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Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanced by negligible expression of its endogenous inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. HAI-1, however, is also involved in matriptase synthesis and intracellular trafficking. The lack of HAI-1 indicates that other related inhibitor, such as HAI-2, might be expressed. Here, we show that HAI-2 is commonly co-expressed in matriptase-expressing neoplastic B-cells. The level of active matriptase shed after induction of matriptase zymogen activation in 7 different neoplastic B-cells was next determined and characterised. Our data reveal that active matriptase can only be generated and shed by those cells able to activate matriptase and in a rough correlation with the levels of matriptase protein. While HAI-2 can potently inhibit matriptase, the levels of active matriptase are not proportionally suppressed in those cells with high HAI-2. Our survey suggests that matriptase proteolysis might aberrantly remain high in neoplastic B-cells regardless of the levels of HAI-2.

BACKGROUND Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors for hepatocyte growth factor activator (HGFA). We attempted to clarify whether serum levels of HAI-1 and HAI-2 could be a useful marker in patients with prostate cancer. METHODS Serum levels of HAI-1 and HAI-2 were measured by enzyme-linked immunosorbent assay in 27 patients with benign prostatic hyperplasia (BPH) and 118 patients with prostate cancer. RESULTS The mean serum levels of HAI-1 in patients with prostate cancer were significantly higher than those in patients with BPH. Furthermore, the serum HAI-1 levels in patients with distant metastasis and hormone resistant prostate cancer were significantly elevated compared with those in patients with organ-confined diseases. There were no significant differences in serum HAI-2 levels among prostate cancer subgroups according to clinical stage. Significantly elevated levels of HAI-1 were detected in 38 patients with prostate cancer before any treatment. CONCLUSIONS HAI-1 may be a potential tumor marker for prostate cancer. Further studies in large groups of patients are needed to define the clinical value of HAI-1. © 2005 Wiley-Liss, Inc.

Hepatocyte growth factor activator inhibitor type-1 (HAI-1) is an integral-membrane proteinase inhibitor. In this study, we examined the effects of HAI-1 on human glioblastoma cells. Two glioblastoma cell lines (YKG-1, U251) were stably transfected with expression plasmid harboring mature membrane-form or truncated secreted-form HAI-1. Culture characteristics were not altered by the expression of HAI-1, whereas in vitro invasiveness of U251 was suppressed. On the other hand, the expression of membrane-form HAI-1 resulted in significantly enhanced tumorigenicity of both cell lines in vivo. In contrast, secreted-form HAI-1 did not promote the tumorigenicity. These results suggest that HAI-1 may play complex roles in progression of glioblastoma cells, and membrane-form HAI-1 may mediate an undefined important signaling in the cells.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), one of the Kunitz-type serine protease inhibitors, has an important role in cancer progression through regulation of the activity of hepatocyte growth factor. HAI-1 is expressed in hepatocellular carcinoma (HCC) to various degrees. Investigation of the relationship between HAI-1 expression and clinicopathological features of HCC may contribute to improved treatment outcomes for HCC through understanding the mechanism of tumor progression or improvement in the prediction of tumor malignancy.The study included 121 HCC patients treated surgically from 1996 to 2005. We performed immunohistological examination for HAI-1 in resected HCC specimens by use of anti-human HAI-1 monoclonal antibody. Clinicopathological features, including postoperative overall survival (OS) and disease-free survival (DFS) rates, were compared between the immunoreaction positive and negative groups.The immunoreaction positive group included 38 patients (31%), and the negative group included 83 patients (69%). OS and DFS rates were significantly higher in the HAI-1 negative group than in the positive group. HAI-1 positivity related to multiplicity, vascular invasion, and characteristics of advanced tumor stage. In multivariate analysis, expression of HAI-1 was a significant independent prognostic tumor factor.Expression of HAI-1 in HCC cells is associated with poor prognosis for HCC patients. HAI-1 may be important in HCC progression and may be a new prognostic factor for HCC.

Hepatocyte growth factor (HGF) is secreted as a single-chain form (proHGF) from mesenchymal cells of the liver, and proHGF is converted to heterodimeric HGF (mature HGF) by serine proteases, including hepatocyte growth factor activator (HGFA), in response to tissue injury. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) have recently been purified from the conditioned medium of a gastric cancer cell line, and their cDNAs have been successfully cloned. The hepatocytes of patients with chronic liver diseases including hepatocellular carcinoma (HCC) were stained with anti-HGFA. HAI-1 was detected in tumorous tissues of HCC, but not in liver tissues of patients with chronic liver diseases. Tumorous tissues in HCC were positive for HAI-1 mRNA. HAI-1 may play an important role in the development or invasiveness of HCC through the mechanism regulating HGF activation.

We used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration.

Dysregulation of pericellular proteolysis has been strongly involved in tumorigenesis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer cell invasion and tumorigenicity within a human prostate cancer progression model (103E, N1, and N2 cells). This model was newly established by a serial of intraprostatic propagation of a human prostate cancer 103E cell line and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was gradually increased after the serial isolations (103E Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A54.