Ewa Sierko

Protease-activated receptors (PARs)—biology and role in cancer invasion and metastasis

Although many studies have demonstrated that components of the hemostatic system may be involved in signaling leading to cancer progression, the potential mechanisms by which they contribute to cancer dissemination are not yet precisely understood. Among known coagulant factors, tissue factor (TF) and thrombin play a pivotal role in cancer invasion. They may be generated in the tumor microenvironment independently of blood coagulation and can induce cell signaling through activation of protease-activated receptors (PARs). PARs are transmembrane G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. They play important roles in vascular physiology, neural tube closure, hemostasis, and inflammation. All of these agents (TF, thrombin, PARs—mainly PAR-1 and PAR-2) are thought to promote cancer invasion and metastasis at least in part by facilitating tumor cell migration, angiogenesis, and interactions with host vascular cells, including platelets, fibroblasts, and endothelial cells lining blood vessels. Here, we discuss the role of PARs and their activators in cancer progression, focusing on TF- and thrombin-mediated actions. Therapeutic options tailored specifically to inhibit PAR-induced signaling in cancer patients are presented as well.

Expression of protein C (PC), protein S (PS) and thrombomodulin (TM) in human colorectal cancer☆

INTRODUCTION Colorectal cancer (CRC) is often complicated by thromboembolic episodes. It has been recognized that blood coagulation proteins play a role in cancer progression. An important inhibitory mechanism is provided by the protein C (PC) system consisting of PC, protein S (PS) and thrombomodulin (TM). Recently, novel biological activities have been ascribed to the PC system that do not relate to their hemostatic functions, eg. in angiogenesis, apoptosis and inflammation. OBJECTIVES The purpose of the study was to elucidate the solid phase interactions between CRC tissue and components of the PC system that may contribute to tumor progression. MATERIAL AND METHODS CRC tissues were obtained at surgical resection during treatment of 66 patients. Immunohistochemical studies were performed using polyclonal antibodies against PC, PS and TM. A semiquantitative analysis of the protein expression was also performed. RESULTS Weak expression of PC was observed in cancer cells of two-thirds of the specimens examined, while in 3/66 cases there was no staining for PC in cancer cells. One fourth of CRCs exhibited strong expression of PC. The presence of PS was demonstrated in 64/66 cases of CRC. However, its expression was irregular in terms of intensity of staining and percentage of cancer cells exhibiting protein expression. Weak expression of TM was demonstrated in two thirds of the cases examined, while a strong TM staining was revealed in one third of colon cancers. CONCLUSION Heterogeneous expression of the PC system components in CRC tissue may point to their biological activity modulating tumor growth.

Protein Z is present in human breast cancer tissue

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com

Protein Z-dependent protease inhibitor (ZPI) is present in loco in human breast cancer tissue.

Protein Z-dependent protease inhibitor (ZPI) is present in loco in human breast cancer tissue -

Thrombin—unique coagulation system protein with multifaceted impacts on cancer and metastasis

The association between blood coagulation and cancer development is well recognized. Thrombin, the pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis, may also trigger cellular events through protease-activated receptors, PAR-1 and PAR-4, leading to cancer progression. Our pioneering findings provided evidence that thrombin contributes to cancer metastasis by increasing adhesive potential of malignant cells. However, there is evidence that thrombin regulates every step of cancer dissemination: (1) cancer cell invasion, detachment from primary tumor, migration; (2) entering the blood vessel; (3) surviving in vasculature; (4) extravasation; (5) implantation in host organs. Recent studies have provided new molecular data about thrombin generation in cancer patients and the mechanisms by which thrombin contributes to transendothelial migration, platelet/tumor cell interactions, angiogenesis, and other processes. Though a great deal is known regarding the role of thrombin in cancer dissemination, there are new data for multiple thrombin-mediated events that justify devoting focus to this topic with a comprehensive approach.

Co-localization of Protein Z, Protein Z-Dependent protease inhibitor and coagulation factor X in human colon cancer tissue: implications for coagulation regulation on tumor cells.

INTRODUCTION Several hemostatic system components, including factor X (FX), contribute to cancer progression. The Protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex directly inhibits factor Xa proteolytic activity. The aim of this study was to determine the antigenic distribution of ZPI and PZ, in relation to FX, as well as indicators of blood coagulation activation (F1+2 and fibrin) in human colon cancer tissue. MATERIALS & METHODS Studies were performed on human colon cancer fragments. Immunohistochemical (IHC) ABC procedures and double staining method employed polyclonal antibodies against PZ, FX, F1+2 and monoclonal antibodies against ZPI and fibrin. In-situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either FX, PZ or ZPI mRNAs. RESULTS Expression of FX, PZ and ZPI in association with colon cancer cells was observed by IHC. Moreover, the presence of both F1+2 and fibrin in association with colon cancer cells was found, which indicates that blood coagulation activation proceeds extravascularly at the tumor site. Furthermore, expression of FX and PZ was visualized in association with endothelial cells. In turn, colon cancer-associated macrophages were characterized by FX , PZ and ZPI presence. The double staining studies revealed strong FX/PZ, FX/ZPI, as well as PZ/ZPI co-localization on colon cancer cells. ISH studies revealed the presence of FX mRNA, PZ mRNA and ZPI mRNA in colon cancer cells indicating induced synthesis of these proteins. CONCLUSIONS The localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. Strong co-localization of PZ/ZPI and FX in cancer cells, and the presence of the mRNAs encoding the proteins, suggests their role in the tumors biology. However, the presence of F1+2 and fibrin at the colon cancer site also suggests that the regulation of FXa by the PZ/ZPI complex at this site is incomplete.

