Volume 395, Issue 9

Human kallikrein-related peptidases (KLKs), including the well-known prostate cancer biomarker, prostate-specific antigen (PSA-KLK3), along with 14 other serine proteinase KLK family members are now known to regulate cells by cleaving and activating members of the G-protein-coupled proteinase-activated receptor (PAR) family. This hormone-like signaling action of the KLKs has provided a new perspective for understanding the biological roles that KLKs may play in normal and pathophysiological settings. This overview summarizes the circumstances leading up to the discovery of this action of the KLKs and provides an overview of the diverse impact on tissue function that may result from KLK-triggered PAR activation.

Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3–13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates.

Netherton syndrome (NS) is an orphan genetic skin disease with a profound skin barrier defect and severe allergic manifestations. NS is caused by loss of function mutations in SPINK5 encoding lympho-epithelial Kazal-type inhibitor (LEKTI), a secreted multi-domain serine protease inhibitor expressed in stratified epithelia. Studies in mouse models and in NS patients have established that unopposed kallikrein 5 activity triggers stratum corneum detachment and activates PAR-2 signaling, leading to the autonomous production of pro-allergic and pro-inflammatory mediators. This emerging knowledge on NS pathogenesis has highlighted a central role for protease regulation in skin homeostasis but also in the complexity of the disease, and holds the promise of new specific treatments.

Most kallikrein-related peptidases (KLKs) are N-glycosylated with N-acetylglucosamine 2 -mannose 9 units at Asn-Xaa-Ser/Thr sequons during protein synthesis and translocation into the endoplasmic reticulum. These N-glycans are modified in the Golgi machinery, where additional O-glycosylation at Ser and Thr takes place, before exocytotic release of the KLKs into the extracellular space. Sequons are present in all 15 members of the KLKs and comparative studies for KLKs from natural and recombinant sources elucidated some aspects of glycosylation. Although glycosylation of mammalian KLKs 1, 3, 4, 6, and 8 has been analyzed in great detail, e.g., by crystal structures, the respective function remains largely unclear. In some cases, altered enzymatic activity was observed for KLKs upon glycosylation. Remarkably, for KLK3/PSA, changes in the glycosylation pattern were observed in samples of benign prostatic hyperplasia and prostate cancer with respect to healthy individuals. Potential functions of KLK glycosylation in structural stabilization, protection against degradation, and activity modulation of substrate specificity can be deduced from a comparison with other glycosylated proteins and their regulation. According to the new concept of protein sectors, glycosylation distant from the active site might significantly influence the activity of proteases. Novel pharmacological approaches can exploit engineered glycans in the therapeutical context.

The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and roles in a range of cellular processes, including proliferation, migration, invasion, differentiation, inflammation and angiogenesis that are required in both normal physiology as well as pathological conditions. These roles require cleavage of a range of substrates, including extracellular matrix proteins, growth factors, cytokines as well as other proteinases. In addition, it has been clear since the earliest days of KLK research that cleavage of cell surface substrates is also essential in a range of KLK-mediated cellular processes where these peptidases are essentially acting as agonists and antagonists. In this review we focus on these KLK-regulated cell surface receptor systems including bradykinin receptors, proteinase-activated receptors, as well as the plasminogen activator, ephrins and their receptors, and hepatocyte growth factor/Met receptor systems and other plasma membrane proteins. From this analysis it is clear that in many physiological and pathological settings KLKs have the potential to regulate multiple receptor systems simultaneously; an important issue when these peptidases and substrates are targeted in disease.

