Abstract 2797: N-linked and O-linked glycosylated RNA enrichment in human prostate cell lines with low/no malignancy

Abstract Our lab is the first to characterize glycosylated RNA in the context of human prostate cancer (PCa). PCa is the 2nd leading cancer-related cause of male deaths in the United States. As high-grade forms of PCa often lead to poor prognoses, it is imperative to improve patient stratification screening tools and treatment options to increase patient survivorship. Noncoding RNAs are widely misexpressed in clinical PCa tissues and many act as key tumor suppressors, oncogenes, and metastatic factors in the prostate. Noncoding RNA harbor a range of post-transcriptional modifications (m6A, pseudouridine, uridine tailing) important for RNA folding, stability, transport, and localization. Dysregulation of these RNA modifications is often associated with malignancy, disease aggressiveness, and drug resistance. Noncoding RNA carrying a newly discovered class of glycosylation modifications was recently discovered in cultured human and mouse cells metabolically labeled using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Nothing is known about the existence/significance of prostate glycoRNA or its role in disease. We hypothesized that RNA requires carbohydrate modifications to maintain prostate homeostasis and predicted that the glycosylated state of RNA would correlate with disease progression. We first confirmed that prostate glycoRNA exists in the human prostate. Our studies employed a stringent RNA isolation workflow and were tested using two independent approaches; metabolic labeling with azide click chemistry for biotin-tag detection & lectin northern blot analysis. Using metabolic azidosugar reagents that preferentially labeled N-linked (Ac4ManNAz) or O-linked (Ac4GalNAz) modifications, we detected small (<200 nt) noncoding RNA (but not large RNA) carrying N-linked and O-linked sugars that showed sensitivity to endoglycosidases (PNGase F, O-glycosidase) and glycosylation inhibitors. GlycoRNA expression correlated with PCa progression with preferential enrichment in human prostate cell lines and syngeneic panels with low/no malignancy compared to metastatic lines. Lectin northern blot studies (WGA, PNA, ConA) verified that prostate RNAs carry N-linked and O-lined sugars. Subcellular fractionation studies indicated glycoRNA localization predominantly at the plasma membrane of prostate cells. Results from our RNAseq and glycomics studies are ongoing and will also be discussed. Furthermore, we verified that C. elegans grown on Ac4GalNAz plates expressed glycoRNA, indicating conservation across species. This work will provide novel insight into how RNA modifications impact PCa progression and identify first-in-class clinical tools to improve patient outcomes. Citation Format: Esther Jones, Samantha McGuire, Spencer Moen, Aurora Esquela Kerscher. N-linked and O-linked glycosylated RNA enrichment in human prostate cell lines with low/no malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2797.