Cellular transformation, tumor growth, and metastasis are complex biochemical processes that involve, among other events, autonomous cell growth and host–tumor interactions. The molecular basis for many of these events is increasingly becoming understood, particularly in understanding deregulation of the cell cycle, programmed cell death, angiogenesis, extracellular matrix remodeling, and evasion of host immune surveillance. In melanoma, some of the mechanisms of cellular transformation have been identified and a portion of the genetics of familial melanoma have been determined (Greene et al, 1983;Halpern et al, 1991;Gruis et al, 1993;Hussussian et al, 1994;Newton-Bishop et al, 1994,2000;Platz et al, 1998,2000). Similarly, the mechanisms of melanoma metastasis are under investigation. The metastatic potential of melanoma contributes to the poor rate of survival following tumor invasion, together with the lack of effective systemic therapies. As in other cancers, matrix-degrading matrix metalloproteinases have been implicated in facilitating melanoma invasion and dissemination (MacDougall et al, 1999;Hofman et al, 2000). Additionally, an increasing body of literature is accumulating that identifies immune evasion as a critical step in melanoma disease progression (Bröcker et al, 1988;Giavazzi et al, 1990;Bottazzi et al, 1992;Kirkwood et al, 1996;Brinckerhoff et al, 2000;Torisu et al, 2000;Fishman et al, 2001). Downregulation of major histocompatibility complex class I expression (Fishman et al, 2001) and modulation of the inflammatory response via cytokines (Balkwill and Mantovani, 2001) have been described. Other recent reports suggest that expression of chemokines and chemokine receptors by melanoma may contribute to the ability to escape tumor surveillance and may partially explain preferential patterns of melanoma metastasis to sites such as lymph nodes, skin, and lungs (Muller et al, 2001).
Chemokines are structurally related, small (8–14 kDa) polypeptide signaling molecules (Zlotnik and Yoshie, 2000) that bind to and activate a family of seven transmembrane G protein-coupled receptors, more specifically, the chemokine receptors (Murphy, 1996). Chemokines were originally characterized by their ability to induce chemotaxis of leukocytes. They have since been shown to act on multiple cell types, including endothelial cells and tumor cells, where they elicit a broad range of cellular signals that may affect cell proliferation and the promotion of angiogenesis. The chemokines and their respective receptors are divided into the CXC, CC, C, and CX3C families, based upon the positions of their conserved two N-terminal cys residues. Their genes are clustered on genomic loci, including chromosome 4q12–q13 (CXC acting mainly on neutrophils), 4q21, and 17q11.2 (CC chemokines acting mainly on monocytes) (Zlotnik and Yoshie, 2000). Although a detailed review of chemokine biology is beyond the scope of this manuscript, comprehensive overviews may be found in recent articles (Belperio et al, 2000). The known human chemokines are listed in Table I with their systematic name, chromosome location and receptor(s).
Chemokine receptor gene clusters exist on chromosomes 2 and 3 (Zlotnik and Yoshie, 2000). There is significant redundancy and promiscuity in chemokine signaling as many chemokines share common receptors and, conversely, bind to multiple receptors. Chemokines and their receptors have been implicated in the regulation of various immune-mediated responses. For example, chemokines serve as attractant cytokines for T cells, and differential expression of chemokine receptors may contribute to the homing and activation of specific T cell subsets. Relevant to autoimmune, allergic, and dermatologic disease, it has been demonstrated that specific chemokine tissue expression profiles function to recruit differentially corresponding T cell subtypes (Sebastiani et al, 2001). Memory T cells differentially express chemokine receptors; T helper 1 lymphocytes expressing CCR5 and CXCR3, and T helper 2 lymphocytes CCR3, CCR4, and CCR8 (Gerber et al, 1997;Sallusto et al, 1997,1998;Bonecchi et al, 1998;D'Ambosio et al, 1998;Zingoni et al, 1998). More recent investigations demonstrate a role for chemokines in T cell differentiation (Luther and Cyster, 2001). In addition, an entirely distinct role for chemokine receptors has been identified with regard to infectious disease processes. An important example are the chemokine receptors CCR5 and CXCR4, present on monocytes and resting T cells, respectively, that bind to their ligands murine macrophage inflammatory protein (MIP) 1-
/
and RANTES (Regulated upon Activation Normal T cell Expressed and Secreted; CCR5) and SDF-1 (CXCR4). Interestingly, these chemokine receptors also act as coreceptors for human immunodeficiency virus, and this differential utilization partially explains the basis for human immunodeficiency virus tropism to specific cell types.
