Thrombin Drives Colorectal Cancer Pathogenesis at the Level of Primary Tumor Growth, Tumor Invasion and Metastasis (2014)

Abstract Cell-associated and circulating hemostatic factors have been identified as powerful determinants of tumor cell metastatis, primarily by supporting the early survival of newly formed micrometastases. However, despite substantial evidence that procoagulants and related signaling molecules can control cell proliferation, angiogenesis and other reparative processes, multiple prior studies unexpectedly demonstrated that hemostatic factors were often dispensable for the growth of established tumors. Here, we report that colorectal cancer represents at least one important exception where hemostatic factors control multiple aspects of cancer pathogenesis. Consistent with clinical findings that patients with prothrombotic mutations (homozygosity for the fVLeiden) have a ~6-fold increased risk of developing colorectal cancer, we have shown previously that mice with even a relatively modest 50% genetically-imposed diminution in circulating prothrombin exhibit a dramatically decreased tumor incidence in a murine model of inflammation-driven colon cancer. In order to determine if thrombin supports the growth of established colorectal cancer, we compared the subcutaneous growth of an aggressive C57Bl/6-derived colonic adenocarcinoma (MC38) in cohorts of wildtype mice and fIILox/- mice carrying just ~10% the normal level of circulating prothrombin. Colon cancer growth was significantly diminished in fIILox/- mice relative to control animals, resulting in a ~3-fold difference in tumor mass between cohorts at the end of the 3 week study period. Complementary studies in which hepatic prothrombin expression and circulating prothrombin levels were diminished using a specific prothrombin antisense oligonucleotide “gapmer” resulted in a similar reduction in colorectal cancer primary tumor growth. Furthermore, prothrombin “gapmer” treatment also slowed the growth of an aggressive, human-derived colorectal adenocarcinoma cell line transplanted into immunodeficient nude mice. Detailed histological analyses revealed no significant prothrombin-dependent differences in angiogenesis, tumor cell apoptosis or the numbers of tumor-associated macrophages. However, histological analyses of colonic adenocarcinoma tumors revealed a significantly lower mitotic index as well as significantly less migration of tumor cells through the panniculus muscle in tumors harvested from fIILox/- mice relative to control mice, suggesting that thrombin promotes tumor cell proliferation and local migration through tissue barriers. Complementary studies of pulmonary metastatic foci formed following intravenous injection of MC38 cells into fIILox/- and control mice further revealed that lowering prothrombin essentially eliminated pulmonary metastases, whereas numerous pulmonary foci were present in animals with normal prothrombin levels. To further characterize the role of thrombin in colorectal cancer growth, we analyzed the potential of a-thrombin to promote MC38 proliferation and invasion in vitro. Here, brief exposure to a-thrombin induced the proliferation of MC38 cells and acted as a chemoattractant when added to the lower chamber in a Boyden chamber assay. Together, these findings suggest thrombin significantly contributes to multiple stages of colon cancer pathogenesis, including tumor growth, invasion, and metastatic seeding, and emphasize the potential utility of targeting prothrombin, thrombin generation or thrombin activity as novel adjunct therapeutic strategies to impede the growth and progression of colorectal carcinoma. Disclosures No relevant conflicts of interest to declare.