review
Coagulation and metastasis: what does the experimental literature tell us? Ana M. Gil-Bernabe, Serena Lucotti and Ruth J. Muschel Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK
Summary Inhibition of coagulation greatly limits cancer metastasis in many experimental models. Cancer cells trigger coagulation, through expression of tissue factor or P-selectin ligands that have correlated with worse prognosis in human clinical studies. Cancer cells also affect coagulation through expression of thrombin and release of microparticles that augment coagulation. In the cancer-bearing host, coagulation facilitates tumour progression through release of platelet granule contents, inhibition of Natural Killer cells and recruitment of macrophages. We are revisiting this literature in the light of recent studies in which treatment of clinical cohorts with anticoagulant drugs led to diminished metastasis. Keywords: coagulation, metastasis, macrophages, aspirin, tissue factor. Over many years cancer biologists have demonstrated that the coagulation pathway is essential for the establishment of metastasis in experimental model systems. This topic has become especially germane with the recent publication of clinical studies that have shown better cancer outcome, and probably diminished metastasis, in patients who had been receiving anticoagulation treatment, in particular those given aspirin prior to cancer diagnosis. Our intention in this review is to examine some of the relevant experimental literature linking coagulation to metastasis, and then consider some of the clinical trials in that light.
Inhibition of coagulation in experimental metastasis assays Agostino et al (1966) showed reduced pulmonary metastasis in rats anticoagulated with a 4-hydroxycoumarin compound. This class of anticoagulants, which includes warfarin, blocks the action of vitamin K, thereby reducing the synthesis of
Correspondence: Professor Ruth J. Muschel, Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK. E-mail:
[email protected] ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
several components of the coagulation cascade. Metastasis in this study was evaluated using intravenous injection of tumour cells, followed by assessment of tumour colonies in the lung (Agostino et al, 1966). This type of experiment is often used as a surrogate assay for lung metastasis. However, while it simulates metastasis through the introduction of circulating tumour cells that then colonize distant organs, this assay does not duplicate other steps in the metastatic process, in particular the entrance of tumour cells into the blood stream (Paweletz et al, 2001). Over the following years, using similar lung colonization assays, a number of drugs that inhibit coagulation, such as heparin and thrombin inhibitors, were also found to block lung colonization (see reviews Borsig et al, 2001; Varki & Varki, 2002; Ekambaram et al, 2011; and Esumi et al, 1991). An early study showed that treatment of mice with neuraminidase led to greatly reduced platelet numbers and diminished lung colonization by cancer cells (Gasic et al, 1968). Given that treatment with neuraminidase could affect the host in many ways, the authors controlled for this possibility by infusing platelets into the treated mice. Reconstitution of normal platelets alone restored metastatic capability. This study demonstrated the importance of platelets in the metastatic response, and showed that their critical time of action was early after introduction of the cancer cells into the circulation. Intravenously introduced tumour cells primarily arrest in the lungs, the first capillary bed that they encounter. Over the next 24 h there is a rapid reduction in the number of surviving tumour cells, a decline that has been termed metastatic inefficiency. This phenomenon was initially documented by injection of tumour cells labelled with radioactive isotopes and following the rapid decline of pulmonary radioactivity, and more recently either by direct counting of tumour cells or by using quantitative polymerase chain reaction (Glaves & Weiss, 1978; Al-Mehdi et al, 2000; Im et al, 2004; Qian et al, 2009). Thus, the early events in metastasis would be the critical platelet-mediated targets, although effects on later events are not excluded by these experiments. More recent experiments also indicate that platelet contributions to metastasis are most pronounced in this early time frame (Coupland et al, 2012). These experiments excluded a role for platelets in lung attachment that would affect tumour cell survival. Although effects on metastasis were First published online 21 May 2013 doi:10.1111/bjh.12381
