Protease-activated receptor-1 impedes prostate and intestinal tumor progression in mice.

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APCMin/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APCMin/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Factor XIII transglutaminase supports hematogenous tumor cell metastasis through a mechanism dependent on natural killer cell function.

BACKGROUND: Multiple studies suggest that the hemostatic and innate immune systems functionally cooperate in establishing the fraction of tumor cells that successfully form metastases. In particular, platelets and fibrinogen have been shown to support metastatic potential through a mechanism coupled to natural killer (NK) cell function. As the transglutaminase that ultimately stabilizes platelet/fibrin thrombi through the covalent crosslinking of fibrin, factor (F) XIII is another thrombin substrate that is likely to support hematogenous metastasis. OBJECTIVE: Directly define the role of FXIII in tumor growth, tumor stroma formation, and metastasis. METHODS: Tumor growth and metastatic potential were quantitatively and qualitatively evaluated in wild-type mice and gene-targeted mice lacking the catalytic FXIII-A subunit. RESULTS: Loss of FXIIIa function significantly diminished hematogenous metastatic potential in both experimental and spontaneous metastasis assays in immunocompetent mice. However, FXIII was not required for the growth of established tumors or tumor stroma formation. Rather, detailed analyses of the early fate of circulating tumor cells revealed that FXIII supports the early survival of micrometastases by a mechanism linked to NK cell function. CONCLUSIONS: Factor XIII is a significant determinant of metastatic potential and supports metastasis by impeding NK cell-mediated clearance of tumor cells. Given that these findings parallel previous observations in fibrinogen-deficient mice, an attractive hypothesis is that FXIII-mediated stabilization of fibrin/platelet thrombi associated with newly formed micrometastases increases the fraction of tumor cells capable of evading NK cell-mediated lysis.

Role of fibrinogen- and platelet-mediated hemostasis in mouse embryogenesis and reproduction.

Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.

Genetic manipulation of fibrinogen and fibrinolysis in mice.

Vascular integrity is maintained by a sophisticated system of circulating and cell associated hemostatic factors that control local platelet deposition, the conversion of soluble fibrinogen to an insoluble fibrin polymer, and the dissolution of fibrin matrices. However, hemostatic factors are likely to be biologically more important than merely maintaining vascular patency and controlling blood loss. Specific hemostatic factors have been associated with a wide spectrum of physiological processes, including development, reproduction, tissue remodeling, wound repair, angiogenesis, and the inflammatory response. Similarly, it has been proposed that hemostatic factors are important determinants of a variety of pathological processes, including vessel wall disease, tumor dissemination, infectious disease, and inflammatory diseases of the joint, lung, and kidney. The development of gene targeted mice either lacking or expressing modified forms of selected hemostatic factors has provided a valuable opportunity to test prevailing hypotheses regarding the biological roles of key coagulation and fibrinolytic system components in vivo. Genetic analyses of fibrin(ogen) and its interacting factors in transgenic mice have proven to be particularly illuminating, often challenging long standing concepts. This review summarizes the key findings made in recent studies of gene targeted mice with single and combined deficits in fibrinogen and fibrinolytic factors. Studies illustrating the role and interplay of these factors in disease progression are highlighted.

Fibrinogen and tumor cell metastasis.

Detailed studies tumor cell-associated procoagulants and fibrinolytic factors have strongly suggested that local thrombin and plasmin generation may be important in tumor progression. Given that one target for both these serine proteases is fibrinogen, a logical extension of this hypothesis is that local fibrin deposition and dissolution may be key determinants of tumor growth and/or dissemination. To directly test this concept, we initiated studies of tumor growth, experimental metastasis, and spontaneous metastasis in C57Bl/6-inbred mice with and without fibrinogen. Using two established C57Bl/6-derived tumor cell lines, Lewis lung carcinoma and B16-BL6 melanoma, fibrinogen deficiency was found to strongly diminish, but not prevent, the development of lung metastases in both experimental and spontaneous metastasis assays. This difference was not a consequence of any obvious difference in tumor stroma formation or the growth of primary or secondary tumors. Rather, tumor cell fate studies argued that there is an important role of fibrin(ogen) in the sustained adhesion and/or survival of tumor cell emboli within the lung. The specific thrombin inhibitor, hirudin, was also shown to strongly diminish metastatic potential, consistent with earlier reports. More importantly, hirudin was found to further diminish the already low metastatic potential of tumor cells in fibrinogen-deficient mice. We conclude that fibrin(ogen) is a critical determinant of metastatic potential, but thrombin appears to contribute to tumor cell dissemination through at least one fibrinogen-independent mechanism. Further, these findings suggest that therapeutic strategies directed at several hemostatic factors might be useful in the suppression of metastatic disease.

Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells.

Detailed studies of tumor cell-associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.

Soft tissue sarcomas of infancy.

Malignancy occurring during the neonatal period (defined as the first 28 days of life) is over 3 times the incidence of other pediatric age groups. Of all neoplasia occurring in infants, benign and malignant, 25% are soft tissue tumors. Differentiating the benign lesions from the 15% that are malignant can be difficult. This article discusses the epidemiology, differential diagnosis, evaluation, and treatment of infants with soft tissue sarcomas. Fibrosarcoma and rhabdomyosarcoma are also discussed at length. The authors review other rare tumors as well. The impact on diagnosis of molecular techniques is included when appropriate. A multidisciplinary team approach for treatment of these infants is recommended.