Proteomic characterisation of clear cell renal cell carcinoma in patients with Von Hippel-Lindau Syndrome

Introduction: The Von Hippel-Lindau (VHL) syndrome describes a familial neoplastic condition, caused by germline mutations of the VHL tumor suppressor gene, resulting in a functional loss of the VHL protein (pVHL). pVHL, as part of a multiunit ubiquitin ligase complex, is involved in the proteasomal degradation of specific target proteins, notably hypoxia‑inducible transcription factors (HIF‑1α/2α). Impaired HIF degradation and the subsequent induction of a pseudo-hypoxic state predispose patients for the development of various tumors, including clear cell renal cell carcinoma (ccRCC). In addition to its role in tumorigenesis, dysregulated HIF signaling in VHL disease affects the extracellular matrix (ECM). We aim to elucidate the molecular alterations linked to hereditary VHL loss in ccRCC, with a focus on matrisomal proteins.

Methods: The present study entails a comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) patient samples (n = 134) from the Freiburg VHL register through LC‑MS/MS. The bead-based single-pot, solid-phase-enhanced sample preparation (SP3) protocol combined with a semi-automated liquid handling platform were utilized for FFPE sample processing. The samples were measured on a timsTOF Flex mass spectrometer coupled to the Evosep One chromatography system. Our investigation involved the proteomic characterization of 54 tumor specimens, 45 pseudo-capsule (PC) specimens, and 35 samples of non-malignant adjacent tissue (NAT).

Preliminary Data: In our study, we employed MS-based proteomics to analyze ccRCC patient samples, achieving a proteome coverage of up to 8500 proteins per sample. To corroborate our findings, we demonstrated the alignment of tumor and NAT samples with those of a Clinical Proteomics Tumor Analysis Consortium (CPTAC) dataset using a principal component analysis (PCA) after batch correction. Differential abundance analysis comparing tumor and NAT and subsequent gene ontology (GO) enrichment analysis unveiled an upregulation of proteins involved in anaerobic glucose metabolism within the tumor, alongside a downregulation of proteins associated with aerobic respiration. Moreover, we analyzed the PC proteome through comparison with both tumor and NAT and identified a predominance of upregulated proteins specifically associated with extracellular matrix (ECM) organization and fibrosis within the PC samples. These findings suggest the PC as a highly intriguing entity in the context of tumor microenvironment dynamics and ECM remodeling. Further, we employed a missingness statistics approach to identify proteins with a significantly higher missingness in either tumor, PC or NAT. In our analysis, we observed that chemokines (CCL14, CXCL12), semaphorins (SEMA3B, SEMA3C), Wnt signaling proteins (WNT9B, SFRP1), and collagens (COL4A3-A6) were significantly depleted in tumor samples. Conversely, angiogenic factors such as VEGF, ANGPTL2, and ANGPTL4 exhibited a lower presence in NAT.