Abstract 2984: Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding

Abstract Glycosylated RNA (glycoRNA) is an emerging field. Carbohydrate modifications are vital for biomolecule (proteins and lipids) stability, transport, and localization. Glycosylation influences cell functions like differentiation, apoptosis, and immune response, potentially impacting human disease. The Bertozzi lab initially discovered that RNAs can carry sugar modifications when they metabolically labeled mammalian cultured cells using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Our lab wanted to investigate the translational potential of glycoRNAs and determine if these modifications could be developed as novel clinical targets for aggressive prostate cancer. (PCa). As advanced forms of PCa often lead to poor prognoses, it is imperative that better screening biomarkers and therapeutic treatment options are developed to increase patient survivorship. We predicted that glycoRNA expression would correlate with malignancy, metastatic status, and hormone sensitivity in prostate cells. Our lab used the sialic acid metabolic precursor Ac4ManNAz and click chemistry to label a panel of human prostate cancer cell lines representing the spectrum of prostate disease followed by rigorous RNA isolation procedures and northern blot analysis. We found that human prostate cell lines with no (RWPE-1) or low malignancy (LNCaP) expressed higher levels of glycoRNA belonging to the small <200 nt (but not large) noncoding RNA class compared to aggressive, metastatic PCa cell lines. Our glycosidase studies and lectin northern blot analysis indicated that RNA carries both N-linked and O-linked modifications. Based on these findings, we hypothesized that glycosylated RNA plays an important role in maintaining prostate homeostasis and in cancer protection. To better profile these glycoRNAs, we aimed to: 1) Test a wider range of metabolic labeling regents to characterize the glycoRNA populations using a panel of human prostate cell lines; 2) Perform cellular fractionation studies to determine localization of glycoRNA in prostate cells; and 3) Explore glycoRNAs in a genetic animal model. These studies focused on LNCaP PCa cell lines, which express high levels of glycoRNA. We found that small glycoRNA expression was noted when LNCaP cells were metabolically labeled with Ac4GalNAz (predominantly O-linked) and Ac4ManNAz (predominantly N-linked), verifying glycoRNA heterogeneity. Cellular fractionation studies indicated glycoRNA localization was enriched in the membrane fraction of LNCaP cells using these metabolic labels. Furthermore, we verified that C. elegans grown on Ac4GalNAz plates expressed small glycoRNA, indicating biological conservation. We are now characterizing these glycoRNAs using RNA sequencing and LC-MS/MS glycomics. Our findings will provide novel insights into cancer-associated glycoRNA, leading to improved clinical tools for PCa. Citation Format: Samantha McGuire, Esther Jones, Spencer Moen, Aurora Esquela-Kerscher. Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2984.