CANCER AND THE CHEMOKINE NETWORK

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1 CANCER AND THE CHEMOKINE NETWORK Fran Balkwill A complex network of chemokines and their receptors influences the development of primary tumours and metastases. New information about the biological role of chemokines in these processes is providing insights into host tumour interactions, such as the role of the leukocyte infiltrate, and into the mechanisms that determine the metastatic potential and site-specific spread of cancer cells. Chemokine-receptor antagonists are showing promise in animal models of inflammation and autoimmune disease. Could manipulating the local chemokine network have therapeutic benefits in malignant disease? CHEMOKINE This term refers to a family of chemotactic cytokines. These small, inducible cytokines are leukocyte-subtype-selective chemoattractants. Some chemokines also have other cytokine-like actions on cells including stimulation of cell proliferation. HYPOXIA This term describes the low levels of oxygen that are found in many areas of advanced cancers and are associated with a poor prognosis. Cancer Research UK Translational Oncology Laboratory, Barts and The London, Queen Mary s Medical School, Charterhouse Square, London EC1M 6BQ, UK. frances.balkwill@ cancer.org.uk doi: /nrc1388 CHEMOKINES are chemotactic cytokines that cause the directed migration of leukocytes, and are induced by inflammatory cytokines, growth factors and pathogenic stimuli 1 3.Chemokine signalling results in the transcription of target genes that are involved in cell invasion, motility, interactions with the extracellular matrix (ECM) and survival 4.Chemokine signalling can coordinate cell movement during inflammation, as well as the homeostatic transport of haematopoietic stem cells (HSCs), lymphocytes and dendritic cells. Directed migration of cells that express the appropriate chemokine receptor occurs along a chemical gradient of ligand known as the chemokine gradient allowing cells to move towards high local concentrations of chemokines (FIG. 1).The small (8 10 kda) chemokine proteins are classified into four highly conserved groups CXC, CC, C and CX3C based on the position of the first two cysteines that are adjacent to the amino terminus. More than 50 chemokines have been discovered so far (FIG. 2) and there are at least 18 human seven-transmembrane-domain chemokine receptors (BOX 1). In general, these receptors, which belong to the G-protein-coupled receptor family, bind to more than one type of chemokine (FIG. 2).However, six receptors bind to only one cytokine: CXCR4, CXCR5, CXCR6, CCR6, CCR9 and CX3CR1. The profile of chemokine-receptor expression on an individual cell is determined by its lineage, stage of differentiation, and microenvironmental factors such as chemokine concentration, the presence of inflammatory cytokines and HYPOXIA. The pattern of chemokinereceptor and ligand expression in a tissue generally correlates with the numbers and types of infiltrating cell that are present. The chemokine gradient that attracts infiltrating cells can be created by different cell populations in a tissue. In infections, the first cells that produce chemokines are probably tissue leukocytes, but fibroblasts, endothelial cells and epithelial cells (both normal and malignant) are all able to produce chemokines and generate a chemokine gradient. Although originally identified on leukocytes, functional chemokine receptors are also found on endothelial cells 5 and on some epithelial cells, particularly those that have been malignantly transformed 6 8.But what is the biological significance of the chemokine receptors that are found on malignant cells? Immune-cell infiltration of tumours the leukocyte infiltrate is a characteristic of cancer, and many human cancers have a complex chemokine network that influences the extent and phenotype of this infiltrate, as well as tumour cell growth, survival, migration and angiogenesis 9.This article explores two aspects of the cancer chemokine network: the role of chemokines in controlling leukocyte infiltration in cancer, and the influence of chemokines on the metastatic potential and site-specific spread of tumour cells. Chemokines and the leukocyte infiltrate in cancer Tumours are not just masses of cancer cells; most solid tumours, whether they are of epithelial, mesothelial or haematopoietic origin, contain many non-malignant 540 JULY 2004 VOLUME 4

2 Summary Chemokines are a subset of cytokines that cause the directed migration of leukocytes along a chemical gradient of ligand, known as the chemokine gradient. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. Many cancers have a complex chemokine network that influences the immune-cell infiltration of a tumour, as well as tumour cell growth, survival and migration, and angiogenesis. Immune cells, endothelial cells and tumour cells themselves express chemokine receptors and can respond to chemokine gradients. Chemokines are a key determinant of the macrophage and lymphocyte infiltrate of human cancers and might contribute to T-helper 2 cell polarization. Malignant cells from different cancer types have different profiles of chemokine-receptor expression, but CXCR4 is most commonly found; at the last count, cells from 23 different cancer types expressed this receptor. Mutations in genes that alter levels of hypoxia-inducible factor, or gene-fusion events, can induce CXCR4 in cells that do not normally express this receptor. CXCR4 is also transiently increased by factors such as hypoxia, vascular endothelial growth factor and oestrogen in the tumour microenvironment. Studies of human cancer biopsy sampler and mouse cancer models show that cancer cell chemokine-receptor expression is associated with increased metastatic capacity. Preliminary laboratory data show that chemokine-receptor antagonists