The DNA adductome of lung carcinogenesis

摘要 肺癌是全球最常见的癌症，2012年有160万人死于肺癌，15.8万人死于肺癌 2015年美国的死亡人数。尽管香烟烟雾在这种流行病中扮演着明确的角色，但其确切的机制 这种癌症是如何发展的仍不清楚。尽管开展了反吸烟运动，但2014年仍有40 美国有100万烟民，全球吸烟者超过10亿。更有效的预防和治疗战略 对于这种疾病，需要更好的工具来了解肺癌的病因和发展机制。 许多研究表明，烟草烟雾中存在的化学物质会引起DNA修饰(DNA 加合物)，如果不修复，可能会导致突变，最终导致失去正常的细胞生长控制 机制和肺癌。其中许多研究使用了相对非特定的技术，如 免疫分析和32P-后标记，清楚地表明DNA加合物在肺中的水平较高 吸烟者多于不吸烟者，这与吸烟者肺部发现的多种突变一致。 然而，这些非特定技术的使用并没有导致任何积极的结构特征 DNA加合物，从而对其形成所涉及的机制进行清晰的鉴定。其他研究 针对特定的DNA加合物，已经导致了一些化学特征的DNA的鉴定 加合物，但这些结果不能解释那些使用更一般的非特定方法发现的结果。一个精准的 精确而全面的肺癌发生过程中DNA损伤的表征 难以捉摸。我们开发了一种新的基于质谱学的DNA加合提取方法 DNA加合物的全面高分辨率分析及其碎片信息 允许结构上的解释。我们的长期目标是确定DNA损伤特征 肺癌发生鉴定DNA加合物组最终可用于早期检测和预防 治疗。我们的假设是，与我们的方法将结合非特定方法的筛选能力 过去与检测到的各种修饰的特定化学特征一起使用，导致 特定的内收侧写。这项应用的目的是：1.表征肺dna加合物组。 烟草特异性亚硝胺NNK诱发肺癌的动物模型及驱动加合物的鉴定 通过共同接触促炎剂脂多糖(LPS)来增强其效果；2. 描述这些模型中DNA加合物随时间的演变，阐明 炎症与内源性过程；3.吸烟者肺DNA中DNA加合物组的特征 将其与非吸烟者以及动物模型中识别的轮廓进行比较。总而言之，我们的结果将 NNK和NNK+内毒素诱导的肺癌相关加合物组的特征及其作用机制 吸烟者肺DNA中的内收子组，为鉴定吸烟者肺DNA的分子特征奠定了基础 人类分子流行病学研究中的癌症病因学研究。 1

SUMMARY Lung cancer is the most common cancer worldwide, accounting for 1.6 million deaths in 2012 and for 158,000 deaths in the US in 2015. Despite the clear role of cigarette smoke in this epidemic, the precise mechanisms through which this cancer develops remain unclear. Despite anti-smoking campaigns, in 2014 there were still 40 million smokers in the U.S. and over 1 billion worldwide. More effective strategies for prevention and treatment of this disease, demand better tools to understand the mechanisms of lung cancer etiology and development. Numerous studies have shown that chemicals present in tobacco smoke induce DNA modifications (DNA adducts) which if not repaired, can lead to mutations ultimately resulting in loss of normal cellular growth control mechanisms and lung cancer. Many of these studies, using relatively non-specific techniques such as immunoassay and 32P-postlabelling, have clearly shown that DNA adduct levels are higher in the lungs of smokers than non-smokers, which is consistent with the multiple mutations found in the lungs of smokers. However, use of these non-specific techniques has not resulted in the positive structural characterization of any DNA adduct, and thus on a clear identification of the mechanisms involved in their formation. Other studies targeted at specific DNA adducts, have resulted in the identification of a few chemically characterized DNA adducts, but these results do not explain those found using the more general non-specific approaches. A precise characterization of the DNA damage during lung carcinogenesis that is both precise and comprehensive remains elusive. We have developed a new mass spectrometry based DNA adductomic approach performing comprehensive high resolution analysis of DNA adducts and providing information on their fragmentation allowing for structural elucidation. Our long-term goal is to determine the DNA damage profile characterizing lung carcinogenesis to identify a DNA adductome that may be ultimately used for early detection prevention and treatment. Our hypothesis is that with our method will combine the screening ability of the non-specific methods used in the past with the specific chemical characterization of the various modifications detected, resulting in a specific adductomic profile. The objectives of this application are: 1. to characterize the lung DNA adductome in animal models using the tobacco specific nitrosamine NNK to induce lung cancer and identify the driver adducts by enhancing its effects by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS); 2. to characterize the evolution of the DNA adductome in these models over time, clarifying the contribution of inflammation and endogenous processes; 3. to characterize the DNA adductome in smokers' lung DNA comparing it to non-smokers and to the profile identified in the animal models. Collectively our results will characterize the adductome associated with NNK and NNK+LPS induced lung carcinogenesis and the adductome in the lung DNA of smokers, setting the stage for the identification of molecular signatures for the investigation of cancer etiology in human molecular epidemiology studies. 1