Review pronounced, tumorigenecity was not, as growth of established primary tumours was often not substantially reduced by anticoagulation (Hu et al, 2004). Initiation of tumourigenicity, however, can be affected by signalling involving coagulation pathways; for example, through tissue factor [TF, also termed factor III (F3)], protease activated receptors (PAR) or thrombin (reviewed in Versteeg & Ruf, 2006; Green & Karpatkin, 2010; Zigler et al, 2011; Schaffner et al, 2012). Additionally, tumour vascular integrity is profoundly affected and reduced by the absence of platelets and may influence tumour growth by this mechanism. Platelets play roles in tumour vascularity and angiogenesis that are critical for limiting intra-tumoral haemorrhage (Ho-Tin-Noe et al, 2008, 2011). Overall, these results have led to the suggestion that coagulation and platelets have special implications for metastasis that are distinct from their effects on tumour growth alone. A summary of these key findings linking coagulation and metastasis can be found in Table I. As detailed above, the initial experiments examining the effect of coagulation on metastasis mainly used lung colonization assays. Subsequent experiments showed that anticoagulation also diminished metastasis in spontaneous metastasis assays, in which metastases are generated from primary tumours. These primary tumours may be induced as allografts, xenografts or from transgenic models of induced primary tumours. Different sites of organ colonization have also been examined. In animals treated with anticoagulants, pulmonary metastasis was diminished after intravenous injection of tumour cells. In addition, liver colonization after intrasplenic or intravenous injection, and brain metastasis in some model systems, in some studies, showed comparable reductions. Furthermore, experimental bone metastasis is reduced by thrombin inhibitors (Asanuma et al, 2004). Nonetheless, the majority of published studies focus on lung colonization assays. Thus, inhibition of coagulation has been shown to diminish metastasis in experimental murine (and a few rat) models in a variety of different assays. The very large literature concerning this topic is the subject of several reviews (Roos & Dingemans, 1979; Ruf & Mueller, 1996; Francis et al, 1998; Palumbo & Degen, 2007; Schaffner & Ruf, 2008).
Tumour cells trigger coagulation These studies raise the question of how metastatic cancer cells might engage the coagulation pathways. They also raise the question of how coagulation might facilitate metastasis. Coagulation is a complex process that can be triggered by different means (Furie & Furie, 2008). In the case of cancers, and as represented in Fig 1, tumour cells express factors that trigger coagulation in different ways (Falanga et al, 2009). One means by which this is achieved is through tumour cell expression of selectin ligands, resulting in their association to platelets through platelet P-selectin binding, and also in endothelial attachment via E-selectin (Laubli & Borsig, 2011; 434
Table I. Experimental review of coagulation and metastasis (we apologise for work that we have not cited). References
Key findings
Agostino et al (1966)
Metastasis reduced in rats given a 4-hydroxicoumarin compound in a lung colonization assay: i.v. tumour cells, analyze lung nodules. Caveat: doesn’t include the first steps of metastasis. Neuraminidase reduced lung colonization by reducing platelet number. Platelets are important at the early stages of metastasis, soon after introduction of tumour cells. Hirudin (thrombin inhibitor) reduces metastasis in a B16F10-C57BL/6 model, analyzed by lung colonization assay and survival assay. Critical within 60 min after introduction of tumour cells. Thrombin has a role in: -Spontaneous metastasis: hirudin inhibits spontaneous metastasis in nude or syngeneic mice s.c. injected tumour cells (human or murine), that metastasize to the lung. -Tumour implantation: hirudin delays s.c. tumour growth, with no effects in already established primary tumours. -Seeding: Hirudin inhibits seeding of tumour cells in the lungs. Coagulation facilitates tumour cell spreading, promoting metastasis. Platelets involved in tumour vascular integrity and angiogenesis, which might influence tumour growth. s.c. implantation of LLC or B16F10 tumour cells followed by platelet depletion causes desestabilization of tumour vessels. Coagulation triggered by tissue factor promotes the recruitment of myeloid cells that support metastasis. Platelets promote metastasis of breast and melanoma cancer cells independently of NK cells in both acute and spontaneous metastasis models. Action of platelets in metastasis is organ-specific. Endothelial-derived and platelet-derived P-selectin are both equally important to promote metastasis.