Such varied utilization of chemokine receptors, their activation and signaling are integrated into, and partially regulate, complex inflammatory and immune responses. Like other G-protein linked signal pathways, activation of chemokine receptors, characteristically by binding of their respective chemokine ligands, initiates a cascade of downstream biochemical events, including hydrolysis of phosphotidylinositol triphosphate, PI-3 kinase, protein kinase C activation, calcium influx, and activation of rac and Rho (reviewed inMukaida, 2000). The latter pathway is involved in cell migration and has recently been implicated through large genomic screens to be a participant in melanoma metastasis (Clark et al, 2000).
Chemokines have also been implicated in the cellular transformation, tumor growth, invasion, and homing of metastasis to distant sites (Figure 1) (Strieter et al, 1995) and in the host–tumor response. The recruitment of leukocytes to the site of a tumor represents a delicate balance between the host–anti-tumor response and the elaboration of inflammatory mediators that may induce or facilitate invasion by the primary tumor. The role of inflammation in cancer has recently been reviewed (Balkwill and Mantovani, 2001), with much evidence supporting the theory that, for many cancers, the presence of leukocytes in a primary tumor negatively impacts prognosis. Relevant to inflammation and tumor biology, some chemokines have an effect on angiogenesis. More specifically, certain CXC chemokines, including CXCL8 [interleukin (IL)-8], contain a three amino acid ELR motif (glutamine–leucine–arginine) (ELR+) between the N-terminus and the first cysteine, and function as potent promoters of angiogenesis (reviewed byBelperio et al, 2000). Other members of this family include Gro, -, and -
(see below), epithelial neutrophil activating protein-78 (ENA-78), granulocyte chemotactic protein-2 (GCP), and platelet basic protein-2 (PBP). CXC chemokines lacking this ELR motif (ELR–) are angiostatic, and include platelet factor-4 (PF-4), interferon- inducible protein (IP-10), and monokine induced by interferon- (MIG). The relevance of the angiogenesis-promoting or angiogenesis-inhibiting properties of these chemokines to melanoma genesis and tumor progression is as yet not established. In fact, the prognostic significance of angiogenesis in melanoma, unlike certain other tumors, is unclear. In one study, macrophage infiltration has been found to correlate with melanoma tumor stage and angiogenesis, with the inflammatory mediators IL-1 and tumor necrosis factor (TNF)- implicated in the pathogenesis of this effect (Torisu et al, 2000). Although the role of chemokines was not specifically addressed in this study, the leukocyte-derived inflammatory cytokine TNF- is angiogenic and is also capable of inducing CXC ELR+ chemokines (Schröder et al, 1990). Their role on angiogenesis aside, given the role of chemokines as potent chemoattractants for inflammatory cells, as well as effectors in other cell types, recent attention has focused upon the expression of chemokines and their receptors in tumorigenesis and metastasis.
Figure 1.
Chemokine role in cellular trans formation, tumor growth, invasion and homing and metastasis to distant preferential organs. Reprinted with permission from: Strieter RM: Chemokines: Not just leukocyte chemo attractants in the promotion of cancer. Nat Immunol 2:285–286, 2001.
