Chemokines: Key players in cancer

Key words: Anticancer agents – Cancer – Chemokines – Cytokines

SUMMARY

Chemokines are a family of low molecular weight (8-10 kDa) pro- inflammatory cytokines, which bind to G-protein coupled receptors. Their primary function is chemoattraction and activation of specific leucocytes in various immuno-inflammatory responses. However, new research suggests that they are key players in cancer being involved in the neoplastic transformation of cells, promotion of aberrant angiogenesis, tumour clonal expansion and growth, passage through the extracellular matrix (ECM), intravasation into blood vessels or lymphatics and the non-random homing of tumour metastasis to specific sites. In view of the increasing significance of chemokines and their receptors in cancers of a variety of types, manipulation of this signalling pathway may be important in the development of new anticancer agents. This review provides an overview of recent research advances in this field and examines the potential therapeutic benefits future developments may bring.

Introduction

Chemokines are a family of low molecular weight (8-10 kDa) pro- inflammatory cytokines, which bind to G-protein coupled receptors. The first chemokine was isolated in 1987 from lipopolysaccharide- stimulated monocytes (later named interleukin-8; IL-8 or CXCL8), and was shown to elicit neutrophil chemotaxis1,2.

Since then more than 40 different chemokines have been isolated, which are structurally divided into four main groups (see Table 1). Their primary function is chemoattraction and activation of specific leucocytes in various immuno-inflammatory responses. It has recently been shown that they are involved at all stages of tumour development, including initiation, growth and progression. A seminal paper has shown that the attraction of breast cancer metastasis to specific visceral organs is influenced by chemokines and chemokine receptors3. This has generated great interest in these small molecules and their role in cancer.

For the purposes of this review, a systematic search of the literature was performed using the Pubmed database (1966 to April 2003) with the search terms ‘chemokines and cancer’, augmented by manual searches and the authors’ personal bibliographic collections.

Chemokine groups and receptors

There are four groups of chemokines, approximately 70-130 amino acids in length, which are defined by the arrangement of the conserved cysteine (C) residues of the mature proteins4,5. The first three classes all have four conserved cysteines whereas the fourth has only two (Figure 1).

Figure 1. Structural classification of the chemokine family. C, cysteine; X, an amino acid other than cysteine

The CXC chemokines have one amino acid residue separating the first two conserved cysteine residues. CX3C chemokines have three amino acid residues between the first and second cysteines. In the CC chemokines group the first two conserved cysteine residues are adjacent and the C chemokines lack two (the first and third) of the four conserved cysteine residues. Unfortunately, as chemokine nomenclature has evolved, it has become extremely confusing (see Table 1)6.

The effects of chemokines on their target cells are mediated by cell surface, G-protein-coupled receptors with seven transmembrane regions7. These chemokine receptors are part of a much bigger superfamily of G-protein-coupled receptors that include receptors for hormones, neurotransmitters, paracrine substances and inflammatory mediators. Chemokine receptors are approximately 350 amino acids in length and are promiscuous in that several different chemokines can bind to a single receptor (although a few monogamous chemokines ligand-receptor relationships do exist – Table 2)8. However, each receptor only binds ligands restricted to one of the four chemokine structural groups. Thus, there are four chemokine receptor classes and currently, 18 human chemokine receptors have been identified: five CXC receptors (CXCR1 – CXCR5); 11 CC receptors (CCR1 – CCR11); one CX3CR1 receptor; one XCR1 receptor (Table 2).

Chemokine involvement in cancer

Chemokines and their receptors have recently been shown to act at all stages of tumour development and progression including the neoplastic transformation of cells, the promotion of aberrant angiogenesis and clonal expansion and growth. Additionally, chemokines are involved in the passage of tumour cells through the extracellular matrix (ECM) followed by their intravasation into blood vessels or lymphatics. Recent research has also demonstrated their key role in the non-random homing of tumour metastases to specific sites. We review the evidence for chemokine involvement in these processes.

Cellular Transformation

There is increasing evidence that chemokines are implicated in the neoplastic transformation of cells. The receptor CXCR2 shares a high degree of homology to the G protein-coupled receptor ORF74 or KSHV-GPCR, encoded by Kaposi’s sarcoma-associated herpesvirus-8(9). This is an agonist-independent receptor whose signalling is further upregulated by binding of CXC chemokines CXCL8 and CXCL1 (GRO[alpha]). Yang et al.10 showed that over-expression of this receptor within haematopoietic cells of transgenic mice led to the development of angioproliferative lesions resembling Kaposi’s sarcoma. Burger et al.11 demonstrated that a point mutation of CXCR2 leads to constitutive signalling of the receptor and cellular transformation of transfected NIH 3T3 cells (a fibroblast cell line derived from mouse embryo) comparable to results seen with KSHV- GPCR. This work with others suggests that CXC chemokines (CXCL8 and CXCL1) continually stimulate certain cells expressing the CXCR2 receptor by autocrine and paracrine mechanisms, ultimately leading to promotion of oncogenic cellular transformation.