Gasic et al (1968)
Esumi et al (1991)
Hu et al (2004)
Im et al (2004) Ho-Tin-Noe et al (2008)
Ho-Tin-Noe et al (2011)
Gil-Bernabe et al (2012)
Coupland et al (2012)
St Hill, 2011). P-selectin ligands are carbohydrate moieties, which can result from modifications of mucins (Borsig et al, 2002). Intravascular tumour cells with these ligands become decorated with platelets and trigger further platelet aggregation. Finally, clotting can be initiated (Francis et al, 1998). ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
Review
Fig 1. Coagulation pathways and cancer metastasis. This figure shows the intersection of the cancer cell with the coagulation pathways. The extrinsic pathway is triggered by tissue factor (TF), expressed either on the surface of the tumour cells, on microparticles or on the tumour stroma, leading to fibrin formation. Platelets are recruited via selectins that interact with their ligands expressed on tumour cells. Furthermore, the production of prostaglandins and thromboxanes by COX expressed on the endothelium and on the tumour cells also triggers platelet aggregation. VTE, venous thromboembolism.
The extrinsic pathway of coagulation is initiated by exposure of TF to the endothelial layer by trauma (Furie & Furie, 2008). Tumour cells can also trigger coagulation by adaptation of this pathway through expression of TF. TF binds and activates factor VIIa (FVIIa), thereby initiating the coagulation cascade and leading to thrombin activation. TF is frequently expressed by cancer cells, as well as by elements of the tumour stroma, such as macrophages, fibroblasts and tumour vascular endothelium (Callander et al, 1992; Contrino et al, 1996; Vrana et al, 1996). Indeed, metastatic cancer cells have been found to express exceptionally high levels of TF, as much as 1000-fold higher than the corresponding non-metastatic cancer cells (Mueller et al, 1992), and it has been suggested that cancer stem cells may express higher levels of TF (Milsom et al, 2007). TF is overexpressed in certain cancer types, its levels correlating with prognosis, and, when expressed, can account for tumour cell procoagulant activity (reviewed in Milsom & Rak, 2008; Kasthuri et al, 2009; van den Berg et al, 2012). More recently, it has become apparent that TF is not only expressed on the surface of endothelial cells, but that it also circulates on the surface of microparticles. Microparticles (MP) are vesicles with a diameter of 01–1 lm, that have been actively shed from cells (Falanga et al, 2012). During the initiation of blood coagulation, MP expressing TF and P-selectin glycoprotein ligand-1 (PSGL-1, SELPLG) accumulate in the platelet thrombus (Falati et al, 2003; Furie & Furie, 2008). Essentially, all cells are capable of the release of MP. Malignant cell transformation and various stresses are both associated with MP release (Ahn, 2005). Platelet-derived MP constitute the majority (>80%) of MP in circulation in healthy individuals. In patients with cancer and in murine cancer models, MP derived from the cancer itself are also commonly found. They express antigens of tumour origin and thereby influence tumour immunology (Zwicker et al, 2009). MP in general contain phosphatidylserine. Some MP in patients with cancer express TF that are ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
derived from the cancer cells. Oncogenic signalling correlates with increased TF expression (see review van den Berg et al, 2012) and can increase not only TF expression by cancer cells, but also enhanced release of TF-expressing MP (Yu et al, 2005). As phosphatidylserine and TF act synergistically to trigger coagulation, the tumour derived TF-bearing MP potentially could create a strongly procoagulant environment (Key & Mackman, 2010). In conclusion, these MP might be of clinical interest in the generation of thrombosis and metastasis in cancer (Falanga et al, 2012). Enhanced cancer cell TF expression can lead to increased tumour growth. This may be due to signalling via proteases from the tumour environment, such as FVIIa, FXa, or thrombin (Yu et al, 2005), or to signalling through the TF cytoplasmic domain (Ruf et al, 2011). While this domain consists of only a few aminoacids, it nonetheless has been demonstrated to contribute to pro-oncogenic signals that may affect the stem cell-like behaviour of the tumour cells expressing it (Garnier et al, 2012; Schaffner et al, 2012). As might be expected, the expression of these procoagulant molecules and microparticles leads to the development of inappropriate coagulation in cancer. While it is not clear whether TF expressing MP affect metastasis, they are implicated in the coagulopathies related with cancer. Cancer is associated with a four-fold increase in the risk of venous thromboembolism (VTE) and chemotherapy further increases this risk to between six- and seven-fold compared with the general population (reviewed in Heit, 2005; Kasthuri et al, 2009). Interestingly, the incidence of thrombosis in patients with tumour types tending to express higher levels of TF, such as brain and pancreatic cancer, is greater than in those with tumours expressing lower levels of TF, such as breast cancer (Kasthuri et al, 2009). The most recognized of these procoagulant disorders is Trousseau syndrome, which is the subject of extensive review (Donati & Lorenzet, 2012). As mentioned above, elevated levels of TF-positive MP have been reported in cancer patients with VTE (Hron et al, 2007; Tilley 435
Review et al, 2008; Manly et al, 2010). In these cases, the elevation was greater in highest risk stages, and showed a correlation with metastasis (Kim et al, 2003). Despite a variety of clinical correlations thus far, determination of the clinical significance of MP as a predictive biomarker for VTE in cancer patients has not been achieved. Some trials are ongoing to evaluate the utility of measuring TF-positive MP to predict VTE in cancer (Zwicker, 2010; Falanga et al, 2012). Nonetheless, these studies make it likely that the release of TF-bearing MP into the circulation contributes to the systemic coagulopathies commonly observed in cancer patients (Milsom & Rak, 2008). Thus, the release of MP by tumours is a common event and raises as yet unaddressed questions about their possible participation in metastasis.
