EPIGENETIC MECHANISM OF THE SYNERGISTIC TUMORIGENIC EFFECT OF ARSENIC AND BENZO[A]PYRENE CO-EXPOSURE

项目摘要/摘要 砷和苯并[a]芘(BaP)是最常见的环境污染物，人类接触到 砷和苯并苯混合物是很常见的。我们对砷和BaP协同作用及其机制的认识 曝光率有限。本研究旨在探讨砷与苯并苯联合作用的机制。 曝光。表观遗传学指的是基因表达模式中的可遗传变化，这些变化不是由变化引起的 在DNA序列中，但由DNA甲基化、组蛋白翻译后修饰和非编码介导 RNA。肿瘤干细胞(CSCs)是具有正常干细胞特征的癌细胞。CSC或CSC- 类细胞被认为是启动癌症的细胞。虽然砷的毒性作用机制还没有明确的定义， 目前认为，BaP的毒性作用主要依赖于其代谢活性和DNA加合物的形成。这个 已报道的砷对DNA加合物水平的影响是有争议的，这表明其他机制可能发挥作用 砷和苯并苯联合暴露的综合效应中的关键作用。我们的初步研究发现：(I)砷和苯并苯 联合暴露在诱导细胞转化、CSC样特性和肺癌方面具有协同作用；(Ii)砷 BaP共暴露对BPDE-DNA加合物水平无协同作用。相比之下，联合曝光显示出 通过增加组蛋白H3K9的水平来证明表观遗传解除调控的协同效应 甲基转移酶SUV39H1和H3抑制甲基化标记H3K9me2。此外，联合曝光也 在降低细胞因子信号转导抑制因子3(SOCS3)水平和提高Akt水平方面显示出协同作用 和ERK1/2激活；(Iii)shRNA敲除SUV39H1降低H3K9me2水平，增加SOCS3水平，降低 AKT和ERK1/2的激活与软琼脂集落形成和CSC样特性；(Iv)稳定地重新表达SOCS3 减少Akt、ERK1/2的激活、软琼脂集落形成和CSC样特性；(V)抑制Akt和Erk/1/2 减少软琼脂集落形成和类似CSC的特性；以及(Vi)砷和BaP共同暴露显著 增加芳香烃受体(AhR)的核定位和siRNA敲除AhR大大降低 砷和苯并苯联合暴露转化细胞中SUV39H1的表达水平。根据我们的初步数据，我们的中央 假说是：(I)砷和BaP共同暴露通过协同激活AhR上调HMTase SUV39H1； (Ii)SUV39H1上调导致SOCS3下调；及。(Iii)SOCS3下调导致 更强的Akt和ERK1/2激活，增强细胞转化和CSC样特性，从而增加肺 肿瘤发生学。提出了三个目标：目标1：砷和苯并苯联合暴露显著增强细胞 通过协同激活AhR上调HMTase SUV39H1的转化和肿瘤发生。目标 2：砷和BaP共同作用上调SUV39H1促进细胞转化和肿瘤发生 通过下调SOCS3导致Akt和ERK1/2更强的激活。目标3：同时失活 AKT和ERK1/2均可减弱砷与BaP的协同作用. 暴露在诱导小鼠细胞转化和肺肿瘤形成中。

PROJECT SUMMARY/ABSTRACT Arsenic and benzo[a]pyrene (BaP) are among the most common environmental pollutants and human exposure to arsenic and BaP mixture is very common. Our knowledge on the effect and mechanism of arsenic and BaP co- exposure is limited. The goal of this study is to determine the mechanism of the effect of arsenic and BaP co- exposure. Epigenetics refers to heritable changes in the pattern of gene expression that are not caused by changes in DNA sequence, but are mediated by DNA methylation, histone posttranslational modifications and noncoding RNAs. Cancer stem cells (CSCs) are cancer cells possessing characteristics of normal stem cells. CSCs or CSC- like cells are considered as cancer initiating cells. While the mechanism of arsenic toxic effect is not clearly defined, it is thought that the toxic effect of BaP mainly depends on its metabolic activation and DNA adduct formation. The reported effects of arsenic on DNA adduct level are controversial, suggesting that other mechanisms may play a key role in the combined effect of arsenic and BaP co-exposure. Our preliminary studies found: (i) Arsenic and BaP co-exposure has a synergistic effect in inducing cell transformation, CSC-like property and lung tumors; (ii) Arsenic and BaP co-exposure has no synergistic effect on BPDE-DNA adduct level. In contrast, the co-exposure shows a synergistic effect in epigenetic deregulations as evidenced by increasing the levels of histone H3K9 methyltransferase SUV39H1 and H3 repressive methylation mark H3K9me2. Moreover, the co-exposure also shows a synergistic effect in reducing the level of suppressor of cytokine signaling 3 (SOCS3) and increasing Akt and Erk1/2 activation; (iii) ShRNA knockdown SUV39H1 reduces H3K9me2 level, increases SOCS3 level, reduces Akt and Erk1/2 activation and soft agar colony formation and CSC-like property; (iv) Stably re-expressing SOCS3 reduces Akt, Erk1/2 activation, soft agar colony formation and CSC-like property; (v) Inhibiting Akt and Erk/1/2 reduces soft agar colony formation and CSC-like property; and (vi) Arsenic and BaP co-exposure significantly increases aryl hydrocarbon receptor (AhR) nuclear localization and siRNA knockdown AhR greatly reduces the level of SUV39H1 in arsenic and BaP co-exposure-transformed cells. Based on our preliminary data, our central hypothesis is: (i) Arsenic and BaP co-exposure up-regulates HMTase SUV39H1 via synergistically activating AhR; (ii) Up-regulation of SUV39H1 leads to SOCS3 down-regulation; and (iii) Down-regulation of SOCS3 causes a stronger Akt and Erk1/2 activation, which enhances cell transformation and CSC-like property thus increasing lung tumorigenesis. Three aims are proposed: Aim 1: Arsenic and BaP co-exposure significantly enhances cell transformation and tumorigenesis by up-regulating HMTase SUV39H1 via synergistically activating AhR. Aim 2: SUV39H1 up-regulation by arsenic and BaP co-exposure enhances cell transformation and tumorigenesis by down-regulating SOCS3 leading to stronger activation of Akt and Erk1/2. Aim 3: Simultaneously inactivating both Akt and Erk1/2 by a natural compound Withaferin A impairs the synergistic effect of arsenic and BaP co- exposure in inducing cell transformation and lung tumorigenesis in mice.