Inflammation and melanoma metastasis

Authors


M. Bar-Eli, e-mail: mbareli@mdanderson.org

Summary

Metastatic melanoma is extremely refractory to existing chemotherapeutic drugs and bioimmune adjuvant therapies, and the life span of patients with metastatic melanoma is often measured in months. Understanding the mechanisms responsible for the development of tumor metastasis is critical for finding successful curative measures. An expending amount of data reveal the importance of inflammatory microenvironment and stroma in cancer initiation and progression, which brings new directions and approaches to cancer treatment. This review will summarize current data on the role of the tumor microenvironment in shaping the metastatic phenotype of melanoma.

Mechanism of tumor metastasis

Cancer metastasis is a sequence of events (Fidler, 2003). Potentially metastatic cells have to exit the primary tumor site by loosening cell to cell contact, adhering to and degrading extracellular matrix (ECM), migrating through the subendothelial basement membrane of local post-capillary veins and lymphatic vessels and intravasating. Once in circulation, tumor cells face severe mechanical and immunosurveillance challenges. Surviving cells can arrest in the peripheral capillary bed of a distant organ, adhere to the subendothelial basement membrane, extravasate, adhere and migrate through the ECM, and form a colony at the new metastatic site. Further induction of neoangiogenesis must occur to assure continuous growth (Fidler, 2003).

Genetic and epigenetic alterations in cancer cells, together with plastic and responsive microenvironment support the metastatic evolution of tumors (Chiang and Massague, 2008; Fidler et al., 2007; Villanueva and Herlyn, 2008; Witz, 2008). Many genes responsible for individual steps of metastasis formation have been identified. They can be classified into three groups: those involved in metastasis initiation, progression and virulence (Chiang and Massague, 2008). Functions associated with initiation of metastasis include those that allow tumor cells to circumvent local hypoxia by inducing local invasion, angiogenesis and epithelial-to-mesenchymal transition. Examples of metastasis initiation genes include Ras homolog gene family, member C, lysyl oxidase (LOX), vascular endothelial growth factor (VEGF), colony-stimulating factor-1 (CSF-1), inhibitor of DNA binding 1, TWIST1, MET, FGF receptors (FGFR), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and neural precursor cell expressed (NEDD9) (Chiang and Massague, 2008). Functions involved in metastatic progression include vascular remodeling, immune evasion and extravasation, which can be mediated, at least in part, by genes like epiregulin, cyclooxygenase 2 (COX-2), MMP-1, CC chemokines 5 (CCL5) and angiopoietin-like 4. Metastatic virulence provides selective advantage to cancer cells during the adaptation and expansion in a distant organ. These organ specific functions can be performed by CXC chemokine receptor 4 (CXCR4), receptor activator of nuclear factor-κB ligand (RANKL), connective tissue growth factor, IL-11 and endothelin-1.

Malignant melanoma has served as an excellent model for studying the molecular changes associated with the metastatic phenotype. This is partly because of well-described sequential steps in the progression of the disease and successful identification of a number of accompanying molecular changes. Following transformation of melanocytes, tumors undergo horizontal or radial initial growth phase followed by a subsequent vertical growth phase corresponding to the infiltration of the dermis (de Braud et al., 2003; Miller and Mihm, 2006). When the lesion enters the vertical growth phase, the tumor penetrates the dermis and acquires the capacity to metastasize (de Braud et al., 2003; Miller and Mihm, 2006). Molecular changes associated with melanoma progression have been extensively reviewed previously (Crowson et al., 2007; Lopez-Bergami et al., 2007; Melnikova and Bar-Eli, 2008; Miller and Mihm, 2006).

Tumor microenvironment consists of tumor, stromal, inflammatory and immune cells, as well as extracellular components including extracellular matrix (Mantovani et al., 2008). Although, it is accepted that inflammation increases the risk of cancer development (Mantovani et al., 2008), normal cells respond to oncogenic/inflammatory stimulation by activating tumor suppressors, which in turn trigger cellular senescence or cell death (Acosta and Gil, 2009; Acosta et al., 2008; Kuilman et al., 2008; Michaloglou et al., 2005). For example, in normal melanocytes, oncogenic stimulation with BRAF induces secretion of inflammatory cytokines including IL-6, IL-8, as well as IL-8 receptor CXCR2, which are instrumental in execution of cellular senescence (Acosta and Gil, 2009; Acosta et al., 2008; Kuilman et al., 2008; Michaloglou et al., 2005). However, following oncogenic transformation, tumor cells, via oncogenic signaling pathways, recruit stromal, inflammatory and immune cells and conduct them to suppress their cytotoxic responses and increase pro-proliferative and proangiogenic activities. This enables, at least in part, the initiation and progression of metastasis (Mantovani et al., 2008). The relationship between the components of the tumor microenvironment and cancer development and progression has been a subject of excellent reviews (Denardo et al., 2008; Li et al., 2003; Mantovani, 2005; Mantovani et al., 2008; Richmond et al., 2009; Robinson and Coussens, 2005; Tlsty and Coussens, 2006). In this article, we review some of the current data that support the involvement of the melanoma tumor microenvironment in the development of melanoma metastasis. A particular attention is paid to the contribution of blood related factors including thrombin and platelet-activating factor (PAF) and their receptors, protease-activated receptor 1 (PAR1) and PAF receptor (PAFR).

Tumor microenvironment and metastasis

Following transformation, the growth of the primary tumor perturbs physical structure of the organ and exposes tumor cells to stresses. Tumor microenvironment is characterized by lack of nutrients or oxygen, low pH and pro-inflammatory mediators. Such environment creates pressure and selects for cells that can circumvent the restrictions. For example, hypoxia induces stabilization of hypoxia-inducible factor 1α (HIF1α). In many tumor types, HIF1α upregulates cell-matrix adhesion, invasion and tumor angiogenesis via a variety of mechanisms. In melanoma, HIF1α is known to stimulate expression of Notch1, which maintains melanoma cell proliferation in xenografts and protects cells from stress-induced cell death (Bedogni et al., 2008). Notch1 expression is found upregulated in melanoma specimens (Bedogni et al., 2008). Notch1 contributes to progression of primary melanoma by activating the β-catenin and the PI3 kinase/Akt and MEK/Erk pathways, and stimulating expression of N-cadherin and melanoma cell adhesion (Liu et al., 2006). In vitro, Notch1 mediates HIF1α-dependent oncogenic transformation of melanocytes by Akt/hypoxia (Bedogni et al., 2005). Further attesting to the role of tumor microenvironment in metastatic progression of melanoma, hypoxia-stimulated HIF1α induces chemokine receptor CXCR4, which, together with its ligand, chemokine stromal-cell-derived factor 1 (SDF-1), directs murine melanoma cells to metastasize to the lung (Murakami et al., 2002).

Innate immunity and melanoma metastasis

In addition to tumor-specific physiological conditions, an expanding amount of data reveals the importance of immune and stromal cells in cancer initiation and progression. Genetic and functional experiments indicated that inflammatory cells such as tumor-infiltrating monocytes/macrophages, neutrophils, mast cells, eosinophils and activated T and B lymphocytes, as well as stromal fibrobalsts contribute to malignancies by releasing growth and survival factors, extracellular proteases and other proangiogenic factors (Denardo et al., 2008; Li et al., 2003; Mantovani, 2005; Mantovani et al., 2008; Richmond et al., 2009; Robinson and Coussens, 2005; Tlsty and Coussens, 2006).

Interleukins

Melanoma cells express a variety of cytokines, chemokines and their receptors, and their spectrum and level of expression undergo changes with melanoma progression (Richmond et al., 2009). Moretti et al. demonstrated that nevi and thin (<1 mm) primary melanomas express low amounts of tumor necrosis factor-α (TNF-α), TGF-β, interleukin-8 (IL-8), TGF-β receptor and c-kit. Marked up-regulation of IL-1α, IL-1β, IL-6 and its receptor, IL-8, TNF-α, TGF-β and its receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, as well as stem cell factor (SCF) was observed in thick primary melanomas (Moretti et al., 1999). Metastases showed similar expression patterns except for SCF, which was absent. It was concluded that IL-6 and IL-8 expression was associated with biologically early malignancy, whereas TGF-β, GM-CSF and IL-1α were highly expressed in late lesions, with TGF-β being marker of metastatic dissemination (Moretti et al., 1999). Frequent co-expression of receptors with their ligands suggest that tumor cells-derived mediators not only affect immune or stromal cells, but also have autocrine functions in cancer cells (Mattei et al., 1994).

Chemokines and their receptors

The cytokine/chemokine spectrum of melanoma cells overlaps significantly with those used by neutrophils and macrophages for their chemoattractant-directed migration, adhesion and accumulation at the site of inflammation. These active mediators often exert similar functions in melanoma cells. In addition to the dynamic accumulation of cytokines with progression that was demonstrated by Moretti et al., a body of works suggests that melanoma cells express chemoattractant receptors CXCR1, CXCR2, CXCR4, CCR2, CCR7 and CCR9 and secret neutrophil chemoattractants CXCL1-3 [growth related oncogene (GRO) or melanoma growth stimulatory activity protein], CCL2 (monocyte chemotactic protein-1), CXCL8 (IL-8) and CCL5 (RANTES) (Balentien et al., 1991; Colombo et al., 1992; Graves et al., 1992; Koga et al., 2008; Mrowietz et al., 1999; Navarini-Meury and Conrad, 2008). CXCR2/CXCL1 receptor-ligand pair exerts chemotactic activity in uveal melanoma cells (Di Cesare et al., 2007; Owen et al., 1997). CXCL1 has been linked to immortalization and transformation of murine melanocytes (Owen et al., 1997). Macrophage-inflammatory protein-2 transgene, a murine analog of CXCL1, potentiates DMBA-induced melanomagenesis in transgenic mice, increasing the number of melanoma-bearing mice to 12%, as compared with 2% in control animals (Yang et al., 2001). Blocking antibody against CXCR2 or CXCL1 inhibit melanoma cell proliferation in vitro (Lawson et al., 1987; Norgauer et al., 1996). Interleukin-8 facilitates melanoma progression through several mechanisms including promotion of chemotaxis, tumor growth, migration and metastatic retention in distant organs (Melnikova and Bar-Eli, 2006).

Macrophage infiltration correlates with tumor stage and angiogenesis in malignant melanoma (Torisu et al., 2000). Both, melanoma cells and resident macrophages produce CCL2 (MCP-1), which is a powerful chemoattractant for monocytes, T-cells and natural killer cells. They also produce CCL5 (RANTES), which mostly activates monocytes/macrophages, mast cells and dendritic cells (Schall et al., 1990). CCL2 and CCL5 might be responsible, at least in part, for the remarkable accumulation of macrophages in melanoma. Indeed, tumor-derived CCL2 and 5 as well as CSF-1 may induce macrophages to produce TNF-α, IL-1α, IL-8, VEGF, MMP-9, cathepsins, FGF, epidermal growth factor receptor (EGFR) ligands and PDGF (Moretti et al., 1999; Navarini-Meury and Conrad, 2008). Strong association has been shown between the recruitment of Tie-2 (angiopoietin receptor)-expressing monocytes and tumor angiogenesis, and between MMP-9-secreting tumor associated macrophages and tumor cell invasion (Coussens et al., 2000; De Palma et al., 2005). In addition, TNF-α and/or IL-1α secreted by activated monocytes/macrophages can upregulate the production of angiogenic IL-8 and VEGF from melanoma cells (Torisu et al., 2000). These mediators play powerful role in angiogensis, tissue remodeling and the release of glycosaminoglycan-bound growth factors. Tumor macrophages also assume an immunosuppressive role by producing IL-10 and TGFβ (Byrne et al., 2008).

TGFβ

Transforming growth factor β is an essential regulator of immunosurveillance, angiogenesis, proliferation, differentiation, migration and cell survival (Elliott and Blobe, 2005). It can function as a potent inhibitor of normal cell proliferation and a tumor suppressor during early stages of carcinogenesis (Elliott and Blobe, 2005). However, in cancer, TGFβ and its isoforms loose their cytostatic functions and promote tumor angiogenesis, invasion and metastasis by stimulating expression of various metastatic markers, including MMPs, VEGF, IL-8 and integrins (Byrne et al., 2008; Javelaud et al., 2008; Lo and Witte, 2008; Padua and Massague, 2009). As shown by genome-wide transcriptome analysis, gene expression of more aggressive melanoma cell lines carry TGFβ signature (Hoek et al., 2006). Indeed, overexpression of the inhibitor of the TGF-β signaling pathway inhibitor, Smad7, blocked the capacity of melanoma cells to form bone metastases (Javelaud et al., 2008). Furthermore, TGFβ2 has been recently shown to home B16 murine melanoma cells to metastasize to the brain parenchyma (Zhang et al., 2009).

TGFβ– is one of the most potent immunosuppressive cytokines (Li et al., 2006; Rubtsov and Rudensky, 2007). It acts as a negative regulator of T-lymphocyte proliferation and activation, and inhibits antigen presentation by antigen-presenting cells. In addition, together with IL-2, it targets and promotes differentiation of the regulatory T cells (Treg), a subset of strongly immunosuppressive T cells found in melanoma and other tumors. This contributes to tumor escape from immunosurveillance.

Advanced and metastatic melanomas also overexpress another member of TGFβ superfamily, Nodal (Postovit et al., 2008). Nodal plays an instrumental role in the maintenance of melanoma tumorigenicity and cell plasticity, which is identified as cellular ability to express differentiation markers and perform functions of other cell lineages, thus promoting metastatic phenotype (Hendrix et al., 2003; Postovit et al., 2008). Interestingly, nodal expression can be observed in tumors as well as mast cells, which accumulate on the periphery of cutaneous melanoma. Mast cells are a rich source of various inflammatory mediators, including histamine and the thickness of mast cell layer correlates directly with melanoma invasiveness (Duncan et al., 1998; Ribatti et al., 2003a,b).

Adaptive immunity and melanoma

Melanoma cells have dual roles on the immune system. As described above, melanoma cells can activate or remodel their environment to secret survival and metastasis-favoring factors. On the other hand, melanoma cells can edit immunoreactive markers, secrete inhibitory immunomodulators and block the recognition and maturation of effector cells of the immune system (Kirkwood et al., 2008; Kusmartsev et al., 2005; Remmel et al., 2001). For example, in melanoma, more immature dendritic cells accumulate locally, and their mobility appears to be impaired by tumor cells (Kusmartsev et al., 2005; Remmel et al., 2001). The ability of tumor cells to conduct immune cells to suppress their cytotoxic responses enables the initiation and progression of metastasis. These interactions, as well as immunomodulatory approaches to melanoma treatment have been extensively reviewed elsewhere (Atkins, 2006; Bedikian et al., 2008; Kirkwood et al., 2008; Weber, 2008).

Stromal cells and keratinocytes in melanoma progression

Another component of melanoma tumor microenvironment is keratinocytes and stromal fibroblasts. As described by Herlyn and his group, alterations in keratinocyte-mediated contact growth inhibition in the skin is a critical step in malignant transformation of melanocytes (Haass and Herlyn, 2005). Melanoma cells escape from control by keratinocytes through several mechanisms, including downregulation of melanocyte–keratinocyte adhesion molecules such as E-cadherin, P-cadherin and desmoglein and upregulation of adhesion molecules involved in homotypic melanoma cell-melanoma cell and melanoma cell–fibroblast interactions such as N-cadherin, MCAM/MUC18 and zonula occludens protein-1. Growth factors like EGF, FGF, insulin-like growth factor-1 (IGF-1) or HGF have been shown to downregulate E-cadherin in tumor cells, thereby promoting tumor cell invasion and metastasis (Chiang and Massague, 2008; Lee and Herlyn, 2007). In addition, when activated by UV, keratinocytes release IL-1, 3, 6 and 8, secrete TNF-α, GM-CSF and generate proinflammatory biolipid PAF, possibly contributing to melanocyte proliferation and tumorigenesis (Marathe et al., 2005; Pei et al., 1998; Schwarz and Luger, 1989; Ullrich, 2005; Walterscheid et al., 2002). Within the tumor microenvironment, a rapid proliferation of fibroblasts is supported by PDGF, bFGF and TGF-β produced by melanoma and the inflammatory cells (Hsu et al., 2002; Lee and Herlyn, 2007). In turn, fibroblasts produce a series of growth factors such as insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), bFGF and TGFβ that further support the growth and proliferation of melanoma cells (Hsu et al., 2002; Lee and Herlyn, 2007). Fibroblasts also have been shown to remodel the matrix and form ‘tracks’ creating a leading edge for tumor cells invasion (Gaggioli et al., 2007).

Role of platelet activation and coagulation in melanoma metastasis

The progression step of metastasis involves lymphatic or vascular dissemination of cancer cells. Cancer-host cell interactions at this stage include formation of platelet-tumor cell aggregates and adhesion to endothelial cells. Platelets and molecular components of the coagulation system have been long recognized for their critical role in metastasis (Gasic et al., 1968; Karpatkin and Pearlstein, 1981; Nierodzik and Karpatkin, 2006; Nierodzik et al., 1998). Tumor cells are capable of coagulating blood and activating platelets. They produce and activate many components of the coagulation/platelet activation pathways – thrombin and its receptor PAR1, tissue factor (TF), fibrinogen, von Willebrand factor (vWF) and PAF and its receptor PAFR (Bromberg et al., 1995; Fischer et al., 1995; Nierodzik and Karpatkin, 2006; Timar et al., 2005). This phenomenon has been termed as platelet mimicry (Timar et al., 2005). The entire machinery assists cancer cells in accomplishing critical steps of vascular dissemination. Central to it, is the production of thrombin. Thrombin is generated on cell surface via reactions initiated by TF. Hypoxic tumor microenvironment greatly stimulates TF expression by endothelial cells, tumor associated macrophages and myofibroblasts, thereby sensitizing them to thrombin production (Ruf, 2007). Tumor cells also aberrantly express tissue factor (Nierodzik and Karpatkin, 2006). On contact with blood, tumor and other cells catalyze the production of thrombin on their surface (Nierodzik and Karpatkin, 2006).

Evidence has suggested that thrombin stimulates the migration of tumor cells into the vasculature, activates tumor cell adhesion to platelets, endothelial cells (EC), and subendothelial matrix proteins, increases tumor cell seeding and spontaneous metastasis, and stimulates tumor angiogenesis (Nierodzik and Karpatkin, 2006). Thrombin-induced activation of platelets plays an essential role in tumor cell extravasation. In addition, platelet–tumor cell interaction triggers early angiogenesis and development of collateral vessels via thrombin-stimulated synthesis and secretion of VEGF, PDGF, angiopoietin-1 and GRO-α, as well as biolipids lysophosphatidic acid and PAF from platelets and/or tumor cells (Boucharaba et al., 2004; Li et al., 2001; Mohle et al., 1997; Nierodzik and Karpatkin, 2006; Ruf and Mueller, 2006).

Thrombin is one of the most prominent angiogenic factors. Among multiple evidence, thrombin has been shown to induce the differentiation of endothelial cells into capillary structures on Matrigel, increase endothelial cell migration in vitro, as well as stimulate angiogenesis in vivo (Haralabopoulos et al., 1997). Recent data from our laboratory indicate that metastatic melanoma cells are better equipped to respond to thrombin and PAF stimuli provided by the tumor microenvironment for their growth advantage. Indeed, we found that human primary melanomas that subsequently develop metastatsis, as well as metastatic melanoma cells overexpress G-protein coupled receptors to thrombin and PAF, PAR1 (Tellez et al., 2007) and PAFR (Bar-Eli M, unpublished data). We will next summarize our recent observation on the role of these receptors in melanoma progression.

The role of thrombin receptor PAR1 in melanoma progression

Thrombin directly activates tumor cells through its seven transmembrane-spanning G-protein coupled receptor PAR1. Because of its serine protease activity, thrombin cleaves the N-terminus of PAR1. The new N-terminal peptide acts as a tethered ligand that binds the receptor and activates signaling via G-proteins. Protease-activated receptor 1 can also be activated by ligands other than thrombin such as factor Xa, granzyme A, trypsin and plasmin (Hansen et al., 2004; O’Brien et al., 2001; Ruf and Mueller, 2006). It has also been reported recently that PAR1 in breast cancer cells can be proteolytically cleaved and activated by MMP-1 (Boire et al., 2005).

In tumor cells, PAR1 stimulates the expression of adhesion molecules such as integrins αIIbβ3, αvβ5 and αvβ3 (Even-Ram et al., 2001; Senger et al., 1996; Wojtukiewicz et al., 1992, 1995). Indeed, thrombin-treated melanoma cells enhance their adhesion to platelets and fibronectin in vitro (Nierodzik et al., 1996). In various cell types, including vascular endothelial cells, PAR1 activation results in upregulation of gene products involved in invasion (MMP-2) (Zucker et al., 1995) and angiogenesis (IL-8, VEGF, bFGF, PDGF) (Cucina et al., 1999; Huang et al., 2001; Shimizu et al., 2000; Ueno et al., 1996). In human melanoma cells, thrombin acts as a growth factor and a weak mitogen (Fischer et al., 1995).

Our tissue analysis from patients demonstrated that PAR1 is overexpressed predominantly in malignant melanoma tumors and in metastatic lesions as compared with common melanocytic nevi (Massi et al., 2005). Furthermore, a significantly higher percentage of PAR1 positive cells was found in metastatic melanoma specimens as compared with both dysplastic nevi and primary melanoma specimens (Tellez et al., 2007). In addition to melanoma, overexpression of PAR1 has been observed in a variety of human cancers such as breast, lung, colon, pancreatic and prostate (Boire et al., 2005; Even-Ram et al., 1998; Kaushal et al., 2006; Rudroff et al., 1998; Wojtukiewicz et al., 1995). We further demonstrated that PAR1 is overexpressed in highly metastatic melanoma cell lines as compared with non-metastatic ones (Tellez and Bar-Eli, 2003; Tellez et al., 2003). Overexpression of PAR1 in the highly invasive and aggressive melanoma cell lines correlates with the loss of the activator protein-2α (AP-2α) transcription factor. Activator protein-2α is a tumor suppressor and a putative partner of p53, and the loss of its expression is a crucial event in the progression of human melanoma (Tellez et al., 2003). An inverse correlation between AP-2α and PAR1 expression was also established using a ‘progressive’ melanoma tissue microarray (Tellez et al., 2007). Stable downregulation of PAR1 expression in metastatic melanoma cells using lentiviral small hairpin RNA or systemic delivery of small interfering RNA (siRNA), incorporated into neutral liposomes, in tumor-bearing mice resulted in a significant decrease in both tumor growth and metastasis (Villares et al., 2008).

Overall, thrombin and PAR1 contribute to the acquisition of the metastatic phenotype of melanoma by facilitating tumor invasion and metastasis through the induction of cell adhesion molecules, matrix degrading proteases and stimulating the secretion of angiogenic factors into the melanoma tumor microenvironment.

The role of platelet-activating factor in melanoma metastasis

Activated platelets release another proangiogenic biolipid – PAF. Platelet-activating factor is a potent proinflammatory mediator and the first bioactive lipid ever identified (Benveniste et al., 1972, 1979; Blank et al., 1979; Demopoulos et al., 1979). It acts through the specific seven transmembrance spanning G-protein-coupled receptor (PAFR) (Bito et al., 1994; Nakamura et al., 1991; Ye et al., 1991). Studies on inflammatory cells, endothelial cells, keratinocytes, and various other types of cells demonstrated that a long lasting consequence of transient stimulation with PAF and PAF-like biolipids is the expression of inflammatory cytokines and mediators such as TNF-α, IL-6, IL-8, IL-10, COX-2, VEGF and inductible nitric oxide synthase (iNOS) (Countryman et al., 2000; Deo et al., 2004; Dy et al., 1999; Pei et al., 1998; Roth et al., 1996; Walterscheid et al., 2002). Platelet-activating factor is required for nuclear factor-κB activation in endothelial cells and macrophages stimulated with critical inflammatory mediators lipopolysaccharides, IL-1 and TNF-α (Seo et al., 2004).

The best described function of PAF in tumors is the induction of angiogenesis (Brizzi et al., 1999; Camussi et al., 1995, 1997; Montrucchio et al., 1994, 1997, 1998; Robert and Hunt, 2001). Numerous reports showed that PAF can activate endothelial cells directly, as well as mediate angiogenesis induced by other angiogenic factors such as bFGF, HGF, TNF-α and NO (Axelrad et al., 2004; Bussolati et al., 2000; Bussolino et al., 1995; Camussi et al., 1995, 1997; Deo et al., 2002; Montrucchio et al., 1997, 1998; Robert and Hunt, 2001). Importantly, VEGF-induced vascular permeability, P-selectin translocation and subsequent neutrophil adhesion requires PAF synthesis (Rollin et al., 2004; Sirois and Edelman, 1997). Angiopoietin-1 and -2 also stimulate PAF synthesis in endothelial cells (Maliba et al., 2006).

In addition to angiogenesis, PAF may play a role in melanomagenesis and metastasis. Platelet-activating factor receptor transgenic mice exhibited skin hyperplasia, hyperpigmentation, increase in the number of dermal melanocytes in the ear and tail (Ishii et al., 1997; Sato et al., 1999). They consequently developed melanocytic tumors late in life (Ishii et al., 1997; Sato et al., 1999). The PAFR transgene expression was detected in keratinocytes but not in melanocytes, suggesting that the progressive recruitment of melanocytes to the dermis was driven by keratinocytes, fibroblasts and mast cells (Ishii et al., 1997; Sato et al., 1999), all of which play a significant role in regulating skin homeostasis, melanoma growth and local malignant invasion (Lee and Herlyn, 2007). Platelet-activating factor was found to mediate systemic UV-induced immunosuppression in experimental animals (Marathe et al., 2005; Walterscheid et al., 2002). In humans, UV-induced immunosuppression is thought to contribute to melanomagenesis (De Fabo et al., 2004; Noonan et al., 2001).

In 1996, Im et al. provided the first evidence that IL-1α- and TNF-α-induced increase in B16F10 melanoma metastasis was augmented by a single intraperitoneal injection of PAF (Im et al., 1996). It was suggested that stimulation of endothelial cell adhesion was the primary mechanism for the observed pro-metastatic effect of PAF (Im et al., 1996). Later on, in a similar set of experiments, Fallani et al. demonstrated that B16F10 lung seeding depends on induction of MMP-9 in mouse lung tissue, which is caused by i.p. injection of PAF (Ko et al., 2007). Notably, stromal MMP-9 appears to be critical for tumor invasion and extracellular growth factor activation in many experimental tumor models including that of squamous carcinogenesis (Coussens et al., 2000). Further supporting its role in melanoma progression, PAF has been shown to promote B16F10 invasion of melanoma cells in vitro (Fallani et al., 2006).

In vivo experiments conducted in our laboratory demonstrated that the PAF receptor antagonist PCA4248 inhibits human melanoma metastasis to the lung (Melnikova et al., 2006). We also demonstrated that in human metastatic melanoma cell lines, PAF-induced phosphorylation of cyclic AMP-response element-binding protein (CREB) and activating transcription factor-1 (ATF-1) transcription factors, and stimulated the secretion and activation of MMP-2 and membrane type 1 MMP (MT1-MMP) (Melnikova et al., 2006). Metastatic melanomas overexpress CREB and ATF-1 (Rutberg et al., 1994). It is therefore possible that metastatic melanoma cells are better equipped to respond to PAF, as well as other cytokines and inflammatory mediators known to activate cAMP signaling pathway. This may amplify the metastatic phenotype whenever melanoma cells come into contact with PAF- and cytokine-secreting platelets, endothelial and inflammatory cells.

Conclusion

Environmental stresses within the genetically unstable primary melanomas result in selection and expansion of malignant cells. Overall, the inflammatory tumor microenvironment contributes to the malignant phenotype of melanoma. By secreting a variety of inflammatory cytokines, chemokines and lipid mediators, melanoma cells attract and co-opt inflammatory, stromal cells and platelets. Tumor- and inflammatory cells-derived bioactive mediators like TGFβ, GRO, thrombin, VEGF and PAF inhibit cytotoxic immune responses, promote tumor angiogenesis and cell proliferation, remodel the extracellular matrix, and facilitate vascular dissemination of cancer cells. In turn, metastatic melanoma cells are better equipped to respond to inflammatory stimuli and use them for their growth advantage (summarized in Figure 1). Consequently, mediators involved in cancer-related inflammation represent a target for innovative diagnostic and therapeutic strategies against metastatic melanoma.

Figure 1.

 Crosstalk between melanoma cells and the tumor microenvironment. Tumor cells release chemoattractants and other active mediators to stimulate migration and activation of macrophages, monocytes, granulocytes as well as other cellular components of innate immunity. In response, inflammatory cells generate an array of cytokines and growth factors as well as inhibitors of metastasis suppressor genes that induce angiogenesis, suppress cytotoxic immunity, stimulate tumor cell invasion and migration toward vessels and growth factors, and prime tumor cells for homing to secondary organs. In a metastatic site, they stimulate growth of metastatic colonies. Many of these functions in melanoma are also performed by mast cells. Activated keratinocytes and fibroblasts support melanomagenesis and/or tumor growth by releasing growth factors and inflammatory mediators. In addition, tumor cells express coagulation proteins and are capable of coagulating blood and activating platelets. Platelets provide catalytic surface for rapid generation of thrombin and release additional angiogenic and growth factors, thereby stimulating angiogenesis, tumor cell survival in circulation, extravasation and growth of metastatic colonies. Metastatic melanoma cells overexpress many pro-iflammatory and growth factor receptors, such as PAR1 and PAFR. They are therefore better equipped to respond to stimuli from the tumor microenvironment.

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