RNA Methylation in Cancer Plasticity and Drug Resistance

项目总结/摘要 癌症耐药性不仅通过选择遗传抗性克隆，而且通过表型 可塑性通过快速诱导转录程序，使一些细胞适应和坚持。在膀胱 在我们实验室建立的模型中观察到表型可塑性，其中两个亚群 肿瘤细胞可逆地和自发地从一种转变到另一种。Hoechst分离并研究 染色和流式细胞术，这些亚群由高度致瘤性的“侧群”（SP）组成， 顺铂抗性干细胞样细胞和缺乏这些特性的细胞的“非侧群”（NSP）。一个潜在 对这种可塑性有贡献的表观遗传因素是N6-甲基腺苷（m6 A）RNA修饰。由m6 A存放 m6 A可动态可逆地调节关键细胞功能， 癌细胞的基本特征。m6 A修饰和m6 A效应器（写入器、擦除器、读取器）的放松管制 与多种癌症的耐药性有关。为了研究m6 A修饰在BC中的作用，我 在本实验室建立并验证了金标准的表位组学分析，甲基-RNA-免疫沉淀 随后进行高通量测序（MeRIP-seq）。我用这种方法比较了SP和NSP 亚群和鉴定的差异甲基化候选转录物。我还发现， 抑制关键的m6 A擦除器、脂肪量和肥胖相关蛋白（FTO）增强了向SP状态的转变。 基于这些初步的数据，我建议测试m6 A修饰调节表达的假设， 在体外和患者来源的样品中促进BC向耐药状态转变的转录物。我会 目的1：我将使用MeRIP-seq和RNA-seq系统地鉴定 差异甲基化和差异表达的转录物，这些转录物驱动耐药基因的转变， 状态我将从基因上调节这些靶点，并测量对顺铂耐药性的影响，SP-NSP 相互转化、菌落形成、迁移和入侵。目标2：我将定义FTO的功能，一个键m6 A 在我们的模型中，它通过遗传和基因调节FTO来影响可塑性。我会衡量 对顺铂耐药性、SP-NSP相互转化、集落形成、迁移和侵袭性的影响，以及 使用MeRIP-seq和RNA-seq鉴定差异甲基化和差异甲基化的转录物， 表达。目的3：我将使用RT-qPCR来测试哪些候选转录本和m6 A效应子与 使用BC患者来源的固体和液体活检样品进行顺铂耐药的临床进展。 表征这种新的表转录组机制将为新的生物标志物和 治疗目标旨在短路BC耐药性。这项研究将提供严格的 在提供一流研究设施的出色环境中进行医学科学家培训， 翻译患者组织资源和多样化的指导专业知识。

PROJECT SUMMARY/ ABSTRACT Cancer drug resistance occurs not only by selection of genetically resistant clones, but also through phenotypic plasticity via rapid induction of transcriptional programs that allow some cells to adapt and persist. In bladder cancer (BC), phenotypic plasticity is observed in a model established in our lab, in which two subpopulations of tumor cells reversibly and spontaneously transition from one to the other. Isolated and studied by Hoechst staining and flow cytometry, these subpopulations consist of a “side population” (SP) of highly tumorigenic, cisplatin-resistant, stem-like cells, and a “non-side population” (NSP) of cells lacking these properties. A potential contributing epigenetic factor to this plasticity is N6-methyladenosine (m6A) RNA modification. Deposited by m6A writers and removed by m6A erasers, m6A dynamically and reversibly regulates key cellular functions and essential features of cancer cells. Deregulation of m6A modifications and m6A effectors (writers, erasers, readers) has been implicated in drug resistance in various cancer types. To study the role of m6A modifications in BC, I set up and validated in our lab the gold standard epitranscriptomic assay, methyl-RNA-immunoprecipitation followed by high-throughput sequencing (MeRIP-seq). I used this assay to compare the SP and NSP subpopulations and identified differentially methylated candidate transcripts. I also found that pharmacological inhibition of a key m6A eraser, fat mass and obesity-associated protein (FTO), potentiates a shift to the SP state. Based on these preliminary data, I propose to test the hypothesis that m6A modifications regulate expression of transcripts that promote transition to a drug-resistant state in BC in vitro and in patient-derived samples. I will accomplish this with the following aims: Aim 1: I will use MeRIP-seq and RNA-seq to systematically identify differentially methylated and differentially expressed transcripts that drive the shift to and from a drug-resistant state. I will genetically modulate these targets, and measure the impact on cisplatin resistance, SP-NSP interconversion, colony formation, migration and invasiveness. Aim 2: I will define the function of FTO, a key m6A eraser that affects plasticity in our model by genetically and pharmacologically modulating FTO. I will measure the impact on cisplatin resistance, SP-NSP interconversion, colony formation, migration and invasiveness, and use MeRIP-seq and RNA-seq to identify transcripts that are both differentially methylated and differentially expressed. Aim 3: I will use RT-qPCR to test which candidate transcripts and m6A effectors are associated with clinical progression to cisplatin resistance using BC patient-derived solid and liquid biopsy samples. Characterizing this novel epitranscriptomic mechanism will provide strong evidence for new biomarkers and therapeutic targets aimed at short-circuiting BC drug resistance. The proposed research study will offer rigorous physician-scientist training in an outstanding environment offering top notch research facilities integrated with translational patient tissue resources and diverse mentoring expertise.