Gene Expression Analysis Of Persistent And Regressive Bronchial Dysplasia Identifies Polo-Like Kinase 1 (Plk1) And Epoxide Hydrolase 3 (Ephx3) As Potential Mediators Of Malignant Progression

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Gene expression microarray analysis was employed to identify alterations that distinguish persistent from regressive bronchial dysplasia (BD). BD is a precursor of squamous cell carcinoma (SCC), and persistent BDs represent a high risk subset of these lesions. A potential role for promotion of genomic instability in persistence of BD is suggested by the overexpression of polo-like kinase 1 (PLK1), which abrogates G2-M checkpoint DNA damage repair, and epoxide hydrolase 3 (EPHX3), which converts tobacco smoke derived pro-carcinogens to mutagens. Methods and Results: Sixty-three frozen baseline biopsies were classified, based on the presence or absence of BD in follow-up biopsies, into persistent BD, regressive BD, progressive non-dysplasia and stable non-dysplasia groups. ANOVA statistical analysis with a false discovery rate of 10% revealed 318 differentially expressed genes between persistent and regressive BD, whereas all other intergroup comparisons except persistent BD versus stable non-dysplasia revealed no or very few (<15) differentially expressed genes. This confirmed the unique biological nature of persistent BD. A pathway analysis using this genelist and employing Ingenuity© software revealed “mitotic roles of PLKs” as having the most significant association with persistence. Based on their potential complimentary contribution to genomic instability, PLK1 and EPHX3 were further studied. Gene expression levels from all specimens with BD at baseline were used to classify biopsies as PLK1 and/or EPHX3 overexpressers based on expression levels greater than the mean expression for the gene. Using a histology score (1-8 for normal-SCC), a significant increase in outcome histology was noted in progressing from groups composed of cases without overexpression either gene, overexpression of one gene or overexpression of both genes (2.58 vs. 3.60 vs. 5.06 respectively). Validational analyses employing quantitative RT-PCR using cultures of 8 persistent BD and 6 regressive BD confirmed increased expression in persistent BD of PLK1 (2.77X, p=0.002) and EPHX3 (2.36X, p=0.081). Treatment of a persistent BD cell line with Volasertib, a specific inhibitor of PLK1, at 100nM showed potent inhibition of cell viability (live cell protease activity, 0.61 fold change vs untreated, p<0.05) and a strong trend toward increased apoptotic activity (caspase 3/7 activity, 1.71 fold change vs. untreated, p=0.076). Volasertib had no effect on these activities in cultured normal bronchial epithelial cells. Conclusion: Gene expression analysis demonstrates the biologically distinct, high risk nature of persistent BD and suggests a potential role for overexpression of PLK1 and EPHX3 in the development of invasive SCC of the lung. Citation Format: Daniel T. Merrick, Michael G. Edwards, Wilbur A. Franklin, Michio Sugita, Micah Friedman, York E. Miller, Lori Dwyer-Nield, Meredith Tennis, Kevin Choo, Greg Hickey, van Bokhoven Adriaan, Lynn Heasley, Paul A. Bunn, Mark Geraci, Robert L. Keith, Raphael Nemenoff. Gene expression analysis of persistent and regressive bronchial dysplasia identifies polo-like kinase 1 (PLK1) and epoxide hydrolase 3 (EPHX3) as potential mediators of malignant progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 231. doi:10.1158/1538-7445.AM2014-231