MicroRNAs (miRNAs) are short RNA nucleotides that negatively regulate their target genes. They are differentially expressed in prostate cancer. Kallikreins are genes that encode serine proteases and are dysregulated in cancer. We elucidated a miRNA-kallikrein network that can be involved in prostate cancer progression. Target prediction identified 23 miRNAs that are dysregulated between high and low risk biochemical failure and are predicted to target five kallikreins linked to prostate cancer; KLK2 , KLK3 , KLK4 , KLK14 and KLK15 . We also identified 14 miRNAs that are differentially expressed between Gleason grades and are predicted to target these kallikreins. This demonstrates that kallikreins are downstream effectors through which miRNAs influence tumor progression. We show, through in-silico and experimental analysis, that miR-378/422a and its gene targets PIK3CG , GRB2 , AKT3 , KLK4 and KLK14 form an integrated circuit in prostate cancer. Our analysis shows that a minisatellite sequence in the kallikrein locus consists of a number of microsatellite repeats that represent predicted miRNA response elements. A number of kallikrein and non-kallikrein prostate cancer-related genes share these microsatellite repeats. We validated some of these interactions in prostate cancer cell lines. Finally, we provide preliminary evidence on the presence of a miRNA-mediated cross-talk between kallikreins, including a kallikrein pseudogene.

Kallikrein-related peptidase 4 (KLK4) is a secreted serine protease that degrades residual enamel proteins to facilitate their removal by ameloblasts, which increases mineralization and hardens the enamel. Mutations in human KLK4 cause hypomaturation amelogenesis imperfecta. Enamel formed by Klk4 null mice is normal in thickness and prism structure, but the enamel layer retains proteins, is hypomineralized, and undergoes rapid attrition following tooth eruption. We searched multiple databases, retrieved Klk4 and Klk5 from various mammalian genomes, and identified Klk4 in 46 boreoeutherian genomes. In non-Boreoeutheria, Klk4 was detected in only one afrotherian genome (as a pseudogene), and not in the other six afrotherian, two xenarthran, or three marsupial genomes. In contrast, Klk5 was detected in both marsupial and eutherian mammals. Our phylogenetic and mutation rate analyses support the hypothesis that Klk4 arose from Klk5 by gene duplication near the divergence of Afrotheria, Xenarthra and Boreoeutheria, and that functionally-differentiated Klk4 survived only in Boreoeutheria. Afrotherian mammals share the feature of delayed dental eruption relative to boreoeutherian mammals. KLK4 shortens the time required for enamel maturation and could have alleviated negative selection following mutations that resulted in thicker enamel or earlier tooth eruption, without reducing enamel hardness or causing dental attrition.

The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.

Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (K iapp =8.6 μ m ). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking coagulation and arterial thrombus formation, without increasing bleeding time.

Kallikreins (KLKs) are a group of 15 serine proteases encoded by the KLK locus on chromosome 19. Certain single nucleotide variants (SNVs) within the KLK locus have been linked to human disease. Next-generation sequencing of large human cohorts enables reexamination of genomic variation at the KLK locus. We aimed to identify all KLK-related SNVs and examine their impact on gene regulation and function. To this end, we mined KLK SNVs across Ensembl and Exome Variant Server, with exome-sequencing data from 6503 individuals. PolyPhen-2-based prediction of damaging SNVs and population frequencies of these SNVs were examined. Damaging SNVs were plotted on protein sequence and structure. We identified 4866 SNVs, the largest number of KLK-related SNVs reported. Fourteen percent of noncoding SNVs overlapped with transcription factor binding sites. We identified 602 missense coding SNVs, among which 148 were predicted to be damaging. Nine missense SNVs were common (>1% frequency) and displayed significantly different frequencies between European-American and African-American populations. SNVs predicted to be damaging appeared to alter tertiary structure of KLK1 and KLK6. Similarly, these missense SNVs may affect KLK function, resulting in disease phenotypes. Our study represents a mine of information for those studying KLK-related SNVs and their associations with diseases.

