N-glycosylation signaling in homeostasis and oral cancer
Yang, Hisao-Ying Candice
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Part one: coordinate regulation of N-glycoosylation gene DP4GT1, Canonical Wnt signaling and E-cadherin adhesion. Protein N-glycosylation influences cell-cell adhesion by affecting the composition and cytoskeletal association of E-cadherin-mediated AJs (Adherens junctions). DPAGT1 gene, a key regulator of the metabolic pathway of protein N-glycosylation, senses the cell density information. On the one hand, E-cadherin is modified with complex N-glycans and forms nascent AJs in cells under sparse density. On the other hand, dense cultures produce hypo-glycosylated E-cadherin with mature AJs. Previous studies from our laboratory demonstrated that DPAGT1 is a target of the canonical Wnt signaling. Here we report that DPAGT1 senses cell density through canonical Wnt signaling. In dense cells, depletion of [beta]一catenin from the DPAGH1 promoter correlated with its diminished cellular abundance, while loss of nuclear [upsilon]-catenin reflected its greater recruitment to AJs. DIAGT1 itself affected canonical Wnt signaling, with forced changes in its expression resulting in corresponding changes in transcriptionally active [beta]-catenin and canonical Wnt activity. Forced overexpression of DPAGT1 by stable transfection resulted in increased N-glycosylation, reduced membrane localization of E-cadherin, and significant changes in cell morphoIogy. Finally, we provide evidence that N-glycosylation status of E-cadherin controIs its inhibitory effect on canonical Wnt signaling. Sparse cells transfected with hypo-glycosylated E-cadherin mutant V 13, had reduced canonical Wnt activit due to depletion of nuclear [beta] and [upsilon]-catenin from its promoter. Our studies show that cells coordinate expression of N-glycosylation gene DPAGT1 with canonical Wnt signaling and E-cadherin mediated adhesion via positive and negative feedback mechanisms. Part two: N-gIycosylation induces the CTHRC1 protein and drives oral cancer cell migration. The search for the mechanisms underlying oral squamous cell carcinoma (OSCC) development and progression has long been one of the most important topics in the dental research field. The canonical Wnt signaling is one of the key pathways that drives early pathogenesis and metastasis of variety of cancers, including OSCC. Our lab previously reported that canonical Wnt signaling functions in a positive feedback loop with the DPAGT1 gene, the regulator of the first step in the synthesis of LLO precursor of the N-glycosylation pathway to drive hyper-glycosylated E-cadherin and subsequently inhibit intercellular adhesion. Here, we show that in OSCC, this positive feedback loop between DPAGT1 and canonical Wnt signaling also converges to up-regulate CTHRC1 (collagen triple helix repeat containing 1), a secreted N-glycoprotein shown to play a role in tumor invasion and metastasis. Our results reveal amplified expression of CTHRC1 in human OSCC specimens and OSCC CAL27 cells. In addition, this increased CTHR 1 level is associated with elevated N-glycosylation via increased expression of the DPAGT1 gene. Indeed, partial knockout of DPAGT1 in OSCC CAL27 cells decreased CTHRC 1 abundance by increasing the protein turnover rate, indicating N-glycosylation helps to stabilize CTHRC 1. Furthemore, Wnt signal enhanced [beta]-catenin/TCF transcriptional activity at the CTHRC 1 promoter further amplified CTHRC1 expression level. Using scratch wound assays and Transwell analyses, we showed that DPAGT1 promoted CAL27 cell migration concomitant with the localization of CTHRC1 to the leading edge of a wound front. We propose that in OSCC, aberrant amplification of canonical Wnt signal along with DPAGTl-dependent N-glycosylation induces CTHRC1 expression to drive OSCC cell migration and tumor spread.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Endodontics).Includes bibliographic references: leaves 96-104.
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