Full figure and legend (210K)CXCL8/IL-8
The first described chemokine, CXCL8, was originally identified as a neutrophil chemotactic and activating peptide isolated from mononuclear cells, a cytokine involved in the acute inflammatory response. It was purified from supernatants of lipopolysaccharide-stimulated human monocyte cultures (Schröder et al, 1987;Walz et al, 1987;Yoshimura et al, 1987;Mrowietz et al, 1999). CXCL8 mRNA is expressed by monocytes, natural killer cells, T lymphocytes, neutrophils, endothelial cells, keratinocytes, fibroblasts, and smooth muscle cells (Baggiolini et al, 1989;Schröder et al, 1987,1990;Walz et al, 1987;Matsushima et al, 1988;Kulke et al, 1998). There is no known mouse homolog. CXCL8 has been shown to induce lysosomal degranulation, generation of the free radical burst, and upregulation of certain adhesion molecules (reviewed inMukaida, 2000); thus acting both as a chemoattractant and upon binding to its receptor, a neutrophil activator. CXCL8 binds with high affinity to two distinct receptors, CXCR1 and CXCR2, primarily expressed on neutrophils (Holmes et al, 1991;Murphy and Tiffany, 1991), but also on other cell types, including keratinocytes (Kulke et al, 1998). CXCR1 (IL8RA) binds CXCL8 (IL-8) and another CXC chemokine NAP-2 (Petersen et al, 1994;Zlotnik and Yoshie, 2000); CXCR2 is a more promiscuous receptor, binding multiple CXC ELR+ chemokines, including CXCL1, and CXCL8 (Zlotnik and Yoshie, 2000). It has been postulated that CXCR2 may, in fact, mediate the angiogenic activity of ELR+ chemokines (Belperio et al, 2000). Like many of the chemokine receptors, binding of ligand to CXCR2 not only leads to receptor activation but may also regulate receptor availability through receptor desensitization (Mueller et al, 1997), clathrin-mediated receptor endocytosis (Yang et al, 1999), and subsequent receptor degradation (Mueller et al, 1995).
Melanoma cells express CXCL8 mRNA (Colombo et al, 1992) and secrete the protein (Förster et al, 1991). In contrast to neutrophils, CXCR2 is the major CXCL8 receptor in melanoma. CXCL8 has been implicated in melanoma progression through several mechanisms, including the promotion of tumor cell growth and migration (Wang et al, 1990;Norgauer et al, 1996). In vitro, CXCL8 has been described as a melanoma cell mitogen (Schadendorf et al, 1993). In one study, inhibition of CXCL8 via anti-sense oligonucleotides or neutralizing CXCL8 monoclonal antibodies decreased melanoma cell proliferation in culture suggesting a role for CXCL8 as a melanoma growth factor (Schadendorf et al, 1993). Separate studies utilizing different melanoma cell lines, however, showed that neutralizing antibodies to another chemokine, CXCL1 (see below), but not CXCL8, similarly inhibited cell proliferation (Fujisawa et al, 1999); this suggested that melanoma may utilize different chemokine ligands, among other proteins, to support growth. CXCL8 mediates the haptotactic migration of melanoma cells (Wang et al, 1990) and induces matrix metalloproteinase-2, facilitating extracellular matrix degradation and migration (Luca et al, 1997).
Chemokine studies in mice may be difficult to translate to the human system. In the murine system, IL-8 does not exist and the most likely functional murine equivalent to human IL-8 is MIP-2 (Hogaboam et al, 1999). Interestingly, however, murine MIP-2 is structurally similar to another chemokine, Gro- (see below) and murine Gro is known as KC (Table I). Nonetheless, CXCL8 expression also correlates with metastatic potential in murine melanoma tumor models using human cell lines (Singh et al, 1994). In a nude mouse model, induction of ultraviolet-induced melanoma cell tumorigenesis and metastasis correlate with CXCL8 mRNA and protein expression (Singh et al, 1995). Using the same model, it was demonstrated that expression of CXCL8 was regulated by the tissue microenvironment (Gutman et al, 1995). CXCL8-expressing human melanoma cells injected into nude mice and harvested after metastasis to the subcutis, spleen, and liver differed in their chemokine expression levels. Melanoma cells metastatic to the skin consistently expressed higher levels of CXCL8 than cells that engrafted in the liver, and cross-over experiments demonstrated that highly expressing metastatic cells isolated from the skin expressed decreased amounts of CXCL8 after reinjection and isolation from liver metastases. In vitro experiments with these cells suggested that these findings were the result of induction of CXCL8 expression by keratinocyte-derived IL-1 and conversely, inhibition of CXCL8 by hepatic-derived transforming growth factor-. These studies suggest a role for paracrine regulation of chemokines in the clinical behavior of melanoma in vivo. In fact, recent related work (Muller et al, 2001) demonstrates that chemokine receptor expression by tumor cells may direct their homing to metastatic sites (Figure 1).