inhibit macrophage infiltrates, can induce tumour growth arrest or apoptosis, and prevent metastatic spread. Research into the cancer chemokine network is revealing parallels between the pathology of inflammation and malignancy, parallels that enhance our understanding of both types of disease and indicate new approaches for treatment. ASCITES An increased accumulation of fluid in the peritoneum owing to cancer, which both increases the accumulation of peritoneal fluid and inhibits its reabsorption. In some cancers, such as ovarian cancer, the ascitic fluid contains variable numbers of tumour cells and inflammatory leuokytes, as well as pico- to nanomolar levels of many cytokines and chemokines. CD8 + T LYMPHOCYTE A T cell bearing the CD8 + cell-surface glycoprotein, which recognizes major histocompatibility complex class I molecules on target cells. CD8 + T cells are usually cytotoxic T cells. REED STERNBERG CELLS A clonal population of transformed germinal-centre B cells that forms the malignant component in Hodgkin s lymphoma. T H 2 CELLS T-helper 2 (T H 2) lymphocytes help B cells make antibody and suppress the action of cytotoxic T cells. Interleukin (IL)-4, IL-5, IL-10 and IL-13 are examples of T H 2-produced cytokines. stromal cells 10.Indeed, stromal cells sometimes outnumber cancer cells. The predominant stromal cells that are found in cancers are macrophages, lymphocytes, endothelial cells and fibroblasts; in addition, eosinophils, granulocytes, natural-killer cells and B cells are also found in some tumour types Many ofthe stromal cells are therefore leukocytes and make up the leukocyte infiltrate. The numbers and types of cell that make up the leukocyte infiltrate in solid tumours are related to the local production of chemokines by both the tumour cells and stromal cells. CC chemokines, for instance, are important determinants of the macrophage and lymphocyte infiltrate in human carcinomas of the breast, cervix and pancreas, as well as sarcomas and gliomas 10,15.In epithelial ovarian cancer, a network of CC and CXC chemokines is found in both the solid tumour (FIG. 3) and the associated ASCITES.The predominant infiltrating cells are macrophages and CD8 + T LYMPHOCYTES 13.Specifically, CCL2 localizes to epithelial areas of the tumour 16, and its levels of expression correlate with the numbers of lymphocytes and macrophages that localize in the same area 13. CCL5 localizes with tumour-infiltrating leukocytes and the CCL5 concentrations reflect the extent of CD8 + T-lymphocyte infiltration 13. Pico- to nanomolar levels of several chemokines CCL2, CCL3, CCL4, CCL5, CCL8, CCL22, CXCL2 and CXCL12 are found in ascitic fluid from this disease, and there is a direct correlation between CCL5 concentration in the ascitic fluid and the number of T cells 17. Another example is Hodgkin s lymphoma, in which the malignant REED STERNBERG CELLS are a minor component of the tumour, and the bulk comprises an inflammatory infiltrate of lymphocytes, eosinophils, fibroblasts, macrophages and plasma cells. The expression of a range of T-HELPER 2 (T H 2) cytokines and chemokines by the Migration MMPs Chemokines Inflammatory cytokines Macrophage Activation Tumour cell Chemokine receptor Cytokines Chemokine bound to receptor Figure 1 Diagram of a chemokine gradient in a cancer. An inflammatory cytokine induces chemokine production by an epithelial tumour cell. A macrophage that expresses the corresponding receptor binds the chemokine and undergoes rapid cytoskeletal rearrangement. This is followed by induction of a transcriptional programme that favours cell migration for example, induction of matrix metalloproteinases (MMPs) and cell survival. The cell migrates towards a higher concentration of chemokine. As the chemokine concentration increases, the chemokine receptor can be downregulated. Alternatively, the chemokine-receptor profile of the cell might change under the influence of other inflammatory cytokines or local conditions, such as hypoxia. This might help to retain the cell at the site of inflammation or to direct it elsewhere. NATURE REVIEWS CANCER VOLUME 4 JULY

3 CCL2,7,8,12,13 CCL5,7,11,13,15,24,26,28 CCL2,3,5,17,22 CCL3,4,5,7,14,15,16,23 CCL3,4,5,8 CXCL9,10,11 CCR1 CCR2 CCR3 CCR4 CCR5 CCL19,21 CXCL1,2,3,5,6,7,8 CXCR3 CCR7 CCL1,4,17 CXCR2 CCR8 CXCL6,8 CXCR1 Shared CCR10 CCL27,28 CCL2,5 CXCL1,8 Duffy Non-signalling XCR1 XCL1,2 CCL2,3, 5,7,17,22 D6 CCR6 CCL20 KSHV Viral Specific CCR9 CXCL1,8, 10,12 E1 CX3CR1 CCL25 CCL11 UL12 CXCR4 CX3CL1 CCL2,3,4,5 US28 ECRF3 CXCR6 CXCR5 CXCL12 CCL2,3,4,5,7 CXCL13 CXCL1,5,7 CXCL16 Figure 2 The chemokine wheel. This illustration explains the ligand-binding patterns of the seven-transmembrane domain G-protein-coupled human chemokine receptors. Receptors CXCR1 CXCR3, CCR1 CCR5, CCR7, CCR8, CCR10 and XCR1 all bind several chemokines. By contrast, CCR6, CCR9, CX3CR1 and CXCR4 CXCR6 bind only one ligand each. Duffy and D6 are considered to be deceptors, as they bind ligands but do not signal, thereby acting as a negative feedback for chemokine responses. T H 1 CELLS T-helper 1 (T H 1) lymphocytes are at the other end of the functional spectrum to T H 2 lymphocytes. They activate macrophages, produce T H 1 cytokines such as interferon-γ and interleukin 2 and help in the production of specific cytotoxic T lymphocytes. Reed Sternberg cells invokes this reactive infiltrate and contributes to the local suppression of T H 1-CELL-mediated cellular immune responses, thereby preventing the host immune system from destroying the tumour because polarized T H 2 cell responses are generally ineffective against tumours and viruses 18. Chemokine-receptor expression on leukocyte infiltrates in malignant disease. Despite the fact that chemokines are abundantly expressed in tumours, there is little information concerning chemokinereceptor expression on tumour-associated leukocytes. In