Introduction

Chemokines are a superfamily of small cytokines (8–12 kDa) with chemotactic properties. They bind to specific receptors and are involved in cell trafficking, activation and differentiation.

More than 50 different chemokines have been identified and are classified into four families based on the position of four conserved cysteine residues. The first group, so-called CC chemokines because of two adjacent cysteines near the amino terminus, has 28 members; the second group, CXC chemokines, include proteins characterized by the presence of a single variable amino acidic residue between the two cysteines and consists of 17 members. The other two chemokine families each have a single member: CX3C, that has three variable amino acidic residues between the cysteines and XC, in which the first and third cysteines are lacking.

Based on the presence or absence of a specific amino acidic motif “glu-leu-arg” (ELR, using the single letter code), CXC chemokines can be subgrouped into ELR ⁺ and ERL ⁻ . CXC–ERL ⁺ chemokines preferentially attract neutrophils and are pro-angiogenic factors (CXCL1, 2, 3, 5, 6, 7, 8,) while ERL ⁻ act on lymphocytes and, with the exception of CXCL12, inhibit the formation of vasculature ( ).

Physiologically, chemokines bind to specific G protein-coupled receptors (GPCRs). Chemokine receptors have a conserved structure with seven transmembrane domains, three extracellular and three intracellular loops with the C-terminus in the cytoplasm. Several chemokines may bind to various receptors and the same receptor may bind different chemokines. At present, we know seven CXC receptors (CXCR1–7), 10 CC receptors (CCR1–10), XCR1 and CX3CR1. In addition, there are decoy receptors that bind multiple chemokines without triggering signal transduction (D6, DARC, CCX–CKR) ( ). Chemokine receptors act through heterotrimeric G-proteins which activate various signal transduction pathways, such as phosphoinositide 3-kinase (PI3K), phospholipase-C (PLC), mitogen-activated protein kinases (MAPK), protein kinase C (PKC) and RAS or Rho GTPases ( ).

Chemokines also interact with glycosaminoglycans (GAGs) that are required for presentation of chemokines by endothelial cells and extracellular matrix, making the formation of a chemotactic gradient that directs leukocyte and tumor cell migration in vivo possible.

Chemokines were initially identified as potent chemotactic agents for inflammatory cells, as leukocytes, monocytes and neutrophils. Their regulatory role in development, homeostasis and various pathological processes was discovered subsequently. Recently, a role in cancer progression and metastasization was proposed for chemokines: tumor cells expressing chemokine receptors could be attracted by chemokines produced by target organs and chemokines produced by tumor cells could recruit endothelial cells and tumor-associated stromal and inflammatory cells ( ).

CXCL12 and its Receptors

Stromal cell-derived factor (SDF-1, CXCL12) is a CXC chemokine widely expressed in many tissue types where it regulates hematopoietic cell trafficking. This role of CXCL12 is crucial not only in adult life, but also in embryo development. Due to the impairment in migration of hematopoietic stem cells from the fetal liver and major defects in brain and heart development, CXCL12 gene knockout in mice is lethal.

CXCL12, produced by osteoblasts, plays an important role in CD34+cell migration to bone marrow and its function has been attributed to the receptor CXCR4.

Due to alternative splicing, CXCL12 has two major isoforms, α and β: CXCL12α is the most diffused and it is secreted by marrow stromal cells and endothelial cells; CXCL12β is expressed by highly vascularized organs such as liver, spleen, and kidneys, it is pro-angiogenic and more resistant than the α isoform to blood proteolysis. Additional splicing variants are CXCL12γ, CXCL12δ, CXCL12ε and CXCL12φ, all with chemotactic actvities.

CXCL12 expression is enhanced in hypoxic or damaged tissues because of elevated levels of HIF-1α that acts on its binding sites on the CXCL12 promoter, leading to chemoattraction of CXCR4-positive cells involved in tissue regeneration ( ).

