ALBERT

Description: Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TMPro mice expressing a mutant form of TM with reduced thrombin affinity and TMLeD mice lacking the N-terminal lectin-like domain. Studies of TMPro mice revealed that TM is a powerful determinant of hematogenous metastasis. TMPro mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TMPro mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Description: Abstract 2237 Chronic inflammation has been recognized as a major factor in the development and progression of multiple cancers. A prime example of this is the strong association between colitis and colon cancer. However, the specific factors that regulate disadvantageous immune processes in the context of inflammation-associated cancers remain poorly defined. Growing evidence suggests that hemostatic system components, traditionally associated with the maintenance of vascular integrity and prevention of blood loss, also directly regulate inflammatory processes. Furthermore, thrombin and several thrombin targets (e.g., PARs, fibrinogen, factor XIII) have been shown to regulate tumor cell proliferation and apoptosis, support metastasis, and protect tumor cells from innate immune surveillance mechanisms in other experimental contexts. A logical extension of these findings is the hypothesis that thrombin, as a master regulator of both inflammatory processes and tumor cell biology, is a major determinant of the progression of inflammation-driven cancers such as colitis-associated colon cancer (CAC). To test this hypothesis, we induced CAC in mice carrying prothrombin levels 50% of normal (fII+/−) and WT mice in parallel using an established two step protocol consisting of azoxymethane (AOM) and dextran sodium sulfate (DSS) exposure. The modest diminution in prothrombin levels imposed by the fII+/− genotype resulted in a dramatic diminution in the number of colonic adenomas formed after AOM/DSS challenge relative to WT mice. In order to determine if the diminution in adenoma formation observed in fII+/− mice was coupled to thrombin function, wildtype mice challenged with AOM/DSS were treated with daily i.p. injections of hirudin, a direct thrombin inhibitor, or saline carrier. Similar to the finding in mice with a genetically-imposed diminution in circulating prothrombin, hirudin treatment significantly blunted adenoma formation. To determine if reduction of thrombin generation improved the inflammation preceding the development of colonic adenomas, we used a novel, highly-specific factor XI antisense oligonucleotide “gapmer” (ISIS Pharmaceuticals) to inhibit hepatic factor XI synthesis prior to DSS challenge. Gapmer-mediated diminution of fXI levels to ∼15% of normal resulted in a dramatic improvement in colitis related symptoms. Gapmer-treated mice had less intestinal bleeding and weight loss associated with DSS challenge relative to mice treated with a control oligonucleotide. Consistent with these gross observations, microscopic analyses of colonic tissue showed that fXI gapmer treatment significantly limited mucosal ulceration. Factor XI gapmer treatment also significantly diminished local levels of several inflammatory cytokines known to play a role in colon cancer progression (i.e., IL-6, IL-1β, IL-12). These results demonstrate that thrombin is a crucial driver of the pathogenesis of colitis-associated colon cancer and suggest that therapies directed at thrombin or thrombin generation could treat or prevent inflammation-driven colon cancer. As pathological inflammation has been estimated to account for as many as 1 in 5 cancer-related deaths, thrombin-directed therapies could have broad applicability to multiple malignancies. Disclosures: Mullins: Baxter: Consultancy. Monia:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. Palumbo:Novo Nordisk: Research Funding.

Description: Abstract 822 A substantial body of evidence indicates that tumor cell-associated (i.e., tissue factor) and circulating hemostatic system components (i.e., thrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. However, the role of endothelial regulators of hemostasis in metastasis remains largely unexplored. Thrombomodulin (TM) is the endothelial thrombin receptor central to thrombin-mediated activation of protein C. To test the hypothesis that thrombin-TM interactions are an important determinant of metastasis, we used mice carrying the Glu387Pro mutation in thrombomodulin (TMPro) known to decrease thrombin affinity ∼100 fold and APC generation ∼1000 fold. TMPro/Pro and control mice were intravenously injected with either a low dose (3 × 104 cells/mouse) or high dose (3 × 105 cells/mouse) of Lewis lung carcinoma cells (LLC) in separate experiments. At the low cell dose, the majority of wild-type mice developed no discernable pulmonary metastases, while TMPro/Pro mice each developed ∼100 grossly apparent pulmonary metastases. At the high cell dose the outcome was possibly even more striking, with few metastatic foci apparent in wild-type mice and fully confluent surface metastases too numerable to count in TMPro/Pro mice. Histological analyses confirmed that lung tissue from TMPro/Pro mice had been largely replaced with tumor. The dramatic augmentation in metastasis observed in TMPro/Pro mice did not appear to be due to genotype dependent differences in tumor growth potential as LLC cells transplanted into the dorsal subcutis of TMPro/Pro and control mice grew at similar rates and were histologically indistinguishable. Rather, tumor cell fate analyses using 125I-radiolabeled LLC cells revealed that the imposition of the TMPro mutation dramatically improved the early survival of tumor cells in the lung. Twenty minutes after tumor cell injection 〉80% of the tumor cells were localized within the lungs regardless of animal genotypes, indicating that the TMPro mutation did not have a major impact on initial tumor cell adhesion/stabilization within the pulmonary vasculature. In contrast, 6 hours post-injection

