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

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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.