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

pVHL/HIF-Regulated CD70 Expression Is Associated with Infiltration of CD27+ Lymphocytes and Increased Serum Levels of Soluble CD27 in Clear Cell Renal Cell Carcinoma

“…Preparation of cell lysates, determination of protein concentration, and Western blotting were performed as described (4). Antibodies used are listed in Supplementary Table S1.…”

“…RNA isolation, cDNA synthesis, and quantitative reverse transcription-PCR (qPCR) were performed as described (4). TaqMan Gene Assays (Applied Biosystems) for CA-IX (Hs00154208_m1),…”

“…No binding was observed for the negative control gene KCNJ5. CD10 HRE2 served as positive control for HIF1a binding (4).…”

“…CD70 is a member of the TNF ligand superfamily and caught our interest because it was one of the ccRCC surface proteins we previously identified by cell surface capturing technology (4). Under normal conditions, CD70 expression is tightly regulated and restricted to a small subset of antigen-stimulated B and T lymphocytes and mature dendritic cells (5,6).…”

Individual approach in choosing second-line targeted therapy for metastatic renal cell carcinoma

“…В случае возникновения соматической мутации происходят инактивация гена VHL, аккумулирование HIF-α и активизация транс-крипции генов, индуцированных гипоксией, что при-водит к гиперэкспрессии различных факторов роста, в первую очередь VEGF, тромбоцитарного -PDGF (Platelet-Derived Growth Factor) и трансформирую-щих -TGF-α и TGF-β (Transforming Growth Factor), которые активируют расположенные вблизи опухо-левой ткани клетки эндотелия для неоангиогенеза [9][10][11]. Мишенью кабозантиниба также являются ре-цепторы киназ MET и AXL, действие которых усили-вается при потере функции гена VHL [12,13]. MET и AXL играют определенную роль в канцерогенезе ПКР, поддерживая альтернативные проангиогенные и пропролиферативные пути, что может способство-вать развитию резистентности к терапии VEGFR-TKI [14,15].…”