#2675 Role of ANXA4 as a modifier in VHL-dependent ccRRC

Abstract

Abstract Background and Aims The von Hippel-Lindau (VHL) syndrome is a rare genetic disease caused by inactivating mutations of the VHL tumor suppressor gene. VHL syndrome patients frequently develop clear cell renal cell carcinomas (ccRCCs). pVHL acts as the substrate recognition subunit of an E3 ligase involved in the ubiquitinylation of HIFs. Consequently, aberrant HIF-signalling results in a pseudo-hypoxic state characterized by pronounced vascularization in respective tumours. Previous studies have indicated VHL-dependent alterations of the extracellular matrix (ECM). However, to date comprehensive description and understanding of VHL-dependent matrisome signatures is missing. Method Proteomics were used to reveal VHL-dependent protein expression in ccRCC cell lines (A498, 786-O) and CRISPR/Cas9-modified human proximal tubular epithelial cells (hRPTECs) as well as syndromic and sporadic ccRCC patient cohorts. For candidate proteins analysis of proteomic and histological datasets was performed. Further functional analysis was conducted using overexpression, CRISPR/Cas9-mediated knockout (KO) and shRNA-mediated knockdown (KD) approaches (assessment of proliferation, migration, invasion, ECM, cellular membrane-integrity, protein localisation studies and transcriptomics). Results Filtering of proteomic datasets (ccRCC tumors and cell lines) for matrisomal proteins highlighted an enrichment of the ANXA-protein family. Interestingly, functional genetic titration of ANXA4 employing knockdown, overexpression or complete ablation using CRISPR/Cas9 genome editing did not translate into major changes of cellular proliferation or migration in renal cancer or tubular cells. Detailed localization studies revealed that ANXA4 is detectable in distinct cellular compartments, namely the cell membrane and nucleus/nuclear lamina. Various cellular stressors (including cellular densities) initiated shuttling of ANXA4 between these different compartments. Remarkably, similar distribution patterns were detectable in situ in tumours tissue of ccRCC patients. Further transcriptomic analysis of ANXA4 knockout cells demonstrated a major impact on ECM- and cell-adhesion-associated gene sets, reflected by impaired invasive migratory capacity in conditions of reduced ANXA4-expression. Conclusion Loss of VHL distinctly alters the ECM composition in ccRCC. We have identified the ECM-associated ANXA4 as a prominently upregulated protein, although canonical hallmarks of cancer were not directly influenced. Based on RNA-sequencing studies and subcellular localization patterns we assume that ANXA4 (co-)regulates matrisome-related transcriptomic signatures in response to various external stressors. These observations highlight the intricate regulation of ECM-associated signalling pathways in the context of VHL-dependent renal cell carcinoma.