Description: Abstract 2221 Increased thrombin generation and hypercoagulability are prominent features of inflammatory colitis and previous studies from our laboratory have suggested that thrombin-mediated proteolysis is a driver of both colitis and colitis-associated colon cancer (CAC). However, the downstream thrombin targets important in these disease processes have not been fully defined. Based on studies showing that the protease activated receptor-1 (PAR-1) can contribute to both inflammatory pathologies and cancer progression in other settings, we hypothesized that PAR-1 is a significant determinant of colitis and CAC. To test this hypothesis, we induced colitis in PAR-1−/− and control mice using intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Consistent with the concept that PAR-1 is a modifier of colitis pathobiology, PAR-1−/− mice lost significantly less weight than WT mice challenged in parallel. Furthermore, multiple inflammatory cytokines known to drive colitis pathology, including IL-6, TNFa, and MIP-1α, were significantly diminished in PAR-1−/− mice. However, histological analyses of colonic tissue revealed similar degrees of inflammatory cell infiltration, crypt abscesses, and mucosal hyperplasia in both genotypes. In order to explore the role of PAR-1 in the more complex process of inflammation-driven colon cancer pathogenesis, we induced CAC in PAR-1−/− and WT mice using a two step protocol consisting of azoxymethane (AOM) and dextran sodium sulfate (DSS) exposure. In contrast to findings in the setting of TNBS challenge, PAR-1−/− mice challenged with DSS developed, not less, but more severe clinical signs of colitis, including wasting and severe diarrhea. More detailed comparative studies of DSS-challenged PAR-1−/− and control mice established that PAR-1-deficient animals developed significantly greater immunological and histopathological evidence of colitis, including elevated IL-6 and MIP-1α levels in colonic tissue and increased edema, ulceration, crypt loss, and inflammatory cell infiltration. Consistent with the more severe antecedent colitis, PAR-1−/− mice challenged with AOM/DSS developed significantly larger adenomas than WT mice challenged and evaluated in parallel. Thus, the impact of PAR-1 on colitis appears to be context-dependent and the distinct outcomes in TNBS- and DSS-challenged mice are likely to stem from the different mechanisms by which these agents induce colitis. TNBS is thought to haptenate colonic mucosal proteins inducing a T cell-mediated colitis akin to human Crohn's disease. In contrast, DSS directly intoxicates colonic crypt epithelia, resulting in loss of barrier function and translocation of colonic microflora, leading to a primarily innate immune-driven colitis sharing many features with ulcerative colitis. A major challenge in dissecting the precise mechanisms coupling PAR-1 to colitis is the fact that PAR-1 is expressed on multiple cell types that can influence colitis and CAC in distinct ways, including immune cells, endothelial cells and colonic mucosa. Therefore, we recently generated mice carrying a conditional “floxed” PAR-1 allele. We interbred these animals with mice expressing Cre recombinase in either colonic epithelia or the hematopoietic/endothelial compartment. Preliminary studies revealed that loss of PAR-1 expression in the hematopoietic/endothelial compartments, but not the colonic epithelia, recapitulates the more severe DSS-induced weight loss and mucosal damage observed in constitutionally PAR-1-deficient mice. These results suggest that PAR-1 activation in either immune cells and/or endothelial cells limits colitis severity in this experimental context. Taken together, these data show that PAR-1 contributes to the pathogenesis of inflammatory colitis and CAC, but the precise contribution is dependent on the underlying insult and disease pathway. Analyses in mice carrying a conditional PAR-1 allele should prove invaluable for dissecting the precise mechanisms coupling PAR-1 to inflammatory bowel disease. Disclosures: Palumbo: Novo Nordisk Corporation: Consultancy.

Description: Abstract 858 Multiple sclerosis (MS) is an inflammatory disease characterized by autoimmune demyelination of neurons and axonal damage within the central nervous system, leading to relapsing/remitting neurological deficits, including sensory and motor deficits. MS plaques are characterized by blood-brain barrier disruption leading to perivascular deposition of fibrin that correlates with areas of microglia activation and myelin damage. Consistent with multiple studies showing that hemostatic factors can serve as important modulators of the inflammatory response in vivo, fibrin(ogen) has been identified as a novel regulator of microglial activation and differentiation. Furthermore, fibrin(ogen) has been found, via the αMβ2 binding motif on the γ chain, to play a role in microglial activation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. However, previous work by our lab, as well as others, suggests that thrombin-mediated proteolysis plays a greater role in driving neuroinflammatory disease than by merely supporting fibrin formation. Our working hypothesis is that thrombin-mediated proteolysis drives autoimmune neuroinflammation through both fibrin(ogen)-dependent and fibrin(ogen)-independent pathways. To test this hypothesis, we have initiated studies of EAE in mice lacking selected thrombin substrates, including protease activated receptors (PARs) and factor XIII (fXIII). Clinical evaluation of loss of motor function in EAE-challenged cohorts of PAR-1-deficient, PAR-4-deficient and WT control mice revealed a similar outcome in all three cohorts, suggesting PARs may have either a modest or a secondary role in EAE-induced neuroinflammatory disease. However, mice with a constitutive deficiency of another thrombin substrate, the fibrin cross-linking transglutaminase fXIII, were found to have a significant amelioration in motor function loss when challenged with EAE. To further explore this phenotype, we have utilized cystamine, a transglutaminase inhibitor, as a pharmacologic complement to the fXIII knockout mice. Mice with cystamine placed in their drinking water during the EAE challenge period also experienced significantly less disease than control mice that did not receive cystamine. This data suggests that (pro)thrombin contributes neuroinflammatory disease by both supporting local fibrinogen polymerization and by activation of fXIII leading to fibrin stabilization. Furthermore, this line of research suggests that more detailed studies of fXIII may reveal novel strategies for limiting or reversing the devastating pathologies associated with multiple sclerosis. Disclosures: Mullins: Baxter: Consultancy. Palumbo:Novo Nordisk Corporation: Research Funding.