epithelial ovarian cancer, the infiltrating leukocytes show a surprisingly limited profile of chemokinereceptor expression. CCR1 is the only CC chemokine receptor that is consistently expressed on leukocytes in solid ovarian tumours 19.This lack of chemokine receptors could be a result of their downregulation by the high levels of chemokine ligands and inflammatory cytokines, such as tumour-necrosis factor-α (TNF-α), which are present in the tumour 20. Physiological conditions, such as hypoxia, could also alter chemokine-receptor profiles and the response to chemokines in the tumour microenvironment 21.In the ascitic disease that is characteristic of advanced ovarian cancer, leukocytes express a wide range of chemokine receptors at levels that are comparable to those found on leukocytes circulating in the peripheral blood stream 17.This observation could reflect differences in the microenvironment between the solid ovarian tumour and the ascitic liquid form of this malignancy. Furthermore, restricted expression of chemokine receptors in solid tumours could allow precise control of the amount and movement of the infiltrating leukocytes in the tumour when several chemokine gradients are present. What is known about the chemokine network of human ovarian cancer is summarized in FIG.3. Chemokines and the cancer leukocyte infiltrate. Chemokines contribute to T H 2 polarization in tumours other than Hodgkin s disease, and this strategy might 542 JULY 2004 VOLUME 4

4 Box 1 The naming of chemokines and their receptors The term chemokine, which is short for chemotactic cytokine, was coined in 1992 to define a large family of proteins with chemotactic activity. Individual members had names such as monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein-1α (MIP-1α), stromal cell-derived factor-1 (SDF1), eotaxin and RANTES (regulated-on-activation normal T-cell expressed and secreted), which reflected the circumstances of their discovery, but not always their most important role. To add to the confusion, some chemokines had several names, which reflected the diversity of their actions. In 1999, when more than 40 different human chemokines had been discovered, a systematic nomenclature was adopted based on a structural classification that related to the number and spacing of conserved cysteines. This distinguished four main groups CXC, CC, C and CX3C with the letter L used to denote a ligand, followed by a number. The chemokines that are discussed in this article include CCL2, CCL5, CCL18, CCL20, CCL21, CXCL1, CXCL2, CXCL8 and CXCL12. The synonyms for the CC chemokines are MCP1 (CCL2), RANTES (CCL5), PARC/MIP4/DCCK1 (CCL18), MIP-3α/LARC/ST38 (CCL20) and 6Ckine/SLC/exodus-2 (CCL21). The CXC chemokines are also known as GRO-α/N51/KC (CXCL1), Gro-β/MIP-2α (CXCL2), IL-8 (CXCL8) and SDF-1α/SDF-1β/PBSF (CXCL12). The naming of chemokine receptors followed the systematic chemokine nomenclature. Although most chemokine receptors recognize more than one chemokine, they are almost always restricted to a single subclass (FIG. 1).Human CC and CXC chemokine receptor names consist of the root CCR or CXCR, respectively, followed by a number. Further information on chemokine nomenclature can be found in REFS 2,3. TYPE-2 MACROPHAGES Also known as M2 macrophages. Type-2 macrophages have an important role in inflammatory circuits that promote tumour growth and progression. They are polarized cells that are induced by T H 2 cytokines and make immunosuppressive cytokines, such as interleukin-10. ANTIGEN-PRESENTING CELLS (APCs). Cells that posses antigen and present antigen fragments to lymphocytes in order to initiate an immune response. Dendritic cells are the most potent APCs. MATRIX METALLOPROTEINASES A family of proteolytic enzymes that degrade the extracellular matrix and have important roles in tissue remodelling and tumour metastasis. PROLIFERATIVE INDEX Describes the numbers of cells that are proliferating in a tissue. Measured by staining cells with antibodies such as Ki67. help the tumour to subvert the immune system by establishing a microenvironment of immune cells and cytokines that suppress any specific anticancer responses 10,18.In Kaposi s sarcoma,for instance, the human Kaposi sarcoma-associated herpes virus (KSHV) encodes three chemokines MIPI, MIPII and MIPIII that selectively attract polarized T H 2 cells 22. Chronic exposure of the leukocytes to high concentrations of chemokines in the tumour microenvironment can activate TYPE-2 MACROPHAGES,which release the immunosuppressive cytokines interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) 23. Type-2 macrophages also release CCL2, which could contribute to T H 2-polarized immunity 10,24.In addition, the tumour microenvironment can inhibit the migration and function of DC1 dendritic cells, which regulate T H 1 differentiation, and this can also suppress specific immune responses. In ovarian cancer, tumour cell production of the chemokine CXCL12 can weaken immunity by attracting and encouraging the survival of CXCR4-expressing dendritic-cell precursor-2 (predc2) cells, and by altering predc1 distribution, immunity and stimulation of fibrosis 25. As a result, the dendritic cells remain immature and ANTIGEN PRESENTATION can not occur. In addition to being immunosuppressive, infiltrating leukocytes might contribute to tumour progression by producing MATRIX METALLOPROTEINASES (MMPs) as well as growth and angiogenic factors 14,26.In fact, CC chemokines, such as CCL2, CCL4 and CCL5, induce MMP9 production in macrophages 27. MMPs, including MMP9, are found at higher levels in many cancers and are important in ECM remodelling. MMPs that are produced by stromal and tumour cells function together to aid tumour cell migration and invasion. However, there is much debate concerning the relevance of infiltrating leukocytes to cancer growth and progression 