Coagulation, angiogenesis and cancer - Chapter 1: General Introduction

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Coagulation, angiogenesis and cancer

Niers, T.M.H.

Publication date

2008

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Citation for published version (APA):

Niers, T. M. H. (2008). Coagulation, angiogenesis and cancer.

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11 General introduction

Coagulation, angiogenesis and cancer

The relationship between cancer and thrombosis is twofold. First, cancer patients have an

increased risk to develop venous thromboembolism (VTE). Second, the coagulation system

affects cancer progression and metastasis. Furthermore, various coagulation factors, like

tissue factor (TF) and thrombin are involved in angiogenesis, new vessel formation, which

facilitates tumor growth and metastasis

_{. This thesis is focused on three major cancer}

progression-stimulating factors: platelets, TF/thrombin and angiogenesis -and the

two-way interactions between cancer and coagulation.

Increased risk of thrombosis in cancer patients

Cancer patients have an increased risk to develop VTE. This was first described by

Bouillaud (1823)

_{and forty years later (1865) interpreted and published by Trousseau}

_{. An}

unprovoked VTE can be the first sign of occult cancer, which was described for the first time

in 1935

_{. According to Virchow, the hallmarks of VTE are pathological changes in blood}

flow, coagulability and the condition of the vessel wall. All three phenomena may occur

in cancer patients. The proposed mechanisms to explain hypercoagulation associated

with cancer include a reaction of the patients body to the tumor such as abnormal protein

synthesis, angiogenesis and necrosis and more specific processes related to

tumor-mediated haemostatic activities (cancer cells interacting with platelets, endothelial cells,

monocytes and with the coagulation and fibrinolytic systems)

_{. Furthermore, cancer}

treatment -irradiation, chemotherapy and surgery- may further upset the balance between

procoagulant and anticoagulant factors

_.

The incidence of VTE is also associated with the use of central venous catheters (CVC).

Cancer patients frequently have to use CVCs for chemotherapy, stem cell infusion, blood

supply, medication, parenteral hyperalimentation and blood sampling. Risk factors for

CVC-related thrombosis include the type of malignancy, chemotherapy and CVC and insertion

sites of the catheter tip

_{. Many studies have addressed the incidence and associated risk}

factors of CVC-related infections and VTE in patients with solid tumors but only few data

are available on haemato-oncological patients. These patients may differ from patients

with solid tumors, because of the more severe and prolonged thrombocytopenia and

leukopenia. Therefore, an important matter of debate is whether haematological cancer

patients should receive thrombosis prophylaxis or not. This issue is dealt with in chapter 9.

The coagulation system affects cancer progression and metastasis

Until the mid nineties of the last century, the initial treatment of VTE consisted of a brief

course of unfractionated heparin (UFH) followed by a course of vitamin K antagonists

for several months. In 1992, Prandoni et al compared the relative safety and efficacy of

12 Chapter 1

low molecular weight heparin (LMWH) and UFH for the treatment of VTE and concluded

that fixed-dose subcutaneous LMWH is at least as effective and safe as UFH for the initial

treatment of VTE

_{. This has been confirmed by others}

_{. Unexpectedly, LWMH showed}

a favourable effect on the survival of cancer patients. At 3 months, 44% (8 of 18) of the

cancer patients died in the UFH group vs. 7% (1 of 15) in the LMWH group (p=0.021)

_.

These findings were confirmed in meta-analyses of 9 studies that compared LMWH with

UFH in the treatment of VTE

_{. These studies initiated clinical trials evaluating the effect}

of anticoagulants on survival of cancer patients without thrombosis

_{. Thus, cancer}

favours thrombosis and the coagulation system promotes cancer as suggested by the

marked survival advantage of patients using anticoagulants.

Various experimental studies showed the inhibitory effects of anticoagulants on

cancer progression and metastasis. The results are reviewed in chapter 2. However, the

mechanisms by which anticoagulants may interfere with tumor growth and metastasis

are diverse, remain poorly defined and seem to be dependent on the type of cancer and

individual anticoagulant

_{. In this thesis, experimental studies are described that focus}

in particular on the phase of haematogenous dissemination when cancer cells are present

in the circulation. These studies are described in chapters 4, 5, 6 and 7. Chapter 3 reviews

validation of noninvasive bioluminescence imaging (BLI) for quantitative assessment of

tumor load in time in small animals, a technique we used in two of our studies.

Platelets

Platelet aggregation on cancer cells takes place rapidly when cancer cells have entered

the circulation. Cancer cells are thus masked for the immune system, protected against

shear stress in the vasculature and adhesion to vessel walls is facilitated. The process starts

with the interaction of activated platelets with cancer cells that express P-selectin ligands,

such as glycoprotein ligand-1, CD24, heparan sulphate proteoglycan (HSPG) and

sialyl-Lewis a/x

_{. The interactions of platelets and cancer cells may also involve ß3-containing}

integrins binding von Willibrand factor (vWF), thrombomodulin and fibrinogen to form

molecular bridges

_{. Then, activation of procoagulant proteins such as TF can occur as is}

described below

_{. These interactions enable rolling of cancer cells or cancer cell-platelet}

complexes along vessel walls where endothelial cells constitutively express low amounts

of P-selectin

_{that facilitates adhesion of cancer cells to the wall and transmigration into}

the subendothelium

_{or protect cancer cells within vessels against mechanical stress and}

the immune system

_.

P-selectin also occurs in a soluble form in blood plasma. Ferroni et al demonstrated that

soluble P-selectin (sP-selectin) plays a pivotal role in the pathogenesis of metastasis by

formation of cancer cell-platelet complexes. sP-selectin is considered to be a marker for

platelet activation and sP-selectin correlated inversely with prognosis in patients with

cancer

_{. It has been suggested that anticoagulants inhibit metastasis by blocking}

13 General introduction

can be explained by its interference in P-selectin-mediated interactions between platelets

and cancer cells. This is described in chapter 8.

