P2Y12 Is Involved in Cancer Metastasis,

Abstract 3356

The platelet ADP receptor P2Y12 plays a prominent role in amplifying platelet activation, aggregation and thrombus formation, and the P2Y12 inhibitor Clopidogrel is widely used clinically to treat coronary artery, peripheral vascular and cerebrovascular diseases. Recently, several publications analyzed the TRITON-TIMI 38 clinical trial (test the efficacy and safety of Prasugrel, a newly FDA approved thienopyridine P2Y12 inhibitor) revealing an increase in multiple types of solid tumors with Prasugrel use, casting doubt on the safety of anti-platelet therapy targeting P2Y12. In this study, we used animal models to investigate the safety of targeting P2Y12, and to elucidate the probable mechanism for the effects of P2Y12 antagonists on metastasis. Tumor growth and metastasis are basic features of malignant cancer. Pulmonary metastasis in the experimental, and spontaneous mouse metastasis models were used to identify the role of P2Y12, and the effects of its inhibitors in tumor growth and metastasis. In the experimental pulmonary metastasis group, one week before tail vein injection of 2×10⁵ melanoma B16 cells, wild type (WT) and P2y12^–/– mice were treated every other day with PBS, Clopidogrel or Prasugrel by oral gavage and continue in whole experiment process. Tumor nodules were counted, and the average nodule sizes measured at 20 days after B16 cell injection. P2y12^–/– mice had fewer lung metastatic loci than the lungs of the WT controls. The experimental and control lungs had similar size nodules. But Clopidogrel dose dependently (1.5mg/kg and 30mg/kg) caused an increase of metastatic loci and loci size in both WT and P2y12^–/– mice. Prasugrel (0.2mg/kg and 4mg/kg) did not affect the number or size of metastatic loci produced in either strain. These results demonstrated that p2y12 deficiency negatively affected cancer metastasis.In contrast, Clopidogrel, but not Prasugrel, promotes metastasis and cancer growth. Importantly, Clopidogrel induced metastasis promotion and growth was not P2Y12 dependent. A plausible explanation of this difference between the two drugs may reside in the fact that both are pro-drugs of thienopyridine family. Presumably, activation of the pro-drug clopidogrel, but not Prasugrel produces one or more metabolites able to promote cancer growth and metastasis in vivo.

This supposition was tested, in part by asking if the effects of Clopidogrel are limited to the B16 model of metastasis. The Lewis lung carcinoma (LLC) spontaneous pulmonary metastasis model was used for this purpose. LLC cells (2.0×10⁶) were implanted intradermally in each mouse. Surgery was done 14 days after cell implantation to completely remove the primary tumors (about 1.0g or 1cm³ in size). One month later, lungs were removed, rinsed with PBS, and pulmonary metastatic nodules counted and measured. P2Y12 deficiency decreased the spontaneous formation of pulmonary metastatic loci, but had no effect on the primary tumor. Also, Clopidogrel, dose dependently promoted lung metastasis.

Next, the mechanism of the effect of P2Y12 absence on tumor metastasis was investigated. In vitro aggregation experiments revealed that B16 melanoma cells directly interact with p2y12^+/+ platelets and cause platelet-cancer cell aggregation. In contrast, P2Y12 deficiency results in significantly less or no cancer cell-platelet aggregation. Interestingly, LLC cells do not cause platelet aggregation. Immunohistochemistry and immunofluorescence analyses of lung samples from the spontaneous metastasis model indicated an obvious attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for metastatic niche formation. Moreover, this effect is transplantable, as wild-type mice reconstituted with p2y12^–/– bone marrow have a similar phenotype as P2y12^–/– mice. These results imply that the mechanism of P2Y12 function on cancer metastasis is complicated, probably mediated through regulation of both cancer cell-platelet direct interaction and metastatic niche formation. Further work is needed to resolve this issue. In summary, it is clear that P2Y12 is a safe drug target for anti-thrombotic therapy. But, metabolic derivatives of the pro-drug Clopidogrel apparently promote cancer metastasis and growth; whereas the newly approved pro-drug Prasugrel lacks that effect in animal models, and hopefully in humans.