The Effects of Increased Oligomannose N-Glycan Expression on Neuroblastoma Progression and Epidermal Growth Factor Receptor Signaling

Abstract

N-glycosylation is an essential post-translational modification with known roles in cancer, including the pediatric cancer neuroblastoma (NB). The folding, stability, regulation, and trafficking of proteins are all dependent on proper N-glycosylation. There are three general classes of N-glycans: oligomannose, hybrid, and complex ranging from the least processed to the most respectively. Changes in metabolism, endoplasmic reticulum (ER) stress, and other cell signaling events can and do exert influence on the N-glycosylation processing pathway to ensure all glycoproteins are processed to fit the current needs of the cell. Pathological diseases are known to exert influence on the types of N-glycans produced. Regarding cancer biology, [beta]1,6 complex N-glycans have often been attributed to the malignant transformation of cells. This work seeks to define the types of N-glycans (oligomannose, hybrid, or complex) that contribute to the progression of neuroblastoma. The link between N-glycosylation and the malignant transformation of cells has often been centered around more processed N-glycans (i.e., complex N-glycans). However, in the body of this work, we find that oligomannose N-glycans are responsible for aggressive neuroblastoma phenotypes, mainly increased invasiveness. By CRISPR/Cas9 knockout of MGAT1, MGAT2, or MGAT3 in human and rat neuroblastoma cell lines, we were able to generate cells with varying levels of N-glycan processing of proteins. MGAT1 encodes N-acetylglucosaminyltransferase-I (GnT-I), a critical glycosyltransferase that converts oligomannose N-glycans to hybrid N-glycans. By loss of GnT-I, cells have reduced synthesis of hybrid N-glycans and therefore also reduced production of complex N-glycans, resulting in cells with increases in oligomannose N-glycan content. MGAT2 encodes GnT-II, which converts hybrid N-glycans to complex N-glycans. Loss of GnT-II prevents the synthesis of complex N-glycans resulting in cells enriched with hybrid N-glycan structures. MGAT3 encodes GnT-III, which produces bisecting hybrid and complex N-glycans and is responsible for terminating N-glycan processing. By knockout of GnT-III, hybrid and complex N-glycans should be more susceptible to additional modifications (e.g., fucosylation, sialylation, galactosylation, etc.). With these various cell lines, we were able to explore how the reduced amounts of various N-glycan modifications influenced neuroblastoma growth, invasion, and cell-cell adhesion; ultimately revealing that increased expression of oligomannose N-glycans leads to neuroblastoma cells that are highly invasive but have decreased proliferation. Furthermore, we examined unmodified human neuroblastoma cells derived from a SK-N-BE(2) cells: BE(2)-C and BE(2)-M17. We found that BE(2)-M17 cells expressed more oligomannose N-glycans and were significantly more invasive but less proliferative relative to BE(2)-C cells, furthering the support that oligomannose N-glycans drive neuroblastoma invasiveness. These studies were instrumental in designating oligomannose N-glycans as perpetrators of aggressive neuroblastoma phenotypes. A primary focus of this work was to better understand how oligomannose N-glycans impacted neuroblastoma progression. Neuroblastoma cells expressing high amounts of oligomannose N-glycans exhibit increased invasiveness but decreased proliferation leading us to examine intracellular signaling, specifically the oncogenic receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Knockout of MGAT1 is a powerful approach to enrich all N-glycosylated proteins of cells with oligomannose N-glycans, including EGFR. This investigation builds upon the towering literature surrounding N-glycosylation and EGFR, in particular how N-glycosylation modification of EGFR impacts the receptor's ability to initiate downstream signaling. The 12-13 N-glycans of EGFR have been characterized as being mostly complex N-glycans and have been shown to be essential to both EGFR's function and regulation; however, very few have attempted to examine how EGFR functions when decorated primarily with oligomannose N-glycans. Here we show BE(2)-C(-MGAT1) cells produce oligomannosylated EGFR (EGFR decorated with oligomannose N-glycans). Further characterization of oligomannosylated EGFR revealed that ligand independent phosphorylation and EGF stimulated phosphorylation are significantly increased in BE(2)-C(-MGAT1) cells. Furthermore, when observing proliferation in response to EGF stimulation in 3D conditions, BE(2)-C cells do not have a major proliferate response whereas BE(2)-C(-MGAT1) cells proliferate robustly likely due to the heightened EGFR phosphorylation of oligomannosylated EGFR. The increased autophosphorylation and sensitization of BE(2)-C to EGF stimulation when expressing oligomannosylated EGFR, due to loss of MGAT1, is novel and further details how changes in N-glycosylation, due to a pathogenic state, could alter a cellular phenotype without genetic mutation in EGFR. Here using both rat and human neuroblastoma cells with selective defects in the N-glycan processing of proteins we further define the role of N-glycosylation in the progression of neuroblastoma. By examining various N-glycosylation mutations as well as unmodified neuroblastoma cell lines we were able to establish that oligomannose N-glycans contribute to neuroblastoma progression through heightened invasiveness. Utilizing newly generated BE(2)-C(-MGAT1) cells we further explored the role of oligomannose N-glycans by examining EGFR signaling in BE(2)-C and BE(2)-C(-MGAT1) cells. Neuroblastoma cells bearing oligomannosylated EGFR respond intensely to EGF stimulation, leading to heightened autophosphorylation and EGF stimulated proliferation. Ultimately, we conclude that neuroblastoma cells with increased oligomannose N-glycan content is consistent with a highly invasive cancer that is readily able to undergo EGF stimulated proliferation.