Dissecting Nrf2-dependent HIF1a activation mechanism in arsenic-induced cancer stem-like cells

长期接触砷，尤其是。无机三价砷 (iAs)，一种人类致癌物 由于工业环境而自然发生在地壳或工作场所，与各种 癌症的类型，特别是。肺癌。几十年来，人们已经知道多种不同的机制 可能参与 iAs 诱导的恶性转化。然而，iAs 是否以及如何诱导 尚未研究来自非干细胞的癌症干细胞样细胞（CSC）。我们之前已经证明了 用 0.125 至 0.25 µM（~9 至 18ppb）iAs 连续处理人支气管上皮细胞 六个月诱导 CSC 的产生。除了较高的干性表达 电路基因，包括 Oct4、Sox2、myc、Klf4 等，这些 iAs 诱导的 CSC 还表现出独特的 以活跃的糖酵解和线粒体氧化减少为特征的代谢模式 磷酸化（OXPHOS）。亲本细胞和 iAs 诱导的 CSC 的 ChIP-seq 分析 揭示组蛋白 H3 赖氨酸 4 三甲基化 (H3K4me3) 的富集显着增加， 基因转录的活性表观遗传标记，在对干性和糖酵解至关重要的基因中 CSC 的成员。了解 iAs 如何诱导这些代谢和表观遗传变化以及 为了生成 CSC，我们最近还研究了 iAs 激活 Nrf2 和 通过生化和 ChIP-seq 方法进行 HIF1α 信号传导。数据表明 Nrf2 和 HIF1α可能作为代谢和表观遗传重编程的启动子，以及获得 CSC 功能。因此，我们假设 iAs 激活的 Nrf2 是 HIF1 的上游激活剂 与糖酵解相关的信号传导，以及随后的表观遗传重编程和生成 CSC。为了检验这一假设，将追求以下三个具体目标： 目标 1，剖析 Nrf2 激活途径，重点是 iAs 诱导的 Keap1 泛素化和降解， Nrf2 内源性抑制剂；目标 2，研究 Nrf2 依赖性 HIF1α 启动机制 细胞中响应 iAs 的信号传导；目标 3，了解 iAs 如何诱导 Nrf2-HIF1 信号传导将代谢从线粒体 TCA 循环转变为与重编程相关的糖酵解 组蛋白甲基化谱和 CSC 的生成。我们预计数据来自 该研究计划的完成将首次揭示 Nrf2 和 HIF1 在 iAs- 中的重要性 诱导的 CSCs，这可能会通过靶向 Nrf2- 转化为癌症治疗的新策略 HIF1α 信号传导和 CSC。

Long-term exposure to arsenic, esp. the inorganic trivalent arsenic (iAs), a human carcinogen that occurs naturally in the earth's crust or work place due to industrial settings, has been linked to various types of cancers, esp. lung cancer. It has been known for decades that multiple different mechanisms might be involved in iAs-induced malignant transformation. However, whether and how iAs induces cancer stem-like cells (CSCs) from non-stem cells hadn’t been studied. We had previously shown that consecutive treatment of the human bronchial epithelial cells with 0.125 to 0.25 M (~9 to 18ppb) iAs for six months induced generation of the CSCs. In addition to the higher expression of the stemness circuit genes, including Oct4, Sox2, myc, Klf4, etc., these iAs-induced CSCs also exhibited a unique metabolic pattern featured with an active glycolysis and diminished mitochondrial oxidative phosphorylation (OXPHOS). ChIP-seq analysis of the parental cells and the iAs-induced CSCs revealed a substantial increase in the enrichment of histone H3 lysine4 trimethylation (H3K4me3), an active epigenetic marker for gene transcription, among genes critical for the stemness and glycolysis of the CSCs. To understand how iAs induces these metabolic and epigenetic changes and the generation of the CSCs, we recently also investigated the capability of iAs on the activation of Nrf2 and HIF1 signaling through both biochemical and ChIP-seq approaches. The data indicate that Nrf2 and HIF1 may serve as initiators for the metabolic and epigenetic reprograming, and the acquisition of the CSC features. Accordingly, we hypothesize that iAs-activated Nrf2 is an upstream activator for HIF1 signaling linked to glycolysis, and the subsequent epigenetic reprogramming and generation of the CSCs. To test this hypothesis, the following three specific aims will be pursued: Aim1, dissecting Nrf2 activation pathways with an emphasis on the iAs-induced ubiquitination and degradation of Keap1, the endogenous inhibitor of Nrf2; Aim 2, investigating mechanisms of Nrf2-dependent initiation of HIF1 signaling in the cells in response to iAs; and Aim 3, understanding how iAs-induced Nrf2-HIF1 signaling shifts the metabolism from mitochondrial TCA cycle to glycolysis that linked to reprogramming of the histone methylation profiles and the generation of CSCs. We expect that the data from the completion of this research plan will be the first to reveal the importance of Nrf2 and HIF1 in iAs- induced CSCs, which may be translated into new strategies of cancer therapy by targeting the Nrf2- HIF1 signaling and the CSCs.