Involvement of the CXCL12/CXCR4/CXCR7 Axis in Brain Metastases

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 ( ).