14.A recent review of the prognostic significance of tumour-associated macrophages (TAMs) cited 15 different studies in a range of cancers 28.Ten of these studies concluded that the greater the macrophage infiltrate, the worse the prognosis; however, three showed a better prognosis and two showed no prognostic correlation with macrophage infiltrate. The balance of opinion would favour a role for TAMs in cancer progression and metastasis, rather than inhibition of tumour growth or destruction of tumour cells 14,26,29.This is supported by experiments in a genetic model of breast cancer,where the macrophage infiltrate was found to promote tumour invasion and metastases 30. As first proposed by Mantovani and colleagues 29, there is probably a balance in tumours between macrophages that promote and inhibit cancer. There is some evidence to indicate that the presence of tumour-infiltrating lymphocytes is a favourable prognostic sign. In ovarian cancer, 5-year survival was significantly increased (38% versus 4.5%) in patients whose tumour biopsy samples contained CD3 + tumourinfiltrating lymphocytes compared with patients whose biopsy samples lacked these cells 31.However,CD4 + T cells increased invasion and disease progression in an experimental model of skin carcinogenesis by recruiting neutrophils and macrophages into premalignant lesions, which promoted a chronic inflammatory microenvironment with the production of MMP9 and the stimulation of angiogenesis 32.As with macrophages, there is probably a balance between tumour-promoting and tumourinhibiting lymphocytes in most malignancies. If tumour-infiltrating leukocytes are able, in some instances, to promote cancer, then the local production of chemokines that attract leukocytes could be a poor prognostic sign. This is the case in human breast cancer, where levels of CCL5 and CCL2 correlate with tumour progression 33,34 and there is a positive correlation between the extent of the macrophage infiltrate, lymph-node metastasis and clinical aggressiveness 34,35. In oesophageal squamous-cell carcinoma, CCL2 expression has been associated with the extent of macrophage infiltration, tumour cell invasion and tumour vascularity 36. In xenografts of melanoma cell lines, tumour formation depends on the level of CCL2 secretion and monocyte infiltration 37. Low-level CCL2 secretion by melanoma cells resulted in modest monocyte infiltration and the cells grew as tumours, whereas cell lines producing high levels of CCL2 invoked a massive macrophage infiltrate and tumour destruction. These results could explain the conclusions from a recent study of chemokine production in pancreatic cancer.similar to breast and ovarian cancer, pancreatic cancer produces the CC chemokine CCL2, which is secreted by tumour cells both in vitro and in vivo. In patients who were undergoing surgical resection of pancreatic tumours, high serum levels of CCL2 were a good prognostic indicator and were associated with a lower PROLIFERATIVE INDEX in the tumour, although there was no association between CCL2, macrophages and dissemination of the cancer 38.Similarly, CCL5 expression by tumour cells in NATURE REVIEWS CANCER VOLUME 4 JULY

5 Chemokine receptor Pre-dendritic cell CD8 lymphocyte Stromal cell Tumour cell CXC chemokines CC chemokines Macrophage Tumour cells have chemokine receptors Infiltrating leukocytes are not the only cells that respond to chemokine gradients in cancers; cancer cells themselves can express chemokine receptors and respond to chemokine gradients 6,41.In fact, organ-specific metastasis might be governed, in part, by interactions between chemokine receptors on cancer cells with metastatic potential and chemokine gradients in target organs. There are similarities, for instance, between the transport of dendritic cells to lymph nodes, which is regulated by chemokine gradients, and the lymphatic spread of cancer cells 42.Malignant cells from different cancer types express different profiles of CC and CXC chemokine receptors. However, the chemokine receptor that is most commonly found on human and murine cancer cells is the CXC receptor CXCR4. The role of this ligand receptor pair in cancer progression is the best understood of all the chemokine receptor interactions in malignant cells. AUTOCRINE A form of bioregulation in which a secretory factor affects the cell from which it was secreted. Figure 3 The chemokine network of human epithelial ovarian cancer. Epithelial tumour cells produce inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), and chemokines, such as CCL2 and CXCL12. Leukocytes bearing the appropriate chemokine receptors are attracted to the malignant cells, and are themselves stimulated to produce more chemokines and inflammatory cytokines. Autocrine and paracrine networks are established that attract more leukocytes, especially T-helper 2 (T H 2) lymphocytes, type-2 macrophages and pre-dendritic cells. The inflammatory leukocytes contribute to tumour growth and progression by producing proteases, angiogenic factors, growth factors and immunosuppressive cytokines. patients with non-small-cell lung cancer was associated with an active lymphocyte response and was a positive predictor of survival 39. The chemokine network of specific cancers Although it seems that chemokine production in cancers has a direct relationship with the nature and extent of the leukocyte infiltrate, we do not have a clear picture of the overall chemokine repertoire of an individual human cancer type. There is even less information on the chemokine-receptor profile of the infiltrate. Tools are now available to conduct a more comprehensive study of the chemokine network of the tumour microenvironment, and levels of circulating chemokines should also be investigated in patients with cancer. Unlike other cytokines, chemokine proteins can be detected at picogram levels in the