Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity

Abstract

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth and metastasis of human breast cancer (MDA-231) cells, and on angiogenesis in vivo. The invasiveness of MDA-231 cells was inhibited by recombinant intact PCI, but not by reactive sitemodified PCI (R354APCI) or by the N-terminal fragment of protease-cleaved PCI (NTPCI). The in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing R354APCI (MDA-R354APCI) or NTPCI (MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA-Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA-PCI, MDA-R354APCI or MDA-NTPCI cells as compared to that containing MDA-Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti-angiogenic activity of PCI was strong as cleaved antithrombin (AT), and slightly stronger than that of plasminogen activator inhibitor (PAI)-1 and pigment epitheliumderived factor (PEDF). Overall, this study showed that, in addition to a reactive site-dependent mechanism, PCI may also regulate tumor growth and metastasis independently of its protease inhibitory activity by inhibiting angiogenesis. ' 2007 Wiley-Liss, Inc.Key words: protein C inhibitor; serine protease inhibitor; invasion; metastasis; angiogenesis Protein C inhibitor (PCI) was originally identified as a plasma inhibitor of the anticoagulant protease, activated protein C. 1,2 PCI is a member of the serine protease inhibitor (SERPIN) family (SERPINA5); it is also able to inhibit other proteases of the blood coagulation and fibrinolysis system including thrombin, 3 thrombin-thrombomodulin complex, 4 factor Xa, 3 factor XIa, 5 plasma kallikrein 5 and urinary plasminogen activator (uPA). 6 Human plasma PCI is mainly synthesized in the liver, 7 but it is also produced in kidneys and reproductive organs, including testes, seminal vesicles and ovaries. 8,9 Unlike humans, mice and rats produce PCI only in reproductive organs including testes and ovaries 10,11 ; thus, rodents are inappropriate for studying the physiological role of PCI in plasma and other organs. We recently developed a human PCI gene transgenic (TG) mouse in which the tissue distribution of PCI is similar to humans. Using this animal model, we demonstrated that PCI is the physiological inhibitor of APC in plasma, and that it promotes blood coagulation and inflammation in animals with endotoxemia. 12 PCI appears to have also a function in fertilization; Uhrin et al. 13 reported that PCI knock-out male mice have infertility due to abnormal spermatogenesis caused by destruc...

Loss of SerpinA5 protein expression is associated with advanced-stage serous ovarian tumors

“…This study supports other publications 19,21 that a decrease in SerpinA5 protein expression is associated with invasion. We hypothesized that SerpinA5 protein expression is decreased or lost in serous carcinomas when compared with serous borderline tumors based on mRNA expression results.…”

“…SerpinA5 expression is decreased in renal, prostate and ovarian cancers. 16,19,20 Overexpression of SerpinA5 results in decreased invasion, metastatic potential and angiogenesis in breast cancer, 21 and elevated levels of SerpinA5 protein was reported to be associated with a better survival in primary breast carcinomas. 22 The aim of this study was to investigate the relation between SerpinA5 protein expression and histopathological characteristics in serous borderline tumors and carcinomas by immunohistochemistry.…”

Expression patterns of protein C inhibitor in mouse development

“…PCI is a multifunctional serpin that can act both as a protease inhibitor of the coagulation (activated protein C, factor Xa, factor XIa, protein Z-dependent protease inhibitor and plasma kallikrein), the fibrinolytic (urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA)), and the fertilization system (acrosin and prostate specific antigen) (Christensson and Lilja 1994; Ecke et al 1992; Espana et al 1989; Geiger et al 1989; Marlar and Griffin 1980; Meijers et al 1988; Suzuki et al 1983; Zheng et al 1994). It also acts as a non-inhibitory serpin by binding retinoic acid (Jerabek et al 2001) and by attenuating breast cancer cell growth, metastasis and angiogenesis (Asanuma et al 2007; Suzuki and Hayashi 2007). A role of PCI in tissue repair and regeneration has been suggested by its inhibitory action on the activator of the hepatocyte growth factor (HGF; Hayashi et al 2007).…”

In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1

“…The concentrations of recombinant PN-1 used in the in vitro experiments were comparable to those used for other antiangiogenic serpins (1,18). Moreover, PN-1 is known to be carried by platelets and released locally in large amounts upon activation.…”