Role Of Immature Cd44 O-Glycosylation In Enrichment Of Cancer Stem Cell Population For Aggressive Pancreatic Cancer

Pancreatic cancer (PC) is an aggressive malignancy expected to be the second leading cause of cancer death by 2030. Accumulating reports demonstrate that truncated o-glycans are largely observed in PC and correlates with poor prognosis and reduced patient survival. We have previously shown that loss of C1GALT1 (Core 1 n-acetylgalactose transferase) expression in PC results in the upregulation of genes associated with aggressive PC and metastasis. To study the involvement of immature O-glycophenotype in pancreatic cancer stem cells (CSCs), we selected the single-transmembrane CD44 glycoprotein based on our unbiased approach using proteomics and RNA-sequence analysis.This highly o-glycosylated protein with diverse cellular activity is essential in cell-cell recognition and is a widely occurring CSC marker identified in several cancers. However, the functional properties of CD44 o-glycan variation in pancreatic CSCs are not well understood. Thus, we hypothesize that truncation of o-glycans on CD44 imparts activation of downstream targets responsible for self-renewal and maintenance of CSC in aggressive PC. Using a proteomics approach of enriched truncated o-glycans, we discovered CD44 as one of the top identified proteins in CRISPR/Cas9 C1GALT1 knockout PC cells. We observed enhanced expression of markers responsible for CSC self-renewal and characteristics as measured by FACS-based side-population analysis and tumor sphere formation as a result of a loss in C1GALT1. To determine a potential mechanism by which truncated o-glycans on CD44 enhances CSC properties, we deleted C1GALT1 and CD44 in PC cells. A loss of CD44 o-glycan truncation significantly reduced expression of the self-renewal CSC marker Nanog and phosphorylated p65, a major component in the NF-κB signaling pathway. Furthermore, pancreatic tumorigenesis was significantly reduced in athymic mice injected with PC cells as a result of C1GALT1 and CD44 deletion. These results provide a novel mechanism by which truncated o-glycans on CD44 is responsible for activating downstream targets for self-renewal and maintenance of CSCs. Our findings begin to contextualize the contributions of aberrant glycosylation on glycoproteins to accelerate metastatic properties by enhanced expression and characteristics of pancreatic CSC. Citation Format: Frank Leon, Rama K. Nimmakayala, Seema Chugh, Rohitesh Gupta, Satyanarayana Rachagani, Surinder K. Batra, Moorthy P. Ponnusamy. Role of immature CD44 o-glycosylation in enrichment of cancer stem cell population for aggressive pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6030.