Thomas Kryza

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor

Abstract Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients. Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.

Kallikrein‐related peptidase 4 induces cancer‐associated fibroblast features in prostate‐derived stromal cells

The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer‐associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein‐related peptidase‐4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF‐like features in the prostate‐derived WPMY1 normal stromal cell line, including increased expression of alpha‐smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease‐activated receptor‐1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient‐derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial‐derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment.

Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

The cleavage preferences of Kallikrein-related peptidase 7 (KLK7) have previously been delineated using synthetic peptide libraries of fixed length, or single protein chains and have suggested that KLK7 exerts a chymotryptic-like cleavage preference. Due to the short length of the peptides utilised, only a limited number of subsites have however been assessed. To determine the subsite preferences of KLK7 in a global setting, we used a mass spectrometry (MS)-based in-depth proteomics approach that utilises human proteome-derived peptide libraries of varying length, termed Proteomic Identification of protease Cleavage Sites (PICS). Consistent with previous findings, KLK7 was found to exert chymotryptic-like cleavage preferences. KLK7 subsite preferences were also characterised in the P2-P2′ region, demonstrating a preference for hydrophobic residues in the non-prime and hydrophilic residues in the prime subsites. Interestingly, single catalytic triad mutant KLK7 (mKLK7; S195A) also showed residual catalytic activity (kcat/KM = 7.93 × 102 s−1M−1). Catalytic inactivity of KLK7 was however achieved by additional mutation in this region (D102N). In addition to characterising the cleavage preferences of KLK7, our data thereby also suggests that the use of double catalytic triad mutants should be employed as more appropriate negative controls in future investigations of KLK7, especially when highly sensitive MS-based approaches are employed.

Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix

Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis – a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12′s proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12′s proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12′s proangiogenic effect is mediated through several molecular mechanisms.