Abstract 3350
Cancer is frequently associated with activation of coagulation, and a procoagulant state facilitates tumor metastasis. Recent studies have suggested that the activated protein C (aPC) ...pathway plays a role in modulating tumor metastasis, and this protection likely requires both the anticoagulant and cytoprotective effects of aPC. Notably, our early work revealed that the inactive precursor, zymogen PC (zyPC), can even more effectively protect against metastasis. The aim of this study was therefore to explore mechanisms through which zyPC could prevent metastatic cancer progression in a murine cancer model.
A liver gene transfer model using viral vectors was utilized to achieve a wide range of sustained expression of wildtype (WT) or mutant murine zyPCs. C57BL/6 experimental mice expressing stable levels of zyPCs and age and gender matched control mice receiving PBS were injected intravenously with 2.5×105 murine melanoma B16F10 cells, which metastasize to the lungs. After 3 weeks the number of pulmonary tumors was determined.
Expression of WT zyPC in C57BL/6s decreased the rates of metastasis in a dose-dependent manner compared to PBS controls (p<0.01; n=8–18/group). These effects were noted even in mice injected with low vector dose (200% zyPC expression). Conversely, when PC-deficient mice (3% of normal, n=7) were administered B16F10s without zyPC-expression, they did not survive the full 3 weeks, while their littermate controls (PC > 50% of normal, n=6) did despite high rates of metastasis. These data clearly demonstrate the protective role of zyPC in tumor progression.
We then tested modified zyPCs to identify the critical functions responsible for our observations in this tumor model. Two mutants with minimal anticoagulant function, R15Q and S195A, were generated. zyPC-R15Q is unable to dock to the thrombin-thrombomodulin complex active site and so cannot be converted to aPC. Compared to PBS controls (n=7), mice expressing zyPC-R15Q still showed a significant decrease in the number of tumor foci (p<0.001; 75–99% reduction; n=13) similar to the WT zyPC (p=0.28; n=8). Mice expressing zyPC-S195A (n=12), which has a mutation in the serine protease active site, also showed a significant decrease in the number of tumor foci compared to PBS controls (n=8; p<0.05; 90–99% reduction). As with the R15Q, mutating the S195 did not affect the ability of zyPC to protect against metastasis (p=0.22).
Next, we tested the importance of the main PC/aPC cellular receptors in our model. Binding to endothelial protein C receptor (EPCR) enhances activation of PC. We inhibited this binding by injecting anti-EPCR blocking antibody 1560 (J Thromb Haemost. 2005 3:1351) intraperitoneally one hour prior to the B16F10 cells. zyPC-expressing mice that received anti-EPCR antibody (n=22) still had a significant reduction in tumor rates compared to PBS controls (n=10; p<0.01; 45–75% reduction). Moreover, mice expressing zyPC had similar levels of protection whether they received the anti-EPCR antibody or an isotype control (n=22–24; p=0.31). EPCR binding not only increases activation of PC, it also mediates the cytoprotective effect by clustering with and facilitating the activation of the signaling protease-activated receptor 1 (PAR1). PAR1 −/− mice expressing zyPC (n=21) challenged with B16F10 cells still had reduced rates of metastasis compared to PAR1 −/− PBS controls (n=15; p<0.01; 67% reduction). The zyPC protection in PAR1 null mice was comparable to that in PAR1 +/− littermate controls (n=10; p=0.619). Collectively, these findings suggest a distinct mechanism by which zyPC modulates tumor progression independent of EPCR and PAR1, both of which are required for aPC-mediated protection.
Despite elevated circulating levels of PC, zyPC-expressing mice did not suffer from increased blood loss following tail clipping or show prolonged activated partial thromboplastin times (aPTTs) compared to hemostatically normal mice.
In conclusion, zyPC protects against metastatic cancer progression in a dose-dependent manner. Our data show for the first time that this zyPC effect is independent of its anticoagulant function. Furthermore, protection is not mediated through EPCR or PAR1, suggesting an alternative pathway by which zyPC limits tumor progression. These findings suggest that WT zyPC and variants with little to no anticoagulant function are safe and efficacious in preventing metastatic cancer progression.
Van Sluis:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. High:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Spek:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Arruda:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties.
Treatment of hemophilia, which involves infusion of the missing clotting factor, is often hindered by the development of neutralizing antibodies to the replaced clotting factor. We utilized ...liver-directed AAV gene therapy to tolerize outbred hemophiliac dogs with pre-existing anti-factor VIII and IX antibodies and to treat their underlying hemophilia. Additionally, we sought to shed light on the immunologic mechanisms responsible for this tolerization. Staining for CD4+CD25+FoxP3 + T cells and cytokine profiles of treated dogs suggest that induced Tregs are at least partially responsible for inducing and maintaining tolerance. The second part of the dissertation attempts to determine the underlying mechanism of the known anti-metastatic effects of activated protein C (aPC). We again utilized liver-directed AAV gene therapy to tease apart the anticoagulation and cytoprotective effects of aPC, as these were the most likely candidates based on available literature, in the B16F10 murine model of metastatic melanoma. Upon finding, however, that neither of these functions were involved in aPC's cancer protection and that zymogen PC is even more protective, we endeavored to find the novel mechanism through which the protein C pathway can modulate tumor progression. While the mechanism has not been found, a number of potential candidates and receptors have been eliminated, including involvement of PAR-4, EPCR, and a potential integrin binding site. We also demonstrate that it is not tumor growth, but rather a step in the metastatic pathway that is inhibited.
