RNA modification and the regulation of partial EMT in head and neck cancer

头颈癌RNA修饰及部分EMT调控

• 批准号: 10428650
• 负责人: Sidharth Venkata Puram
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428650
• 关键词: Binding; Biochemical; Biology; Cancer Etiology; Cell Line; Cells; Clinical; Complex; Critical Pathways; Data; Decision Making; Diagnosis; Disease; Drug Design; Epithelial; Failure; Future; Genes; Genetic Transcription; Goals; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Immunoprecipitation; In Vitro; Investigation; Maps; Mass Spectrum Analysis; Measures; Mesenchymal; Messenger RNA; Methods; Methyltransferase; Modeling; Modification; Neoplasm Metastasis; Nodal; Nuclear; Nuclear Export; Nucleotides; Operative Surgical Procedures; Organoids; Outcome; Pathway interactions; Patient-Focused Outcomes; Patients; Play; Post-Translational Protein Processing; Process; Proteins; RNA; Radiation; Reader; Regulation; Research Proposals; Resistance; Ribonucleosides; Role; Signal Transduction; Smoking Status; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Transcriptional Regulation; Translations; Tumor Cell Invasion; Validation; Writing; alcohol exposure; attributable mortality; base; chemotherapy; crosslink; epithelial to mesenchymal transition; epitranscriptomics; genetic regulatory protein; improved; insight; lymph nodes; mortality; new therapeutic target; novel; patient derived xenograft model; polysome profiling; pre-clinical; prognostic value; programs; single-cell RNA sequencing; small molecule; therapeutic target; tobacco exposure; transcription factor; treatment response; tumor; tumor heterogeneity; tumorigenesis

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of HNSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA- sequencing (scRNA-seq) in HNSCC and identified a partial epithelial-to-mesenchymal (p-EMT) transcriptional program that is predictive of poor clinical outcomes including nodal metastasis and diminished survival (Puram et al., Cell). Understanding the regulatory factors that control the p-EMT program in HNSCC is of critical importance as targeting multiple genes in a complex pathway such as p-EMT is particularly challenging, yet has the potential to significantly improve HNSCC outcomes and treatment decision-making. RNA modification proteins, which can directly read/write nucleotide marks on RNA, have emerged as one exciting class of such regulatory proteins. Because these proteins modulate multiple RNA transcripts, targeting RNA modification proteins may disrupt expression of multiple disease-related genes and make resistance less likely to emerge. In HNSCC, we have found that METTL3, an RNA methyltransferase which catalyzes the N6-methyladenosine (m6A) modification, is a key regulator of the p-EMT program in HNSCC. In preliminary studies, we have discovered that perturbation of METTL3 in HNSCC disrupts p-EMT signaling and reduces invasion in vitro. However, the precise mechanisms by which METTL3 and other RNA modification proteins exert their function are poorly understood, but may depend on changes in transcript stability and/or translation. Thus, a better understanding of how METTL3 modulates p-EMT in HNSCC is likely to improve rational drug design and future small molecule- and biologically-based screens in search of effective epitranscriptomic therapeutics. We hypothesize that METTL3 antagonizes p-EMT in HNSCC by disrupting the translation of critical p-EMT target genes. To test which domains in METTL3 are essential for its function, we will first perform sophisticated structure-function analyses in HNSCC cell lines and patient-derived xenograft organoid (PDXOs) (Aim 1). We will determine which domains are required for m6A function using state-of-the-art mass spectrometry methods, while also mapping the binding partners of the critical domains. To determine if METTL3 controls the transcription or translation (or both) of p-EMT genes, we will utilize advanced biochemical techniques including meRIP-seq to study the stability of p-EMT RNA transcripts and polysome profiling and PAR-CLIP to investigate the effects of METTL3 on translation of p-EMT genes (Aim 2). These studies will provide indispensable insight into the mechanism by which METTL3 directs HNSCC tumorigenesis, specifically focusing on its regulation of p-EMT signaling, and thereby revealing METTL3 domains that could be targeted by new therapeutics to more effectively and specifically treat HNSCC.