Several members of the family of tissue kallikrein and kallikrein-related peptidases have been suggested as promising tumor biomarkers with important prognostic significance. However, only one ( KLK11 ) has already been studied in laryngeal squamous cell carcinoma (LSCC) as a potential biomarker for LSCC diagnosis and/or prognosis. Our study investigated the prognostic value of kallikrein-related peptidase-4 ( KLK4 ) mRNA expression as a molecular tissue biomarker in LSCC. For this purpose, KLK4 mRNA expression analysis was performed in 116 cancerous and 74 paired non-cancerous laryngeal tissue specimens obtained from patients that had undergone surgical treatment for primary LSCC. A remarkable downregulation of KLK4 mRNA expression was discovered in laryngeal tumors, compared to non-cancerous laryngeal tissue specimens. KLK4 mRNA expression was also shown to distinguish LSCC from non-cancerous laryngeal tissues. Furthermore, low KLK4 mRNA expression was shown to predict poor disease-free survival, independently of the histological grade and size of the malignant tumor as well as patient TNM stage. According to Kaplan-Meier survival analysis, low KLK4 mRNA expression predicts short-term relapse even among patients with well-differentiated tumors or those at an early TNM stage. Thus, KLK4 mRNA positivity could be regarded as a novel independent indicator of favorable prognosis for the disease-free survival of LSCC patients.

Recent studies provide a functional link between kallikrein 6 (Klk6) and the development and progression of disease in patients with multiple sclerosis (MS) and in its murine models. To evaluate the involvement of additional kallikrein family members, we compared Klk6 expression with four other kallikreins (Klk1, Klk7, Klk8, and Klk10) in the brain and spinal cord of mice infected with Theiler’s murine encephalomyelitis virus, an experimental model of progressive MS. The robust upregulation of Klk6 and Klk8 in the brain during the acute phase of viral encephalitis and in the spinal cord during disease development and progression points to their participation in inflammation, demyelination, and progressive axon degeneration. More limited changes in Klk1, Klk7, and Klk10 were also observed. In addition, Klk1, Klk6, and Klk10 were dynamically regulated in T cells in vitro as a recall response to viral antigen and in activated monocytes, pointing to their activities in the development of adaptive and innate immune function. Together, these results point to overlapping and unique roles for multiple kallikreins in the development and progression of virus-mediated central nervous system inflammatory demyelinating disease, including activities in the development of the adaptive and innate immune response, in demyelination, and in progressive axon degeneration.

Emerging evidence indicates that serine proteases of the tissue kallikrein-related peptidases family ( KLK ) are implicated in tumorigenesis. We recently reported the ectopic expression of KLK4 and KLK14 in colonic cancers and their signaling to control cell proliferation. Human tissue kallikrein-related peptidase 7 (KLK7) is often dysregulated in many cancers; however, its role in colon tumorigenesis has not yet been established. In the present study, we analyzed expression of KLK7 in 15 colon cancer cell lines and in 38 human colonic tumors. In many human colon cancer cells, KLK7 mRNA was observed, which leads to KLK7 protein expression and secretion. Furthermore, KLK7 was detected in human colon adenocarcinomas, but it was absent in normal epithelia. KLK7 overexpression in HT29 colon cancer cells upon stable transfection with a KLK7 expression plasmid resulted in increased cell proliferation. Moreover, subcutaneous inoculation of transfected cells into nude mice led to increased tumor growth that was associated with increased tumor cell proliferation as reflected by a positive Ki-67 staining. Our results demonstrate the aberrant expression of KLK7 in colon cancer cells and tissues and its involvement in cell proliferation in vitro and in vivo . Thus, KLK7 may represent a potential therapeutic target for human colon tumorigenesis.

The prognosis of patients following surgery for gastric cancer is often poor and is estimated using traditional clinicopathological parameters, which can be inaccurate predictors of future survival. Kallikreins are a group of serine proteases, which are differentially expressed in many human tumors and are being investigated as potential cancer biomarkers. This study assessed the prognostic utility of human tissue kallikrein-like peptidases 6 and 10 (KLK6 and KLK10) and correlated their expression with histopathological and clinical parameters in gastric cancer. We constructed a gastric tumor tissue microarray from 113 gastrectomy specimens and quantified KLK6 and KLK10 expression using immunohistochemistry. To overcome the problem of inter-observer variability and subjectivity in immunohistochemistry interpretation, a whole-slide scanned image of the tissue microarray was analyzed using an automated algorithm to quantify staining intensity. KLK6 expression was positively correlated with nodal involvement ( p =0.002) and was predictive of advanced-stage disease ( p <0.05). Kaplan-Meier survival curves revealed that tumors expressing high levels of KLK6 were significantly associated with significantly lower overall survival ( p =0.04). KLK10 overexpression was also a predictor of advanced-stage disease ( p <0.01), but was not significantly correlated with lymph node involvement or survival period. Our results show the potential ability of KLK6 as a prognostic marker for gastric cancer.

