Translational studies of syndecan-1 as angiogenesis inhibitor : from basic research to clinical applications

Abstract: Malignant mesothelioma (MM) is a highly aggressive primary tumor of the pleura, associated with poor prognosis. MM is mainly related to exposure to mineral fibers such as asbestos. The diagnosis of MM, despite multiple diagnostic tools, is challenging and treatment options are limited. Previous studies have shown that angiogenesis plays an important role in MM progression thus, anti-angiogenic agents show promising use in MM therapy. In addition to that, detection of new soluble diagnostic or prognostic biomarkers may improve patients’ outcomes and therapeutic options. Syndecan-1 (SDC-1) is a membrane proteoglycan which regulates various biological processes in tumor cells by acting as a co-receptor for growth factors. SDC-1 is also a significant mediator of cell-cell and cell-matrix interactions. Cell membrane SDC-1 can be shed by sheddases and its Heparan Sulfate (HS) degraded by heparanase-1 (HPA-1). Additionally, syndecan-1 can translocated to the nucleus through a tubulin-dependent mechanism. Loss of cell membrane SDC-1 is associated with epithelial-mesenchymal transition (EMT) and worse prognosis, while nuclear translocation of SDC-1 seems to have the opposite effect. The purpose of this thesis work was to investigate the role and potential value of syndecan-1 as an angiogenic factor and as a diagnostic biomarker for MM. We showed the inhibitory effect of SDC-1 overexpressing mesothelioma cells on migration, proliferation, and tube formation capacity in endothelial cells. We also found that silencing of SDC-1in MM cells promoted experimental wound closure but had no effect on tube formation in endothelial cells (paper I). These effects were mediated by angiogenic factors comprising Angiopoietin-1, FGF-4, HGF, TGF-β1, TIMP-1, TSP-1, and TRG1-β1 which were significantly up- or down regulated by SDC-1 overexpression, as well as IL8 which was significantly up-regulated by SDC-1 silencing. In the same study, we evaluated the expression level of SDC-1 and VEGF in pleural effusion and showed the prognostic value of VEGF in malignant mesothelioma patients. We furthermore studied the potential value of Angiopoietin-1, HGF, and TIMP-1 among other angiogenic-related proteins as diagnostic and prognostic biomarkers in patient material. We found that Galectin-1, Mesothelin, Osteopontin, VEGF, HGF, shed SDC-1, MMP-7, NRG1-β1, and TIMP-1 were significantly higher in malignant pleural mesothelioma patients. Additionally, we showed that shed SDC-1, MMP-7, Mesothelin, and Galectin-1 significantly discriminated MM from metastatic adenocarcinoma patients (paper III). We further verified our result in paper I and showed that shed SDC-1 and VEGF were prognostic in MM patients. The role of nuclear SDC-1 on epithelial-mesenchymal plasticity of tumor cells – a key event in tumor spread and metastasis – was studied in paper II. Using tumor cell lines, we found that loss of nuclear SDC-1 was associated with cellular elongation and induced EMT in human lung adenocarcinoma cells. Further investigation revealed that nuclear SDC-1 reduce mesenchymal properties and invasiveness of fibrosarcoma cells. Extracellular vesicles have recently gained much interest for their ability to mediate cell-to-cell communication. In paper IV we characterized extracellular vesicles including, apoptotic bodies, microvesicles, and exosomes, derived from pleural effusion from malignant pleural mesothelioma, metastatic lung adenocarcinoma, and benign patients. We found that the size of exosomes was in accordance with previous studies, but their concentration varied between individuals in the same patients group. Additional characterization showed that the presence of CD2, CD8, CD9, CD81, CD24, CD44, CD105, CD146, CD133, MCSP, and ROR1 were higher in the exosome fraction compared with microvesicles and apoptotic bodies whereas, CD40 and CD45 were lower. Furthermore, we demonstrated the presence of the angiogenesis -related proteins which we studied in paper III in extracellular vesicles.