blood of healthy individuals. If chemokine production is higher in cancers than in normal tissues, it is possible that circulating chemokines could be useful tumour or prognostic markers. In addition to the pancreatic cancer study reported above, one recent report indicated that higher levels of the chemokine CCL18 might be a useful marker for acute lymphoid leukaemias 40. CXCR4 CXCL12 and tumour cells Tumour cells from at least 23 different types of human cancers of epithelial, mesenchymal and haematopoietic origin express CXCR4 (REF. 8). Not all cancerous cells in the primary tumour are CXCR4 positive. In ovarian and non-small-cell lung cancer, for instance, only a sub-population of cells expresses this receptor 7,43.When it has been possible to study freshly isolated tumour cells for example from leukaemias and cells that have been isolated from ovarian cancer ascites the CXCR4 receptor is functional and various signalling pathways are activated. CXCL12 is the only known ligand for CXCR4. It is found in primary tumour sites in lymphoma and glioma, and ovarian and pancreatic cancer 44 47, and at sites of metastasis in breast and thyroid cancer, neuroblastoma and haematological malignancies 6,48,49. However, CXCL12 is also constitutively expressed in many normal organs, including the bone marrow. Most importantly, interactions between CXCR4 and CXCL12 have a crucial role in the migration and patterning of many embryonic cell lineages 50,51, the mobilization of HSCs 52 and the transport of naive lymphocytes 53,54. Expression of CXCR4 by tumour cells. CXCR4 expression is low or absent on normal breast, ovarian and prostate epithelia 6,7,55, although it is present on normal colonic epithelium 56.Therefore, CXCR4 expression is generally a characteristic of the malignant epithelial cell and not its normal counterpart. But, how are the malignant cells programmed to express functional receptors? Recent studies have indicated a range of mechanisms. As part of its AUTOCRINE action on breast cancer cells, vascular endothelial growth factor (VEGF) can induce the expression of CXCR4 (REF. 57). In addition, CXCL12 is also activated in human ovarian and breast cancer cell lines that are positive for oestrogen receptor-α 58 ;thishormone upregulates CXCR4 expression, and the mitogenic effects of oestradiol are neutralized by the addition of CXCL12 antibody. Furthermore, in breast cancer, the transcription factor nuclear factor-κb (NF-κB), which controls cell motility, upregulates CXCR4 (REF. 59). 544 JULY 2004 VOLUME 4

6 a c b d a poor prognosis. Re-expression of wild-type VHL in these cells downregulates CXCR4. Further proof of the link between hypoxia and CXCR4 came from a study that showed positive regulation of CXCR4 by hypoxia in a range of normal and malignant cells 61.Increased CXCR4 expression and increased migration to CXCL12 was dependent on HIF activation and CXCR4-transcript stabilization. Gene-fusion events have also been associated with the induction of CXCR4 expression, as transfer of the PAX3 FKHR fusion gene into embryonal RHABDOMYOSARCOMA cells activates CXCR4 expression 62. Therefore, in malignant tumours, mutations in genes that alter levels of HIF or, alternatively, gene-fusion events can induce CXCR4 in cells that do not normally express this receptor. In addition, CXCR4 might be transiently increased by factors such as hypoxia, VEGF and oestrogen in the tumour microenvironment. Tumour cell Tumour-associated macrophage Blood vessel Chemokines Normal cells RHABDOMYOSARCOMA A highly lethal mesenchymal cancer of early childhood, which is most commonly found in the eye, head and neck region, and the genitourinary tract. Stromal cell CD8 lymphocyte Chemokine receptor Other chemokines and cytokines Figure 4 The significance of chemokine-receptor expression on cancer cells. a Cancer cells in a primary tumour have metastatic potential, but do not always express chemokine receptors. b Some cancer cells acquire chemokine-receptor expression by gene mutation, gene fusion or local conditions, such as hypoxia. c If local levels of the specific chemokine ligand are low, chemokine-receptor-expressing cancer cells can now respond to high levels of ligand at sites of metastasis and migrate towards the chemokine gradient. Alternatively, the acquisition of chemokine receptor might make tumour cells more likely to invade and spread. d Chemokine ligand at the metastatic site can deliver anti-apoptotic and proliferative signals, and induce tumournecrosis factor-α. This cytokine can initiate a pro-tumour inflammatory network in the surrounding stroma. Hence, the chemokine ligand encourages the tumour cells to survive and grow. In renal-cell carcinoma, the mechanism of CXCR4 upregulation involves mutation of the von Hippel- Lindau (VHL) tumour-suppressor gene 60.CXCR4 expression can be regulated by hypoxia-inducible factor (HIF) and wild-type VHL suppresses CXCR4 expression by targeting HIF for degradation in normoxic conditions. VHL mutations are characteristic of clear-cell renal carcinoma and increased expression of CXCR4, which is found in tumours with mutated VHL, is associated with Migration and invasion. Hypoxia and VHL mutations are poor prognostic signs in cancer, and are associated with advanced and metastatic disease. Activation of CXCR4 stimulates directed migration of cancer cells and increases their invasion through Matrigel and monolayers of endothelial cells, bonemarrow stromal cells and fibroblasts 8,46,47,62.IfCXCR4 is associated with metastatic activity in vivo,expression of CXCR4 and/or its receptor CCL12 might be higher in metastases compared with primary tumours. This has been reported to be the case in two different cancer types. In a comprehensive series of more than 600 prostate cancer specimens 55,CXCR4 protein expression increased with tumour aggressiveness and levels of CXCL12 