After binding with its receptor CXCR4, CXCL12 forms a complex with the Gαi-subunit resulting in Gβγ-subunit dissociation. The Gαi-subunit acts by inhibiting cAMP production by adenylyl cyclase and stimulating intracellular calcium mobilization. The Gβγ-subunit in turn activates PI3K and PLC leading to activation of downstream pathways such as MAPK, ERK1/2, JNK and AKT ( ). These effectors induce actin polymerization and cytoskeletal rearrangements responsible for chemotaxis and expression and activation of integrins involved in adhesion to endothelial cells. These processes are essential for leukocyte activities in the immune response, but they are also implicated in the metastatic process and in local invasion of tumor cells.

CXCR4 was extensively studied as co-receptor for HIV infecting CD4+T cells.

As CXCL12, CXCR4 plays an essential role in hematopoiesis, and in brain and heart development. The role in hematopoiesis is maintained in adult life: the CXCR4 inhibitor AMD3100 was recently approved as an immunostimulant to induce hematopoietic stem cell mobilization in hematological diseases ( ).

While CXCR4 expression is low or absent in many healthy tissues, it was demonstrated to be upregulated in over 23 types of cancer, including melanoma, breast, ovarian and prostate cancer, and gliomas. CXCR4 expression is induced by vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF1α), thus, in hypoxic regions of tumor, the CXCL12/CXCR4 axis may promote survival and metastasization.

Until recently, CXCR4 was believed to be the unique CXCL12 receptor. However, a second receptor was identified recently: the orphan receptor RDC1, now called CXCR7. CXCR7 binds CXCL12 with higher affinity than CXCR4, and it also binds another chemokine, CXCL11/I-TAC, previously known as a CXCR3 ligand, even if with lower affinity than CXCL12 ( ).

CXCR7 gene maps on chromosome 2 in humans, as CXCR4. It is expressed and plays an important role in the hematopoietic system, heart, bone, kidney and brain ( ). Like CXCR4, CXCR7 was found expressed in a variety of tumors, such as lymphoma, breast, lung and prostate cancer. It is involved in the inhibition of apoptosis and in the adhesion to endothelial cells and metastasis. CXCR7 is also expressed in activated endothelial cells and in tumor vasculature stimulated by hypoxia ( ).

Initially, CXCR7 was considered a decoy receptor because it does not elicit typical signaling pathways of G-protein-coupled receptors such as CXCR4. It was shown that it causes CXCL12 sequestration generating the chemokine gradient essential for proper migration of CXCR4-positive primordial germ cells in embryos ( ). Another mechanism of action proposed for CXCR7 is regulation of CXCR4 activity through the formation of functional CXCR4/CXCR7 heterodimers. Recently, it has been demonstrated that CXCR4/CXCR7 heterodimers can recruit β-arrestin activating ERK1/2, p38 MAPK, and SAPK and enhancing cell migration in response to CXCL12 stimulation ( ).

CXCL12 Axis in Brain

CXCL12 and its receptor CXCR4 are expressed in embryonic life and play a major role in development of the cerebellum, neocortex and hippocampal dentate gyrus: CXCL12 acts as a chemoattractive cue for CXCR4 positive precursor cells regulating neuronal migration ( ). CXCR4 is also expressed in neural stem cells in adult life: its mRNA is abundant in the subventricular zone neural stem cells in adult mice. More recently, CXCR7 has also been investigated for its role in neurogenesis: both CXCR4 and CXCR7 are involved in protecting neural progenitor cells from apoptosis in response to CXCL12 ( ).

In the central nervous system (CNS), CXCR4 is expressed in astrocytes, endothelial cells, neurons and microglia. CXCL12 is known to promote astrocyte proliferation via CXCR4 activation and MAPK and Erk1/2 signaling. More recently, Ödemis and colleagues showed that CXCR7 also activates traditional G-protein transduction pathways, such as Erk and Akt, involved in cell proliferation and migration, in astrocytes ( ). This observation is in contrast with previous works that excluded CXCR7-mediated G-protein activation, suggesting different mechanisms of action of this receptor depending on cell type.

CXCL12 is also involved in repair processes after CNS trauma ( ).

