Identification and Functional Characterization of pVHL-Dependent Cell Surface Proteins in Renal Cell Carcinoma

Abstract

The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renalcell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable. A hallmark of most RCCis the loss-of-function of the von Hippel–Lindau (pVHL) protein by mutation of its gene (VHL). Using the cell surfacecapturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive786-O cells. One hundred six cell surface N-glycoproteins were identified. Stable isotope labeling with amino acidsin cell culture–based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed tochange in a pVHL-dependent manner. Targeted validation experiments using transcriptional profiling of primary RCCsamples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcrip-tional regulation. Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correla-tion between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation ofglycoproteins may influence the biologic behavior of RCC. Functional characterization of the metalloprotease CD10 incell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment withthe CD10-specific inhibitor thiorphan. Our proteomic surfaceome screening approach in combination with transcrip-tional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnosticmarkers for therapeutic targeting and RCC patient monitoring.