项目概要 头颈鳞状细胞癌 (HNSCC) 是癌症相关死亡的第六大原因， 大多数死亡归因于肿瘤转移和治疗失败。因为大多数 HNSCC 病例 由于暴露于烟草和酒精，这些肿瘤具有高度异质性，极大地复杂化 该疾病的诊断、治疗和生物学研究。我们最近进行了单细胞RNA- 对 HNSCC 进行测序 (scRNA-seq)，并鉴定出部分上皮间质 (p-EMT) 转录 预测不良临床结果的计划，包括淋巴结转移和生存率降低（Puram 等人，细胞）。了解控制 HNSCC p-EMT 程序的监管因素至关重要 重要性，因为在复杂的途径（例如 p-EMT）中靶向多个基因特别具有挑战性，但 显着改善 HNSCC 结果和治疗决策的潜力。 RNA修饰 蛋白质可以直接读取/写入 RNA 上的核苷酸标记，已成为此类令人兴奋的一类蛋白质 调节蛋白。因为这些蛋白质调节多个 RNA 转录物，靶向 RNA 修饰 蛋白质可能会破坏多种疾病相关基因的表达，从而降低出现耐药性的可能性。在 HNSCC，我们发现 METTL3，一种催化 N6-甲基腺苷的 RNA 甲基转移酶 (m6A) 修饰是 HNSCC 中 p-EMT 程序的关键调节因子。在初步研究中，我们有 发现 HNSCC 中 METTL3 的扰动会破坏 p-EMT 信号传导并减少体外侵袭。 然而，METTL3 和其他 RNA 修饰蛋白发挥其功能的精确机制 人们对此知之甚少，但可能取决于转录稳定性和/或翻译的变化。因此，更好的 了解 METTL3 如何调节 HNSCC 中的 p-EMT 可能会改善合理的药物设计和 未来基于小分子和生物的筛选，寻找有效的表观转录组 疗法。我们假设 METTL3 通过破坏 HNSCC 的翻译来拮抗 p-EMT 关键的 p-EMT 靶基因。为了测试 METTL3 中的哪些域对其功能至关重要，我们将首先执行 HNSCC 细胞系和患者来源的异种移植类器官 (PDXO) 的复杂结构功能分析 （目标 1）。我们将使用最先进的质谱法确定 m6A 功能所需的域 方法，同时还绘制了关键域的结合伙伴。确定 METTL3 是否控制 p-EMT 基因的转录或翻译（或两者），我们将利用先进的生化技术，包括 meRIP-seq 研究 p-EMT RNA 转录本和多核糖体分析的稳定性，并使用 PAR-CLIP 进行研究 METTL3 对 p-EMT 基因翻译的影响（目标 2）。这些研究将提供不可或缺的见解 深入研究 METTL3 指导 HNSCC 肿瘤发生的机制，特别关注其对 p-EMT 信号传导，从而揭示 METTL3 结构域，可以通过新疗法靶向更多 有效且特异性地治疗 HNSCC。

项目成果

Single-cell RNA sequencing identifies a paracrine interaction that may drive oncogenic notch signaling in human adenoid cystic carcinoma.

• DOI: 10.1016/j.celrep.2022.111743
• 发表时间: 2022-11-29
• 期刊: Cell reports
• 影响因子: 8.8

Immune Cell Deconvolution Reveals Possible Association of γδ T Cells with Poor Survival in Head and Neck Squamous Cell Carcinoma.

• DOI: 10.3390/cancers15194855
• 发表时间: 2023-10-05
• 期刊: CANCERS
• 影响因子: 5.2
• 作者: Parikh, Anuraag S.;Li, Yize;Mazul, Angela;Yu, Victoria X.;Thorstad, Wade;Rich, Jason;Paniello, Randal C.;Caruana, Salvatore M.;Troob, Scott H.;Jackson, Ryan S.;Pipkorn, Patrik;Zolkind, Paul;Qi, Zongtai;Adkins, Douglas;Ding, Li;Puram, Sidharth V.
• 通讯作者: Puram, Sidharth V.