Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

The role of TMPRSS6 gene polymorphism in iron resistance iron deficiency anaemia (IRIDA): a systematic review.

Iron resistance iron deficiency anaemia is a rare autosomal recessive disorder characterized by hypochromic microcytic anaemia, low transferrin saturation and inappropriately high hepcidin levels. The aetiology of this condition is rooted in genetic variations within the transmembrane serine protease 6 (TMPRSS6) genes, responsible for encoding matriptase-2, a pivotal negative regulator of hepcidin. We conducted a systematic search across four electronic databases, yielding 538 articles in total out of which 25 were finally included and were preceded further, aiming to prognosticate prevalent single nucleotide polymorphisms (SNPs) and detrimental genetic alterations. This review aims to elucidate the effects of various SNPs and pathogenic mutations on both haematological and biochemical parameters, as well as their potential interethnic correlation. Employing bioinformatics tools, we subjected over 100 SNPs to scrutiny, discerning their potential functional ramifications. We found rs1373272804, rs1430692214 and rs855791 variants to be most frequent and were having a significant impact on haematological and biochemical profile. We found that individuals of European ancestry were more prone to have these variants compared to other ethnic groups. In conclusion, this review not only sheds light on the association of TMPRSS6 polymorphism in iron resistance iron deficiency anaemia (IRIDA), but also highlights the critical need for further investigations involving larger sample size and more diverse ethnic groups around the globe. These future studies will be vital for gaining a stronger and more reliable understanding of how these genetic differences are linked to the development of IRIDA.

Anaemia and iron deficiency associate with polymorphism TMPRSS6 rs855791 in Brazilian children attending day care centres.

Fe-deficiency anaemia is a major public health concern in children under 5 years of age. TMPRSS6 gene, encoding matriptase-2 protein, is implicated in Fe homoeostasis and has been associated with anaemia and Fe status in various populations. The aim of this cross-sectional study was to investigate the associations between the single nucleotide polymorphism (SNP) TMPRSS6 rs855791 and biomarkers of anaemia and Fe deficiency in Brazilian children attending day care centres. A total of 163 children aged 6-42 months were evaluated. Socio-economic, demographic, biochemical, haematological, immunological and genotype data were collected. Multiple logistic and linear regressions with hierarchical selection were used to assess the effects of independent variables on categorised outcomes and blood marker concentrations. Minor allele (T) frequency of rs855791 was 0·399. Each copy of the T allele was associated with a 4·49-fold increased risk of developing anaemia (P = 0·005) and a 4·23-fold increased risk of Fe deficiency assessed by serum soluble transferrin receptor (sTfR) (P < 0·001). The dose of the T allele was associated with an increase of 0·18 mg/l in sTfR concentrations and reductions of 1·41 fl and 0·52 pg in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), respectively. In conclusion, the T allele of SNP TMPRSS6 rs855791 was significantly associated with anaemia and Fe deficiency assessed by sTfR in Brazilian children attending day care centres. The effect was dose dependent, with each copy of the T allele being associated with lower MCV and MCH and higher concentrations of sTfR.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Insufficiency of hepatocyte growth factor activator inhibitor-1 confers lymphatic invasion of tongue carcinoma cells.

Hepatocyte growth factor (HGF) activator inhibitor type-1 (HAI-1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane-anchored serine proteases, particularly matriptase. Here, we explored the role of HAI-1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI-1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI-1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI-1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI-1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase-type plasminogen activator, which induced activation of HGF/MET signaling in the co-culture with pro-HGF-expressing fibroblasts and plasminogen-dependent plasmin generation, respectively. The enhanced plasminogen-dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI-1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor-C (VEGF-C), a lymphangiogenesis factor, into the mature form in a plasminogen-dependent manner. Furthermore, HGF significantly stimulated VEGF-C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI-1KO TSCC cells compared to control cells. Our results suggest that HAI-1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease-dependent growth factors, such as HGF and VEGF-C, in a tumor microenvironment to contribute to TSCC progression.

Proteolytic cleavage of Podocin by Matriptase exacerbates podocyte injury.

Podocyte injury is a critical step toward the progression of renal disease and is often associated with a loss of slit diaphragm proteins, including Podocin. Although there is a possibility that the extracellular domain of these slit diaphragm proteins can be a target for a pathological proteolysis, the precise mechanism driving the phenomenon remains unknown. Here we show that Matriptase, a membrane-anchored protein, was activated at podocytes in CKD patients and mice, whereas Matriptase inhibitors slowed the progression of mouse kidney disease. The mechanism could be accounted for by an imbalance favoring Matriptase over its cognate inhibitor, hepatocyte growth factor activator inhibitor type 1 (HAI-1), because conditional depletion of HAI-1 in podocytes accelerated podocyte injury in mouse model. Matriptase was capable of cleaving Podocin, but such a reaction was blocked by either HAI-1 or dominant-negative Matriptase. Furthermore, the N terminus of Podocin, as a consequence of Matriptase cleavage of Podocin, translocated to nucleoli, suggesting that the N terminus of Podocin might be involved in the process of podocyte injury. Given these observations, we propose that the proteolytic cleavage of Podocin by Matriptase could potentially cause podocyte injury and that targeting Matriptase could be a novel therapeutic strategy for CKD patients.

