Angiogenesis (the development of new blood vessels from established vasculature) is essential for tumor growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) is a receptor tyrosine kinase (RTK) involved in angiogenic signaling in normal and pathological conditions. Tumors frequently express high levels of hypoxia-induced VEGF ligands that target VEGFR-2 to support tumor vascularization and growth. While VEGFR-2 inhibitors including bevacizumab, sorafenib and sunitinib are used for cancer treatment, the development of resistance to these agents poses an obstacle to effective management of cancer. Recently, impaired trafficking and ligand-independent VEGFR-2 activation and signaling in the Golgi have been demonstrated. Intriguingly, disruption of N-glycosylation affects VEGFR-2 trafficking and may play a role in this process. However, the impact of VEGFR-2 glycosylation on trafficking and receptor activation is largely unexplored. We hypothesize that altered protein glycosylation in pathological circumstances contributes to the sustained localization of VEGFR-2 to the Golgi apparatus by affecting trafficking of VEGFR-2 and leads to ligand-independent activation and signaling from the Golgi. Therefore, we propose to study the impact of VEGFR-2 glycosylation on VEGFR-2 trafficking and activation. We plan to explore this hypothesis by (A) establishing the glycosylation status of membrane and subcellular fractions of VEGFR-2 and examining their impact on receptor activation, (B) screening for glycosylation-dependent VEGFR-2 protein interactions in cell membrane and Golgi fractions, and (C) investigating the role of VEGFR-2 glycosylation in protein recycling, turnover and degradation. A major goal of this proposal is to explore the role of glycosylation in VEGFR-2 trafficking, signaling and aberrant tumor angiogenesis. I will analyze VEGFR-2 glycopeptides and released N-glycans and screen for VEGFR-2 glycosylation-dependent protein interactions using UV-activated crosslinking sugars via mass spectrometry. Results from this study will illuminate the role of glycosylation in VEGFR-2 trafficking and signaling, including: characterization of differences in glycosylation between Golgi and extracellular fractions, assessment of changes in receptor activation and signaling associated with changes in glycosylation, identification of glycosylation-specific VEGFR-2 interacting partners that modulate VEGFR-2 trafficking, and determination of the impact of receptor glycosylation on protein recycling and degradation.

Public Health Relevance

Vascular endothelial growth factor receptor-2 (VEGFR-2) mediates angiogenesis (the development of new blood vessels from established vasculature) in normal and pathological conditions and is essential for tumor growth and metastasis. While VEGFR-2 inhibitors including bevacizumab, sorafenib and sunitinib are used for cancer treatment, the development of resistance to these agents poses an obstacle to effective management of cancer. A major goal of this work is to explore how altered protein glycosylation of VEGFR-2 in cancer contributes to localization of VEGFR-2 to the Golgi apparatus and leads to ligand-independent activation and signaling that contribute to aberrant tumor angiogenesis and drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32CA196157-03
Application #
9215653
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcguirl, Michele
Project Start
2015-04-01
Project End
2018-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D et al. (2017) Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2. J Proteome Res 16:677-688
Chandler, Kevin Brown; Costello, Catherine E (2016) Glycomics and glycoproteomics of membrane proteins and cell-surface receptors: Present trends and future opportunities. Electrophoresis 37:1407-19