were higher in metastatic lesions than in the primary tumour. High CXCR4- expressing breast tumours also produced more extensive nodal metastasis compared with low CXCR4-expressing tumours, but there was no significant correlation with blood-borne metastasis 63. Results from mouse cancer models also support the hypothesis that CXCR4 expression is associated with metastatic capacity. Human ovarian cancer cell lines that were engineered to express high levels of CXCR4, or those that naturally expressed high levels of the receptor, established extensive local and distant metastases when grown intraperitoneally in nude mice (H. Kulbe and J. Wilson, manuscript in preparation). Prostate cancer cells expressing high levels of CXCR4 produce larger subcutaneous xenografts, with increased blood-vessel density and muscle invasion, compared with wild-type cells 64.In a mouse model of melanoma, CXCR4-transfected cells showed a tenfold increase in lung metastases compared with CXCR4-negative cells 65, and there was increased adhesion of CXCR4-expressing cells to dermal and pulmonary microvascular endothelial cells. Furthermore, in this model, the β1-integrins were essential for the CXCR4-mediated metastasis of melanoma cells to the lungs 66. Human cancer cells that have metastasized in nude mice also show increased CXCR4 expression. Sub-populations of breast cancer cells with increased NATURE REVIEWS CANCER VOLUME 4 JULY

7 SCID Severe combined immunodeficiency. Mice with this defect do not make T cell or antibody responses. Tumour cells from another species can be grown in these mice without rejection. LEUKOCYTOSIS The state of increased numbers of leukocytes circulating in the peripheral blood. metastatic abilities in nude mice are characterized by the overexpression of four genes, one of which is CXCR4 (REF. 67). CXCR4 expression and CXCL12- mediated migration are increased in tumour cells that grow in the mammary fat pad compared with parental cells that are grown in culture 59.Finally, when non-small-cell lung cancer cells were grown in severe combined immunodeficiency (SCID) mice, 99% of the cells in metastases expressed CXCR4, compared with only 35% of the cells in the primary tumour 68.Taken together, these experiments indicate that cancer cells with high levels of CXCR4 are more likely to form metastases. However, they do not prove that CXCR4-expressing cancer cells in primary tumours spread only because they migrate towards gradients of CXCL12 in other organs. The ability of tumour cells to use CXCR4 CXCL12 during the process of metastasis might depend on chemokine gradients in the primary tumour, as well as common sites of spread, levels of functional receptor, and the presence of other cytokines and proteases that can cleave ligand and receptor (FIG. 4). The detailed studies that have been carried out on human and murine HSC mobilization and homing should guide future research in this area. Cancer cell survival. CXCL12 might also be involved in tumour cell growth and survival. In several types of cancer including glioma, melanoma, ovarian, smallcell-lung, renal and thyroid cancers CXCL12 can stimulate the proliferation and/or survival of CXCR4-expressing cancer cells when they are grown under suboptimal conditions, such as low serum concentrations 45,47,55,69. This adaptation might allow tumour cells to grow in distant sites that would normally be less favourable. For instance, when cancer cell CXCR4 expression was inhibited in a colon cancer metastasis model, CXCR4-deficient cells colonized the lungs to the same extent as control cells, but did not proliferate in the distant location 70. CXCL12 stimulation of ovarian cancer cell lines and primary cells that were isolated from ascitic ovarian disease also induced the pro-inflammatory cytokine TNF-α 47,which is implicated in tumour stromal communication, establishment of tumour-cytokine networks and promotion of tumour growth 71,72. A metastatic cancer cell arriving in a new location could therefore initiate cytokine and/or chemokine signalling in the surrounding stroma by producing this important inflammatory cytokine. Taken together, these data indicate that CXCR4-expressing metastatic cells might spread to many sites in the body, but only become established at metatastatic locations where high levels of CXCL12 are found. However, CXCL12 ligand can be detected in several primary tumours 8, which raises doubts about this hypothesis. The significance of CXCL12 expression at the primary tumour site. In follicular lymphoma, pancreatic cancer, ovarian cancer, glioma and astrocytoma, protein and/or mrna for the chemokine have been detected in the tumour cells themselves (reviewed in REF. 8). The significance of this is not fully understood. By analogy with its role in the bone marrow 52,high levels of CXCL12 should retain the tumour cells in situ rather than encourage their dissemination. CXCL12 is a survival factor for CXCR4-positive HSCs, and high levels of localized CXCL12 inhibit HSC movement out of the bone marrow 73.Preclinical and clinical studies with stem-cell-mobilizing agents, such as granulocyte colony-stimulating factor and cyclophosphamide, cause a profound decrease in bone-marrow levels of CXCL12, owing to its degradation by neutrophil elastase, cathepsin G and MMPs 74,75. HSCs that are mobilized to move out of the bone marrow generally also express decreased levels of CXCR4, although the data are conflicting. Low CXCR4 levels could maintain stem cells in the systemic circulation, whereas increased CXCR4 levels would mediate attraction to other tissues in which CXCL12 levels are increased owing to tissue injury or infection, as well as homing back to the bone marrow and re-engraftment 76. Direct evidence of the role of CXCR4 CXCL12 in the transport of CD34 + HSC progenitors in humans comes from clinical trials with the CXCR4 