Protein Z/protein Z-dependent protease inhibitor system in human non-small-cell lung cancer tissue.

INTRODUCTION NSCLC progression is often associated with VTE. Activation of factor X is an important step in blood coagulation activation in cancer patients. PZ)/ZPI contribute to direct factor Xa inhibition, and ZPI - attenuates factors IXa and XIa activity. The role of the PZ/ZPI in NSCLC is obscure. The aim of the study was to localize ZPI and PZ in NSCLC tissue in relation to factors X, IX and XI, as well as indicators of blood coagulation activation: prothrombin fragment F1+2 (F1+2) and fibrin. MATERIAL & METHODS Immunohistochemical studies were performed on surgical NSCLC specimens employing antibodies against ZPI, PZ, coagulation factors X, IX, XI, as well as fibrinogen, F1+2 and fibrin. A semiquantitative analysis (acc. to immunoreactive score-IRS) was conducted. RESULTS Medium expression of ZPI(IRS=6.5), together with weak expression of PZ(IRS=4), was observed in cancer cells. Strong or medium staining for factors IX, X, and XI(IRS=8-9) was revealed in cancer cells. Fibrinogen(IRS=10) and fibrin(IRS=8) were demonstrated in tumor stroma and cancer cells. F1+2(IRS=10) was localized in NSCLC cells. Endothelial cells (ECs) and tumor infiltrating macrophages (TAMs) were characterized by a positive staining for ZPI and PZ. CONCLUSIONS ZPI and PZ expression in NSCLC cells, ECs and TAMs may suggest a role for PZ/ZPI in the anticoagulant mechanisms at the tumor site. The presence of F1+2 and fibrin, along with a disproportional expression of ZPI and PZ, might point to impaired function of the coagulation inhibitory system in NSCLC tissue.

Platelets and cancer angiogenesis nexus

There has been remarkable insight into the importance of platelets in a wide range of pathophysiologic events, including inflammation and cancer progression. Thrombocytosis in cancer patients is a common finding. Tumor cells induce platelet activation and subsequent aggregation through direct and indirect mechanisms. Platelets are recognized to contribute to metastatic dissemination. There is plenty of evidence that components of the hemostatic system contribute to the process of angiogenesis. Furthermore, there are accumulated data on the substantial influence of blood platelets in the process of blood vessel formation during malignancy. Platelets appear to be the main physiologic transporters of proangiogenic and antiangiogenic factors. Moreover, they influence the process of angiogenesis through platelet-derived microparticles, microRNA, lipids, and variety of surface receptors. Platelets contribute to early and late stages of angiogenesis. Available data support the overall stimulatory effect of platelets on tumor angiogenesis. It raises the possibility that interfering with platelet function may be an effective antineoplastic treatment strategy.

Antiplatelet agents for cancer treatment: a real perspective or just an echo from the past?

The association between coagulation and cancer development has been observed for centuries. However, the connection between inflammation and malignancy is also well-recognized. The plethora of evidence indicates that among multiple hemostasis components, platelets play major roles in cancer progression by providing surface and granular contents for several interactions as well as behaving like immune cells. Therefore, the anticancer potential of anti-platelet therapy has been intensively investigated for many years. Anti-platelet agents may prevent cancer, decrease tumor growth, and metastatic potential, as well as improve survival of cancer patients. On the other hand, there are suggestions that antiplatelet treatment may promote solid tumor development in a phenomenon described as “cancers follow bleeding.” The controversies around antiplatelet agents justify insight into the subject to establish what, if any, role platelet-directed therapy has in the continuum of anticancer management.

Protein Z/protein Z-dependent protease inhibitor system in loco in human gastric cancer

In gastric cancer, hemostatic system components contribute to cancer progression, as activation of factor X (FX) was observed. The protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex inhibits factor Xa proteolytic activity. The purpose of this study was to determine the distribution of ZPI and PZ in relation to FX, and prothrombin fragment (F1 + 2), a standard marker for blood coagulation activation, in human gastric cancer tissue. ABC procedures and a double staining method employed polyclonal antibodies against PZ, FX, and F1 + 2 and a monoclonal antibody against ZPI. In situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either PZ or ZPI mRNAs. FX and components of PZ/ZPI coagulation inhibitory system were observed in cancer cells. F1 + 2 was observed in gastric cancer cells as well. Double staining studies revealed FX/PZ, FX/ZPI, and PZ/ZPI co-localization on gastric cancer cells. ISH studies demonstrated the presence of PZ mRNA and ZPI mRNA in gastric cancer cells indicating induced synthesis of these proteins. The co-localization of PZ/ZPI and FX in gastric cancer cells indicates in loco that these proteins may play a role in anticoagulant events at the tumor tissue.