Tumour Growth Factors

Growth factors induce cells to enter and proceed through the cell cycle12,13. Autocrine and paracrine growth regulation has been observed in a number of tumour systems, suggesting that cells respond to the same growth factors they produce.

Schadendorf et al.14 found CXCL8 to be an essential autocrine growth factor for some human melanoma cell lines. This chemokine binds to CXCR1 or CXCR2 receptors, which have been shown to be present in melanoma cell lines15,16. Metzner et al.17 demonstrated that proliferation of human epidermoid carcinoma cell lines A431 and KB could be induced by CXCL8 and constitutive proliferation could be inhibited by neutralizing antibodies against CXCL8 ligand or CXCR2 receptor. This indicated that constitutive CXCL8 and CXCR2 protein expression enabled an autocrine growth mechanism in these cells17.

Other tumour cell lines in which CXCL8 acts as an autocrine growth factor are those obtained from human cancers of the colon, stomach, liver, pancreas, skin and lung18-22. Also, in biopsy tissue obtained from human ovarian carcinomas, neuroblastomas and squamous cell carcinomas of the head and neck, both CXCL8 and its receptor, CXCR2 are expressed by malignant cells. This suggests CXCL8 could also function in an autocrine pathway for these tumours23-25.

Table 1. Chemokine nomenclature*

CXCL1 (GRO[alpha]) was initially identified and purified from serum-free culture supernatants of a malignant melanoma cell line, Hs294T, and characterized as an autocrine growth factor for melanoma cells26,27. Two highly related CXC chemokmes CXCL2 (GRO[beta]) and CXCL3 (GRO[gamma]) have additionally been shown to be involved in melanocyte transformation and tumour growth28. All three share the receptor CXCR2.

CXCL1 has also been shown to act as an autocrine growth factor for some human adenocarcinoma cell lines derived from the lung and stomach18 and in human malignant pancreatic cell lines21.

CXCR4 expression has been observed to be upregulated in human glioblastomas and inhibition of this receptor blocks tumour cell proliferation29,30. Bajetto et al.31,32 demonstrated the concomitant expression of the CXCL12 ligand (SDF-1) with its receptor CXCR4 leads to autocrine and paracrine regulation of cell growth in cultured astrocytes.

Table 2. The four classes of chemokine receptors and some of their ligands*

Modulation of Angiogenesis/Angiostasis

Angiogenesis is a normal physiological process that takes place during embryonic development and wound healing. It is also required for solid tumours to grow beyond 2 mm in diameter and for their subsequent rapid growth33,34. CXCL4 (PF-4) and CXCL8 are members of the CXC chemokine subfamily, but they differ in that CXCL8 contains the ELR motif (ELR+; Glu-Leu-Arg) at its NH2 terminus. It has been demonstrated that CXCL4 has angiostatic properties. For example, it inhibits endothelial cell proliferation, angiogenesis in the chick chorioallantoic membrane assay and tumour growth in immunodeficient mice35. CXCL8 was the first chemokine shown to stimulate endothelial cell chemotaxis, proliferation and in vivo angiogenesis (using a rat corneal micropocket assay)36.

Strieter et al.37 hypothesised that the presence of the ELR motif is critical for determining the effect a CXC chemokine has on angiogenesis. They showed that substitution of the ELR motif in CXCL8 with the amino acids TVR (Thr-Val-Arg) or DLQ (Asp-Leu-Gln) resulted in an ELR- mutated CXCL8 that was unable to stimulate endothelial chemotaxis or in vivo angiogenesis. The mutated CXCL8 actually inhibited angiogenesis. In contrast, the addition of ELR to CXCL9 (MI\G), an ELR- chemokine, resulted in its conversion from an angiostatic to an angiogenic agent.

Clinically, serum CXCL8 (ELR+) is elevated in prostate cancer patients38. Antibody to CXCL8 secreted by the cell line PC-3 (prostate cancer) has been shown to reduce tumour growth and tumour- related angiogenesis in a SCID mouse model39.

Luan et al.40 tested the biological consequence of overexpression of CXCL1, CXCL2, CXCL3 (GRO[alpha], GRO[beta], GRO[gamma]: all ELR+) chemokines following their transfection of non-tumourigenic immortalised mouse melanocytes. This resulted in the formation of highly vascular tumours in nude mice. Antibodies to these three proteins slowed or inhibited the formation of tumours in the SCID mouse model (accompanied by a reduction in the number of viable endothelial cells in tumours) and blocked the angiogenic response to conditioned medium from tumourigenic transfectants in the rat corneal micropocket assay.

