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

在我们以前的研究中，我们已经证明了砷（As 3+），一种环境类金属，能够 以诱导人支气管上皮细胞的转化。额外的实验数据显示， 在As 3+诱导的转化细胞中，癌干细胞样细胞（CSCs）的比例最高。其他初步数据显示， 结果表明：（1）低浓度As 3+处理人支气管上皮细胞， 细胞表现出显着减少的活性氧化物质（ROS），由于严重抑制的 线粒体氧化磷酸化（OXPHOS）;（ii）As 3+诱导的JNK和STAT 3（pSTAT 3S 727） 线粒体中的磷酸化沿着线粒体转录因子A（TFAM）的减少;（iii） 综合转录组学和代谢组学分析表明，糖酵解速率较高， 线粒体DNA（mtDNA）缺失导致的线粒体代谢; 和（iv）一种独特的糖酵解特征，不同于幼稚胚胎干细胞（ESC）和癌细胞， 在这些As 3+诱导的CSC中发现。ESC和癌细胞都直接糖酵解产生乳酸。在 相反，As 3+诱导的CSC显示糖酵解中间体转化为次级代谢产物的增加。 产生N-乙酰葡糖胺的途径，对干性基因的O-GlcNAc化很重要， S-腺苷甲硫氨酸（SAM），有助于DNA和组蛋白甲基化。因此，这一目标 （1）As 3+诱导的JNK依赖性pSTAT 3S 727是否负责抑制 （2）如果是这样，As 3+诱导的JNK依赖性pSTAT 3S 727信号通路如何损害线粒体的功能。 线粒体的完整性或功能，如mtDNA复制，转录，OXPHOS等;（3）如何 线粒体功能受损有助于As 3+诱导的CSC的产生。我们假设 As 3+诱导的JNK依赖性pSTAT 3S 727信号通过抑制CSCs的形成， 线粒体OXPHOS和ROS的产生，以及随后的细胞糖酵解的增强。到 为了验证这一假设，提出了以下三个具体目标：具体目标1：确定As 3 + 激活线粒体JNK，使BEAS-2B和其他肺细胞中的STAT 3 S727（pSTAT 3S 727）磷酸化， CSCs的形成。我们将重点关注JNK上游激酶的激活， 对As 3+的反应。将研究JNK依赖的其他线粒体蛋白的磷酸化 通过线粒体磷酸化蛋白质组;具体目标2：了解As 3+诱导的JNK依赖性 pSTAT 3S 727通过解决pSTAT 3S 727在mtDNA结合中的作用，其与线粒体的相互作用， 线粒体转录因子A（TFAM）及其对线粒体ROS产生、蛋白质组和细胞凋亡的影响。 As 3+处理的细胞和As 3+诱导的CSC中的细胞代谢组学;具体目标3：利用我们独特的小鼠 原位肺癌模型，以及临床相关的靶向药物，以进行临床前探索 JNK、STAT 3和糖酵解抑制剂在CSC产生的小鼠肺癌模型中的治疗潜力。 将测试JNK、STAT 3和糖酵解抑制剂的短期和长期全身方案。我们 预期所提出的研究结果将揭示As 3+诱导的JNK依赖性 pSTAT 3S 727对CSC产生的影响，并导致As 3+致癌新概念的出现， 强调了As ~（3+）诱导CSC的能力。此外，我们认为，完成日期 这一项目将是真实的价值，在确定一些新的和直接的目标点，可能有助于 加速其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.