Understanding chemokine and chemokine receptor regulation may contribute to our understanding of the metastatic patterns of melanoma. As indicated above, CXCL8 is constitutively expressed by some, but not all, melanoma cells in vitro (Schadendorf et al, 1993). CXCL8 expression is upregulated by the inflammatory cytokines IL-1 and TNF- via AP-1 and NF-
B response elements in its promoter (Singh et al, 1995;Mohler et al, 1996). Conversely, interferons and inhibit this response (Singh and Varney, 1998). Potentially relevant to tumorigenesis, the CXCL8 promoter also contains an Oct1 repressor element (Wu et al, 1997) that is deactivated through retinoblastoma protein expression (Zhang et al, 1999). Parallel mechanisms of chemokine receptor expression regulation are similarly beginning to be understood (Mueller et al, 1997;Nieto et al, 1997;Sica et al, 1997;Sozzani et al, 1998;Romagnani et al, 2001;Zella et al, 1991)
CXCL1–3/MGSA-
–
/GRO-–
CXCL1 protein was originally purified as an autocrine growth factor MGSA (melanoma growth stimulatory activity protein) from supernatants of Hs29T melanoma cell cultures (Richmond et al, 1985;Richmond and Thomas, 1986;Bordoni et al, 1990) and described as the product of the growth-related oncogene (gro) locus (Anisowicz et al, 1987;Richmond et al, 1988), which is identical to the CXCL1–3 gene cluster on chromosome 4q12–q13. Later, it was purified from lipopolysaccharide-activated monocytes as a neutrophil chemoattractant (Schröder and Christophers, 1989) and from similarly stimulated human umbilical vein endothelial cells (Schröder et al, 1990). CXCL1 mRNA expression has also been demonstrated in keratinocytes of psoriatic skin (Kulke et al, 1998). CXCL1 is clustered on three homologous genes (, , and ; CXCL1, 2, and 3, respectively) on chromosome 4 (Haskill et al, 1990). Some background on nomenclature regarding this group of related chemokines is necessary to understand the literature. CXCL1, 2, and 3 are also known as MGSA-/Gro-, MGSA-/Gro and MGSA-/Gro-, respectively. As previously described, the murine homolog is Gro/KC (Zlotnik and Yoshie, 2000). Suggesting a role in melanocyte transformation, overexpression of CXCL1, CXCL2, or CXCL3 in immortalized melanocytes results in their ability to form tumors (Balentien et al, 1991;Owen et al, 1997).
Whether there are independent biologic roles for the three ligands (CXCL1–3) is uncertain. In addition to its role as a purported autocrine growth factor for melanoma (Richmond et al, 1988), CXCL1 promotes angiogenesis in the rat cornea model, with a less robust angiogenic response generated by CXCL2 and 3 (Strieter et al, 1995). CXCL1–3 promote neutrophil chemotaxis with similar efficacy (Baggiolini et al, 1994), but demonstrate differing levels of potency with respect to calcium mobilization (an important indicator of receptor activation), which is dependent upon cell type (Geiser et al, 1993). CXCL1–3 bind with high affinity to a common receptor, CXCR2 (Haskill et al, 1990;Mueller et al, 1994), with CXCL1 having the highest affinity (Hammond et al, 1996). Conversely, CXCL1 binds with a lower affinity to CXCR1 (CXCL8 receptor, see above) (Lee et al, 1992). The biologic significance of multiple receptors for CXCL1 is unclear, as blocking antibodies to either CXCL1 or CXCR2 inhibit melanoma cell growth in vitro (Lawson et al, 1987;Norgauer et al, 1996), indicating that CXCR2 may be necessary and sufficient for melanoma growth signaling by CXCL1 in these cells. Mice lack expression of CXCR1, but do express CXCR2 that bind CXC chemokines (Lee et al, 1995). As previously discussed, human CXCR1 transcripts are primarily expressed in neutrophils, melanoma cells express transcripts for CXCR2 (Muller et al, 2001).
