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R.L. Keith
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MO04 - Lung Cancer Biology I (ID 86)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 1
- Moderators:G. Sozzi, D.C. Lam
- Coordinates: 10/28/2013, 16:15 - 17:45, Bayside 103, Level 1
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MO04.05 - Tracheal basal cells in the lung squamous dysplasia: moving proximal. (ID 1700)
16:35 - 16:40 | Author(s): R.L. Keith
- Abstract
- Presentation
Background
Squamous cell carcinoma (SCC) is the second most common form of lung cancer, a disease primarily observed in smokers. Studies have shown that preneoplastic dysplasias are the precursors for SCC. However, only a subset of these lesions progress to invasive carcinoma and predicting the fate of individual lesions is difficult. Understanding the processes associated with the development of dysplasia would therefore have significant impact on preventative therapy for high-risk lesions. To this goal we have studied N-nitroso-tris chloroethyl urea (NTCU) model of premalignant murine squamous dysplasia. Bi-weekly topical application of NTCU for 32-weeks caused endobronchial dysplasias that were pathologically similar to dysplasias encountered in human smokers. In the current study we used NTCU model to identify the events that precede dysplastic changes in murine airways.Methods
Immunofluorescence analysis of NTCU treated airways showed abundant expression of keratin 5 positive (K5+) basal cells. In normal mouse respiratory epithelium K5+ cells are confined only in the trachea and are absent in the bronchial epithelium. Ectopic expressions of K5+ cells in the mouse bronchial epithelium therefore suggested a role for tracheal basal cells in the pathogenesis of NTCU-induced dysplasia. To explore this possibility we analyzed tracheal and bronchial histology after vehicle and 20mM NTCU treatment at 4, 8, 12, 16, 25 and 32 weeks.Results
Low-grade dysplasia of the tracheal epithelium was observed as early as 4 weeks of NTCU exposure. On the contrary, squamous metaplasia or low-grade dysplasia of the bronchial epithelium was not seen until 25 weeks. Morphometric analysis of immunostained tracheal tissue showed a time-dependent increase in the numbers of K5+ cells and a concomitant loss of cells expressing Clara cell secretory protein (CCSP) and ciliated cells. We have shown previously that injury to the trachea upregulates expression of keratin 14 (K14) in basal cells. Consistent with this phenomenon, NTCU exposure showed a time-dependent increase in the number of K5/K14 dual-positive basal cells. To understand the mechanism by which NTCU treatment mediates ectopic expression of basal cells in the lung, we used flow-cytometry to analyze cell-surface markers expressed by tracheal basal cells. These studies revealed a time-dependent decrease in the level of integrin a6b4 (CD49f). CD49f is a hemidesmosomal protein that facilitates attachment of basal cells to the basement membrane. These findings suggested that NTCU treatment might promote migration of tracheal basal cells by destabilizing their adherence to the basement membrane.Conclusion
Collectively, our results showed an early involvement of tracheal basal cells in the generation of dysplastic lung lesions in a murine model of SCC. By moving the focus proximal, we have discovered a realm of biological developments in the trachea that had not been previously examined. Further research of these processes will aid in the elucidation of dysplastic development in SCC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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O12 - Lung Cancer Biology II (ID 87)
- Event: WCLC 2013
- Type: Oral Abstract Session
- Track: Biology
- Presentations: 1
- Moderators:Y. Nakanishi, B. Solomon
- Coordinates: 10/29/2013, 10:30 - 12:00, Parkside 110 A+B, Level 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)
10:40 - 10:50 | Author(s): R.L. Keith
- Abstract
- Presentation
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.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
