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

开发糖基化 RNA 作为侵袭性前列腺癌的新临床靶点。

• 批准号: 10651206
• 负责人: Aurora Esquela Kerscher
• 金额: $ 21.04万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651206
• 关键词: Animals; Apoptosis; Area; Azides; Benign; Biological; Biological Markers; Biological Process; Biotinylation; Cancer Biology; Cancer Etiology; Cancer Patient; Carbohydrates; Cell Line; Cell surface; Cells; Cellular Membrane; Cessation of life; Chemicals; Chemistry; Classification; Clinical; Colon; Comparative Study; Cultured Cells; Diagnosis; Disease; Disease Progression; Distant; Ensure; Enzymes; Extracellular Matrix; Fractionation; Glycolipids; Glycoproteins; Golgi Apparatus; Health; Homeostasis; Hormones; Human; Immune System Diseases; Immune response; Immunologic Surveillance; Inosine; Invaded; Knockout Mice; Label; Lectin; Link; Lipids; Liquid substance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Mediating; Metabolic; Methods; Modification; Molecular; Monitor; Morphologic artifacts; Mus; Mutant Strains Mice; Neoplasm Metastasis; Non-Malignant; Northern Blotting; Nucleic Acids; Oncogenic; Organ; Pancreas; Pathology; Pathway interactions; Patient-Focused Outcomes; Patients; Pattern; Pharmaceutical Preparations; Play; Polysaccharides; Population; Post-Transcriptional RNA Processing; Post-Translational Protein Processing; Process; Prognosis; Prostate; Prostate Cancer therapy; Prostatic Intraepithelial Neoplasias; Prostatic Neoplasms; Protein Glycosylation; Proteins; Pseudouridine; RNA; Recurrent disease; Role; Screening procedure; Sialic Acids; Signal Transduction; Site; Survival Rate; Tail; Testing; Therapeutic; Time; Tissues; Tumor Suppressor Genes; United States; Untranslated RNA; Uridine; Work; advanced disease; angiogenesis; biomarker development; cancer biomarkers; cancer prevention; cancer therapy; cell growth; curative treatments; exosome; extracellular vesicles; glycosylation; hormone sensitivity; human disease; improved; in vivo; inhibitor; innovation; insight; intercellular communication; male; men; mouse model; novel; nucleocytoplasmic transport; patient stratification; posttranscriptional; prostate cancer cell; prostate cancer cell line; prostate cancer model; prostate cancer progression; response; screening; sialylation; sugar; survivorship; tool; transcriptome sequencing; translational potential; treatment strategy; tumor growth; tumor progression; tumorigenic; virtual

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.

项目总结 这项探索性的建议将是第一个在人类前列腺背景下表征糖基化RNA的方案 癌症(PCA)。前列腺癌是男性最常见的非皮肤癌形式，是 男性中与癌症相关的死亡。20%被诊断为前列腺癌的男性将进展为快速增长的晚期 疾病。对于已经扩散到远处的前列腺癌，目前尚无根治方法，其5年存活率 对于转移性疾病来说只有30%。由于高级别的PCa往往导致不良的预后，因此必须更好地 了解这些病理，改进患者分层方法和治疗方法 提高患者存活率的选择。非编码RNA在前列腺癌患者中广泛错误表达，并发挥关键作用 肿瘤抑制基因，前列腺癌中的致癌因子。非编码RNA含有大量的POST- 转录修饰(m6A、肌苷、伪尿苷、2‘-O-Me、A-to-I编辑、3’-尿苷拖尾) 对RNA的成熟、折叠、表达和核运输非常重要。这些后的失调- 转录修饰与肿瘤生长、侵袭、血管生成、免疫反应和 疾病复发。偶然发现携带糖基化修饰的非编码RNA 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 通常用于糖化蛋白质和脂肪的浓缩。糖基化是一个复杂的过程， 通常包括碳水化合物随着生物分子的移动而共价附着到蛋白质和脂类上 通过分泌途径。异常的蛋白质/脂肪糖基化有助于肿瘤的生长、转移 和免疫监视逃避，并被用作癌症生物标记物。我们假设RNA 需要细胞信号的糖基化来维持前列腺内环境的稳定和预防癌症，因此 RNA的糖基化状态将与前列腺癌的发生相关。为了支持这一理由，我们 用点击化学和Northern blotting证实了糖RNA的存在，并且具有不同的丰度 人类前列腺细胞。具体目标：该提案将使用两种独立的方法，代谢标记 叠氮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 转移潜能和激素敏感性不同的前列腺细胞系。目标1将不偏不倚地决定 如果小的非编码RNA与大的非编码RNA在前列腺中优先糖基化，如果这些糖RNA共享 N-或O-连接的糖的特征，并使用RNAseq鉴定这些物种。在目标2中，分馏方法 化学抑制物研究将确定与PCA相关的糖RNA的亚细胞定位，这是可能的 外切体富集，并通过质谱学表征这些碳水化合物部分。在目标3中， Ptenpc-/-Smad4 PC-/-双基因敲除小鼠模型将用于鉴定活体糖核糖核酸 动物在前列腺癌进展的整个时间过程中。这项工作将导致对如何 RNA修饰影响前列腺癌的进展，并确定一流的临床工具来改善患者的预后。