CXCR4 and CXCL12 are overexpressed in brain tumors, such as gliomas ( ), medulloblastomas ( ) and meningiomas ( ). In glioblastoma, CXCL12 and CXCR4 are associated to areas of necrosis and angiogenesis ( ), promote tumor proliferation and chemotaxis ( ), and angiogenesis ( ). CXCL12 was found to be associated with poor prognosis in glioblastomas and in low-grade gliomas ( ). Recent studies have shown that CXCR7 is also expressed in gliomas and a role in promoting survival of tumor cells has been proposed ( ).

Overall, the complex interactions between CXCL12 and its two receptors may be implicated in tumor cell survival and in the mechanisms of tumor invasion. Their role in brain tumor metastasis remains so far less clearly defined.

CXCL12/CXCR4/CXCR7 and Metastasis

The chemokine CXCL12 and its receptors CXCR4 and CXCR7 have been described to play a pivotal role in tumor growth, in metastatic dissemination and in the cross-talk between neoplastic and support cells in the tumor microenvironment (reviewed in ). It has been suggested by recent works that the two receptors could make different contributions to tumor progression ( ).

It is well known that expression of specific adhesion molecules, chemokines and receptors on tumor cells as well as on target organs are essential for organotropism in metastasis.

In physiological conditions, CXCL12 is constitutively expressed in a variety of tissue types and organs, including bone marrow, liver, lung and lymph nodes that represent common organs for metastasis homing. Moreover, both tumor cells and tumor-associated stromal cells contribute to create a microenvironment able to promote the metastatic process. Orimo and co-workers showed that, in breast cancer, high levels of CXCL12, released by tumor cells and tumor-associated stromal cells, directly stimulated the proliferation and invasiveness of cancer cells by autocrine and paracrine mechanisms ( ). In in vitro experiments, high amounts of CXCL12 released by CAFs (cancer associated fibroblasts lines, isolated from non-small cell lung cancer) induced an increased colony-forming capacity and extracellular signal-regulated kinase phosphorylation in cancer cells ( ). The CXCR4/CXCL12 axis has been also reported to regulate immune cell infiltrates into tumors which, in turn, may promote neoangiogenesis and tumor progression.

Interestingly, antineoplastic agents commonly used in tumor treatment, such as irradiation, cyclophospamide and 5-fluorouracil, may increase CXCL12 expression in stem cells. However, CXCL12 production in the tumor context may exert an antimetastatic effect. As a matter of fact, experiments performed in mammary cancer cells leading to an epigenetic silencing of CXCL12, showed an increase of metastatic potential of colorectal and melanoma tumor cells and exogenous administration of CXCL12 increased survival of tumor-bearing mice because of a reduced metastatic ability of tumor cells ( ). The authors speculated that CXCL12 production by tumor cells could have an antimetastatic effect shielding the primary tumor by distant chemokine gradient.

Enhanced CXCR4 expression is reported in many different types of cancers ( ), including breast, ovarian and prostate cancer, pancreatic adenocarcinoma, small cell lung cancer (SCLC), melanoma, neuroblastoma and glioma ( ), while CXCR4 expression is low or absent in most normal tissues. CXCR4 expression on tumor cells is correlated with a poor prognosis in cancer patients.

Hypoxia-induced HIF-1α activation and VEGF release have been described among the mechanisms inducing the increase in CXCR4 expression in glioma cells, while stimulation with interleukin-5 (IL5) and interferon-γ (IFN-γ), produced by stromal cells, increases CXCR4 expression in neuroblastoma cells. In addition, radiotherapy has also been reported to produce the undesirable side effect of increasing CXCR4 expression ( ).

After CXCL12 binding, CXCR4 signaling promotes actin polymerization, pseudopodia formation and chemotactic response in breast cancer cells. In SCLC cells, CXCL12 elicited an activation of phospho-p44/42 mitogen-activated protein kinase and induced tumor cell invasion into extracellular matrix and firm adhesion to marrow stromal cells, suggesting that CXCR4 can induce integrin activation on SCLC cells ( ).

The central role of the CXCR4/CXCL12 pathway in metastasis was also demonstrated in preclinical studies in melanoma, pancreatic, thyroid, prostate, and colon cancer models, showing that directed metastasis of cancer cells is mediated by CXCR4 activation and migration of tumor cells towards CXCL12 expressing organs.

