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: 10643865
• 负责人: Emmanuelle Hodara
• 金额: $ 5.52万
• 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/10643865
• 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; Flow Cytometry; Gene Expression; Genes; Genetic Transcription; 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; Patients; Phenotype; Physicians; Plasma Cells; Population; Property; RNA; RNA methylation; Reader; Resistance; Role; Sampling; Scientist; Side; Small Interfering RNA; Solid; Stains; Testing; Training; Transcript; Transcriptional Activation; cancer cell; cancer drug resistance; cancer therapy; cancer type; differential expression; drug-sensitive; epitranscriptomics; fat mass and obesity-associated protein; knock-down; liquid biopsy; migration; neoplastic cell; new therapeutic target; notch protein; novel; pharmacologic; 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.

项目摘要/摘要 癌症耐药性不仅通过选择具有遗传抗性的克隆来发生，还通过表型来发生 通过快速诱导转录程序使某些细胞适应并持续存在而产生的可塑性。在膀胱里 癌症(BC)，在我们实验室建立的模型中观察到表型可塑性，在该模型中，两个亚群 肿瘤细胞可逆地自发地从一种转移到另一种。由Hoechst分离和研究 染色和流式细胞术，这些亚群由高度致瘤性的“侧群”(SP)组成， 耐顺铂的干细胞样细胞，以及缺乏这些特性的“非侧群”(NSP)细胞。一种潜力 这种可塑性的表观遗传因素是N6-甲基腺苷(M6A)RNA修饰。由M6A存放 写入并被m6A橡皮擦去除，m6A动态和可逆地调节关键的细胞功能和 癌细胞的基本特征。解除对m6A修改和m6A效应器(写入器、擦除器、读取器)的管制 与各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 本实验室建立并验证了甲基核糖核酸免疫沉淀金标准表位转录分析方法 然后是高通量测序(MERIP-SEQ)。我用这个方法比较了SP和NSP 亚群和鉴定差异甲基化的候选转录本。我还发现从药理上讲 抑制关键的m6A橡皮擦，脂肪质量和肥胖相关蛋白(FTO)，加强了向SP状态的转变。 基于这些初步数据，我建议检验m6A修饰调控基因表达的假设。 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。这就做 目标1：我将使用MERIP-SEQ和RNA-SEQ系统地识别 差异甲基化和差异表达的转录本推动耐药基因的转变 州政府。我将从基因上调整这些靶点，并衡量其对顺铂耐药的影响 相互转化、殖民地形成、迁徙和入侵。目标2：我将定义FTO的功能，这是M6A的关键 在我们的模型中，橡皮擦通过遗传和药物调节FTO来影响可塑性。我会量一量 对顺铂耐药性、SP-NSP相互转化、克隆形成、迁移和侵袭性的影响 使用MERIP-seq和RNA-seq来识别差异甲基化和差异转录本 表达。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患者衍生的固体和液体活检样本进行顺铂耐药的临床进展。 表征这一新的表位转录机制将为新的生物标志物和 针对短路BC耐药性的治疗靶点。拟议的研究性研究将提供严格的 在卓越的环境中进行医生-科学家培训，提供与 可翻译的患者组织资源和多样化的指导专业知识。