(PQC1) PLK1 And EPHX3 Promotion Of Genomic Instability In Bronchial Dysplasia

描述(申请人提供)：支气管发育不良(BD)是一种可进展为鳞状细胞癌(SCC)的癌前病变。识别具有高进展风险的病变对于帮助识别潜在的恶性转化的细胞机制以及帮助识别将从预防措施中受益的患者是重要的。我们通过基因表达阵列分析比较了高危持续性BD和退行性BD，并鉴定了300多个在持续性BD中差异表达的基因。通过通路分析，我们发现了两个与持久性有密切关系的基因，即Polo-like Kinase(PLK1)和环氧化物水解酶3(EPHX3)，这两个因子与持久性存在协同关系，并提示在促进BD基因组不稳定性方面存在潜在的相互作用。EPHX3可将香烟烟雾中的致癌多环芳烃(PAHs)转化为致突变的代谢物。PLK1通过G2-M检查点促进细胞周期进程。当存在遗传损伤时，这一检查点通常用于阻止细胞周期，并随后诱导具有广泛损伤的细胞凋亡。我们假设，突变能力的增加与G2-M检查点的取消和随后的细胞周期进展相结合，促进并确立了介导进展为浸润性肺癌的遗传损伤。此外，可以获得这些因子的特定抑制剂，这表明了预防肺鳞状细胞癌发展的潜在机制。我们建议进行研究，以确定PLK1和EPHX3抑制对进展相关细胞活动的影响，并评估PLK1和EPHX3过表达在促进基因组不稳定性中的作用，这些细胞培养自持续性BD和正常支气管上皮。该提案的第一个目标将扩大初步研究，这些研究表明，在正常的上皮细胞培养中，用Volasertib(勃林格-英格尔海姆)抑制PLK1显著减少培养的持续性BD来源的上皮细胞的增殖和诱导凋亡活性，而不影响这些特性。同样，我们已经证明了EPHX3抑制剂Auda可以降低SCC细胞系中EPHX3的活性，并将研究其在BD来源的培养中的作用。第二个目标将评估基因组不稳定性在持续性BD中的作用，方法是表征改变的类型并量化 与致癌物质接触有关的损害程度。烟草烟雾冷凝物处理持续性BD和正常支气管培养所引起的遗传不稳定性将通过在PLK1和EPHX抑制剂存在和不存在的情况下培养的细胞系的全基因组测序和甲基化阵列分析来衡量，以验证我们的假设。PLK1和EPHX3依赖的基因组不稳定性的证明将促进长期目标包括建立一个重要的细胞机制，通过该机制介导与进展相关的活动，并为开发针对这些酶的潜在有效的化学预防疗法提供理论基础。

DESCRIPTION (provided by applicant): Bronchial dysplasia (BD) is a precancerous lesion that can progress to squamous cell carcinoma (SCC). Identification of lesions with high risk for progression is important to help identify cellular mechanisms underlying malignant transformation and to help identify patients that would benefit from preventive measures. We have compared high risk persistent BD to regressive BD by gene expression array analysis and identified more than 300 genes that are differentially expressed in persistent BD. Using a pathway analysis to identify genes with a strong relationship to persistence, we have identified two factors, polo-like kinase (PLK1) and epoxide hydrolase 3 (EPHX3), which demonstrate a synergistic relationship with persistence and suggest a potential interaction in promoting genomic instability in BD. EPHX3 converts cigarette smoke associated pro- carcinogenic polycyclic aromatic hydrocarbons (PAHs) to mutagenic metabolites. PLK1 promotes cell cycle progression through the G2-M checkpoint. This checkpoint normally operates to arrest the cell cycle when genetic damage is present with the subsequent induction of apoptosis in cells with extensive damage. We have hypothesized that the combination of increased mutational capacity coupled with G2-M checkpoint abrogation and subsequent cell cycle progression promotes and establishes genetic damage that mediates progression to invasive lung cancer. Furthermore, specific inhibitors of these factors are available suggesting a potential mechanism for preventing the development of lung SCC. We propose studies to establish the effect of PLK1 and EPHX3 inhibition on progression associated cellular activities and to assess the role of PLK1 and EPHX3 overexpression in promoting genomic instability using bronchial cell cultures established from persistent BD and normal bronchial epithelium. The first aim of the proposal will expand on preliminary studies that indicate PLK1 inhibition with Volasertib (Boehringer-Ingelheim) significantly reduces proliferation and induces apoptotic activity in cultured persisten BD derived epithelial cells without affecting these properties in normal epithelial cultures. Similarly, we have shown that the EPHX3 inhibitor, AUDA, reduces EPHX3 activity in SCC cell lines and will study its effects in BD derived cultures. The second aim will evaluate the role of genomic instability in persistent BD by characterizing the types of alterations and quantifying the amount of damage that are associated with carcinogen exposure. Genetic instability induced by tobacco smoke condensate treatment of persistent BD and normal bronchial cultures will be measured by genome-wide sequencing and methylation array analyses of cell lines cultured in the presence and absence of PLK1 and EPHX inhibitors to test our hypothesis. Long term goals that would be facilitated by the demonstration of PLK1 and EPHX3 dependent genomic instability include the establishment of an important cellular mechanism through which progression associated activities are mediated and the provision of rationale for the development of potentially effective chemopreventive therapy targeting these enzymes.