The importance of CXCR4/CXCL12 axis in tumor growth and metastatic process was confirmed by data showing that CXCR4 antagonists or neutralizing antibodies affect tumor growth and invasiveness both in in vitro and in experimental models.

Small interfering RNA (siRNA)-mediated inhibition of CXCR4 expression in breast cancer cells decreased both their invasion capacity in in vitro invasion assay and metastasis development in a murine model ( ). In an animal model of prostatic cancer, neutralizing antibody against CXCR4 has been reported to reduce the extent of bone metastasis ( ).

In a murine model, D’Alterio et al. studied the effect of genetic CXCR4 reduction on the development of lung metastasis of melanoma cells ( ). After injection of murine melanoma B16 cells in CXCR4 ^+/+ and CXCR4 ^+/− C57Bl/6 mice, they observed smaller metastasis in the latter. The authors suggested that the reduction may be partially due to a significant decrease in LY6G myeloid cells in the tumor context in CXCR4 ^+/− mice when compared to CXCR4 ^+/+ mice, leading to a reduction of p38 MAPK activation. The administration of AMD3100 (plerixafor), a CXCR4 antagonist, further reduced the size and the number of lung metastases, but it was more effective in CXCR4 ^+/− mice.

CXCR7 has been described to be expressed on fetal liver cells, activated endothelial cells and a variety of tumor cells ( ), while its expression on T lymphocytes is controversial ( ). CXCR7 is highly expressed on tumor-associated vasculature and it may be upregulated on endothelial cells by tumor necrosis factor α (TNFα), IL1β ( ) and by hypoxia.

Transfection-induced CXCR7 expression has been described to increase cell proliferation in the human breast cancer cell line MDA-MB-435. Moreover, the anti-apoptotic effect exerted by CXCR7 was abrogated in a dose-dependent manner by the CXCR7 antagonist CCX754.

In vitro adhesion experiments on cytokine-stimulated human umbilical vein endothelial cells demonstrated an increased adherence of CXCR7-transfected tumor cells ( ). Similar results on cell survival, as well as on adhesion ability, were obtained on CXCR7-expressing prostate cancer cell lines. Moreover, CXCR7 overexpression has been reported to increase the invasive abilities of cancer cells in in vitro invasion assays ( ). Strong CXCR7 expression, observed both on cancer and endothelial cells in brain metastasis of different solid tumors, suggests an important role of this receptor in promoting migration of tumor cells through the blood–brain barrier ( ). In addition, CXCR7 was described to increase the expression of VEGF and IL-8, supporting its relevant contribution to the growth of tumor vasculature ( ). In experimental models, CXCR7 expression enhanced the ability of breast cancer cells to produce lung metastasis ( ).

These findings on CXCR4 and CXCR7, in some cases apparently contradictory, need further studies, aimed to clarify the individual contribution of the two receptors and especially their reciprocal interactions.

In an interesting study, investigated the roles of the two receptors in the different steps of metastasis. The authors evaluated the effect of overexpression of CXCR4, CXCR7 and both receptors (double transductant) in the rat mammary adenocarcinoma cell line MTLn3, analyzing invasiveness, entry into the bloodstream and lung metastasis development. While CXCR4 overexpression enhanced the chemotactic and invasive ability of the tumor cells in response to CXCL12, as well as intravasation (associated also to an increase in MMP12 production), CXCR7 overexpression further increased chemotactic response only if associated with high CXCR4 expression. The authors suggested that CXCR7 binds monomeric CXCL12, leaving dimeric CXCL12 to bind CXCR4 and mediate signalling through β-arrestin, as reported by other studies ( ).

On the other hand, CXCR7 overexpression has been reported to inhibit invasion and intravasation of tumor cells, probably following a decreased expression of the metalloprotease MMP12, leading to an inhibition of the ability to degrade extracellular matrix ( ). However, unexpectedly, in this mouse model both CXCR4 and CXCR7 overexpression was not reported to increase the number of lung metastases.