Contribution of coagulation pathways to metastasis Coagulation has been shown to contribute to metastasis through several mechanisms, as detailed in Table II. Inhibition or downregulation of TF in tumour cells in many experimental settings reduces metastasis (Mueller et al, 1992; Mueller & Ruf, 1998; Amirkhosravi et al, 2002; Milsom et al, 2007; Ngo et al, 2007; and reviewed in Rickles, 2006; Schaffner et al, 2012). Similarly, downregulation of P-selectin has
Table II. Inhibition of coagulation reduces metastasis. Mechanism that leads to reduced metastasis Downregulation of TF
Downregulation of P-selectin Haemophilila-like syndromes Inhibition of thrombin
Inhibition of PAR
Deficiency in fibrinogen Deficiency in prothrombin Deficiency in platelets
436
References Mueller et al (1992) Mueller and Ruf (1998) Amirkhosravi et al (2002) Milsom et al (2007) Ngo et al (2007) Reviewed in Rickles et al (2006) and in Schaffner et al (2012) Borsig et al (2002) Kozlowski et al (2011) Coupland et al (2012) Bruggemann et al (2008) Bobek and Kovarik (2004) Esumi et al (1991) Hu et al (2004) Im et al (2004) Camerer et al (2004) (PAR-4, but not PAR-1 or PAR-2 in the host reduces metastasis) Nierodzik et al (1998) (PAR-1 in tumour cells enhances metastasis) Camerer et al (2004) Palumbo et al (2000) Palumbo et al (2007) Camerer et al (2004) Palumbo et al (2005)
the same effect (Borsig et al, 2002; Kozlowski et al, 2011; Coupland et al, 2012). Both TF and P-selectin can lead to platelet aggregation and the concomitant conversion of fibrinogen to fibrin, resulting in clot formation. Fibrinogen also enhances metastasis, but tumour growth is not reduced in fibrinogen-deficient mice (Palumbo et al, 2000; Camerer et al, 2004). Mice with haemophilia-like syndromes also have diminished metastasis (Bruggemann et al, 2008). Activation of thrombin is a central hub of the coagulation cascade. Both TF activation and platelet aggregation result in thrombin activation, and inhibition of thrombin also inhibits coagulation and results in reduced metastasis (Esumi et al, 1991; Bobek & Kovarik, 2004; Hu et al, 2004; Im et al, 2004). Thrombin signals to endothelial cells and platelets through cleavage of protease activated receptors (PAR). PAR-1 and PAR-2 are expressed on endothelial cells and on some cancer cells. PAR-4 is expressed on platelets. Mice deficient in prothrombin (Palumbo et al, 2007), platelets (Camerer et al, 2004; Palumbo et al, 2005), fibrinogen (Palumbo et al, 2000; Camerer et al, 2004), or PAR-4 (Camerer et al, 2004) also showed reduced metastasis. Deficiency in PAR-1 or PAR-2 did not impair the ability of the host to support metastasis (Camerer et al, 2004). On the other hand, using tumour cells expressing PAR-1 and in which PAR-1 signalling led to increased proliferation, pretreatment with thrombin before intravenous injection of the tumour cells enhanced metastasis (Nierodzik et al, 1998). Tumour cell signalling induced by thrombin enhances metastasis by altering tumour cell transcription and expression of the metastasis suppressor gene SERPINB5 (Maspin) (Villares et al, 2011; Zigler et al, 2011). Thus, thrombin affects metastasis through two separate mechanisms: firstly, by acting on platelets and leading to fibrin formation; and secondly, by triggering tumour cell signalling (Green & Karpatkin, 2010; Gay & Felding-Habermann, 2011).