CXCL1 regulation occurs at both the transcriptional and translational level, although again, in a cell-type-specific manner. Relevant to cells of melanocyte lineage, CXCL1 mRNA is constitutively expressed in cultured nevocytes from benign and "dysplastic" nevi as well as melanoma cells, but is not detectable in cultured primary melanocytes (Bordoni et al, 1990). This tight regulation of CXCL1 mRNA expression in "normal" melanocytes, along with deregulated expression in transformed cells (melanoma) in vitro is analogous to the expression profile of cellular oncogenes (Campisi et al, 1984). In cells of melanocyte lineage, CXCL1 protein, unlike CXCL1 mRNA, is constitutively expressed only by melanoma cells (through activation of NF-B;Shattuck-Brandt and Richmond, 1997) and protein release can be induced in nevocytes as well as melanocytes by exogenous growth factors (Bordoni et al, 1990). One proposed explanation for these findings is that CXCL1 mRNA contains 3' regulatory sequences that, in a nontransformed cell, normally signal for rapid turnover, making steady-state detection in normal melanocytes difficult (Bordoni et al, 1990). It has also been proposed that the increased level of mRNA observed in melanoma cells is due not only to this increased mRNA stability but also to transcriptional regulation in response to exogenous cytokines and growth factors, including CXCL1 itself (Anisowicz et al, 1987;Richmond et al, 1988). Finally, translational regulation of CXCL1 has also been demonstrated, where mRNA–polyribosome association is dependent upon specific growth factors, exogenously derived for melanocytes and endogenous in melanoma cells (Bordoni et al, 1990). When multiple melanoma cell lines were examined, it was similarly found that regulation of chemokine protein expression involves both transcription and mRNA stability, as well as mechanisms affecting translation and secretion (Yang and Richmond, 2001). In the context of these findings, the pattern of expression of the chemokine CXCL1, in the cellular transformation of the melanocyte presents a multitiered process that appears to involve a stepwise regulation of transcriptional and translational control. Stimulation by exogenous factors such as ultraviolet irradiation, as well as those yet to be determined, may serve as initiating events in the circumvention of normal growth control regulatory mechanisms in the melanocyte (Singh et al, 1995). If one proposes that melanocyte transformation progresses from either the melanocyte directly to melanoma or, in certain instances, the melanocyte to nevus cell to melanoma, determining such cellular and molecular characteristics that accompany this transformation may aid our understanding of the process. In fact, G protein-coupled receptor overexpression has previously been linked to cellular transformation in another cell type. Overexpression of the Kaposi's sarcoma virus chemokine-like G protein-coupled receptor (KSHV-G protein-coupled receptor), having similarities to CXCR2, in hematopoietic cells in mice leads to the spontaneous development of angioproliferative lesions resembling Kaposi's sarcoma (Yang et al, 2000).
CCL5/RANTES
Human and murine CCL5, or RANTES, share sequence homology and chromosomal location (17q11.2) (Zlotnik and Yoshie, 2000). RANTES, similar to the other chemokines, was originally identified as a leukocyte chemoattractant protein (Schall et al, 1990;Schall, 1999). More recently, however, its role in binding to one of its receptors, CCR5, one of the human immunodeficiency virus cell entry receptors on CD4+ T cells (Ward and Westwick, 1998) has received much attention. RANTES is produced by CD8+ T cells, platelets, epithelial cells, and fibroblasts, typically in response to inflammatory mediators (Appay and Rowland-Jones, 2001). Its receptors, CCR1, 3, 4, and 5 may be found on a variety of cell types, including T cells, monocytes, dendritic cells, and mast cells (Schall, 1999), and upon binding its ligand, mediate the effects of this chemokine at nanomolar concentrations (Nieto et al, 1997). The expression of multiple receptors on one cell type (i.e., the macrophage) may potentially explain the rapid receptor activation, that is, in fact, a characteristic of this chemokine. Interestingly, at micromolar concentrations, RANTES acts as a T cell mitogen that is independent of chemokine binding to its G protein-coupled receptor (Appay et al, 2000). Also unique to RANTES is its ability to self-aggregate on the cell surface, having potential implications in cell–cell cross-linking (Appay and Rowland-Jones, 2001).
