Zymogen Protein C as a Novel Modulator of Cancer Progression In Murine Models

Abstract 718

Recent evidence on the role of the protein C (PC) pathway in tumor progression of the experimental mouse melanoma model has revealed that inhibition of the cytoprotective effects of endogenous activated PC (aPC) enhances tumor cell extravasation, whereas exogenous administration of recombinant human APC has a protective effect. Moreover transgenic mice overexpressing endothelial PC receptor (EPCR) in tissue endothelium exhibit low rates of tumor metastasis. Here we report our findings in C57Bl/6 mice expressing murine forms of APC or zymogen PC by viral-mediated gene transfer. Vector-injected mice resulted in continuous expression of murine APC (mAPC) or PC (mPC), which reached plateau levels after week 2. On week 3, we administered B16F10 murine melanoma cells (3.5×10^5) intravascularly and analyzed the rates of lung metastasis 21 days later compared to age and gender matched saline-injected groups (control cohort=26 mice). We observed a dose-dependent protective effect of mAPC. Mice expressing mAPC at levels of 7.3 ± 1.5 ng/ml (n=8) or lower (determined by a functional ELISA-capture assay) did not differ from saline injected mice (that had baseline mAPC levels < 3 ng/ml). By increasing the vector dose, mAPC levels of 25.6 ± 4.8 ng/ml (n=16) to 118 ±6 ng/ml (n=10) reduced the numbers of lung metastasis compared to saline injected mice (p<0.05). To investigate the contribution of the cytoprotective/anticoagulant role of mAPC, we injected mice with a variant form of mAPC with reduced anticoagulant but intact cytoprotective activity (mAPC-5A). Following melanoma cell infusion, animals expressing levels of mAPC-5A ranging from 15.2 ± 3.2 ng/ml (n=16) to 80.4 ± 4.7 ng/ml (n=10) exhibited rates of lung metastasis similar to controls. To further explore the anticoagulant pathway in this metastasis model, we injected mice with AAV expressing zymogen mPC. There was a dose-dependent increase in the mPC levels measured by a chromogenic assay resulting in 3–4 fold of normal levels. However, this was not associated with increased levels of mAPC compared to saline-injected mice. Notably, in the mPC expressing mice (n=26), the rates of tumor metastasis were significantly reduced compared to controls (p<0.005). The protective effect of zymogen mPC remained even in the absence of protease-activated receptor (PAR-1), one main cellular receptor for the APC-mediated cytoprotective effect. In particular, the lung metastasis rates in PAR-1 null mice expressing mPC (n=21) were lower than PAR-1 null mice injected with saline (n=15) (p<0.01). Lastly, the hemostatic effects of the expressed transgenes (mPC, mAPC and mAPC-5A) in all mice were investigated. Prolongation of the activated partial prothrombin time and increase blood loss following tail clipping assay was restricted to animals expressing APC-WT in a dose-dependent manner but not in APC-5A or zymogen PC compared to controls. These findings support a novel and important role of zymogen PC in modulating tumor progression with minimal risk of bleeding.