Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. METHODS: To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. RESULTS: Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. CONCLUSIONS: Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.

CDK10 in Gastrointestinal Cancers: Dual Roles as a Tumor Suppressor and Oncogene.

Cyclin-dependent kinase 10 (CDK10) is a CDC2-related serine/threonine kinase involved in cellular processes including cell proliferation, transcription regulation and cell cycle regulation. CDK10 has been identified as both a candidate tumor suppressor in hepatocellular carcinoma, biliary tract cancers and gastric cancer, and a candidate oncogene in colorectal cancer (CRC). CDK10 has been shown to be specifically involved in modulating cancer cell proliferation, motility and chemosensitivity. Specifically, in CRC, it may represent a viable biomarker and target for chemoresistance. The development of therapeutics targeting CDK10 has been hindered by lack a specific small molecule inhibitor for CDK10 kinase activity, due to a lack of a high throughput screening assay. Recently, a novel CDK10 kinase activity assay has been developed, which will aid in the development of small molecule inhibitors targeting CDK10 activity. Discovery of a small molecular inhibitor for CDK10 would facilitate further exploration of its biological functions and affirm its candidacy as a therapeutic target, specifically for CRC.

Inhibition of pericellular plasminogen activation by apolipoprotein(a): Roles of urokinase plasminogen activator receptor and integrins αMß2 and αVß3.

BACKGROUND AND AIMS: Lipoprotein(a) (Lp(a)) is a causal risk factor for cardiovascular disorders including coronary heart disease and calcific aortic valve stenosis. Apolipoprotein(a) (apo(a)), the unique glycoprotein component of Lp(a), contains sequences homologous to plasminogen. Plasminogen activation is markedly accelerated in the presence of cell surface receptors and can be inhibited in this context by apo(a). METHODS: We evaluated the role of potential receptors in regulating plasminogen activation and the ability of apo(a) to mediate inhibition of plasminogen activation on vascular and monocytic/macrophage cells through knockdown (siRNA or blocking antibodies) or overexpression of various candidate receptors. Binding assays were conducted to determine apo(a) and plasminogen receptor interactions. RESULTS: The urokinase-type plasminogen activator receptor (uPAR) modulates plasminogen activation as well as plasminogen and apo(a) binding on human umbilical vein endothelial cells (HUVECs), human acute monocytic leukemia (THP-1) cells, and THP-1 macrophages as determined through uPAR knockdown and overexpression. Apo(a) variants lacking either the kringle V or the strong lysine binding site in kringle IV type 10 are not able to bind to uPAR to the same extent as wild-type apo(a). Plasminogen activation is also modulated, albeit to a lower extent, through the Mac-1 (αMß2) integrin on HUVECs and THP-1 monocytes. Integrin αVß3 can regulate plasminogen activation on THP-1 monocytes and to a lesser extent on HUVECs. CONCLUSIONS: These results indicate cell type-specific roles for uPAR, αMß2, and αVß3 in promoting plasminogen activation and mediate the inhibitory effects of apo(a) in this process.

IL-10 correlates with the expression of carboxypeptidase B2 and lymphovascular invasion in inflammatory breast cancer: The potential role of tumor infiltrated macrophages.

Pro-carboxypeptidase B2 (pro-CPB2) or thrombin-activatable fibrinolysis inhibitor (TAFI) is a glycoprotein encoded by the CPB2 gene and deregulated in several cancer types, including breast cancer. Thrombin binding to thrombomodulin (TM), encoded by THBD, is important for TAFI activation. CPB2 gene expression is influenced by genetic polymorphism and cytokines such as interleukin 10 (IL-10). Our previous results showed that tumor infiltrating monocytes/macrophages (CD14+/CD16+) isolated from inflammatory breast cancer (IBC) patients' secrete high levels of IL-10. The aim of the present study is to test genetic polymorphism and expression of CPB2 in healthy breast tissues and carcinoma tissues of non-IBC and IBC patients. Furthermore, to investigate whether IL-10 modulates the expression of CPB2 and THBD in vivo and in-vitro. We tested CPB2 Thr325Ile polymorphism using restriction fragment length polymorphism, (RFLP) technique in healthy and carcinoma breast tissues. The mRNA expression of CPB2, THBD and IL10 were assessed by RT-qPCR. Infiltration of CD14+ cells was assessed by immunohistochemistry. In addition, we investigated the correlation between infiltration of CD14+ cells and expression of IL10 and CPB2. Furthermore, we correlated IL10 expression with the expression of both CPB2 and THBD in breast carcinoma tissues. Finally, we validated the role of recombinant IL-10 in regulating the expression of CPB2 and THBD using different breast cancer cell lines. Our results showed that CPB2 genotypes carrying the high-risk allele [Thr/Ile (CT) and Ile/Ile (TT)] were more frequent in both IBC and non-IBC patients compared to control group. CPB2 genotypes did not show any statistical correlation with CPB2 mRNA expression levels or patients' clinical pathological properties. Interestingly, CPB2 and IL10 expression were significantly higher and positively correlated with the incidence of CD14+ cells in carcinoma tissues of IBC as compared to non-IBC. On the other hand, THBD expression was significantly lower in IBC carcinoma versus non-IBC tissues. Based on molecular subtypes, CPB2 and IL10 expression were significantly higher in triple negative (TN) as compared to hormonal positive (HP) carcinoma tissues of IBC. Moreover, CPB2 expression was positively correlated with presence of lymphovascular invasion and the expression of IL10 in carcinoma tissues of IBC patients. Furthermore, recombinant human IL-10 stimulated CPB2 expression in SUM-149 (IBC cell line) but not in MDA-MB-231 (non-IBC cell line), while there was no significant effect THBD expression. In conclusion, carcinoma tissues of IBC patients are characterized by higher expression of CPB2 and lower expression of THBD. Moreover, CPB2 positively correlates with IL10 mRNA expression, incidence of CD14+ cells and lymphovascular invasion in IBC patients. IL-10 stimulated CPB2 expression in TN-IBC cell line suggests a relevant role of CPB2 in the aggressive phenotype of IBC.

Activated thrombin-activatable fibrinolysis inhibitor attenuates the angiogenic potential of endothelial cells: potential relevance to the breast tumour microenvironment.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen present in blood plasma. Proteolytic activation of TAFI by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin, or plasmin results in an enzyme (TAFIa) that removes carboxyl-terminal lysine residues from protein and peptide substrates, including cell-surface plasminogen receptors. TAFIa is therefore capable of inhibiting plasminogen activation in the pericellular milieu. Since plasminogen activation has been linked to angiogenesis, TAFIa could therefore have anti-angiogenic properties, and indeed TAFIa has been shown to inhibit endothelial tube formation in a fibrin matrix. In this study, the TAFI pathway was manipulated by providing exogenous TAFI or TAFIa or by adding a potent and specific inhibitor of TAFIa. We found that TAFIa elicited a series of anti-angiogenic responses by endothelial cells, including decreased endothelial cell proliferation, cell invasion, cell migration, tube formation, and collagen degradation. Moreover, TAFIa decreased tube formation and proteolysis in endothelial cell culture grown alone and in co-culture with breast cancer cell lines. In accordance with these findings, inhibition of TAFIa increased secretion of matrix metalloprotease proenzymes by endothelial and breast cancer cells. Finally, treatment of endothelial cells with TAFIa significantly inhibited plasminogen activation. Taken together our results suggest a novel role for TAFI in inhibiting tumour angiogenic behaviors in breast cancer.