Gene-associated methylation status of ST14 as a predictor of survival and hormone receptor positivity in breast Cancer.

BACKGROUND: Genomic profiles of specific gene sets have been established to guide personalized treatment and prognosis for patients with breast cancer (BC). However, epigenomic information has not yet been applied in a clinical setting. ST14 encodes matriptase, a proteinase that is widely expressed in BC with reported prognostic value. METHODS: In this present study, we evaluated the effect of ST14 DNA methylation (DNAm) on overall survival (OS) of patients with BC as a representative example to promote the use of the epigenome in clinical decisions. We analyzed publicly available genomic and epigenomic data from 1361 BC patients. Methylation was characterized by the ß-value from CpG probes based on sequencing with the Illumina Human 450 K platform. RESULTS: A high mean DNAm (ß > 0.6779) across 34 CpG probes for ST14, as the gene-associated methylation (GAM) pattern, was associated with a longer OS after adjusting age, stage, histology and molecular features in Cox model (p value < 0.001). A high GAM status was also associated with a higher XBP1 expression level and higher proportion of hormone-positive BC (p value < 0.001). Pathway analysis revealed that altered GAM was related to matrisome-associated pathway. CONCLUSIONS: Here we show the potential role of ST14 DNAm in BC prognosis and warrant further study.

Insights into the regulation of the matriptase-prostasin proteolytic system.

The membrane-associated prostasin and matriptase belonging to the S1A subfamily of serine proteases, are critical for epithelial development and maintenance. The two proteases are involved in the activation of each other and are both regulated by the protease inhibitors, HAI-1 and HAI-2. The S1A subfamily of serine proteases are generally produced as inactive zymogens requiring a cleavage event to obtain activity. However, contrary to the common case, the zymogen form of matriptase exhibits proteolytic activity, which can be inhibited by HAI-1 and HAI-2, as for the activated counterpart. We provide strong evidence that also prostasin exhibits proteolytic activity in its zymogen form. Furthermore, we show that the activity of zymogen prostasin can be inhibited by HAI-1 and HAI-2. We report that zymogen prostasin is capable of activating zymogen matriptase, but unable to activate its own zymogen form. We propose the existence of an unusual enzyme-enzyme relationship consisting of proteolytically active zymogen forms of both matriptase and prostasin, kept under control by HAI-1 and HAI-2, and located at the pinnacle of an important proteolytic pathway in epithelia. Perturbed balance in this proteolytic system is likely to cause rapid and efficient activation of matriptase by the dual action of zymogen matriptase and zymogen prostasin. Previous studies suggest that the zymogen form of matriptase performs the normal proteolytic functions of the protease, whereas excess matriptase activation likely causes carcinogenesis. HAI-1 and HAI-2 are thus important for the prevention of matriptase activation whether catalysed by zymogen/activated prostasin (this study) or zymogen/activated matriptase (previous studies).

Inhibition of an active zymogen protease: the zymogen form of matriptase is regulated by HAI-1 and HAI-2.

The membrane-bound serine protease matriptase belongs to a rare subset of serine proteases that display significant activity in the zymogen form. Matriptase is critically involved in epithelial differentiation and homeostasis, and insufficient regulation of its proteolytic activity directly causes onset and development of malignant cancer. There is strong evidence that the zymogen activity of matriptase is sufficient for its biological function(s). Activated matriptase is inhibited by the two Kunitz-type inhibitor domain-containing hepatocyte growth factor activator inhibitors 1 (HAI-1) and HAI-2, however, it remains unknown whether the activity of the matriptase zymogen is regulated. Using both purified proteins and a cell-based assay, we show that the catalytic activity of the matriptase zymogen towards a peptide-based substrate as well as the natural protein substrates, pro-HGF and pro-prostasin, can be inhibited by HAI-1 and HAI-2. Inhibition of zymogen matriptase by HAI-1 and HAI-2 appears similar to inhibition of activated matriptase and occurs at comparable inhibitor concentrations. This indicates that HAI-1 and HAI-2 interact with the active sites of zymogen and activated matriptase in a similar manner. Our results suggest that HAI-1 and HAI-2 regulate matriptase zymogen activity and thus may act as regulators of matriptase trans(auto)-activation. Due to the main localisation of HAI-2 in the ER and HAI-1 in the secretory pathway and on the cell surface, this regulation likely occurs both in the secretory pathway and on the plasma membrane. Regulation of an active zymogen form of a protease is a novel finding.

Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors.

Matriptase is a member of the type-II transmembrane serine protease (TTSP) family and plays a crucial role in the development and maintenance of epithelial tissues. As all chymotrypsin-like serine proteases, matriptase is synthesized as a zymogen (proform), requiring a cleavage event for full activity. Recent studies suggest that the zymogen of matriptase possesses enough catalytic activity to not only facilitate autoactivation, but also carry out its in vivo functions, which include activating several proteolytic and signaling cascades. Inhibition of zymogen matriptase may therefore be a highly effective approach for limiting matriptase activity. To this end, here we sought to characterize the catalytic activity of human zymogen matriptase and to develop mAb inhibitors against this enzyme form. Using a mutated variant of matriptase in which the serine protease domain is locked in the zymogen conformation, we confirmed that the zymogen form of human matriptase has catalytic activity. Moreover, the crystal structure of the catalytic domain of zymogen matriptase was solved to 2.5 Å resolution to characterize specific antibody-based matriptase inhibitors and to further structure-based studies. Finally, we describe the first antibody-based competitive inhibitors that target both the zymogen and activated forms of matriptase. We propose that these antibodies provide a more efficient way to regulate matriptase activity by targeting the protease both before and after its activation and may be of value for both research and preclinical applications.

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin.

Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an iron-regulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2-/- mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

Specifically targeting cancer proliferation and metastasis processes: the development of matriptase inhibitors.

Matriptase is a type II transmembrane serine protease, which has been suggested to play critical roles in numerous pathways of biological developments. Matriptase is the activator of several oncogenic proteins, including urokinase-type plasminogen activator (uPA), hepatocyte growth factor (HGF) and protease-activated receptor 2 (PAR-2). The activations of these matriptase substrates subsequently lead to the generation of plasmin, matrix metalloproteases (MMPs), and the triggers for many other signaling pathways related to cancer proliferation and metastasis. Accordingly, matriptase is considered an emerging target for the treatments of cancer. Thus far, inhibitors of matriptase have been developed as potential anti-cancer agents, which include small-molecule inhibitors, peptide-based inhibitors, and monoclonal antibodies. This review covers established literature to summarize the chemical and biochemical aspects, especially the inhibitory mechanisms and structure-activity relationships (SARs) of matriptase inhibitors with the goal of proposing the strategies for their future developments in anti-cancer therapy.

Cell surface-anchored serine proteases in cancer progression and metastasis.

Over the last two decades, a novel subgroup of serine proteases, the cell surface-anchored serine proteases, has emerged as an important component of the human degradome, and several members have garnered significant attention for their roles in cancer progression and metastasis. A large body of literature describes that cell surface-anchored serine proteases are deregulated in cancer and that they contribute to both tumor formation and metastasis through diverse molecular mechanisms. The loss of precise regulation of cell surface-anchored serine protease expression and/or catalytic activity may be contributing to the etiology of several cancer types. There is therefore a strong impetus to understand the events that lead to deregulation at the gene and protein levels, how these precipitate in various stages of tumorigenesis, and whether targeting of selected proteases can lead to novel cancer intervention strategies. This review summarizes current knowledge about cell surface-anchored serine proteases and their role in cancer based on biochemical characterization, cell culture-based studies, expression studies, and in vivo experiments. Efforts to develop inhibitors to target cell surface-anchored serine proteases in cancer therapy will also be summarized.

An investigation of the relationship between TMPRSS6 gene expression, genetic variants and clinical findings in breast cancer.

Breast cancer is one of the most common types of cancer among women worldwide. The TMPRSS6 (Transmembrane Serine Protease 6) gene encodes matriptase-2, which plays an important role in iron hemostasis as the hepcidin regulator and may play a role in breast cancer susceptibility. In this study, we examined the expression levels of the TMPRSS6 gene in healthy tissues and tumor tissues of breast cancer patients; and the relationship between these levels and pathological findings. The relationship between TMPRSS6 polymorphisms (rs733655, rs5756506, rs2413450, rs855791, rs2235324, rs4820268) and patients' hematological parameters. The gene expression study encompassed 47 breast cancer patients and the gene polymorphism study consisted of 181 breast cancer patients and 100 healthy controls. Gene expression analysis was performed by qRT-PCR. The genotyping of TMPRSS6 polymorphisms was performed by RT-PCR. TMPRSS6 gene expression levels in tumor tissues were found to be 1.88 times higher than the expression levels in the control tissues. We examined the relationship between TMPRSS6 gene expression levels and pathological data, statistically significant relationship was found between patient's estrogen receptor (ER) and HER2 findings and TMPRSS6 gene expression (respectively p = 0.02, p = 0.002). When the relationship between TMPRSS6 gene polymorphisms related genotypes distributions and hematological findings was investigated, a significant relationship was identified between mean corpuscular hemoglobin concentration (MCHC) parameter and the polymorphism of only the rs733655. According to our findings, the increase in TMPRSS6 gene expression in cancerous tissues shows that matriptase-2 may be effective in the cancer process. Thus TMPRSS6 gene polymorphisms may affect the disease process by affecting the blood parameters of patients.

