Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

Abstract

The von Hippel-Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL-defective RCC cell line UMRC2 by transfection with vector control or VHL (-/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin-affinity chromatography and analysis by GeLC-MS/MS, VHL-associated changes in plasma membrane proteins were analysed. Comparative analysis of -/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the alpha 3 and beta1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL-defective cells. Western blotting confirmed these changes and also revealed VHL-dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient-matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL-dependent proteins that may be important in tumorigenesis.

Plasma Membrane Proteomics and Its Application in Clinical Cancer Biomarker Discovery

“…Enrichment of PM proteins using whole cell protein tagging is often based on a membrane-impermeable biotin labeling reagent followed by cell lysis and affinity purification using streptavidin-coated beads (18,19,21,73). Other systems, including coating with cationic silica beads and purification through a density gradient, have also been used (82).…”

Mutations of the von Hippel–Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma

“…The major consequence of the loss of VHL function is continuous activation of hypoxia-inducible factors (HIFs), resulting in accumulation of HIF effectors and increased angiogenesis, cell growth, low oxygen survival and ultimately metastasis (Linehan et al, 2009). VHL regulates the expression of genes coding for molecules involved in cell-cell contact (Aggelis et al, 2009) and that are implicated in the recognition and activation of immune cells, particularly natural killer (NK) cells.…”

Alterations of the serum peptidome in renal cell carcinoma discriminating benign and malignant kidney tumors

“…identify RCC-specific biomarkers have investigated at gene and protein levels either in cells, primary culture [10] or stable lines [11], or in tissues [12][13][14]. These candidate markers identified by these strategies may not be present or their profile in biological fluids (e.g., urine, serum and plasma) may not be altered, as would be necessary for screenings based on biological material collected with noninvasive or less invasive methods.…”

“…Highly sensitive profiling studies require a combination of MS and separation technologies [40]. RCC-specific biomarkers have been searched in primary culture, stable lines, and in solid biological material (primary cell cultures, stable cell lines and tissues) with classical proteomics approaches [10][11][12][13]. Sample fractionation can be used to increase the number of proteins detectable with MS (e.g., enriches for the low abundant proteins) such as by bi-dimensional liquid chromatography (MudPIT) or to reduce the sample complexity by activated surfaces.…”