Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression

Abstract

Background: The 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 findings: We 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 significance: Pathway 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.

Figure 1. Ingenuity Pathway Analyses (IPA).

Proteins with significant differential expression regulate a network of cell-to-cell signaling and interaction, cellular growth and proliferation, as well as cellular development. Red shading: upregulated in VHL-mut RCC; green shading: downregulated in VHL-mut RCC. Protein–protein interactions from the network diagram are represented by single lines and proteins/compounds that regulate another protein are indicated by arrows. Solid or dashed lines indicate direct or indirect interactions, respectively. The various shapes represent different protein functions.

Figure 2. Assessment of cell-survival by MTT assay.

The survival of Caki-2 (VHL-wt) and 786-O (VHL-mut) RCC cell lines was assessed after treatment with increasing concentrations of the glutaminolytic inhibitor, aminooxyacetate (AOA) at 24 h (a), 48 h (b) and 72 h (c) and in glutamine-depleted medium (d). VHL-mut 786-O RCC cells were more susceptible to inhibition of glutaminolysis or glutamine depletion at all time-points (* indicates statistically significant difference from VHL-wt control by the t-test, P<0.05, n = 4).

Figure 3. Summary of Ingenuity Pathways Analyses (IPA) revealing differentially regulated molecular and cellular functions in VHL-mut RCC compared to VHL-wt RCC.