RNA Methylation in Cancer Plasticity and Drug Resistance

RNA 甲基化在癌症可塑性和耐药性中的作用

• 批准号: 10316114
• 负责人: Emmanuelle Hodara
• 金额: $ 5.1万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10316114
• 关键词: Adenosine; Affect; Biological Assay; Biological Markers; Biopsy Specimen; Blood specimen; CRISPR/Cas technology; Cancer Patient; Cancer cell line; Candidate Disease Gene; Cell physiology; Cells; Chromatin; Cisplatin; Clinical; DNA Methylation; DNA Sequence Alteration; Data; Deposition; Disease Progression; Drug resistance; Environment; Epigenetic Process; Fatty acid glycerol esters; Flow Cytometry; Gene Expression; Genes; Genetic Transcription; Gold; High-Throughput Nucleotide Sequencing; Immunoprecipitation; In Vitro; Knock-out; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Measures; Mediating; Mentors; Messenger RNA; Methylation; Modeling; Modification; Neoadjuvant Therapy; Obesity; Patients; Pharmacology; Phenotype; Physicians; Plasma Cells; Play; Population; Property; Proteins; RNA; RNA methylation; Reader; Resistance; Role; Sampling; Scientist; Side; Small Interfering RNA; Solid; Stains; Testing; Training; Transcript; Transcriptional Activation; base; cancer cell; cancer drug resistance; cancer therapy; cancer type; differential expression; drug-sensitive; epitranscriptomics; knock-down; liquid biopsy; migration; neoplastic cell; new therapeutic target; notch protein; novel; programs; research facility; research study; stem-like cell; therapeutic target; tissue resource; transcriptome sequencing; tumor; tumor progression; tumorigenic

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.

项目概要/摘要 癌症耐药性不仅是通过选择遗传抗性克隆而发生的，而且还通过表型来发生 通过快速诱导转录程序来实现可塑性，使某些细胞能够适应并持续存在。在膀胱内 癌症（BC），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、自发地从一种细胞转变为另一种细胞。赫斯特进行分离和研究 通过染色和流式细胞术，这些亚群由高度致瘤性的“侧群”（SP）组成， 顺铂耐药的干细胞样细胞，以及缺乏这些特性的“非侧群”（NSP）细胞。有潜力 造成这种可塑性的表观遗传因素是 N6-甲基腺苷 (m6A) RNA 修饰。 m6A 存入 m6A 动态地、可逆地调节关键细胞功能和 癌细胞的基本特征。 m6A 修改和 m6A 效应器（写入器、擦除器、读取器）的放松管制 与多种癌症类型的耐药性有关。为了研究 m6A 修饰在 BC 中的作用，我 在我们的实验室中建立并验证了金标准表观转录组测定，甲基-RNA-免疫沉淀 随后进行高通量测序 (MeRIP-seq)。我用这个测定法来比较 SP 和 NSP 亚群并鉴定出差异甲基化的候选转录本。我还发现药理 抑制关键的 m6A 擦除器、脂肪量和肥胖相关蛋白 (FTO)，可增强向 SP 状态的转变。 基于这些初步数据，我建议检验 m6A 修饰调节表达的假设 在体外和患者来源的样本中促进 BC 向耐药状态转变的转录本。我会 通过以下目标来实现这一目标： 目标 1：我将使用 MeRIP-seq 和 RNA-seq 来系统地识别 差异甲基化和差异表达的转录本驱动耐药性的转变 状态。我将对这些目标进行基因调节，并测量对顺铂耐药性的影响，SP-NSP 相互转化、菌落形成、迁移和侵袭。目标2：我将定义FTO的功能，一键m6A 通过遗传和药理学调节 FTO 来影响我们模型的可塑性的橡皮擦。我会测量 对顺铂耐药性、SP-NSP 相互转化、集落形成、迁移和侵袭的影响，以及 使用 MeRIP-seq 和 RNA-seq 来识别差异甲基化和差异的转录本 表示。目标 3：我将使用 RT-qPCR 来测试哪些候选转录本和 m6A 效应子与 使用 BC 患者来源的固体和液体活检样本观察顺铂耐药性的临床进展。 表征这种新颖的表观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。拟议的研究将提供严格的 在一个优秀的环境中接受医生科学家的培训，提供一流的研究设施 转化患者组织资源和多样化的指导专业知识。