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M. Sato



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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O12.05 - Defining the role of ZEB1 in the pathogenesis of non-small cell lung cancer (NSCLC) using immortalized human bronchial epithelial cells (HBECs) (ID 1139)

      10:30 - 12:00  |  Author(s): M. Sato

      • Abstract
      • Presentation
      • Slides

      Background
      To study the role of common lung cancer mutations in transforming lung epithelial cells in an appropriate cellular context we used cdk4/hTERT-immortalized normal HBECs. We developed an isogenic series of HBECs by introducing genetic manipulations representing common lung cancer mutations (such as p53, KRAS[V12], cMYC, and LKB1). This defined in vitro system allows characterization of specific tumorigenic contributions as well as identification of acquired changes, likely representing tumor acquired vulnerabilities and novel therapeutic targets (Mol Cancer Res 2013). One acquired change observed with oncogenic transformation of HBECs is a spontaneous epithelial-to-mesenchymal transition (EMT), an important biologic process in cancer. This study sought to characterize the role of EMT in driving tumorigenesis in HBECs and, in turn, lung cancer to identify novel therapeutic targets.

      Methods
      Genetic manipulations were introduced into cell lines using siRNA/shRNA or over-expression constructs. Tumorigenicity was measured using in vitro (anchorage-dependent and -independent colony formation, proliferation, migration and transwell Matrigel invasion assays) and in vivo (subcutaneous or intravenous injection into NOD/SCID mice) methods. Genome-wide mRNA expression data from five independent datasets was obtained either in-house using Illumina HumanHT-12v4 BeadChips or from publicly available databases.

      Results
      Analysis of EMT-promoting transcription factors in our isogenic series of oncogenically-manipulated HBECs found ZEB1 expression highly correlated with mesenchymal-like HBECs. Functional studies confirmed ZEB1 was a significant driver of tumorigenic phenotypes in both oncogenic HBECs and human lung cancer cell lines where loss of ZEB1 resulted in decreased colony formation, migration and invasion in vitro and subcutaneous tumor growth and intravenous colonization in vivo. A set of ZEB1-associated genes was identified from analyzing five independent mRNA microarray datasets comprising both cell lines and lung adenocarcinomas. From this gene set we found ZEB1 directly represses ESRP1 by binding to its promoter, which leads to increased mesenchymal splicing of the ESRP1 target CD44. The mesenchymal isoform of CD44, CD44s, conferred a CD44[hi] flow cytometry profile which, in turn, could be used to select for a highly tumorigenic subpopulation in partially transformed HBECs. To identify candidate ZEB1-activated targets we screened ZEB1-upregulated genes in a siRNA invasion assay. Several genes including PMP22 and CD70 could phenocopy ZEB1 where siRNA-mediated loss of expression led to decreased invasiveness in multiple NSCLC cell lines. CD70 (also called TNFSF7, tumor necrosis factor ligand superfamily member 7) may represent a prime therapeutic target for anti-metastatic growth in lung cancer. The ligand for CD27, it is involved in immune regulation, upregulated in some cancers and is being studied as a potential target for antibody therapeutics. Importantly, an anti-CD70 monoclonal antibody inhibited invasion of NSCLC cell lines comparably to siCD70 and siZEB1.

      Conclusion
      We demonstrate in vitro models of defined oncogenic HBEC transformation provide an invaluable tool to study lung cancer progression where EMT is an important mediator. ZEB1 is spontaneously expressed with malignant transformation of HBECs and is a significant driver of oncogenic progression in both HBECs and NSCLC cells. Identification of CD70 and PMP22 as downstream targets of ZEB1 may represent novel therapeutic targets for lung cancer.

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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.01-002 - Clinicopathological and biological significance of epiregulin expression in non-small cell lung cancer (ID 755)

      09:30 - 16:30  |  Author(s): M. Sato

      • Abstract

      Background
      KRAS mutations are one of the most common driver mutations in non-small cell lung cancer (NSCLC) and efficient therapeutic stratergies for oncogenic KRAS-driven NSCLC are urgently needed. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the present study, we assessed clinicopathological and biological significance of EREG expression in NSCLC.