Mechanisms leading to enhanced metastasis through coagulation How does coagulation manifest itself during metastasis? Despite the evidence that inhibition of coagulation decreases metastasis, our understanding of the mechanisms involved is limited. Tumour cells circulate in the blood stream and are deposited at metastatic sites. These tumour cells can trigger platelet aggregation depending on their expression of coagulation initiating factors, as previously mentioned. Even in tissue culture, tumour cells can stimulate aggregation and collection of platelets on their surface. In some cases, expression of TF or the endogenous PSGL-1 has been shown to be the trigger and associated platelets can be found surrounding these tumour cells in the circulation in mice (Kim et al, 1998). Table III describes some mechanisms by which coagulation supports metastasis. Im et al (2004) showed that metastatic tumour cells attached to the pulmonary endothelium immediately after intravenous infusion were surrounded by aggregates of platelets. However, these clots, that contained both ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
Review Table III. Coagulation promotes metastasis: mechanisms involved. Mechanism that leads to enhanced metastasis
References
Key findings
Tumour cell spreading
Im et al (2004)
Clot formation on the tumour cells facilitates tumour cell spreading, improving the attachment to the endothelium and enhancing tumour cell survival and metastasis. Integrin a3b1 in the tumour cells interacts with laminin-5 in the basement membrane to enhance tumour cell attachment to the endothelium in the lung. Inhibition of plasmin-mediated fibrinolysis increases clot retention by tumour cells and tumour cell survival. Clot elements form molecular bridges between the tumour cells and the vascular beds. In vitro studies show a decrease in tumour cell lysis by Natural Killer (NK) cells when the tumour cells are surrounded by a clot on their surface. In vivo studies show that tumour cells evade the immune surveillance by physical means or by mechanisms involving signalling (NK quiescence).
Wang et al (2004)
Kirstein et al (2009) Protection of tumour cells from vascular shear stress Protection from NK cells
Konstantopoulos and McIntire (1996) Bobek et al (2005)
Platelets directly stimulate tumour growth and angiogenesis.
Nieswandt et al (1999) Palumbo et al (2005) Palumbo et al (2007) Kopp et al (2009) Stewart et al (2006) Gay and Felding -Habermann (2011)
Epithelial-mesenchymal transition
Labelle et al (2011)
Recruitment of monocytes/macrophages
Gil-Bernabe et al (2012)
Endothelial and platelet-derived P-selectin
Coupland et al (2012)
platelets and fibrin(ogen), were delimited and did not block the vessels to flow. Only tumour cells associated with clots appeared to persist. After several hours, these clots dissolved, coincident with the increase in tumour cell spreading on the endothelium. The retention of clots increases if plasmin-mediated fibrinolysis is inhibited soon after the introduction of the tumour cells, resulting in enhanced metastatic colonization (Kirstein et al, 2009). Thus, the timing of the microscopic clots found soon after introduction of tumour cells correlates with the timing of platelet action on metastasis. Multiple mechanisms have the potential to contribute to metastasis enhancement by coagulation and platelet aggregation by tumour cells. One of them is the ability of platelet aggregation to reduce shear stress and to enhance survival of tumour cells under flow. Platelets, thrombin and fibrin could help to support metastasis by serving as cellular or molecular bridges between the tumour cells and the vascular beds and protect them from the intravascular shear forces (Konstantopoulos & McIntire, 1996). This mechanism clearly is operative in experimental model systems. Proof in vivo is still tenuous. The aggregation of platelets around tumour cells in tissue culture greatly diminishes the ability of Natural Killer (NK) cells to lyse cancer cells in culture (Bobek et al, 2005). ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
Platelets surrounding tumour cells release growth factors and chemokines (VEGF, ILGF1, TGF-b) that enhance tumour cell proliferation and angiogenesis. Direct interaction between platelets and tumour cells triggers EMT through platelet derived TGF-b/SMAD and NF-jB pathways. Clot formation on tumour cells triggers the recruitment of a subset of monocytes/macrophages, promoting tumour cell survival. Platelets promote metastasis in lung, but not in liver, in a mechanism that is chronologically independent of NK cells and in which P-selectin from the endothelium and from the platelets are equally important.