antagonist AMD3100 (REFS 77,78). This antagonist frees HSCs from the influence of CXCL12, as a single dose produces a rapid generalized LEUKOCYTOSIS that is associated with an increase in peripheral blood CD34 + cells. At the highest dose of AMD3100, the number of circulating progenitors increases 15- to 20-fold. Expression of CXCL12 in primary tumours could therefore be expected to retain the tumour cells at this site, which would encourage their growth and survival, but discourage invasion and metastasis (FIG. 4). However, this is likely to be a dynamic process. In some areas, protease production could degrade CXCL12 and local conditions could increase expression of the receptor. Therefore, CXCL12 CXCR4 interactions might encourage sub-populations of tumour cells to leave the primary site, even when CXCL12 is present. Finally, in kidney cancer, levels of CXCL12 mrna were actually lower in the tumour areas compared with adjacent normal tissue 79,which indicated that CXCL12 might function as a tumoursuppressor-like molecule. More detailed studies of the localization of CXCL12 mrna, and measurements of protein levels in primary tumours and metastases, are required. Other chemokine receptors on cancer cells In most, but not all, of the cancer types studied, CXCR4 is co-expressed with other CC or CXC chemokine receptors on malignant cells (see TABLE 1 for some examples from the published literature) and some of these receptors have also been implicated in cancer progression. The CC chemokine receptor CCR7 has been found in breast, gastric, non-small-cell lung and oesophageal squamous cancer, and chronic lymphocytic leukaemia (CLL) 6, CCR7 expression correlates with metastatic potential and poor prognosis, and its ligand CCL21 is found at high levels in the lymph nodes that drain many cancers. 546 JULY 2004 VOLUME 4

8 Table 1 Some of the chemokine receptors that are expressed on cancer cells Chemokine Cancer cell expression Normal-cell expression receptor CXCR4 23 different haematopoietic and solid cancers* HSC, thymocytes, T cells, B cells, immature and mature dendritic cells, some endothelium, macrophages and neutrophils CCR3 T-cell leukaemia T cells, basophils, eosinophils and plasma cells CCR4 T-cell leukaemia Thymocytes, NK cells, immature dendritic cells, skin-homing T cells and T H 2 T cells CCR5 Breast cancer cell lines Thymocytes, B lymphocytes, immature and mature dendritic cells, and macrophages CCR7 Breast cancer, CLL, gastric cancer, B cells, T cells and mature dendritic cells non-small-cell lung and oesophageal cancer CCR10 Melanoma Plasma cells and skin-homing T cells CXCR2 Melanoma Macrophages, eosinophils and neutrophils *Breast cancer, ovarian cancer, glioma, pancreatic cancer, prostate cancer, acute myeloid leukaemia, B-chronic lymphocytic leukaemia, B-lineage acute lymphocytic leukaemia, non-hodgkin s lymphoma, intraocular lymphoma, follicular centre lymphoma, chronic myelogenous leukaemia, multiple myeloma, thyroid cancer, colorectal cancer, squamous-cell carcinoma, neuroblastoma, renal cancer, astrocytoma, rhabdomyosarcoma, small-cell lung cancer, melanoma and cervical cancer. CLL, chronic lymphocytic leukaemia; HSC, haematopoietic stem cells; NK, natural killer; T H 2, T-helper 2. CCR3 has been implicated in the recruitment and retention of malignant T cells in the skin 84.The chemokine receptor CCR4 is frequently expressed in adult T-cell leukaemia and its ligand is found on cutaneous endothelium 85.Human melanoma cells express CCR10, which binds to CCL27 and CCL28; these chemokines are expressed at high levels in skin, which is a common site of metastasis for this cancer 6.Pancreatic cancer cells express CCR6 and proliferate in response to its ligand, which is the CC chemokine CCL20 (REF. 86). Some breast cancer cell lines, such as MCF-7, express the chemokine receptor CCR5 (REF. 87). Stimulation of these cells with the CCR5 ligand CCL5 caused the transcriptional activation of p53 target genes, such as CDKN1A (which encodes WAF1, also known as p21) and MDM2,by p38 mitogen-activated protein kinase (MAPK) activation. Approximately 1% of the Caucasian population is homozygous for the CCR5* 32 polymorphism that renders this receptor non-functional. The allelic frequency and genotype of CCR5* 32 was investigated in 547 patients with primary non-metastatic breast cancer, and patients with the largest tumour size at presentation showed a significant increase in 32 allelic frequency. Furthermore, disease-free survival was shorter in individuals whose tumours expressed wild-type p53 and carried the 32 allele. The CXC chemokines CXCL1 and CXCL8 are constitutively produced by melanoma cells, but not by untransformed melanocytes 88.Melanoma cells also show higher levels of the CXCR2 receptor for these chemokines, and autocrine chemokine stimulation increases survival, proliferation and tumour cell migration. The ability of tumour cells to proliferate in response to CXC chemokines seems to have been exploited by KSHV. The KSHV genome encodes a G-protein-coupled receptor that signals constitutively and is structurally similar to CXCR2. Cells that were transfected with a mutated CXCR2 that signalled constitutively were transformed. Transgenic mice that overexpressed the KSHV G-protein-coupled receptor, under the control of the CD2 promoter, developed lesions that were similar to Kaposi s sarcoma 89. Although few studies have comprehensively characterized the range of chemokine receptors found on cancer cells, it is apparent that a single chemokine receptor can influence the direction of spread of a cancer cell. It is certainly possible that different chemokine receptors are involved in metastases of the same cancer to different sites. This has been shown experimentally in the B16 mouse melanoma model, in which sites of metastasis can be controlled by transfection of the cancer cells with