      Methods
      Seventy-eight lung cancer cell lines (23 small cell lung cancers [SCLCs] and 35 NSCLCs), five noncancerous bronchial epithelial cell lines and 174 surgical specimens from NSCLC patients (136 adenocarcinomas and 38 squamous cell carcinomas) were used for EREG expression analysis by real-time RT-PCR methods. In vitro cell growth was evaluated by MTT assay, colony-formation assay in liquid culture and soft agar assay. Apoptosis was evaluated by the DNA fragment detection method and the annexin-V-fluorescein staining method. The Kaplan-Meier method was used for analysis of disease-free survival (DFS) and overall survival (OS) and log-rank test was used for survival differences. Cox proportional hazards model was used to identify independent prognostic factors for PFS and OS.

      Results
      EREG is predominantly expressed in NSCLC lines harboring KRAS, BRAF or EGFR mutations whereas most SCLC lines lack EREG expression. Small interfering RNAs (siRNAs) targeting against these mutations resulted in down-regulation of EREG expression in NSCLC cells. EREG expression was significantly reduced by treatments with the inhibitors of MEK or ERK in EREG-overexpressing NSCLC cell lines, irrespective of mutation status of KRAS, BRAF and EGFR. EREG expression significantly correlated with KRAS copy number in KRAS-mutant NSCLC cell lines whereas EREG expression significantly correlated with EGFR copy number in NSCLC cell lines with wild-type KRAS/BRAF/EGFR. In the analysis of surgical specimens from NSCLC patients, EREG was predominantly expressed in lung adenocarcinomas. In a subgroup of adenocarcinomas, EREG expression was significantly higher in the tumors from elderly patients (≥70-year-old), males and smokers and was higher in the tumors with pleural involvement-, lymphatic permeation- or vascular invasion-positive. EREG was highly expressed in lung adenocarcinomas with KRAS mutation compared to those with EGFR mutation or wild-type EGFR/KRAS. Lung adenocarcinoma patients with high EREG expression had significantly shorter DFS and OS compared to those with low EREG expression. When the patients were divided into four groups according to EREG expression levels and KRAS mutation status, DFS and OS were significantly shorter in the patients with KRAS-mutant/EREG-high than those with wild-type KRAS/EREG-low. Cox regression analysis demonstrated that elevated EREG expression was an unfavorable prognostic factor. siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis in KRAS-mutant and EREG-overexpressed lung adenocarcinoma cells.

      Conclusion
      Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and unfavorable prognosis in lung adenocarcinoma patients, and EREG could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-010 - Evaluation of the oncogenic ability of EML4-ALK to transform human bronchial epithelial cells (HBECs) (ID 1503)

      09:30 - 16:30  |  Author(s): M. Sato

      • Abstract

      Background
      Lung cancer is a highly lethal disease, and is believed to develop through a multistep carcinogenic process, which involves numerous genetic and epigenetic alterations. Among these alterations, mutations in “driver genes” such as KRAS and EGFR are found in non-small cell lung cancer (NSCLC) and they are demonstrated to contribute to a phenomenon, oncogene addiction. Recently, the EML4-ALK (echinoderm microtubule-associated protein–like 4 anaplastic lymphoma kinase) fusion gene has been discovered as a novel driver gene in a subset of NSCLC. We evaluated the oncogenic transformation ability of EML4-ALK by using an hTERT/CDK4-immortalized normal human bronchial epithelial cell (HBEC) model.

      Methods
      We used two HBEC lines, HBEC3 and HBEC4. Mutant KRAS[V12]-expressing HBEC was used as a positive control for oncogenic transformation. A lentiviral vector system was used to generate HBECs stably expressing EML4-ALK. EML4-ALK protein expression was confirmed by westernblotting, and downstream pathways were analyzed by westernblotting with phospho-specific antibodies. Malignant phenotypes of EML4-ALK-expressing HBECs were examined by WST-1 proliferation assay and liquid and soft agar colony formation assays.

      Results
      Westernblotting analysis showed that EML4-ALK was expressed in HBECs. Analysis of downstream pathways did not show significant differences between EML4-ALK-expressing and control HBECs. Introduction of EML4-ALK in HBECs increased the number of soft agar colonies but its effect was not as strong as KRAS[V12].Figure 1 A. Soft agar colony formation assay showing that EML4-ALK increased the number of colonies compared to control cells to a lesser extent than did KRAS[V12]. B. Cell proliferation assay (MTS-1) showing no significant difference between EML4-ALK-expressing and control HBECs.

      Conclusion
      EML4-ALK alone did not induce dramatic oncogenic changes in HBECs. To acquire more malignant phenotype, additional genomic alterations may be required and this is now under investigation.