Dysregulation of Type II Transmembrane Serine Proteases and Ligand-Dependent Activation of MET in Urological Cancers.

Unlike in normal epithelium, dysregulated overactivation of various proteases have been reported in cancers. Degradation of pericancerous extracellular matrix leading to cancer cell invasion by matrix metalloproteases is well known evidence. On the other hand, several cell-surface proteases, including type II transmembrane serine proteases (TTSPs), also induce progression through activation of growth factors, protease activating receptors and other proteases. Hepatocyte growth factor (HGF) known as a multifunctional growth factor that upregulates cancer cell motility, invasiveness, proliferative, and anti-apoptotic activities through phosphorylation of MET (a specific receptor of HGF). HGF secreted as inactive zymogen (pro-HGF) from cancer associated stromal fibroblasts, and the proteolytic activation by several TTSPs including matriptase and hepsin is required. The activation is strictly regulated by HGF activator inhibitors (HAIs) in physiological condition. However, downregulation is frequently observed in cancers. Indeed, overactivation of MET by upregulation of matriptase and hepsin accompanied by the downregulation of HAIs in urological cancers (prostate cancer, renal cell carcinoma, and bladder cancer) are also reported, a phenomenon observed in cancer cells with malignant phenotype, and correlated with poor prognosis. In this review, we summarized current reports focusing on TTSPs, HAIs, and MET signaling axis in urological cancers.

HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase.

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

3-Cl-AHPC inhibits pro-HGF maturation by inducing matriptase/HAI-1 complex formation.

Matriptase is an epithelia-specific membrane-anchored serine protease, and its dysregulation is highly related to the progression of a variety of cancers. Hepatocyte growth factor activator inhibitor-1 (HAI-1) inhibits matriptase activity through forming complex with activated matriptase. The balance of matriptase activation and matriptase/HAI-1 complex formation determines the intensity and duration of matriptase activity. 3-Cl-AHPC, 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid, is an adamantly substituted retinoid-related molecule and a ligand of retinoic acid receptor γ (RARγ). 3-Cl-AHPC is of strong anti-cancer effect but with elusive mechanisms. In our current study, we show that 3-Cl-AHPC time- and dose- dependently induces matriptase/HAI-1 complex formation, leading to the suppression of activated matriptase in cancer cells and tissues. Furthermore, 3-Cl-AHPC promotes matriptase shedding but without increasing the activity of shed matriptase. Moreover, 3-Cl-AHPC inhibits matriptase-mediated cleavage of pro-HGF through matriptase/HAI-1 complex induction, resulting in the suppression of pro-HGF-stimulated signalling and cell scattering. Although 3-Cl-AHPC binds to RARγ, its induction of matriptase/HAI-1 complex is not RARγ dependent. Together, our data demonstrates that 3-Cl-AHPC down-regulates matriptase activity through induction of matriptase/HAI-1 complex formation in a RARγ-independent manner, providing a mechanism of 3-Cl-AHPC anti-cancer activity and a new strategy to inhibit abnormal matriptase activity via matriptase/HAI-1 complex induction using small molecules.

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein.

Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.

Overexpression of matriptase in tumor stroma is a poor prognostic indicator of extrahepatic bile duct cancer.

Bile duct cancer is known to contain numerous fibroblasts, and reported to recruit cancer- associated fibroblasts by secreting platelet-derived growth factor-D (PDGF-D) which needs serine proteases, such as matriptase, to behave as a ligand. However, their expression pattern, and prognostic value have not been clarified. In this study, we investigated the clinicopathological significance of PDGF-D and matriptase expression in patients with extrahepatic bile duct cancer. The samples were obtained from 256 patients who underwent the surgical resection between 1991 and 2015, and the expression levels of PDGF-D and matriptase were evaluated immunohistochemically. Staining intensities and distribution were scored, and finally classified into low and high expression groups in cancer cells and stroma respectively. High expression of matriptase in the cancer stroma was detected in 91 tumors (40%). The high stromal matriptase expression was significantly associated with shorter recurrence-free survival (RFS) and overall survival (OS) (P = 0.0027 and 0.0023, respectively). Multivariate analyses also demonstrated that the stromal matriptase expression level was an independent influential factor in RFS (P = 0.0050) and OS (P = 0.0093). Our findings suggest that the high stromal matriptase expression was strongly associated with tumor progression, recurrence and poor outcomes in patients with extrahepatic bile duct cancer.