Dokument

Summary:
The expression of the membrane-bound protein ADAM8 is associated with various diseases such as respiratory diseases, liver injury, neuroinflammation, and cancer. Despite its low expression level in distinct cell types, ADAM8 can be upregulated to significant levels upon specific stimuli. Unlike inflammatory processes, ADAM8 is constitutively overexpressed in cancer diseases as it has been described for brain, breast, lung, and pancreatic cancer, and correlates almost exclusively with poor survival. Most importantly, PDAC demonstrated that ADAM8 plays an essential role in tumor cell migration, invasion, and metastasis in vitro and in vivo. The treatment of PDAC is impeded by its high metastatic ability and the lack of specific symptoms leading to late detection of the tumor disease and a high mortality rate. Therefore, there is a high urgency to identify biomarkers for early detection as well as specific therapeutic targets. The first aim of this thesis was to detect ADAM8 in tumor tissue sections of the Marburg PDAC patient cohort. DAB staining of all patients revealed a positive detection of ADAM8 in all sections, suggesting an essential role for ADAM8 in PDAC tumor and disease progression. Nevertheless, ADAM8 expression did not correlate with patient overall survival. ADAM8 expression was also detected in tumor-associated macrophages, NK cells, and neutrophils, indicating that ADAM8 is involved in the recruitment of immune cells to the tumor microenvironment and thus might promote tumor progression. However, the quantification and subsequent analysis of the neutrophil density in post-capillary venules within the tumor areas disclosed the significant negative correlation of neutrophils positive for ADAM8 with overall survival, emphasizing the potential of ADAM8 for diagnosis and prognosis of PDAC. In the second part of this study, ADAM8 was identified as an active protease in exosomes derived from sera of PDAC patients by applying a bead-coupled FACS analysis. Elevated ADAM8 levels in exosomes correlated with disease progression. In addition, two miRNAs, miR-451 and miR-720, that are reported to exert tumor-promoting functions, were dysregulated in exosomes derived from PDAC patients compared to healthy individuals. The validation of these two miRNAs in exosomes from pancreatic cancer cell lines Panc89 control and Panc89 ADAM8 knockout cells revealed the ADAM8-dependent upregulation of miR-720 and downregulation of miR-451. Based on these results, the detection of ADAM8, miR-451, and miR-720 in exosomes may facilitate diagnosis, as this regulatory profile allows the differentiation between different stages of PDAC disease. The final part of this work investigates the regulatory function of ADAM8 in the tumor microenvironment. As a protease involved in tumor cell invasion and metastasis, MMP-9 was previously described to be regulated by ADAM8 via MAPK/CREB signaling. Based on this finding, MMP-9 expression was validated in Panc89 control and ADAM8 knockout cells. However, ADAM8 was shown to regulate MMP-9 activity but not MMP-9 expression. Further analysis of other regulatory proteins revealed the ADAM8-dependent downregulation of LCN2, a protein that promotes PDAC progression and is involved in the regulation of MMP-9 activity. These data suggest that ADAM8 regulates LCN2 expression levels to control MMP-9 activity, promoting invasion and metastasis in PDAC. In addition, the described regulatory effect of ADAM8 on LCN2 and MMP-9 was enhanced by the co-culture of Panc89 cell with THP-1 derived macrophages demonstrating the essential role of ADAM8 in the PDAC tumor microenvironment and thus underlining the potential as a therapeutic target. The functional analysis of exosomes derived from Panc89 control and ADAM8 knockout cells demonstrated an ADAM8-dependent release of LCN2 and revealed that ADAM8 is sorted as a proteolytically active enzyme in exosomes. The presence of the ADAM8 in extracellular vesicles and the ability to exert specific regulatory functions in the tumor microenvironment by either cleavage of essential proteins or regulating particular genes involved in tumor progression offers the possibility to develop advanced treatment strategies and to improve the early detection of PDAC disease.

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten