Chapter 1 Regulation of Melanoma Progression by the Tumor Microenvironment: The Roles of PAR-1 and PAFR Gabriel J. Villares and Menashe Bar-Eli Abstract The interaction of tumor cells and the host stroma (microenvironment) is essential for tumor progression and metastasis. The melanoma tumor microen- vironment has emerged within the last decade as a significant player in melanoma progression from the radial growth phase to the vertical growth phase by providing the necessary elements for growth, invasion and survival. Two receptors involved in this transition that are not only activated by factors from the tumor microenvironment but also in turn secrete factors into the microenvironment are the Protease Activated Receptor 1 (PAR-1) and the Platelet Activating Factor Receptor (PAFR). Throm- bin, which is abundant in the microenvironment milieu, activates PAR-1 causing cell signaling via G-proteins resulting in upregulation and secretion of gene products in- volved in adhesion (integrins), invasion (MMP-2) and angiogenesis (IL-8, VEGF, PDGF, bFGF). PAF, which is secreted by platelets, macrophages, neutrophils, en- dothelial cells and keratinocytes within the tumor microenvironment, will activate PAFR and signal through p38 MAPK to phosphorylate the CREB/ATF-1 transcrip- tion factors. Phosphorylation of CREB/ATF-1 results in overexpression and secretion of MMP-2 and MT1-MMP. Since only metastatic melanoma cells express activated CREB/ATF-1, we propose that they are better equipped to respond to PAF than their non-metastatic counterparts. These two G-protein coupled receptors that play major roles in melanoma progression highlight the crucial interactions between the tumor microenvironment and melanoma cells in the acquisition of the metastatic phenotype. Keywords Melanoma progression · Metastasis · Invasion · Angiogenesis · Thrombin · Protease activated receptor-1 · Platelet activating factor · Tumor microenvironment · Transcription factors · Metalloproteinase · G-protein coupled receptor Melanomas, as with all other cancers, are not comprised of a group of stand-alone cells with similar characteristics or capabilities. They are, however, comprised of M. Bar-Eli Department of Cancer Biology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA e-mail: [email protected] M. Bar-Eli (ed.), Regulation of Gene Expression in the Tumor Environment , C © Springer Science+Business Media B.V. 2008 1 2 G.J. Villares, M. Bar-Eli a group of heterogeneous cells that co-exist and interact with an infrastructure of other cells(keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and stromal components, all together known as the tumor microenvironment [1]. The tumor microenvironment is comprised of diverse cell types and elements such as extracellular matrix components (lamin, collagen), growth factors (VEGF, bFGF, thrombin), proteases and interleukins involved in invasion (MMP-2, IL-8, uPA) as well as varying concentrations of oxygen [2]. Furthermore studies have shown that inflammatory cells within the tumor microenvironment contribute to malignancies by releasing growth factors and chemokines [3]. It seems evident that the interac- tion of tumor cells and the host stroma (microenvironment) is, therefore, essential for tumor progression and, eventually metastasis. Following these same lines, the melanoma tumor microenvironment has emerged within the last decade as a key player in melanocyte transformation and transdifferentiation by providing these nec- essary elements for growth, invasion and survival [2]. In melanoma, there are several cell types within the tumor microenvironment that influence melanoma progression. For example, keratinocytes, which are found within normal skin, form interactions with melanocytes that are mediated by E-cadherins. Keratinocyte-regulated expression of E-cadherins affects the phenotypic behavior of melanocytes [1]. Disturbances in normal keratinocyte–melanocyte adhesion may con- tribute to malignant transformation by releasing melanocytes from contact-mediated regulatory controls leading to the advancement of melanoma [4]. Furthermore, ker- atinocytes induce several pro-angiogenic interleukins (IL-6, IL-8) as well as pro- inflammatory factors (PAF), which may also lead to melanoma progression [3]. Fibroblasts, once thought to play a minimal role in tumorigenesis, have been found to play an important role in potentiating tumor growth. A bi-directional model between melanoma cells and fibroblasts has been proposed in which melanoma cells first produce growth factors such as PDGF, bFGF and TGF-  to activate fi- broblasts and endothelial cells and, subsequently, fibroblasts produce a series of growth factors (IGF-1, HGS/SF, bFGF, TGF-  ) that further supports the growth and proliferation of melanoma cells [1, 4]. These paracrine signaling loops act to create an environmental niche conducive to tumor growth [1]. As can be seen, transformed melanocytic cells will recruit and interact with host cells in the microenvironment. These cells will then become activated and in turn elicit survival, proliferation and invasion signals [4]. The progression of melanoma from radial growth phase to vertical growth phase is accompanied by a myriad of molecular changes that are involved in this transition. Two of the factors involved in this transition that are not only activated by the tumor microenvironment but also in turn affect the microenvironment are the thrombin receptor (PAR-1) and the Platelet Activating Factor Receptor (PAFR). 1.1 PAR-1 Thrombin is a serine protease abundant in the tumor microenvironment milieu, which not only plays a crucial role in blood coagulation but also initiates various 1 The Roles of PAR-1 and PAFR 3 cellular responses through the activation of the thrombin receptor, PAR-1 [5]. In fact, activation of coagulation factors have been implicated in tumor growth and are hallmarks of advanced cancers [5, 6]. Studies have also demonstrated that tissue factor (TF) is constitutively expressed in melanoma cells and can activate throm- bin in a coagulation independent manner, thereby promoting melanoma metasta- sis [7, 8]. In fact, the hypoxic tumor microenvironment also induces TF expression by endothelial cells, tumor associated macrophages and myofibroblasts, thereby also augmenting thrombin in the tumor microenvironment [6]. Furthermore, thrombin-treated tumor cells (including melanoma) enhance their adhesion to platelets and fibronectin in vitro [9]. Thrombin also promotes endothe- lial cell alignment in Matrigel in vitro and angiogenesis in vivo [10]. It induces the differentiation of endothelial cells into capillary structures in a dose-dependent manner on Matrigel [10]. Furthermore, in the in vivo Matrigel system of angiogen- esis, there is a 10-fold increase in endothelial cell migration infiltration in response to thrombin. In lung epithelial cells, thrombin was also found to stimulate the ex- pression of PDGF [11]. Blocking of the coagulation pathways at the level of tissue factor, factor Xa, or thrombin, inhibits metastasis of human melanoma cells in SCID mice [8]. Thrombin can also activate several signal transduction pathways through its receptor. The thrombin receptor is a 7-pass transmembrane G-protein coupled re- ceptor. Unlike typical ligand-receptor interactions, thrombin does not activate PAR- 1 upon binding. Rather, it cleaves the N-terminus of PAR-1 at serine 42. Upon cleavage, the new amino terminal peptide acts as a tethered ligand that will now bind to the body of the receptor thereby causing cell signaling via G proteins resulting in upregulation of gene products involved in adhesion (  IIb  3 ,  v  5 ,  v  3 integrins) [12–14], invasion (MMP-2) [15], and angiogenesis (IL-8, VEGF, bFGF, PDGF) [11, 16–18]. This suggests that activation of the thrombin receptor may facilitate tumor invasion and metastasis through the induction of cell adhe- sion molecules, matrix degrading proteases, and stimulating the secretion of angio- genic factors into the melanoma tumor microenvironment, thus contributing to the metastatic phenotype of melanoma. In human melanoma cells, thrombin acts as a growth factor and is mitogenic, suggesting that signaling by PAR-1 is involved in the biological response of these cells [8]. PAR-1 can also be activated by ligands other than thrombin such as fac- tor Xa, granzyme A, trypsin and plasmin [19–21]. In addition to melanoma, over- expression of PAR-1 has been observed in a variety of human cancers, such as breast, lung, colon, pancreatic and prostate [5, 22–26]. It has also been recently reported that PAR-1 in breast cancer cells can also be proteolytically cleaved and activated by membrane metalloprotease-1 (MMP-1) [27]. Our laboratory has pre- viously demonstrated that PAR-1 is differentially expressed in melanoma cell lines with overexpression being found in highly metastatic cells as compared to non- metastatic melanoma cell lines [5, 28]. Moreover, we found that the overexpression of PAR-1 correlates with the loss of the activator protein-2  (AP-2  ), which is a crucial event in the progression of human melanoma [28]. In fact, we observed an inverse correlation between AP-2 and PAR-1 from primary melanoma cell lines 4 G.J. Villares, M. Bar-Eli Transcription Factors PDGF IL-8 uPA VEGF integrins MMP-2 MAPK PKB Thrombin Thrombin Thrombin G-proteins G-proteins Facilitation of tumor cell invasion and angiogenesis Fig. 1.1 Schematic representation of molecules involved in cell invasion and angiogenesis via activation of PAR-1, which is overexpressed in metastatic melanoma cells. Thrombin from the microenvironment cleaves the N-terminus of PAR-1 to activate the receptor. The tumor-promoting signals transduced by PAR-1 through G-proteins upregulate molecules involved in angiogenesis and invasion up to highly invasive and aggressive melanomas [28]. Overexpression of PAR-1 is predominantly seen in patients with malignant melanoma tumors and in metastatic lesions as compared to common melanocytic nevi and normal skin [29]. Further- more, our laboratory has found a significantly higher percentage of PAR-1 positive cells in metastatic melanoma specimens as compared to both dysplastic nevi and primary melanoma specimens [30] attesting to the role of PAR-1 in regulating tumor growth and metastasis of melanoma. As can be seen, activation of PAR-1 in melanoma cells through different ligands present in the tumor microenvironment will subsequently cause activation of the angiogenic and invasive gene products that are released into the tumor microen- vironment (Fig. 1.1). This will also cause activation of fibroblasts and endothelial cells that subsequently forms a more pro-invasive and proliferative environment for melanoma growth and metastasis. 1.2 PAFR As mentioned previously, it has been shown through genetic and functional exper- iments that inflammatory cells such as tumor-infiltrating monocytes/macrophages, neutrophils, mast cells, eosinophils, and activated T-lymphocytes contribute to