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M. Friedman



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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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      O12.02 - Pathway Analysis of Gene Expression Profiles that Distinguish Persistent from Regressive Bronchial Dysplasia Indicate Synergistic Role for Polo-Like Kinase 1 (PLK1) and Epoxide Hydrolase 3 (EPHX3) in Malignant Progression. (ID 3334)

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

      • Abstract
      • Presentation
      • Slides

      Background
      160,000 Americans die from lung cancer annually and the prognosis for invasive lung cancer is poor. Prevention of cancer represents an approach with high potential for significant reduction in mortality. Bronchial dysplasia (BD) is a precursor lesion of squamous cell carcinoma (SCC) of the lung, and persistent BDs represent a high risk subset of these lesions. Genomic instability is an important process underlying malignant progression. Gene expression microarray analyses were used to identify potential mediators of genomic instability in persistent BD and study their activity in these high risk lesions. Two genes, PLK1, which abrogates G2-M checkpoint DNA damage repair, and EPHX3, which converts tobacco smoke derived pro-carcinogens to mutagens, were selected for further analysis.

      Methods
      Sixty-three frozen baseline biopsies were classified into persistent/progressive BD, regressive BD , progressive non-dysplasia and stable non-dysplasia groups according to the presence or absence of BD on follow-up biopsies. H&E staining was performed on frozen sections to confirm histology, and RNA was harvested for global gene expression microarray analysis. Intergroup comparisons employed ANOVA statistical analysis with a false discovery rate of 10% to identify differentially expressed genes associated with persistence and gene expression alterations related to baseline histology used Spearman correlation coefficient cutoff of r= +/- 0.5. A pathway analysis (Ingenuity) using the persistence related genelist was performed to identify active pathways associated with persistence of BD. Validational studies were performed by quantitative RT-PCR in cell lines established from persistent and regressive bronchial sites. Inhibitors of persistence associated enzymes were used in tissue culture based assays of cellular proliferation.

      Results
      Gene expression analyses support the unique biological nature of persistent BD. Intergroup comparisons showed significant numbers of differentially expressed genes only in the comparisons of persistent BD with regressive BD (318 genes) or stable non-dysplasia (6254 genes). 831 genes showed differential expression associated with increasing baseline dysplastic grade regardless of outcome. While approximately half of these genes also differentiated persistent from regressive BD, the presence of numerous persistence related genes that are independent of histology further substantiates the unique high risk nature of persistent BD. A pathway analysis revealed “mitotic roles of PLKs” as having the most significant association with persistence. Quantitative RT-PCR using cultures of 8 persistent BD and 6 regressive BD validated increased expression in persistent BD of PLK1 (2.77X, p=0.002) and EPHX3 (2.36X, p=0.081). Using a classification of dysplastic specimens as high or low expressers of PLK1 and/or EPHX3 (high > mean), we found a significant direct relationship with increased level of outcome diagnosis score: low expression of both genes (2.58); high expression of only one gene (3.60); and high expression of both (5.06). The baseline diagnosis did not differ between groups. Culture of the SCC cell line H2009 with EPHX inhibitor revealed a non-significant trend toward decreased proliferation (80.4% vs untreated).

      Conclusion
      Gene expression data confirms the biologically distinct nature of persistent BD. PLK1 and EPHX3 overexpression demonstrate a cooperative effect in respect to increased outcome histology suggesting a potential role for these enzymes in persistence/progression of BD via promotion of genomic instability.

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    P2.03 - Poster Session 2 - Technology and Novel Development (ID 151)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.03-007 - Changes in pulmonary immune cell recruitment and function during cigarette smoke-induced lung carcinogenesis in mice (ID 3370)

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

      • Abstract

      Background
      Smoking-mediated lung cancer will cause at least 134,000 deaths in the USA this year. Even when detected at the earliest stages (stage IA and IB), five year survival is just slightly more than 50%. Chronic pulmonary inflammation is an established risk factor for lung cancer. Increased lung tumor macrophage infiltration is associated with poor prognosis, and murine lung tumor models exhibit increased macrophage infiltration. We hypothesize that chronic cigarette smoke exposure causes changes in inflammatory cell numbers and phenotypes creating a permissive environment for tumor formation. Chemopreventive agents such as the prostacyclin analog iloprost, may mitigate these changes.

      Methods
      FVB mice were given control AIN-76A chow or AIN-76A with 0.015% pioglitazone two weeks prior to smoke exposure. Mice were exposed to 70-90 mg/m[3] particulate level cigarette smoke for 6 hours/day, 5 days/week. Lungs were harvested 1 and 22 weeks after smoke exposure and after 22 weeks smoke/20 weeks ambient air (standard protocol for tumor induction). Bronchoalveolar cells were collected for qRT-PCR analysis of macrophage markers. Tumors were harvested and dissected from uninvolved tissue. Uninvolved lung tissue was digested and immune cell content analyzed by flow cytometry.

      Results
      Cigarette smoke exposure induced changes in macrophage phenotype after 1 week as indicated by CD11b exression (flow cytometry) and phenotypic markers (qRT-PCR). Macrophage infiltration increased in 22 week smoke/20 week ambient air exposed mice. Neutrophil numbers were increased at one week of smoke exposure. CD4 and CD8 positive T cell numbers decreased during smoke exposure but returned to control levels after smoking cessation. Smoke exposure differentially affected macrophage programming at each time point.

      Conclusion
      Cigarette smoke exposure affects pulmonary inflammatory cell numbers and function as early as 1 week after smoking initiation. Some of these changes are transient (increases in neutrophil numbers), while others are consistent but return to normal after smoking cessation (decreases in T cell populations). Smoke exposure resulted in early functional effects on macrophages and alveolar macrophage infiltration was still increased 20 weeks after smoking cessation, indicating that the effects of cigarette smoking on innate immunity are long lasting.