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

长期接触砷，特别是无机三价砷（iAs），一种人类致癌物质， 自然发生在地壳或工作场所，由于工业环境，已被链接到各种 癌症类型，特别是肺癌。几十年来，人们已经知道， 可能参与iAs诱导的恶性转化。然而，iAs是否以及如何诱导 从非干细胞中提取癌症干细胞样细胞（CSCs）还没有被研究过。我们之前已经证明， 用0.125至0.25 μ M（~9至18 ppb）iAs连续处理人支气管上皮细胞 诱导CSCs的产生。除了更高的干性表达外， 回路基因，包括Oct 4、Sox 2、myc、Klf 4等，这些iAs诱导的CSC也表现出独特的 代谢模式以活跃的糖酵解和减少的线粒体氧化为特征 磷酸化（OXPHOS）。亲本细胞和iAs诱导的CSC的ChIP-seq分析 显示组蛋白H3赖氨酸4三甲基化（H3 K4 me 3）的富集显著增加， 在对干性和糖酵解至关重要的基因中，基因转录的活性表观遗传标记 的CSC。为了了解iAs如何诱导这些代谢和表观遗传变化， 在CSCs的产生中，我们最近还研究了iAs对Nrf 2活化的能力， 通过生物化学和ChIP-seq方法的HIF 1 α信号传导。数据表明，Nrf 2和 HIF 1 α可能作为代谢和表观遗传重编程的启动子，并获得了转录因子。 CSC功能。因此，我们假设iAs激活的Nrf 2是HIF 1 α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 CSC。为了检验这一假设，将追求以下三个具体目标：目标1，解剖Nrf 2 激活途径，重点是iAs诱导的泛素化和Keap 1的降解， Nrf 2的内源性抑制剂;目的2，研究Nrf 2依赖的HIF 1 α启动机制， 目标3：了解iAs诱导的Nrf 2-HIF 1 α表达的机制。 信号传导将代谢从线粒体TCA循环转移到与重编程相关的糖酵解 组蛋白甲基化和CSC的产生。我们预计， 这项研究计划的完成将首次揭示Nrf 2和HIF 1 α在iAs中的重要性- 诱导的CSCs，这可能会转化为新的癌症治疗策略，通过靶向Nrf 2- HIF 1 α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.