Thrombin

When TF is expressed on the plasma membrane of cancer cells, it activates circulating

liver-derived coagulation factors VII, V and X that leads to the generation of thrombin

from prothrombin (Figure 1). Thrombin has distinct effects on cells. Intracellular effects

of thrombin are mediated by protease activated receptors (PARs), members of the family

of G-coupled receptors

_{. Four PARs have been described: PAR-1, -2, -3 and -4. Thrombin}

seems to be the major physiological activator of PAR-1

_{and PAR-4}

_{, but it can also}

activate PAR-3

_{, that functions as a cofactor of PAR-4. PAR-2 is not directly activated by}

thrombin but via trypsin

_{, coagulation factor VIIa and factor X}

_{. TF and PARs play an}

important role in cancer progression

_.

PAR signalling upregulates adhesion molecules on endothelial surfaces and triggers

production of chemokines by activating neutrophils and monocytes. This leads to binding,

rolling and attachment of platelets and leukocytes on the surface of endothelium. High

Figure 1: Coagulation cascade. HWMK=High molecular weight kinogen; PK=Prekallikrein; TFPI=Tissue factor pa-thway inhibitor. Grey arrows, functions of thrombin. Mediators studied or discussed in this thesis (TF, Factor Xa, thrombin, Factor V, Factor VIII, Fibrin and activated protein C).

14 Chapter 1

concentrations of locally-produced thrombin may lead to direct release of P-selectin

stored in Weibel bodies in endothelial cells through PAR-4-dependent signalling, resulting

in increased platelet aggregation and cancer cell-platelet binding. Local aggregates of

cells expressing TF

_{along with procoagulant activity of platelets}

_{may trigger further}

thrombin formation

_{and increased permeability of the endothelium}

_{. Thrombin also}

activates platelets to release growth factors that may sustain tumor development and may

aid angiogenesis by production of platelet derived growth factor (PDGF), basic fibroblast

growth factor (bFGF) and vascular endothelial growth factor (VEGF)

_{. Furthermore,}

thrombin converts fibrinogen into fibrin, the end product of the coagulation cascade.

Fibrin depositions have been found in and around many types of tumors, providing a

scaffold for angiogenesis and possibly also protecting the cancer cells against the host

defence system

_{. Thus, possible mechanisms by which anticoagulants prolong survival}

of cancer patients may also be reduction of thrombin and fibrin formation. The relationship

between thrombin and cancer is described in the chapters 5 and 7.

Angiogenesis

Several coagulation factors, such as TF and thrombin play a role in angiogenesis, that is

required for tumor growth and metastasis

_{. First, thrombin can activate angiogenesis}

by reduction of endothelial cell attachment to lamina basalis proteins and activation of

matrix metalloproteinases

_{. Second, thrombin has chemotactic and apoptotic effects on}

endothelial cells and upregulates expression of VEGF receptors (VEGFR). Third, thrombin

upregulates expression of αvβ3 integrin, the marker of the angiogenic phenotype of

endothelial cells

_{. Platelets may contribute to this process because they also release}

angiogenic factors like VEGF upon activation by thrombin via PAR-1

_.

VEGF is one of the most important angiogenic factors. It binds to specific tyrosine kinase

receptors on the surface of endothelial cells including VEGFR-1 (Flt-1) and VEGFR-2 (KDR/

flk-1) on vascular endothelium and VEGFR-3 (Flt-4) expressed on lymphatic endothelium,

resulting in cell migration, proliferation and survival

_{. Clinical research on angiogenesis}

has two major directions in cancer patients. First, quantification of angiogenesis for

diagnosis, prognosis as well as for the monitoring of responses. Second, the inhibition of

angiogenesis to halt tumor growth

_{. However, serum, plasma and whole blood have been}

indiscriminately used to determine VEGF levels in the body. Because coagulation results

in the release of VEGF from platelets, serum VEGF levels include plasma-derived VEGF and

platelet-derived VEGF

_{. Therefore, VEGF levels in serum do not reflect the true circulating}

levels of VEGF. In citrate or EDTA plasma, where less platelet activation and subsequent

VEGF release occurs than in serum, VEGF levels were found to be higher in cancer patients

than in controls and this was interpreted as a reflection of the higher levels of VEGF in the

circulation of cancer patients

_{. However, the release of VEGF from platelets may contribute}

to increased VEGF levels in plasma as well under these conditions. Therefore, the effects

of the different blood collection protocols on the measurement of circulating VEGF and

15 General introduction

their impact on VEGF release from platelets by in vitro platelet activation is described in

chapter 10.

VEGF expression is regulated by a number of factors. In renal cell carcinoma (RCC),

VEGF expression is a consequence of inactivation of the von Hippel-Lindau (VHL) tumor

suppressor gene, resulting in a remarkable overexpression of VEGF. Because of the

high levels of VEGF occurring in RCC, VEGF may be identified as a critical component of

angiogenesis in RCC and as a potential therapeutic target to treat RCC. The strategy to

inhibit the activity of VEGF includes binding of the VEGF protein and blockade of VEGFR.

Besides the classical prognostic markers for advanced RCC

_{, novel validated biomarkers}

are needed to predict the outcome of targeted therapy and the development of drug

resistance. Circulating levels of VEGF, placenta growth factor (PlGF), sVEGFR-1 and -2 and

bFGF are potential candidates to predict outcome of the various therapies. We correlated

base line levels and changes in the levels during treatment of these potential markers

with disease outcome and the development of resistance during therapy in chapter 11.

16 Chapter 1

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20 Chapter 1

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