Consistent with these findings, activation of platelets and the presence of fibrinogen have been shown to help tumour cells to evade immune surveillance mechanisms, protecting them from killing by NK cells (Nieswandt et al, 1999; Palumbo et al, 2005, 2007), by both physical means and through signalling that leads to NK quiescence (Stewart et al, 2006; Kopp et al, 2009). However, while the genetic or immunological elimination of NK cells completely abrogated the role of more distal haemostatic system components, like fibrinogen, platelets and FXIII, in metastasis, TF expression by tumour cells remained a significant determinant of metastasis and early tumour cell survival even in mice lacking NK cells (Palumbo et al, 2007). Further, in studies detailing the timing of NK depletion and restoration of NK cells and platelets, it has been suggested that they may act independently, especially at the earliest times when platelets have a clear effect (Coupland et al, 2012). Even in the absence of NK cells, coagulation still affected the extent of experimental metastasis, suggesting that the initial recruitment of platelets by tumour cells was highly supportive of metastasis (Coupland et al, 2012). Platelets directly stimulate tumour growth and influence angiogenesis by releasing a variety of growth factors and chemokines 437
Review stored in their granules (Gay & Felding-Habermann, 2011; Sabrkhany et al, 2011). Many of the individual factors in the granules, such as vascular endothelial growth factor (VEGF, VEGFA), insulin-like growth factor 1 (IGF1), and transforming growth factor-b (TGFB), are highly bioactive and have been implicated in cancer and metastasis progression. Coagulation activation not only generates a protective barrier against immune surveillance, but also facilitates the initial steps of tumour cell extravasation, such as cell adherence at the distant organ and spreading, and epithelial-mesenchymal transition (Im et al, 2004; Wang et al, 2004; Labelle et al, 2011). Thus, coagulation has a potential role in facilitating immune system evasion. Evidence for the direct link between platelet aggregation and macrophage recruitment comes from Gil-Bernabe et al (2012), who showed that recruitment of monocytes/macrophages by TF-induced platelet aggregates on tumour cells played an important role in sustaining metastasis. In this study, formation of clots by tumour cells was shown to recruit a subset of monocytes/macrophages that expressed CD11b and F4/80. Conditional elimination of these cells using a CD11b-DTR (diphtheria toxin receptor) system did not alter clot formation, but reduced metastasis and was deleterious for survival by the tumour cell in the pulmonary circulation after adherence to the pulmonary vasculature (Gil-Bernabe et al, 2012). Thus, part of the facilitating effect of the clot was not directly due to the platelets themselves but was exerted by the recruited monocytes/macrophages. The importance of macrophage CD11b in this recruitment was highlighted because using mice genetically deficient in CD11b (Mac1 knockout mice) as recipients for tumour cells led to clot formation around the tumour cells, but failed to recruit macrophages and had diminished tumour cell colonization. These results open the question of the function of these macrophages recruited to the clots formed on the tumour cells. Different populations of macrophages have been shown to facilitate metastasis in a variety of systems, but in many cases their function is not known (Qian et al, 2009; Qian & Pollard, 2010). Qian et al (2011) showed that tumour cell metastasis in mice with macrophages deficient in VEGF failed to extravasate in the lung, but in other systems the deficiency occurs before extravasation while tumour cells are still intravascular and in other cases, like in the liver, the macrophages act after extravasation has occurred (Zhao et al, 2013). Extravasation of tumour cells has not been thoroughly investigated as a potential coagulation affected event.
