Investigating tRNA biology as a prognostic and oncogenic feature in pancreatic adenocarcinoma

项目摘要 胰腺导管腺癌（PDAC）是一种独特的难以治疗的疾病，由于诊断晚， 为大多数患者提供治疗选择。尽管有大量关于疾病驱动因素的分水岭研究， 在过去的十年中，机制，强调需要替代方法来研究这种疾病。是 现在清楚的是，PDAC呈现沿着的转录组亚型的双峰连续体， 研究人员，以及对先进的PDAC模型的额外研究发现，这些肿瘤，尽管高度 化学抗性，通过代谢失调对外部氨基酸供应敏感。的转录物 定义已知的PDAC分子亚型存在独特的密码子偏好，这表明PDAC受到 生物决定性的密码子水平选择压力。我们实验室和其他人的工作表明， 丰富的适当氨酰化的转移RNA（tRNA），高度结构化和化学修饰的非- 编码RNA，是mRNA半衰期的高度确定性。该机制通过密码子-反密码子起作用 识别和核糖体延伸率，以及功能性tRNA丰度的改变可以决定细胞的 通过伴随的mRNA稳定性调节发挥作用。此外，大量的文献证据表明， 跨物种的细胞类型表明，tRNA调节可以是疾病特异性的。因此，tRNA可能 在PDAC亚型表达和氨基酸敏感性中起关键调节作用， 在蛋白质生产过程中桥接密码子-氨基酸配对。 作为概念的证明，我有强有力的初步证据表明，tRNA表达可以高度预测疾病 在原发性结直肠肿瘤样本的有限队列中进行分期。此外，PDAC的试点研究显示， 广泛的tRNA失调，半胱氨酸解码转录物的使用增加，与现有文献相匹配 PDAC对这种氨基酸的缺失特别敏感。在这个提议中，我试图审问 在PDAC中，tRNA作为mRNA和营养可利用性表型的调节剂的关联作用。我的核心假设是 PDAC特异性调节tRNA表达以赋予细胞存活和增殖优势， tRNA分析可以揭示用于临床决策的新生物标志物。我将阐述这个假设 目的1：表征原发性PDAC患者中tRNA的表达和修饰; 目的2：研究PDAC tRNA调节作为肿瘤细胞存活的驱动因素。这些目标将通过 使用存档的患者样本的生物化学和高通量测序方法的组合， 建立体外细胞系。除了生物审讯，这项建议还涉及新的技术发展， 在实验和分析应用的tRNA测序的大患者样本队列。这个项目 将是第一个分析癌症中tRNA基因特异性调控的人，并且将具有重要意义，因为它可能揭示 PDAC维持的新靶向机制和一组有效的疾病相关临床特征， 很可能为基础癌症生物学提供信息。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is uniquely difficult to treat due to late diagnosis and limited medical management options for a majority of patients. This is despite a wealth of watershed studies on disease driver mechanisms over the past decade, highlighting a need for alternative approaches to studying this disease. It is now clear that PDACs present along a bimodal continuum of transcriptomic subtypes that exhibit distinct prognoses, and additional work on advanced PDAC models has uncovered that these tumors, though highly chemoresistant, are sensitive to external amino acid supply through metabolic dysregulation. Transcripts that define known PDAC molecular subtypes present unique codon biases, suggesting that PDACs are subject to biologically deterministic codon-level selective pressures. Work from our lab and others has shown that the abundance of properly aminoacylated transfer RNAs (tRNAs), highly structured and chemically modified non- coding RNAs, is highly deterministic of mRNA half-life. This mechanism acts through codon-anticodon recognition and ribosome elongation rate, and alterations of functional tRNA abundance can dictate cellular functions via concomitant regulation of mRNA stability. Furthermore, a wealth of literature evidence in diverse cell types across species demonstrates that tRNA regulation can be disease-specific. Thus, tRNAs may likely serve a key regulatory role in PDAC subtype expression and amino acid sensitivity, given their function in bridging codon-amino acid pairings during protein production. As proof of concept, I have strong preliminary evidence that tRNA expression can be highly predictive of disease stage in a limited cohort of primary colorectal tumor samples. Furthermore, a pilot study of PDACs revealed widespread tRNA dysregulation, with increased use of cysteine-decoding transcripts, matching existing literature that PDACs are specifically sensitive to deprivation of this amino acid. In this proposal, I seek to interrogate linked roles of tRNAs as regulators of mRNA and nutrient availability phenotypes in PDAC. My central hypothesis is that PDACs specifically regulate tRNA expression to confer cell survival and proliferation advantages and that tRNA profiling can reveal novel biomarkers for use in clinical decision-making. I will address this hypothesis through the following aims: Aim 1: Characterize tRNA expression and modifications in primary patient PDACs; Aim 2: Investigate PDAC tRNA regulation as a driver of tumor cell survival. These aims will be achieved through a combination of biochemical and high-throughput sequencing approaches using archived patient samples and established in vitro cell lines. Beyond biological interrogation, this proposal involves novel technical development in the experimental and analytical application of tRNA sequencing for large patient sample cohorts. This project would be the first to analyze tRNA gene-specific regulation in cancer, and will be significant in that it may reveal novel targetable mechanisms of PDAC maintenance and a potent set of disease-associated clinical features that are likely to inform fundamental cancer biology.