Role of activated protein C and its receptor in inhibition of tumor metastasis

Abstract

IntroductionMetastasizing cancer cells are thought to overcome the endothelial barrier through mechanisms commonly observed in leukocyte trafficking, including the up-regulation of adhesion molecules, apoptosis induction, cytoskeletal reorganization, and disruption of normal endothelial intercellular contacts. 1-5 Both cancer cell transmigration and leukocyte trafficking are increased by proinflammatory cytokines, such as interleukin-1␤ and tumor necrosis factor (TNF), which up-regulate adhesion molecules, including P-selectin, 5-7 and E-selectin. 1,8 Given the parallels between these processes, we hypothesized that a therapy directed at the endothelium to reduce leukocyte trafficking might also be beneficial in preventing tumor cell metastasis. The serine protease activated protein C (aPC) has been used to treat severe sepsis. 9 It possesses anti-inflammatory and cytoprotective activities mediated through engagement of its receptor, endothelial protein C receptor (EPCR). [10][11][12] This interaction down-regulates endothelial proinflammatory and proapoptotic gene expression and decreases vascular permeability. 13,14 In this report, we assessed the ability of melanoma cells to adhere to and transmigrate through endothelial monolayers treated with recombinant human aPC (rhaPC). Furthermore, we investigated the role of the aPC/EPCR pathway in a murine model of hematogenous B16-F10 melanoma metastasis by characterizing liver and lung metastases in EPCR-overexpressing mice and by administering rhaPC to wild-type (WT) mice. Study design Animals and cell linesFemale Tie2-EPCR transgenic mice were generated on a C57BL/6 background as previously described. 15 Wild-type C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME) and transgene-negative littermates were used as controls. Experiments were performed in accordance with the Canadian Council on Animal Care guidelines. B16-F10 mouse melanoma and bEnd.3 mouse brain microvascular endothelial lines were purchased from ATCC (Manassas, VA), and rhaPC was purchased from SigmaAldrich (Oakville, ON). Research ethics approval was obtained for all animal studies from the Dalhousie University Committee on Laboratory Animals in accordance with the Canadian Council on Animal Care. Adhesion and transendothelial migration assaysFor adhesion assays, confluent bEnd.3 monolayers grown in 24-well polystyrene plates (BD Biosciences, Mississauga, ON) were pretreated with rhaPC (0, 5, 10, 25, 100 nM) for 3 hours then washed. B16-F10 cells (2 ϫ 10 5 ) labeled with CFDA-SE (Invitrogen, Burlington, ON) were added to each well. 4 One hour later, wells were washed and adherent fluorescent cells were counted. For transmigration assays, bEnd.3 monolayers on polycarbonate Transwell inserts (8-m pore-size, 6.4-mm diameter; CorningCostar, Corning, NY) were pretreated with rhaPC. To induce chemotaxis, DMEM supplemented with 15% FCS was added to the lower chambers. CFDA-SE-labeled B16-F10 cells (2 ϫ 10 5 ) in DMEM were added to the upper chambers. After 16 hours at 37°C/5% CO 2 , fluorescent cell...

Receptor of Activated Protein C Promotes Metastasis and Correlates with Clinical Outcome in Lung Adenocarcinoma

“…In apparent contrast to our findings, in a model of melanoma, APC decreased lung metastasis by the ability to prevent tumor transmigration through the endothelial cells, by the EPCR/PAR1/ S1P1 axis (32,33). This difference is explained by the fact that the melanoma cell line did not expressed EPCR, and therefore APC effects were exclusively mediated by the EPCR expressed in endothelial cells.…”

The innate immune receptor Dectin-2 mediates the phagocytosis of cancer cells by Kupffer cells for the suppression of liver metastasis

“…We first examined the contribution of Dectin-2 to antitumor immunity by evaluating wild-type (WT) and Dectin-2-deficient (Dectin-2 KO) mice for subcutaneous tumor growth, lung metastasis, and liver metastasis of colon carcinoma cell line SL4, Lewis lung carcinoma cell line 3LL, and melanoma cell line B16F1 and B16F10, all of which have been demonstrated to undergo metastasis in mice (15)(16)(17)(18). We found that Dectin-2 KO mice showed more metastatic nodules in the liver compared with WT mice at 14 d after the intrasplenic inoculation with SL4, B16F1, or B16F10 cells, but not after inoculation with 3LL cells (Fig.…”

Anti-Inflammatory Actions of the Anticoagulant, Activated Protein C

“…EPCR on the vascular wall inhibits cancer cell adhesion and transmigration (Bezuhly, 2009;Lindahl, 1993). Finally, significant resistance to APC was found in women with a lymph-node-positive breast carcinoma (Bezuhly, 2009;Lindahl, 1993;Nijziel, 2003). In a mouse model, endogenous APC has been found to limit cancer cell extravasation via sphingosine-1-phosphate receptor-1 and VE-cadherin-dependent vascular barrier enhancement.…”

“…For example, EPCR is expressed on human breast cancer cells, with an extremely high frequency (Tsuneyoshi, 2001). EPCR on the vascular wall inhibits cancer cell adhesion and transmigration (Bezuhly, 2009;Lindahl, 1993). Finally, significant resistance to APC was found in women with a lymph-node-positive breast carcinoma (Bezuhly, 2009;Lindahl, 1993;Nijziel, 2003).…”