Reduced Reactive Oxygen Species and Oxidative Phosphorylation in Arsenic-Induced Cancer Stem Cells

在我们之前的研究中，我们已经证明了砷(As3)，一种环境中的类金属，能够 诱导人支气管上皮细胞转化。更多实验数据显示存在 在As3诱导的转化细胞中，肿瘤干细胞(CSCs)的数量最多。其他初步数据显示 结果：(1)连续低浓度As3处理人支气管上皮细胞诱导的CSCs 由于对细胞活性氧化物种(ROS)的严重抑制，细胞内ROS显著减少 线粒体氧化磷酸化(OXPHOS)；(Ii)As3诱导的JNK和STAT3(PSTAT3S727) 线粒体的磷酸化和线粒体转录因子A(TFAM)的减少； 综合转录和代谢分析显示糖酵解率较高，水平较低。 As3诱导的CSCs中线粒体DNA(MtDNA)耗竭所致的线粒体代谢的改变； 和(Iv)不同于原始胚胎干细胞(ESCs)和癌细胞的独特的糖酵解特征是 在这些As3诱导的CSCs中发现。胚胎干细胞和癌细胞都直接通过糖酵解产生乳酸。在……里面 相反，As3诱导的CSCs显示糖酵解中间产物向辅助物的转化增加 N-乙酰氨基葡萄糖的产生途径对茎基因的O-GlcN酰化和 有助于脱氧核糖核酸和组蛋白甲基化的S-腺苷蛋氨酸(SAM)。因此，这一目标是 应用于确定：(1)是As3诱导的JNK依赖的pSTAT3S727负责抑制 (2)如果是这样的话，As3诱导的依赖JNK的pSTAT3S727信号通路如何损害 线粒体的完整性或功能，如mtDNA复制、转录、氧合酶等；以及(3)如何 线粒体功能受损参与了As3诱导的CSCs的产生。我们假设 As3诱导的依赖JNK的pSTAT3S727信号通过抑制CSCs的形成促进CSCs的形成 线粒体OXPHOS和ROS的产生，以及随后细胞糖酵解的增强。至 检验这一假设，提出了以下三个具体目标：具体目标1：确定AS3 激活线粒体JNK，使BEAS-2B和其他肺细胞中的STAT3 S727(PSTAT3S727)磷酸化 CSCs的形成。我们将重点研究线粒体定位的JNK上游激酶的激活。 对AS3的响应。依赖JNK的额外线粒体蛋白的磷酸化将被研究 通过线粒体磷酸蛋白质组；特定目标2：了解As3如何诱导JNK依赖 PSTAT3S727通过解决pSTAT3S727在线粒体DNA结合中的作用，它与线粒体的相互作用来抑制线粒体 线粒体转录因子A(TFAM)及其对线粒体ROS产生、蛋白质组和蛋白质组的影响 As3处理细胞和As3诱导的CSCs的细胞代谢组学；特定目标3：利用我们独特的小鼠 原位肺癌模型及临床相关靶向药物的临床前探索 JNK、STAT3和糖酵解抑制剂对CSC诱发小鼠肺癌模型的治疗作用 JNK、STAT3和糖酵解抑制剂的短期和长期全身方案都将接受测试。我们 预期拟议研究的结果将揭开As3诱导的JNK依赖的重要性 PSTAT3S727对肿瘤干细胞的产生和导致As3致癌的新概念的出现 重视As3在CSC诱导中的作用。此外，我们认为，完工所产生的日期 在定义一些新的和直接的目标点方面将具有真正的价值，这些点可能有助于 加快它们在临床环境中的使用。

In our previous studies we had demonstrated that arsenic (As3+), an environmental metalloid metal, was able to induce transformation of the human bronchial epithelial cells. Additional experimental data revealed presence of cancer stem-like cells (CSCs) among the transformed cells induced by As3+. Other preliminary data showed that: (i) The CSCs induced by consecutive low-concentration As3+ treatment of the human bronchial epithelial cells exhibited significant decrease of reactive oxidative species (ROS) due to severe inhibition of the mitochondrial oxidative phosphorylation (OXPHOS); (ii) As3+ induced JNK and STAT3 (pSTAT3S727) phosphorylation in mitochondria along with a diminish of the mitochondrial transcription factor A (TFAM); (iii) integrated transcriptomic and metabolomic analyses demonstrated a higher rate of glycolysis and lower levels of mitochondrial metabolism due to mitochondrial DNA (mtDNA) depletion among these As3+-induced CSCs; and (iv) a unique glycolytic feature that is different from naïve embryonic stem cells (ESCs) and cancer cells was found in these As3+-induced CSCs. Both ESCs and cancer cells direct glycolysis for lactate production. In contrast, the As3+-induced CSCs show increased conversion of the glycolytic intermediates into the subsidiary pathways for the generation of N-acetylglucosamine important for O-GlcNAcylation of the stemness genes and the S-adenosyl methionine (SAM) that contributes to DNA and histone methylation. Accordingly, the goal of this application is to determine: (1) is As3+-induced JNK-dependent pSTAT3S727 responsible for the inhibition of mitochondria; (2) if so, how this JNK-dependent pSTAT3S727 signaling pathway elicited by As3+ impairs the integrity or function of mitochondria, such as mtDNA replication, transcription, OXPHOS, etc; and (3) how the impaired function of mitochondria contributes to the generation of the CSCs induced by As3+. We hypothesize that As3+-induced JNK-dependent pSTAT3S727 signaling promotes formation of the CSCs by inhibiting mitochondrial OXPHOS and ROS generation, and the subsequent enhancement of glycolysis of the cells. To test this hypothesis, the following three specific aims are proposed: Specific Aim 1: determine how As3+ activates mitochondrial JNK that phosphorylates STAT3 S727 (pSTAT3S727) in BEAS-2B and other lung cells for the formation of CSCs. We will focus on the activation of mitochondrial-localized upstream kinases of JNK in response to As3+. The JNK dependent phosphorylation of additional mitochondrial proteins will be investigated through mitochondrial phosphoproteome; Specific aim 2: understand how As3+-induced JNK-dependent pSTAT3S727 inhibits mitochondria by addressing the role of pSTAT3S727 in mtDNA binding, its interaction with the mitochondrial transcription factor A (TFAM), and its effects on mitochondrial ROS production, proteome and the cellular metabolomics in the As3+-treated cells and As3+-induced CSCs; Specific Aim 3: utilize our unique mouse orthotopical lung cancer model, together with clinically relevant targeting agent, to preclinically explore therapeutic potential of inhibitors of JNK, STAT3 and glycolysis in CSC-generated lung cancer model in mice. Both short- and long-term systemic regimens of JNK, STAT3 and glycolysis inhibitors will be tested. We anticipate that the results from the proposed studies will unravel importance of As3+-induced JNK-dependent pSTAT3S727 on the generation of CSCs and lead to emerging of new concepts of As3+ carcinogenesis by emphasizing the ability of As3+ in CSC induction. Moreover, we believe that the date generated from completion of this project will be of real value in defining some new and straightforward targeting points that may help accelerate their use in clinical settings.