different chemokine receptors: transfection with CCR10 caused metastasis to the skin 90,transfection with CCR7 caused cells to spread to the lymph nodes 91 and transfection with CXCR4 caused lung metastasis 65. Implications for new cancer treatments It is clear that chemokines and their receptors are involved in malignant progression and that a better understanding of chemokine signalling in this process could lead to new therapeutic strategies for cancer. With this in mind, it is possible that drugs that are being tested in inflammatory and autoimmune disease that target the chemokine network 92 could also be useful as cancer biotherapies. As the chemokine network is complex, it is unlikely that an individual chemokine antagonist would have a powerful action in cancer, and inhibitors of chemokine-inducing cytokines, such as TNF-α,could also be useful. Chemokine and cytokine antagonists have the potential to inhibit tumour-promoting leukocyte infiltrate, metastatic spread and angiogenesis 72,93. Recent preclinical studies have reported anticancer activity of chemokine-receptor antagonists in several murine cancer models. In the mouse model of breast cancer, tumour cells express CCL5, and the CCL5 receptors CCR1 and CCR5 are expressed by the leukocyte infiltrate. Daily NATURE REVIEWS CANCER VOLUME 4 JULY

9 treatment of tumour-bearing mice with an antagonist of CCR1 and CCR5 Met-CCL5 (REF. 94) led to modest anticancer effects at doses similar to those that have activity in animal models of inflammatory disease. Specifically, the total number of inflammatory cells and the proportion of infiltrating macrophages were decreased in tumours treated with Met-CCLS 95. Inhibiting chemokine-receptor signalling on tumour cells has the potential to induce growth arrest or apoptosis, and prevent invasion and metastasis. Proof of this principle came from a series of experiments in which cancer cells, transfected with CXCL12 that contained an endoplasmic-reticulum retention sequence, bound CXCR4 in the endoplasmic reticulum, thereby preventing its surface expression 96.When T-hybridoma cells were transfected with this construct and injected intravenously into mice, they no longer metastasized to distant organs 96.Furthermore, when similar experiments were carried out with colorectal cancer cells, lung and liver metastases were greatly reduced 70.However,CXCR4-deficient cells colonize the lungs to the same extent as control cells they survive, but do not proliferate. Anti-CXCR4 antibodies can also inhibit the spread of breast cancer xenografts to the lymph nodes 6.In addition, pre-incubation of lymphoblastoid cells with anti-cxcr4 or anti-cxcl12 antibodies delayed tumour growth in non-obese diabetic (NOD)/SCID mice and reduced tumour mass 97.Ifcells that are preincubated with CXCR4 antibodies are injected intravenously, they are present in the circulation for much longer than control cells, which points to a role for CXCR4 in tissue extravasation. Antibodies to CXCL12 also inhibit organ metastases of non-small-cell lung cancer cells when they are administered from the time of tumour cell injection into the mice 68. CXCL12 and CXCR4 regulate the proliferation and migration of neural precursor cells, and are also expressed at high levels in brain tumours of both neuronal and astrocytic lineage 45.Systemic administration of the CXCR4 antagonist AMD3100 inhibited the growth of intracranial glioblastoma and medulloblastoma xenografts, and increased tumour cell apoptosis within 24 hours 98.Therefore, CXCR4 is a potential therapeutic target in human cancer, although more extensive studies using established tumours and antagonists of other receptors are required. Where there is high expression of CXCL12 at the site of the primary tumour, it will be important to ensure that antagonists, such as AMD3100, do not encourage tumour cell release, thereby increasing, rather than decreasing, the risk of metastasis. Radiolabelled chemokines have recently been used for in vivo imaging of inflammatory conditions, and there might be a rationale for using such nuclearmedicine reagents for both cancer imaging and therapy 99.Radiolabelled CXCL12 would be particularly interesting to use in such a context. Finally, as has been shown in many experimental cancer studies 100, an alternative approach is to manipulate the cancer chemokine network to encourage the influx and activation of host cells that have general or specific tumour-destructive capacities. Conclusion Inappropriate and increased cell invasion is a hallmark of cancer; however, there is still much to learn about chemokines and malignant disease. Most cancers abound with chemokine ligands, but we have no idea why there are so many different chemokine gradients. Are some chemokines and chemokine receptors more important than others? Is overexpression of some of these for example CCL2, CCL5 and CXCR4 common to many cancers? The comprehensive study of chemokines and receptors in primary tumours, metastatic lesions and corresponding normal tissues will be crucial to further understanding of the cancer chemokine network. The idea that metastases occur because cancer cells follow chemokine gradients at distant sites is probably too simplistic. Chemokines are equally, if not more, important in aiding the survival of metastatic cancer cells, but how do they interact with other growth- and survival-factor signalling pathways, and why is the chemokine ligand sometimes expressed in the primary tumour? We can learn much from the elegant studies on CXCR4/CXCL12 and the homing/migration of germ cells and haematopoietic stem cells. The importance of CXCR4 in stem-cell biology has been well demonstrated 52, and CXCR4 expression by a sub-population of cells in a primary cancer indicates that these cells might be cancer stem cells 101. 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