Clinical evidence The accumulation of information that inhibition of platelet activation through inhibition of coagulation could reduce metastasis in murine models led to a variety of clinical trials. In these trials different anticoagulants were administered to cancer patients, followed by evaluation of outcome. Many of these trials were based on administration of low molecular 438
weight heparins, with a few using warfarin or similar compounds (Zacharski et al, 2005). In most cases these trials failed to demonstrate any prolongation of survival. However, it should be considered that the pleiotropic effects of heparins complicate the conclusions of these studies. Additionally, most of these trials enrolled patients with already advanced disease, although without evident metastases at presentation. The experimental evidence is that anticoagulation treatment was mainly effective in reducing metastasis in the lung when administered soon after tumour cell arrival and arrest in the pulmonary endothelium. These patients might be expected to already have occult, disseminated disease (Erpenbeck & Schon, 2010; Chan et al, 2012). More recent trials have included populations in which the study cohorts were comprised of patients already receiving aspirin or anticoagulation treatment for non-oncological medical conditions, such as atherosclerotic disease with cardiac or cerebral occlusive events. In these populations an epidemiological approach might reveal whether metastasis is reduced by anticoagulation treatment, when administered before cancer dissemination. Aspirin causes irreversible inhibition of cyclooxygenases (COX), the enzymes responsible for the synthesis of prostaglandin H2, which is the precursor of the highly biologically active eicosanoids prostaglandins, thromboxanes and prostacyclin. While high dose aspirin is an effective inhibitor of both isoforms of this enzyme, COX1 (PTGS1) and COX2 (PTGS2), low doses mainly inhibit COX1, for which aspirin has higher affinity. COX1 is expressed in platelets, and consequently low doses of aspirin act to reduce platelet aggregation. While COX1 is constitutively expressed, COX2 expression is inducible upon inflammation. A variety of studies have been published examining the effect of low dose aspirin on cancer prevention and outcome. Some of these studies have shown a favourable effect, while others fail to find any significant decrease in cancer incidence. Colorectal cancer incidence, however, appears to be substantially reduced by prophylactic aspirin use (Rothwell et al, 2010, 2011, 2012a,b; Algra & Rothwell, 2012; Chan et al, 2012). These findings are consistent with the molecular basis for COX2 involvement in this disease in mice (Oshima & Oshima, 2012). However, in other cancer types the evidence for reduction in tumour incidence is less striking, with no effect seen, or inconsistent results. For example, some studies find a reduction in prostate cancer in patients receiving a low dose of aspirin, but not other non-steroideal anti-inflammatory drugs (Shebl et al, 2012; Veitonmaki et al, 2012), while other studies found a modest inverse association (Bosetti et al, 2012). However, in another study, treatment with anticoagulants, and particularly with aspirin, reduced the risk of prostate cancer mortality (Choe et al, 2012). Analyses of large patient cohorts receiving aspirin or other anticoagulants for outcome of cancer and metastasis suggested that the administration of aspirin is likely to improve patient outcome not only by reducing cancer incidence, but also by reducing metastasis (Algra & ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 433–441
Review Rothwell, 2012; Rothwell et al, 2012b). This is certainly consistent with the wide body of preclinical literature considered above, relating platelets and the coagulation system to metastasis. Some experimental studies in mice have focused upon aspirin specifically in regards to its effect on metastasis. Aspirin effectively inhibits metastasis in lung colonization models (Ekambaram et al, 2011). Interestingly, aspirin also inhibits intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) expression, and could thereby also affect the recruitment of immune cells (Weber et al, 1995). Whilst Weber et al (1995) suggested that this reduction might prevent cancer cell attachment at the distant site, work in our laboratory indicates that VCAM1 is also important in metastasis due to its action in enabling the coagulation-dependent homing of macrophages
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to the sites of early metastasis (Ferjancic et al, 2013). Thus, the concept that interference with coagulation might reduce early metastasis led to clinical studies that are partly consistent with the experimental work. One notable inconsistency remains, in that warfarin reduced metastasis in murine models, but did not result in a reduction in metastasis in the clinical data sets. Most importantly, the preclinical work has not established the mechanisms of action through which platelets and coagulation precisely affect metastasis. Understanding these mechanisms should help to stratify patients who are likely to respond to anti-metastatic strategies and also to develop alternative pharmacological approaches, including those to prevent metastasis. Thus, this clinical work leads to important questions for the laboratory researchers to investigate.
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