Nicotine/NNK Signaling in Human Lung Cancer Cells

描述（由申请人提供）：肺癌是全球癌症死亡的主要原因，与吸烟有很强的病因学关联。尼古丁和亚硝胺4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）是香烟烟雾中的两种重要成分。尼古丁可以激活促进生长的途径，从而促进肺癌的发展。NNK是由尼古丁的亚硝化作用形成的，被认为是香烟烟雾中最有效的致癌物。我们最近证明，尼古丁通过激活ERK1/2诱导Bcl2丝氨酸70位点的磷酸化，这与延长小细胞肺癌（SCLC）细胞的存活有关。因此，尼古丁诱导的细胞存活结果，至少在一定程度上，来自一个涉及Bcl2磷酸化的机制。c-Myc是一种主要的致癌蛋白，可与Bcl2在细胞增殖、转化、凋亡和致瘤性中发挥协同作用。我们的初步数据表明，尼古丁和NNK同时诱导Bcl2和c-Myc磷酸化，与SCLC细胞增殖增加有关，但细胞内机制尚不清楚。Bcl2和c-Myc在正常肺上皮细胞和肺癌细胞中均有表达。这两种癌蛋白可能是尼古丁或nnk激活信号通路的致瘤靶点。NNK可以直接诱导单链DNA断裂，增加引起DNA氧化损伤的活性氧（ROS）。由于Bcl2和c-Myc都具有促进基因组不稳定性的功能，Bcl2可能克服c-Myc的凋亡效应，并与c-Myc协同减弱DNA修复并保留存活细胞中nnk诱导的DNA损伤，这可能有助于肿瘤的发生。为了严格验证这些假设，我们确定了两个特定的目标：(1)确定尼古丁或NNK诱导的Bcl2和c-Myc磷酸化是否以及如何促进它们在调节人类肺癌细胞存活和增殖中的合作。研究将确定Bcl2和c-Myc之间直接相互作用的机制；(2)确定尼古丁或nnk诱导的正常肺上皮细胞和肺癌细胞的存活、增殖和维持nnk诱导的DNA损伤和遗传不稳定是否需要Bcl2和c-Myc之间的功能合作。研究将确定Bcl2在核和线粒体水平上通过磷酸化与c-Myc合作的新机制。将采用最先进的分子和生化方法，包括定点基因诱变、RNA干扰或基因沉默。该结果有望填补我们在尼古丁和NNK调节正常肺上皮细胞和肺癌细胞中Bcl2、c-Myc、增殖和遗传不稳定性的信号传导和致癌机制方面的基本知识空白。这些研究的结果有望对烟草相关癌症（特别是肺癌或其他表达Bcl2和c-Myc的恶性肿瘤）的治疗具有潜在的临床意义，并可能为开发新的策略做出重大贡献，这些策略专门针对在功能上破坏Bcl2和c-Myc之间的致癌合作。

DESCRIPTION (provided by applicant): Lung cancer is the predominant cause of cancer deaths worldwide and has a strong etiological association with cigarette smoking. Nicotine and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are two important components in cigarette smoke. Nicotine can activate the growth-promoting pathways to facilitate the development of lung cancer. NNK is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. We have recently demonstrated that nicotine induces Bcl2 phosphorylation exclusively at serine70 through activation of ERK1/2 in association with prolonged survival of small cell lung cancer (SCLC) cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. c-Myc is a major oncogenic protein that can functionally cooperate with Bcl2 in cell proliferation, transformation, apoptosis and tumorigenicity. Our preliminary data indicate that nicotine and NNK simultaneously induces Bcl2 and c-Myc phosphorylation in association with increased proliferation of SCLC cells but the intracellular mechanism(s) remains unclear. Bcl2 and c-Myc are expressed in both normal lung epithelial and lung cancer cells. These two oncoproteins may function as the oncogenic targets of nicotine or NNK-activated signal pathways. NNK can directly induce single strand DNA breaks and increases reactive oxygen species (ROS) that cause oxidative DNA damage. Since both Bcl2 and c-Myc function to promote genomic instability, Bcl2 may overcome the apoptotic effect from c-Myc and synergize with c-Myc to attenuate DNA repair and retain NNK-induced DNA damage in surviving cells, which may contribute to tumorigenesis. To critically test these hypotheses, we have identified two specific aims: (1) To determine if and how nicotine- or NNK- induced phosphorylation of Bcl2 and c-Myc can promote their cooperation in regulating survival and proliferation of human lung cancer cells. Studies will identify the mechanism(s) for the direct interaction between Bcl2 md c-Myc; (2) To determine whether the functional cooperation between Bcl2 and c-Myc is required for nicotine- or NNK-induced survival, proliferation and retaining NNK-induced DNA damage and genetic instability in both normal lung epithelial and lung cancer cells. Studies will identify novel mechanism(s) by which Bcl2 may functionally cooperate with c-Myc through phosphorylation at both the nuclear and mitochondrial levels. State of the art molecular and biochemical methodologies will be employed including site-directed gene mutagenesis, RNA interference or gene silencing. The results are expected to fill in fundamental gaps in our knowledge regarding the signaling and oncogenic mechanisms by which nicotine and NNK regulate Bcl2, c-Myc, proliferation and the genetic instability in both normal lung epithelial and lung cancer cells. Results from these studies are expected to have potential clinical relevance for the treatment of tobacco-related cancer specifically lung or other Bcl2 and c-Myc expressing malignancies and may contribute significantly to the development of novel strategies specifically aimed at functionally disrupting the oncogenic cooperation between Bcl2 and c-Myc.