With respect to melanoma, a subset of melanoma cells has been shown to express CCL5/RANTES (Mrowietz et al, 1999). In these cells, expression of CCL5 was 5–50-fold higher than CXCL8 and was upregulated by TNF-. CCL5 expressing melanoma cells formed increasingly aggressive tumors in nude mice in a concentration-dependent fashion. CCL5-expressing tumors were highly chemotactic for leukocytes, and transplantation experiments demonstrated that CCL5 expression favored tumor progression.
CCL2/MCP-1
Monocyte chemotactic protein-1 (MCP-1, CCL2) is another highly conserved CC chemokine (Yoshimura et al, 1989). CCL2 is a potent monocyte chemoattractant protein, isolated from cultured smooth muscle vascular cells (Valente et al, 1988) that also recruits natural killer cells (Allavena et al, 1994) and certain T lymphocyte populations (Carr et al, 1994). CCL-2 is expressed in a variety of "inflammatory" conditions (Kuziel et al, 1997), including atherosclerosis (Yla-Herttuala et al, 1991), and its role in tumorigenesis and progression, including melanoma, has been widely investigated (Salcedo et al, 2000;Sica et al, 2000;Ueno et al, 2000;Nesbit et al, 2001). The predominant chemokine receptor is CCR2 (Kurihara et al, 1997). Mice deficient in this receptor demonstrate a severe reduction in leukocyte adhesion, monocyte extravasation (Kuziel et al, 1997) and recruitment (Boring et al, 1997). In ovarian carcinoma, tumor-associated macrophages isolated from ascites or solid tumors demonstrated defective CCR2 expression and the investigators propose that this may represent one mechanism of the defective immune response seen in advanced cancers (Sica et al, 2000).
Early studies demonstrated the expression of monocyte chemotactic protein (CCL2/MCP-1) by melanoma in vivo (Graves et al, 1992), a finding of some interest as macrophages are commonly found in melanoma as the quantitatively dominant leukocyte type (Bröcker et al, 1988;Van Ravenswaay-Claasen et al, 1992). Tumor-associated macrophages elaborate cytokines and growth factors, some of which are angiogenic and angiostatic, which can either promote or inhibit tumor progression (Mantovani et al, 1992) Along these lines, previous studies on gene transfer of human CCL2 into murine melanoma cells demonstrated that CCL2-producing melanoma clones showed a 2-fold increase in the percentage of tumor-associated macrophages in vivo as well as a 2-fold decrease in the rate of tumor growth, associated with increased survival (Bottazzi et al, 1992). This relationship is not straightforward, however, as inoculation of smaller numbers of CCL2-expressing cells resulted in increased tumorigenicity. As previously noted, one group has found that macrophage infiltration in human melanoma correlates with tumor stage and angiogenesis (Torisu et al, 2000). Interestingly, we have found that CCL2, as well as certain other chemokines, induce matrix-degrading matrix metalloproteinase secretion in macrophages, potentially relevant to tumors having macrophage infiltration (LAC, unpublished observations). Clearly, these relationships are complex, and further studies are needed to determine the role of these factors in the host immune response that is specific to melanoma.
