Developing glycosylated RNAs as novel clinical targets for aggressive prostate cancer.

项目摘要 这一探索性的提议将是第一个在人类前列腺背景下表征糖基化RNA的提议。 癌（PCa）。前列腺癌是男性中最常见的非皮肤癌形式，也是导致前列腺癌的第二大原因。 与癌症有关的男性死亡人数。20%被诊断患有PCa的男性将发展为快速增长， 疾病对于已经扩散到远处的PCa没有根治性治疗方法， 只有30%。由于高等级的PCa往往导致不良反应，因此必须更好地 了解这些病理，改善患者分层方法和治疗方法 提高患者生存率的选择。非编码RNA在PCa患者中广泛错误表达， 肿瘤抑制基因，前列腺中的促癌因子。非编码RNA含有大量的后- 转录修饰（m6A、肌苷、假尿苷、2'-O-Me、A-to-I编辑、3 '尿苷加尾）， 对于RNA成熟、折叠、表达和核转运很重要。这些后调节失调- 转录修饰与肿瘤生长、侵袭、血管生成、免疫应答和 疾病复发。偶然发现非编码RNA携带糖基化修饰 当使用生物正交化学方法对培养的人和小鼠细胞进行代谢标记时， 通常用于糖基化蛋白和脂质富集。糖基化是一个复杂的过程， 典型地涉及当生物分子移动时碳水化合物共价连接到蛋白质和脂质上 通过分泌途径。异常的蛋白质/脂质糖基化有助于肿瘤生长、转移， 和免疫监视逃避，并被用作癌症生物标志物。我们假设RNA 需要糖基化用于细胞信号传导以维持前列腺稳态和癌症预防， RNA的糖基化状态将与前列腺肿瘤发生相关。为了支持这一理论，我们 使用点击化学和北方印迹证实了糖RNA的存在，并且在细胞中具有不同的丰度。 人类前列腺细胞具体目的：该提案将使用两种独立的方法，代谢标记， 叠氮化物点击化学和基于凝集素的纯化，以分类来自一组人的糖RNA分布 前列腺细胞系的转移潜能和激素敏感性不同。目标1将无偏地确定 如果小的非编码RNA相对于大的非编码RNA在前列腺中优先糖基化，如果这些糖RNA共享 N-或O-连接糖的特征，并使用RNAseq鉴定这些种类。在目标2中， 化学抑制剂研究将确定PCa相关的糖RNA亚细胞定位， 外泌体富集并通过质谱法表征这些碳水化合物部分。在目标3中， 将采用Ptenpc-/-Smad4 pc-/-双敲除小鼠模型来表征来自活细胞的糖RNA。 在前列腺癌进展的整个时间过程中的动物。这项工作将导致新的见解如何 RNA修饰影响PCa进展并确定一流的临床工具以改善患者结局。

PROJECT SUMMARY This exploratory proposal will be the first to characterize glycosylated RNAs in the context of human prostate cancer (PCa). PCa is the most prevalent form of non-skin cancer in males and second leading cause of cancer-related deaths among men. 20% of men diagnosed with PCa will progress to fast-growing, advanced disease. There are no curative treatments for PCa that has spread to distant sites and the 5-year survival rate for metastatic disease is only 30%. As high-grade PCa often leads to poor prognoses, it is imperative to better understand these pathologies and improve patient stratification methods as well as therapeutic treatment options to increase patient survival. Noncoding RNAs are widely misexpressed in PCa patients and act as key tumor suppressor genes, pro-oncogenic factors in the prostate. Noncoding RNAs harbor a large range of post- transcriptional modifications (m6A, inosine, pseudouridine, 2'-O-Me, A-to-I editing, 3’ uridine tailing) that are important for RNA maturation, folding, expression, and nuclear transport. Dysregulation of these post- transcriptional modifications are associated with tumor growth, invasion, angiogenesis, immune response and disease recurrence. Noncoding RNAs were serendipitously discovered to carry glycosylation modifications when cultured human and mouse cells were metabolically labeled using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Glycosylation is an intricate process that typically involves the covalent attachment of carbohydrates onto proteins and lipids as the biomolecules move through the secretory pathway. Aberrant protein/lipid glycosylation contributes to tumor growth, metastasis, and immunosurveillance evasion and is being utilized as cancer biomarkers. We hypothesize that RNAs require glycosylation for cell signaling to maintain prostate homeostasis and cancer prevention and therefore the glycosylated state of RNA will correlate with prostate tumorgenicity. In support of this rationale, we confirmed using click chemistry and northern blotting that glycoRNAs exist and with differing abundance in human prostate cells. Specific Aims: This proposal will use two independent methods, metabolic labeling with azide click chemistry and lectin-based purification, to classify glycoRNA distribution from a panel of human prostate cell lines differing in their metastatic potential and hormone sensitivity. Aim 1 will unbiasedly determine if small versus large noncoding RNAs are preferentially glycosylated in the prostate, if these glycoRNAs share features for N- or O-linked sugars, and identify these species using RNAseq. In Aim 2, fractionation methods and chemical inhibitor studies will determine the PCa-associated glycoRNA subcellular localization, possible exosome enrichment and characterize these carbohydrate moieties via mass spectrometry. In Aim 3, the Ptenpc-/- Smad4 pc-/- double knockout mouse model will be employed to characterize glycoRNAs from living animals throughout a prostate cancer progression time course. This work will lead to novel insights into how RNA modifications impact PCa progression and identify first-in-class clinical tools to improve patient outcome.