter CpG island microarray analysis. By comparative studies, we found distinct gene expression and promoter DNA methylation profiles associated with acquired resistance to fulvestrant versus tamoxifen. Fulvestrant resistance was characterized by pronounced up-regulation of multiple growth-stimulatory pathways, resulting in estrogen receptor alpha (ERalpha)-independent, autocrine-regulated proliferation. Conversely, acquired resistance to tamoxifen correlated with maintenance of the ERalpha-positive phenotype, although receptor-mediated gene regulation was altered. Activation of growth-promoting genes, due to promoter hypomethylation, was more frequently observed in antiestrogen-resistant cells compared with gene inactivation by promoter hypermethylation, revealing an unexpected insight into the molecular changes associated with endocrine resistance. In summary, this study provides an in-depth understanding of the molecular changes specific to acquired resistance to clinically important antiestrogens. Such knowledge of resistance-associated mechanisms could allow for identification of therapy targets and strategies for resensitization to these well-established antihormonal agents.

High serum interleukin-6 level predicts future hepatocellular carcinoma development in patients with chronic hepatitis B.

Increased interleukin‐6 (IL‐6) production is implicated in the pathogenesis of hepatocellular carcinoma (HCC) in animal models. Although previous studies showed that HCC patients had higher serum IL‐6 level at the time of diagnosis, it is unclear if the cytokine contributes to the development of HCC or is just a reaction to cancer. To address this question, we performed a nested case‐control study. Consecutive chronic hepatitis B patients were recruited from 1997 to 2000 and followed till 2008. Profiling of 27 cytokines, chemokines and growth factors was performed at baseline, date of peak alanine aminotransferase (ALT) level and the last visit. Thirty‐seven patients developed HCC at a median follow‐up of 62 months (interquartile range: 41–110). Serum IL‐6 was higher in patients with HCC than controls both during peak ALT and at the last visit (both p = 0.02). Patients with IL‐6 above 7 pg/ml during peak ALT had increased risk of HCC or death (adjusted hazard ratio 3.0; 95% confidence interval 1.2, 7.8; p = 0.02). The sensitivity, specificity, positive and negative predictive values of this cutoff to predict future HCC development were 70%, 73%, 72% and 71%, respectively. Combination of IL‐6 and AFP improved the sensitivity in diagnosing HCC or predicting future HCC development. In conclusion, high serum IL‐6 level predates the development of HCC in chronic hepatitis B patients, and has moderate accuracy in predicting future cancer. This may assist clinicians in selecting high‐risk patients for HCC surveillance program.

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes β-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/β-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 β-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 β-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/β-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and β-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/β-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor.

Epithelial progeny of estrogen-exposed breast progenitor cells display a cancer-like methylome.

Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non-estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer.

NF-κB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors

Cigarette smoke is one of the risk factors for gastric cancer and nicotine has been reported to promote tumor growth. Deregulation of microRNA (miRNA) and cyclooxygenase-2 (COX-2) expressions are hallmarks of many cancers including gastric cancer. Here, we used an miRNA array platform covering a panel of 95 human miRNAs to examine the expression profile in nicotine-treated gastric cancer cells. We found that miR-16 and miR-21 were upregulated upon nicotine stimulation, transfection with anti-miR-16 or anti-miR-21 significantly abrogated cell proliferation. In contrast, ectopic miR-16 or miR-21 expression exhibited a similar stimulatory effect on cell proliferation as nicotine. Nicotine-mediated IkappaBα degradation and nuclear factor-kappa B (NF-κB) translocation dose-dependently. Knockdown of NF-κB by short interfering RNA (siRNA) or specific inhibitor (Bay-11-7085) markedly suppressed nicotine-induced cell proliferation and upregulation of miR-16 and miR-21. Interestingly, NF-κB-binding sites were located in both miR-16 and miR-21 gene transcriptional elements and we showed that nicotine enhanced the binding of NF-κB to the promoters of miR-16 and miR-21. Furthermore, activation of COX-2/prostaglandin E₂ (PGE₂) signaling in response to nicotine was mediated by the action of prostaglandin E receptors (EP2 and EP4). EP2 or EP4 siRNA or antagonists impaired the nicotine-mediated NF-κB activity, upregulation of miR-16 and miR-21 and cell proliferation. Taken together, these results suggest that miR-16 and miR-21 are directly regulated by the transcription factor NF-κB and yet nicotine-promoted cell proliferation is mediated via EP2/4 receptors. Perhaps this study may shed light on the development of anticancer drugs to improve the chemosensitivity in smokers.

Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis

In cancer cells, glucose is often converted into lactic acid, which is known as the ‘Warburg effect’. The reason that cancer cells have a higher rate of aerobic glycolysis, but not oxidative phosphorylation, remains largely unclear. Herein, we proposed an epigenetic mechanism of the Warburg effect. Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonize glycolysis was downregulated through NF-kappaB pathway in Ras-transformed NIH3T3 cells. Restoration of FBP1 expression suppressed anchorage-independent growth, indicating the relevance of FBP1 downregulation in carcinogenesis. Indeed, FBP1 was downregulated in gastric carcinomas (P<0.01, n=22) and gastric cancer cell lines (57%, 4/7). Restoration of FBP1 expression reduced growth and glycolysis in gastric cancer cells. Moreover, FBP1 downregulation was reversed by pharmacological demethylation. Its promoter was hypermethylated in gastric cancer cell lines (57%, 4/7) and gastric carcinomas (33%, 33/101). Inhibition of NF-kappaB restored FBP1 expression, partially through demethylation of FBP1 promoter. Notably, Cox regression analysis revealed FBP1 promoter methylation as an independent prognosis predicator for gastric cancer (hazard ratio: 3.60, P=0.010). In summary, we found that NF-kappaB functions downstream of Ras to promote epigenetic downregulation of FBP1. Promoter methylation of FBP1 can be used as a new biomarker for prognosis prediction of gastric cancer. Such an important epigenetic link between glycolysis and carcinogenesis partly explains the Warburg effect.

Xenoestrogen-induced epigenetic repression of microRNA-9-3 in breast epithelial cells.

Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.