Chemokine receptors and melanoma
The coordinated secretion of chemokines, and their binding to receptors on the cell surface, directs leukocyte cell homing to specific tissue sites. The secretion of chemokines and their receptor expression by dendritic cells during maturation, migration, and antigen presentation in the lymph node provides an elegant example of this homing process (Dieu et al, 1999;Förster et al, 1999;Zlotnik and Yoshie, 2000) as reviewed byZlotnik and Yoshie (2000). Similar processes have been suggested in the homing of transformed cells to specific sites (Figure 1). In fact, a role for chemokine receptors has recently been demonstrated in the homing of metastatic tumor cells in breast cancer and melanoma (Muller et al, 2001). Investigators demonstrated that malignant cells express distinct and nonrandom patterns of chemokine receptors that guide their metastatic destination determined by the expression levels of chemokines by target organs. Specifically, melanoma cells were found to express high levels of CXCR4, CCR7, and CCR10 mRNA as compared with normal primary melanocytes. The respective ligands for these receptors, CXCL12/SDF-1, CCL21/6Ckine, and CCL27/CTACK, exhibit peak levels of expression in lymph node, lung, liver, bone marrow, and skin, in accordance with the primary metastatic destinations of melanoma. CCL27/CTACK is a novel skin-specific chemokine (Morales et al, 1999) whose expression can be upregulated by the inflammatory cytokines IL-1 and TNF- (Homey et al, 2000). The receptor for CCL27, CCR10, is expressed by melanocytes, melanoma tumor cells, dermal fibroblasts, endothelial cells, T cells, and Langerhans cells (Homey et al, 2000), supporting the role for CCL27/CCR10 interaction in this, as well as other, immune-mediated processes of the skin (Rottman et al, 2001).
In the above breast cancer model of metastasis, signaling through CXCR4 or CCR7 induces actin polymerization, pseudopod formation, and chemotactic migration in tumor cells. Treatment of SCID mice with anti-CXCR4 neutralizing monoclonal antibody following intravenous or orthotopic injection of human breast cancer tumor cells significantly inhibited lymph node and lung metastases (Muller et al, 2001). In these investigations, however, it was unclear whether this effect was achieved by disruption of metastatic homing and/or primary inhibition of tumor cell growth. Prior studies using human melanoma cells in nude mice have demonstrated that treatment with a synthetic peptide that competitively inhibits CXC chemokine binding can inhibit pulmonary metastases in vivo (Fujisawa et al, 1999). Similarly, preliminary studies in a murine melanoma model using CCR-7-transduced B16 melanoma cells demonstrated a 200-fold increase in draining lymph node tyrosinase-related protein-1 mRNA (indicative of B16 tumor cell homing) than their vector-transduced littermates (Gonzolez et al, 2001). Given the multiple steps in the pathway toward metastasis, including tumor growth, cell migration, and engraftment, the actual mechanism for chemokine receptor dependent homing is undoubtedly more complex than mere ligand recognition and most likely includes the local induction of proteases and adhesion molecules, as well as other factors.
Chemokines and melanoma therapy
In addition to melanoma chemokine receptor expression and potential homing to preferential metastatic sites, the increasing data on the association of chemokines with dendritic cell maturation and lymph node homing (Zlotnik and Yoshie, 2000) is in itself also relevant to melanoma therapy. As previously cited, dendritic cell expression of chemokine receptors such as CCR7 has been shown to mediate dendritic cell migration to lymph nodes, an essential event for antigen-specific T cell activation (Förster et al, 1999;Sallusto et al, 2000) and an important mechanism for mounting an immune response to certain tumors. Dendritic cells also express specific chemokines such as CCL17 whose receptor CCR4 has been associated with the T helper 2 phenotype, suggesting that the particular chemokine secreted determines the subset of T cells targeted for recruitment and expansion (O'Garra et al, 1998;Imai et al, 1999). Separate investigations have determined that this recruitment of dendritic cells to tumor tissue involves chemokines and receptors that are distinct from those employed in leukocyte recruitment (tumor-associated macrophages) (Hillibrand et al, 1999). Further research into mechanisms of dendritic chemokine expression and receptor regulation as it relates to tumor cell antigen presentation may have significant clinical benefit relevant to melanoma vaccine adjuvant therapy (Brinckerhoff et al, 2000).
In the area of infectious disease, chemokine receptor blockade strategies are currently under investigation in human immunodeficiency virus chemokine receptor recognition and cell entry (Moore and Trkola, 1997;Hadida et al, 1998;Berger et al, 1999). Although this does not represent an entirely analogous biologic system, further investigations linking tissue and tumor cell chemokine and receptor expression to cellular transformation and tumor metastasis may provide a scientific basis for similar blocking strategies to be investigated in the area of cancer biology and metastasis.
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