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Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. Key messages: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Testisin, a serine protease abundantly expressed only in normal testes, is thought to be a tumor suppressor gene silenced by aberrant methylation in testicular germ cell tumors (TGCT). This study aimed at identifying the CpG sites relevant for testisin gene silencing when methylated and evaluating the potential of aberrant methylation as a biomarker in TGCT. Bisulfite sequencing and subsequent real-time RT-PCR were carried out in germ cell tumor cell lines and a cervical carcinoma cell line to reveal CpG sites implicated in testisin gene silencing. The normalized index of methylation (NIM) and the relative gene expression (RGE) were calculated in 33 TGCT and 19 normal testicular tissue samples by quantitative methylation-specific PCR (QMSP) and real-time RT-PCR. CpG sites in the 5′untranslated region proved to be relevant for testisin gene silencing when methylated. Targeting these CpG sites by QMSP, we demonstrated that the median NIM was 8.6 times higher in the TGCT group than in normal testicular samples. This was associated with a median 23.6 times lower RGE in the TGCT probes. Non-seminomas had a significantly higher NIM than seminomas, but RGE was downregulated to comparable levels in both subgroups. QMSP was highly sensitive and distinguished TGCT from normal samples with excellent specificity. Our results demonstrate for the first time that aberrant methylation of specific CpG sites near the transcription initiation site is an important factor in testisin gene silencing in TGCT. [ABSTRACT FROM AUTHOR]

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 5' CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis. [ABSTRACT FROM AUTHOR]

Objective:In a continued effort to identify and characterized secreted proteases that are overexpressed in ovarian carcinomas, we discovered the testisin protease as such a candidate. When this discovery was originally made, no data existed in the literature or in the GenBank database that identified such a gene. Our main objective was to determine whether this gene was overexpressed exclusively in ovarian tumor tissues compared with normal ovary and whether it was expressed in any other normal tissues.Methods:mRNA was isolated and cDNA was prepared from 34 ovarian tumors (four adenomas, three low malignant potential tumors, and 37 carcinomas) and seven normal ovaries. The testisin mRNA expression level relative to internal control, β-tubulin, was determined by Northern blot analysis and semiquantitative polymerase chain reaction (PCR).Results:Northern blot hybridization showed that the testisin transcript was abundant in ovarian carcinoma but was not detected in normal ovary. On examination of Northern blots from normal fetal and adult tissues, only adult testis showed abundant transcripts of testisin. Semiquantitative PCR examination showed that the testisin mRNA levels in ovarian tumors of low malignant potential and in ovarian carcinomas were significantly higher than in normal ovaries (P < .01). Testisin mRNA level in ovarian carcinomas was also significantly higher than in ovarian adenomas (P < .05). Testisin overexpression rates in advanced stage (stage 2 or 3) diseases were significantly higher than that in early stage disease (stage 1) in ovarian carcinoma samples (P < .05).Conclusions:The induction of the testisin transcript might contribute to the development, progression, and invasive or metastatic capacity of ovarian carcinomas. [ABSTRACT FROM PUBLISHER]