Amongst the angiostatic chemokines the most-studied has been CXCL10 (IP-10), which is ELR-. This molecule inhibits growth of new blood vessels stimulated by either vascular endothelial growth factor (VEGF) or angiogenic CXC chemokines in the rat corneal micropocket assay41. Using a mouse matrigel neovascularisation model, CXCL10 was shown to inhibit angiogenesis stimulated by basic fibroblast growth factor in vivo42. Burkitt’s lymphoma cells transfected to overexpress CXCL10 had reduced ability to form subcutaneous tumours in nude mice. This effect of CXCL10 was attributed to its ability to decrease tumour angiogenesis43.

Interestingly, only one ELR- CXC chemokine actually stimulates, rather than inhibits, angiogenesis: SDF-1 or CXCL12. Gupta et al.44 demonstrated that the CXCR4 receptor is expressed on endothelial cells and that its ligand CXCL12 was an efficacious chemoattractant for these cells. Additionally, CXCL12 induces angiogenesis from cross-sections of leukocyte-free rat aorta in vitro45 and the formation of capillary-like structures by endothelial cells in culture46.

These studies suggest the presence of both stimulators and inhibitors of angiogenesis among the CXC chemokine subfamily. It is postulated that CXC chemokines form a balanced network of angiogenic and angiostatic regulators that are disrupted in cancer. Importantly, the balance of ELR+ and ELR-chemokines produced by a tumour and its stroma may determine the degree of angiogenesis surrounding the tumour and thus, the consequent invasiveness of the tumour47.

Local Tumour Invasion

The ability of tumour cells to secrete metalloproteinases (MMPs) and other protease enzymes aids invasiveness through the extracellular matrix48. Chemokines are important in inducing tumour cell production and secretion of several of these enzymes. CXCL8 expression by human melanoma cells induces transcriptional activation of the gene encoding MMP-2, which results in increased invasiveness through the ECM49,50. Also, CXCL8 over-expression in androgen-independent prostate cancer cells induces expression of MMP- 9, which leads to increased local invasion of tumours in a nude mouse model51.

Chemokines in Cancer Metastasis

Metastasis is not a random process and different cancer types have specific metastatic sites. Breast cancer favours regional lymph nodes, bone marrow, lung and liver. Malignant melanoma has a similar pattern but also has a high incidence of skin metastasis. Prostate cancer also favours the bone marrow. Several explanations for these metastatic patterns have been proposed.

In 1889, Paget52 described the concept of ‘seed’ (tumour cell) and ‘soil’ (specific organ), for the non-random metastasis of breast cancer to specific organs. This theory states that different organs provide growth conditions optimised for specific cancers. A second concept is that endothelial cells in the vascular beds of certain organs express adhesion molecules that specifically trap circulating tumour cells53. The ‘homing’ theory, on the other hand, states that different organs have special abilities to arrest or attract through chemotactic factors specific for types of cancer cells53,54.

There is evidence to support all three theories, but the underlying molecular mechanisms have not previously been elucidated. In a recent publication by Muller et al.3, they researched into the mechanisms involved in the metastasis of breast cancer to specific organs. They have found that amongst 17 different chemokine receptor genes, CXCR4 and CCR7 were highly expressed in human breast cancer cells lines, malignant breast tumours and metastases relative to the levels in normal mammary epithelial cells. They then screened a panel of normal human organs for the ligands of these receptors, CXCL12 and CXCL21 respectively, and found they exhibited peak levels of expression in organs preferred for breast cancer metastases. In vitro, using breast cancer cell lines, these ligands stimulated pseudopodia formation and directional migration in cells in addition to local invasion through extracellular matrix and basement membrane. Also, extracts of organs targeted by breast cancer (lung, liver, bone marrow and lymph node) had chemotactic activity for breast cancer cells that could be neutralised by anti-CXCR4 antibody, thus suggesting CXCL12 was the active agent. Neutralising anti-human CXCR4 monoclonal antibody suppressed lymph node and lung metastases in a metastatic model of human breast cancer (MDA-MB-231 cell line injected either orthotopically into the mammary fat pad or intravenously in immunodeficient SCID mice). The same group found that melanoma cell lines express receptors CCR7 and CCR10 and that skin and lymph nodes, the two major sites of metastatic melanoma, selectively express ligands for both these receptors.

In prostate cancer, CXCR4 is expressed in certain tumour cell lines and its ligand CXCL12 was shown to promote tumour cell transendothelial chemotaxis and adhesion to osteoclastic cells55.

These findings support the hypothesis that certain chemokine ligands and their receptors are involved in the homing of metastatic tumour cells to specific organs.

Clinical implications

In view of the importance of chemokines and their receptors in cancers of a variety of types, manipulation of this signalling pathway may be of therapeutic benefit.

