Proteome profiling of clear cell renal cell carcinoma in von Hippel-Lindau patients highlights upregulation of Xaa-Pro aminopeptidase-1, an anti-proliferative and anti-migratory exoprotease

Abstract

Patients of the von Hippel-Lindau (VHL) disease frequently develop clear cell renal cell carcinoma (ccRCC). Using archived, formalin-fixed, paraffin-embedded (FFPE) samples, we sought to determine global proteome alterations that distinguish ccRCC tissue from adjacent, non-malignant kidney tissue in VHL-patients. Our quantitative proteomic analysis clearly discriminated tumor and non-malignant tissue. Significantly dysregulated proteins were distinguished using the linear models for microarray data algorithm. In the ccRCC tissue, we noticed a predominant under-representation of proteins involved in the tricarboxylic acid cycle and an increase in proteins involved in glycolysis. This profile possibly represents a proteomic fingerprint of the “Warburg effect”, which is a molecular hallmark of ccRCC. Furthermore, we observed an increase in proteins involved in extracellular matrix organization. We also noticed differential expression of many exoproteases in the ccRCC tissue. Of particular note were opposing alterations of Xaa-Pro Aminopeptidases-1 and -2 (XPNPEP-1 and -2): a strong decrease of XPNPEP-2 in ccRCC was accompanied by abundant presence of the related protease XPNPEP-1. In both cases, we corroborated the proteomic results by immunohistochemical analysis of ccRCC and adjacent, non-malignant kidney tissue of VHL patients. To functionally investigate the role of XPNPEP-1 in ccRCC, we performed small-hairpin RNA mediated XPNPEP-1 expression silencing in 786-O ccRCC cells harboring a mutated VHL gene. We found that XPNPEP-1 expression dampens cellular proliferation and migration. These results suggest that XPNPEP-1 is likely an anti-target in ccRCC. Methodologically, our work further validates the robustness of using FFPE material for quantitative proteomics.

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

“…This number is in the range of several other FFPE-based proteomic studies [30], [31], [33]. Limited proteome overlap between biological and technical replicates continues to be a characteristic limitation of explorative shotgun proteomics [26], [27].…”

Proteomic distinction of renal oncocytomas and chromophobe renal cell carcinomas

“…FFPE tissue specimens of six ROs and six chRCCs were used as described previously [ 15 , 19 ], including microscopically controlled macrodissection to remove areas of necrosis, fibrosis, hemorrhage, and inflammation. For quantitative comparison, triplex isotopic dimethylation of primary amines was employed [ 20 ], distinguishing RO, chRCC, and a pooled mix that serves as a standard similar to the Super-SILAC approach [ 21 ].…”

“…LC–MS/MS was performed using a Q-Exactive plus (Thermo Scientific) mass spectrometer coupled to an Easy nanoLC 1000 (Thermo Scientific) as described previously [ 18 ]. MS data were analyzed by MaxQuant version 1.5.28 [ 23 ] as described previously [ 19 ]. Proteins were only further considered if they were identified and quantified in at least four RO samples and four chRCC samples.…”

“…However, proteomic profiling is gaining interest for the investigation of malignancies due to the limited correlation between mRNA and protein levels [ 13 , 14 ]. Formalin-fixed, paraffin-embedded (FFPE) samples are a valuable resource for proteomic profiling [ 15 – 17 ], enabling retrospective profiling of clinic-pathologically annotated specimens [ 18 , 19 ]. In the present study, we employed “FFPE proteomics” for the differential proteomic profiling of RO and chRCC cases for which we find noticeable differences.…”

High‐throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

“…Third, the ability to analyze a histological region of small FFPE samples remains challenging. Most published studies analyzed tissue micrometer sections with each tissue containing multiple histological types (Drendel et al ., ; Holfeld et al ., ; Piehowski et al ., ). Laser capture microdissection has been used to analyze multiple regions of a tissue section; however, experimental complexities preclude application to large‐scale analysis.…”