A large number of prostate cancer (PCa) patients receive treatment without significant benefits, strengthening the need for accurate prognosis, which can be supported by the study of miRNAs. In silico specificity analysis was performed for the identification of miRNAs able to regulate KLK2 and KLK4 expression. Total RNA was extracted from prostate tissues obtained from PCa and benign prostate hyperplasia patients. Thereafter, RNA was polyadenylated and reverse transcribed to cDNA, which was used for qPCR analysis. miR-378 was predicted to target both KLK2 and KLK4 and downregulated levels detected in PCa patients ( p =0.050). The reduction of miR-378 was correlated with higher Gleason score ( p =0.018), larger diameter tumors ( p =0.034), and elevated serum PSA ( p =0.006). Regarding prognosis, miR-378 was able to improve risk stratification according to Gleason score or tumor stage, while higher risk to recur highlighted for the patients expressing lower miR-378 levels. Finally, the loss of miR-378 was able to predict the short-term relapse of ‘high’- and ‘very high’-recurrence-risk patients, independent of Gleason score, tumor stage, PSA, and age as indicated by Kaplan-Meier survival curves ( p =0.030) and multivariate Cox regression analysis ( p =0.018). In conclusion, loss of miR-378 expression increases the risk for PCa progression and relapse, despite active treatment.

KLK6 is a secreted trypsin-like serine protease. KLK6 mRNA expression and its association with colon cancer (CC) progression was studied using quantitative real-time PCR. We examined the expression of KLK6 in 232 colon tissues (cancerous, non-cancerous, and adenomatous). We proved that KLK6 expression in CC behaves as a continuous variable, as its expression correlates significantly with increasing tumor stage ( p =0.004) and histological grade ( p =0.007). Interestingly, the expression of KLK6 in adenomas was significantly higher than that in the cancerous or non-cancerous tissues examined ( p <0.001). Cox proportional hazard regression model using univariate analysis revealed that positive KLK6 expression is a significant factor for disease-free survival (DFS) ( p =0.017) and overall survival (OS) ( p =0.002) of patients. Kaplan-Meier survival curves demonstrated that KLK6- negative expression is significantly associated with longer DFS ( p =0.009) and OS ( p =0.001). ROC analysis showed that KLK6 expression has significant discriminatory power in distinguishing cancerous from non-cancerous colon tissues ( p <0.001), or cancerous from adenoma tissues ( p =0.001), or adenoma from non-cancerous colon tissues ( p <0.001). Additionally, strong KLK6 immunostaining was seen in the cancer cells of selected CC sections, as well as in glandular cells and inflammatory cells of adenomas. In conclusion, KLK6 may represent a potential unfavorable prognostic biomarker for CC.

We have raised monoclonal antibodies against KLK6 and constructed a ‘sandwich’ type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 μg/l±1.37) than in controls (3.39 μg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257–965 μg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.

We assessed whether alternative transcripts (using KLK2 , KLK3 and KLK4 as models) are differentially regulated by androgens and anti-androgens as an indicator of prostate cancers as they acquire treatment resistance. Using RNAseq of LNCaP cells treated with dihydrotestosterone, bicalutamide and enzalutamide, we show that the expression of variant KLK transcripts is markedly different to other variant transcripts at those loci. We also reveal that KLK variants are also over 2-fold more highly expressed in prostate cancers compared to their corresponding normal prostate. We propose that androgens and anti-androgens can activate specific variant transcripts of critical prostate cancer genes during treatment resistance.