Abstract
BackgroundThe loss of von Hippel–Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α).Methods and Principal FindingsWe used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography–mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335.Conclusions and SignificancePathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.
Highlights
Loss of the tumor suppressor von Hippel-Lindau (VHL) gene is an established genetic risk factor for renal cell carcinoma (RCC) and the affected individuals are at higher risk to develop tumors including pheochromocytomas, retinal angioma, pancreatic cysts and central nervous system hemangioblastomas [1,2]
Our method of choice for this first study in RCC varying in HIF-2a expression was based on our earlier comparison of 2DLC-mass spectrometry (MS)/MS results with those obtained from shotgun liquid chromatography (LC)-MS/ MS analyses demonstrating that most molecular functions and biological processes were represented via gene ontology (GO) analysis using either methodology [17]
In the context of downregulation of key glycolytic and pentose phosphate pathway enzymes, especially Glucose-6-phosphate isomerase (GPI) and UDP-glucose 6-dehydrogenase (UGDH), revealed by proteomic analysis of von Hippel–Lindau (VHL)-mut RCC, we further investigated the importance of glutaminolysis
Summary
Loss of the tumor suppressor von Hippel-Lindau (VHL) gene is an established genetic risk factor for renal cell carcinoma (RCC) and the affected individuals are at higher risk to develop tumors including pheochromocytomas, retinal angioma, pancreatic cysts and central nervous system hemangioblastomas [1,2]. Loss-of-function mutations in the VHL gene upregulate hypoxia-inducible factor-1a (HIF1a) and hypoxia-inducible factor-2a (HIF2a), but advanced stages of RCC have predominantly increased expression of HIF2a [4,5]. Overexpression of HIF2a in RCC leads to enhanced angiogenesis and tumor progression even in the absence of HIF1a [6,7]. In this regard, proteomic analyses have focused on molecular binding partners of VHL and interactions of VHL-related binding proteins [8,9]. We aimed at comparing the global cellular proteome in 786-O (VHL-mut, over-expressing HIF2a) and Caki-2 (VHL-wt, lacking HIF2a) cell lines thereby focusing on HIF2a-dependence of RCC in this study. Loss of VHL in renal tumors differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2a (HIF2a) instead of hypoxia-inducible factor-1a (HIF1a)
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