Immunotherapy

Chemokines may have great potential as agents in cancer immunotherapy. As they function physiologically as immunostimulatory molecules (i.e. promotion of chemotaxis and the effector function of leucocyte subpopulations), these proteins may be used to enhance antitumour immunity in the host. Also, as previously discussed, they can be angiostatic and thus inhibit tumour growth. Several approaches have been used in the delivery of chemokines into the tumour microenvironment.

1. Transduction of tumour cells with chemokines genes – Braun et al.56 showed that rejection of C3L5 murine breast carcinomas was achieved after CCL19-transduction of tumour cells. This rejection was mediated by immunostimulation (natural killer cells and CD4+ T lymphocytes). Alternatively, angiostatic chemokine genes may be transduced into cancer cells. An advantage of using chemokines is that, unlike tumour suppressor genes, it is theoretically not necessary for every tumour cell to be transduced. Thus, if a proportion of tumour cells secrete an angiostatic molecule, this might alter the balance of angiogenic factors in the tumour microenvironment sufficiently to prevent neovascularisation and further tumour growth57.

2. The injection of recombinant chemokine proteins into tumour sites – Vicari et al.58 showed that mouse CCL21 exerts strong antitumour effects by inducing angiostatic and T lymphocyte- mediated tumour resistance mechanisms. Furthermore, injection of recombinant CCL21 directly into the tumour induced potent antitumour responses and led to complete eradication of alveolar carcinoma (L1C2) and Lewis lung carcinoma59.

3. The administration of tumour vaccine (combining tumour antigen and chemokines) – CXCL10 or CCL7 fusion to lymphoma immunoglobulin resulted in variable regions eliciting leucocyte chemotactic responses in vitro and inducing inflammatory responses in vivo. Also, they enhanced protection against tumour challenge60.

Chemokine Receptor Antagonists

One of the first non-peptide small molecule receptor antagonists described, [N-(2-hydrexy-4-nitrophenyl)- N'-(2-bromophenyl) urea, is an inhibitor of ELR+ chemokine-receptor binding61, It has high (150x) selectivity fer CXCR2 compared to CXCR1, despite the sequence similarity of the two receptorg, In animal models administration inhibited migration of neutrophils in a dose-dependent manner61, The role of this receptor antagonist in inhibiting tumour growth and progression has not yet been elucidated, However, the hexapeptide antileukinate, which antagonises binding of ELR+ angiogenic chemokines to their CXCR2 receptor, has been shown to inhibit the growth of adenocarcinomas effectively in vitro as well as in animal models18. The bicyclams, in particular AMD3100, are highly potent and specific antagonists of CXCR4 - this is also known to be a co- receptor used by the HIV-1 virus for entry into cells62,63, AMD3100 has been particularly studied in the prevention of T-tropic HIV=1 infection of target cells64, and has been injected into human volunteers. It is well tolerated, but it is unlikely to be approved for drug use owing to pharmacokinetic parameters65.

Other CXCR4 antagonists include T22 ([Tyr5,12, Lys]-Polyphemusin II) – an 18-residue peptide amide”66,67, T134 – a shortened version of T22(68), and ALX40-4C (N-[alpha]-acetyl-nona-D-arginine)69. All have been investigated for their anti-HIV activity in a similar manner to AMD3100(66-69). They may be useful in the manipulation of the chemotactic responses of metastatic cancer cells, however, the clinical application of such inhibitors must be approached with caution due to the importance of \many of these pathways in normal lymphohaematopoiesis.

Conclusion

Chemokines are unique chemotactic cytokines, which are increasingly being recognised as key players in cancer. They have been implicated in tumour initiation, growth, angiogenesis, local invasion and metastasis. All of these mechanisms are important points for cancer intervention and thus chemokine production and action are obvious targets for drug development with the potential for new, effective anti-cancer agents being introduced. With ongoing successful preclinical models we can expect these agents to be entered into phase 1 trials in the treatment of human cancers in the near future.

CURRENT MEDICAL RESEARCH AND OPINION(R)

VOL. 19, NO. 6, 2003, 557-564

(C) 2003 LIBRAPHARM LIMITED

CrossRef links are available in the online published version of this paper: http://www.cmrojournal.com

Paper CMRO-2386, Accepted for publication: 16 June 2003

Published Online: 22 July 2003

doi:10.1185/030079903125002216

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M. Arya1, H. R. H. Patel2 and M. Williamson2

1 Institute of Urology and Nephrology, University College London and Royal Free Hospital London, UK

2 Institute of Urology and Nephrology, University College London, UK

Address for correspondence: Mr M. Arya, MBChB, FRCS, Specialist Registrar, Department of Urology, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel: 07930-548021; Fax: 0208-5021762; email: manit_arya@hotmail.com

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