Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that is involved in inflammatory pathways in skin and central nervous system. Additionally, its overexpression was detected in several tumor entities including pancreatic ductal adenocarcinoma (PDAC), thereby rendering it as a potential cancer biomarker candidate. However, distinct substrates of KLK6 are only sparsely identified and the extent of its pleiotropic biological effects in PDAC remains to be fully understood.This study aims to elucidate biological mechanisms of KLK6 in a MiaPaCa2 knockdown model. For that, cell conditioned medium was generated and a mass spectrometry-based secretome and degradome analysis was performed. By using an isobaric labeling approach (TMT 16-plex), a quantitative analysis and comparison between control and KLK6 knockdown condition was achieved. The secretome analysis revealed over 1400 proteins classified as “extracellular space” or “secreted” by Gene Ontology (GO). An upregulation of the TGF-receptor binding proteins TGFB2 and THBS1 could be detected in the knockdown condition, aligning with previously reported effects of KLKs on the TGFβ-signalling pathway. Effects on the extracellular matrix (ECM) assembly by KLK6 can be suggested due to the upregulation of several ECM structure proteins such as VCAN, FN1, SPON1, COL3A1, COL5A2 and COL6A1. On the opposite, COL1A2 and PCOLCE2 were found downregulated, indicating an effect on ECM structure assembly or a compensatory feedback mechanism. A downregulation of MHC class I (HLA-A, HLA-B, HLA-C, HLA-E) and a higher abundance of secreted MICA and MICB indicate an influence on immune escape and NK-cell and T-cell recognition, caused by a suppression of expression of the immunoproteasome (PSMB8, PSMB9, PSMB10). Interestingly, many interferon-stimulated genes were found downregulated in the KLK6 knockdown condition, suggesting an effect on IFN signaling by KLK6. For the degradome analysis, neo-N-termini were labelled with TMT 16-plex before tryptic digestion and analyzed by LC-MS/MS. The analysis revealed 2871 neo-N-termini classified as secreted by GO, providing a deeper insight into the effects of KLK6 on ECM remodelling: Differential processing of the C-terminal sides of COL18A1, COL3A1 and COL1A2 as well as LOXL2 highlight the biological importance of proteolytic processing as a posttranslational modification and the value of mass spectrometry-based proteomics.Altogether, this study represents a first mass spectrometry-based secretome and degradome analysis to unravel the biological effects of KLK6 in PDAC. Given that KLK6, along with other KLKs, is already considered as potential biomarker or therapeutic target, our findings promise to furnish crucial and valuable insights into the physiological mechanisms that could be influenced by KLK6 inhibition.AcknowledgmentsThe Schilling laboratory acknowledges funding by the Deutsche Forschungsgemeinschaft SFB1479-Oncoescape (project ID: 441891347)