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PDF file - 57K, SuperArray Analysis of Oncogenes, Tumor Suppressors, and Stem Cell-Related Genes in MPM Lines Following Selective PRC2 Knockdown or 5 �M DZNep.

PDF file - 661K, TaqMan Primers, shRNA, and Antibodies.

PDF file - 102K, Effects of DZNep treatment on cultured mesothelioma cell lines relative to normal mesothelia cells. Pyrosequencing analysis of NBL2 DNA repeat in normal mesothelia (LP9) and MPM cell lines cultured in the presence or absence of DZNep.

PDf file - 63K, qRT-PCR and immunoblot analysis of EZH2 and EED levels in MPM lines following transduction with shRNA targeting EZH2, EED, or sham sequences. Effects of knock-down of EZH2 and EED on proliferation of MPM lines.

PDF file - 63K, Genes Simultaneously Modulated by DZNep in MES1 and H28.

PDF file - 661K, Photomicrographs of NMES1 and NMES2 normal pleura cultures, and MES1 through MES4 mesothelioma cell lines established at the NCI.

PDF file - 97K, Supplementary Materials and Methods.

PDF file - 62K, Demographic Data Pertaining to the Patients from whom NCI-SB-MES 1-4 lines Were Established.

PDF file - 245K, EZH2 expression in tumors correlates with cell line expression.

Purpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).Experimental Design: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.Results:EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy. Clin Cancer Res; 18(1); 77–90. ©2011 AACR.

PDF file - 65K, Top 20 Gene Sets Enriched in MES1 and H28 Cells following DZNep Treatment.

PDF file - 54K, Top 20 Gene Ontologies Modulated by DZNep in MES1 and H28 Cells.

PDF file - 66K, Summary of percentages of mesothelioma or normal mesothelia specimens expressing EZH2 in figure 2D.

PDF file - 52K, ABCG2 Expression in Side Populations (sp) and Non-side Populations (nsp) of A549 and Calu-6 Cells Cultured with or without CSC

PDF file - 87K, Bar graph depicting total number of differentially expressed genes, as well as relative numbers induced or repressed under various treatment conditions

PDF file - 116K, qRT-PCR analysis of ABCG2 expression in untreated NSCLC and SCLC lines relative to SAEC, and in NSCLC and SCLC lines following CSC exposure

PDF file - 111K

PDF file - 363K, Ingenuity pathway analysis demonstrating two common networks targeted by mithramycin in lung cancer cells in vitro and in vivo

PDF file - 76K, TaqMan and ChIP Primers, and Antibodies

PDF file - 346K, Ingenuity pathway analysis depicting representative intracellular networks modulated by mithramycin lung cancer xenografts

Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell–related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies. Cancer Res; 72(16); 4178–92. ©2012 AACR.

Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of thoracic malignancies. In order to examine this issue, lung and esophageal cancer cells (A549, Calu-6, NCI-SB-ESC1 and NCI-SB-ESC2), were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that five day CSC exposure significantly up-regulated ABCG2, encoding a xenobiotic pump highly expressed in cancer stem cells. Quantitative reverse transcription-PCR (qRT-PCR), western blot, and immunohistochemistry experiments confirmed up-regulation of ABCG2 in cultured cancer lines, but not normal small airway epithelial cells (SAEC) or immortalized esophageal squamous cells (HET1A) exposed to CSC. Flow cytometry experiments demonstrated that CSC increased the side population (SP) of cultured cancer cells. Transient transfection experiments using ABCG2 promoter reporter constructs revealed that deletion of xenobiotic response elements as well as SP-1 sites markedly attenuated ABCG2 induction by CSC. Chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated induction of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor (AHR), SP-1, and Nrf2, as well as increased levels of RNA pol II and H3K9Ac within the ABCG2 promoter. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AHR, SP-1, and Nrf2 levels within the ABCG2 promoter, down-regulated ABCG2 expression, decreased S P, inhibited in-vitro proliferation of lung and esophageal cancer cells, and markedly diminished growth of established tumor xenografts. Microarray analysis revealed significant down-regulation of numerous stem-cell related pathways in lung cancer cells following mithramycin exposure. Collectively, these findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical evaluation of mithramycin for targeting cancer stem cells in thoracic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A54.

Objective: Previously, we reported that cigarette smoke condensate dramatically increases tumorigenicity of lung cancer cells via epigenetic silencing of Dickkopf-1(Dkk-1), which encodes an inhibitor of Wnt stem cell signaling. Furthermore, we demonstrated that knock-down of Dkk-1 up-regulates Wnt5A in lung cancer cells. Expression of this non-canonical Wnt ligand correlates with adverse outcome in lung cancer patients. The present study was undertaken to ascertain if Wnt5A directly modulates the malignant phenotype of lung cancer cells. Methods: Calu-6, H358, A549, and H841 lung cancer lines were transduced with lentiviral vectors expressing Wnt5A or control sequences. Cell count, scratch, and matrigel assays were used to evaluate in-vitro proliferation, migration, and invasion of transduced cells. Similar experiments were performed with exogenous recombinant Wnt5A (rWnt5A). Nude mouse xenograft experiments were used to assess tumorigenicity of lung cancer cells transduced with Wnt5A or vector controls. Canonical and non-canonical Wnt signal pathways were evaluated using western blot, promoter-reporter, and focused quantitative RT-PCR array techniques. Results: Wnt5A transduced lung cancer cells exhibited significantly increased proliferation, migration, and invasion relative to vector controls (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5156.

Polycomb group (PcG) proteins, which are global epigenetic gene silencers, play key roles in the maintenance of stem cell pluripotency, multicellular development, and tumorigenesis. EZH2, a core component of PcG and H3K27 methyltranferase, is highly expressed during embryogenesis and malignant transformation. Presently the mechanism by which EZH2 mediates tumorigenesis and metastasis are unclear. Our recent studies indicate that cigarette smoke condensate (CSC) increases EZH2 expression and global levels of H3K27Me3 in immortalized human bronchial epithelial cells (HBEC). In the present study, we sought to further investigate the role of EZH2 in modulating the malignant phenotype of lung cancer cells. Western blot experiments demonstrated that EZH2 was significantly over-expressed in lung cancer cell lines but not in normal human bronchial epithelial cells (NHBE) or small airway epithelial cells (SAEC). EZH2-shRNA lentiviral vectors were used to knockdown EZH2 in A549 and Calu-6 lung cancer cells as well as CSC-treated HBEC. Knockdown of EZH2 decreased H3K27Me3 levels, and diminished proliferation and clonogenicity of these cells in soft agar. These observations coincided with down-regulation of Cyclin D1. Additional western blot and flow cytometry experiments are underway to examine the impact of EZH2 knockdown on cell cycle progression and apoptosis in lung cancer cells. Illumina gene expression array experiments are also in progress to identify PcG target genes upregulated by knockdown of EZH2 that correlate with reduced proliferation and decreased clonogenicity of these cells. Collectively, these data indicate that knockdown of EZH2 inhibits the malignant phenotype of lung cancer cells, and suggest that EZH2 is a promising target for lung cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4886.