Review
7 May 2024
Endocrinology and Nutrition Department, University and Politecnic Hospital La Fe (Valencia), 46026 Valencia, Spain
Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics, Health Research Institute La Fe, 46026 Valencia, Spain
Nuclear Medicine Department, University and Politecnic Hospital La Fe (Valencia), 46026 Valencia, Spain
Patholoy Department, University and Politecnic Hospital La Fe (Valencia), 46026 Valencia, Spain
Neuroendocrine neoplasms are a heterogeneous group of malignant tumors that originate from the diffuse endocrine system. They generally have a slow course and somatostatin receptor-targeted based management is the first line of treatment. However, high-grade tumors and neuroendocrine carcinomas have a poor prognosis and somatostatin receptor-targeted therapy is not effective. The membrane receptor CXCR4 has been studied in several neoplasms and it is known to be overexpressed in aggressive tumors and associated with a worse prognosis. However, there is a lack of evidence of its use in neuroendocrine neoplasms. For that reason, this review describes the significance of CXCR4 and its possible clinical applications in the diagnostic and therapeutic management of neuroendocrine neoplasms.
There are several well-described molecular mechanisms that influence cell growth and are related to the development of cancer. Chemokines constitute a fundamental element that is not only involved in local growth but also affects angiogenesis, tumor spread, and metastatic disease. Among them, the C-X-C motif chemokine ligand 12 (CXCL12) and its specific receptor the chemokine C-X-C motif receptor 4 (CXCR4) have been widely studied. The overexpression in cell membranes of CXCR4 has been shown to be associated with the development of different kinds of histological malignancies, such as adenocarcinomas, epidermoid carcinomas, mesenchymal tumors, or neuroendocrine neoplasms (NENs). The molecular synapsis between CXCL12 and CXCR4 leads to the interaction of G proteins and the activation of different intracellular signaling pathways in both gastroenteropancreatic (GEP) and bronchopulmonary (BP) NENs, conferring greater capacity for locoregional aggressiveness, the epithelial–mesenchymal transition (EMT), and the appearance of metastases. Therefore, it has been hypothesized as to how to design tools that target this receptor. The aim of this review is to focus on current knowledge of the relationship between CXCR4 and NENs, with a special emphasis on diagnostic and therapeutic molecular targets.
Chemokines are a group of small molecules (~8–10 k Da) that belong to the cytokine family together with angiogenic factors, growth factors, or interferons and are secreted not only by neoplastic cells but also by macrophages, lymphocytes, or dendritic cells. Their main function is to stimulate chemotaxis of immune system cells as part of the inflammatory response through the interaction with fibroblasts or endothelial cells, while in neoplastic status they induce angiogenesis and sustain cell growth [1]. This action is exerted by binding the N-terminal domain, which is rich in the amino acid cysteine, to its specific receptor [2]. Depending on the distribution of this amino acid, four subtypes of cytokines are identified: CXC, CX3C, CC, and C [3].
Among the 50 types of chemokines known today, the chemokine CXCL12, which is also recognized as stromal cell-derived factor-1 (SDF-1) [4], has some characteristics that make it different from the rest of its family. Firstly, it is the only cytokine whose mRNA can be subjected to a process known as differential splicing, which is why up to six variants of this molecule have been recognized in humans (α to ϕ) [5,6]. Secondly, it is a chemokine with an almost exclusive affinity for a single receptor, with nothing to do with the promiscuity of the rest of the cytokines [7]. Until a few years ago, CXCR4 was recognized as the only natural receptor for CXCL12, although it has been discovered that it can also mediate its action through interaction with the atypical chemokine receptor type 3 (ACKR3), previously known as chemokine C-X-C motif receptor 7 (CXCR7) [8,9].
CXCL12 is probably the most important cytokine that binds to CXCR4 but this receptor does not only bind to this type of molecule. Different ligands for CXCR4 have been recognized, most notably macrophage inhibitory factor (MIF) [10] and ubiquitin [11,12].
CXCR4 is a molecular structure that has also presented different names throughout history. It was initially called leukocyte-derived seven-transmembrane receptor (LESTR) when it was isolated from a human blood monocyte cDNA library [13]. It has also been known as cluster of differentiation 184 (CD184) or fusin. The latter name refers to the ability of the human immunodeficiency virus 1 (HIV-1) to infect human cells by the process of fusion following the binding of its glycoprotein 120 (gp120) [14]. Although its natural ligand is the chemokine CXCL12 (as mentioned before), there is greater evidence that it has a wider spectrum of interactions with other molecules. In fact, it also recognizes ligands as small proteins like ubiquitin or the macrophage migration inhibiting factor (MIF) [15,16]. This receptor belongs to the family of G protein-coupled receptors (GPCRs), which are characterized by the presence of seven membrane-spanning α-helical segments separated by alternating intracellular and extracellular loop regions [17]. The intracytoplasmic domain of the receptor remains in contact with a heterotrimeric G protein that is composed of a G α, G β, and G γ subunits and, when the interaction between CXCL12 and CXCR4 occurs, the exchange of guanosine diphosphate (GDP) for triphosphate (GTP) leads to a complex process in which a GTP-bound G α monomer and a G βγ dimer are released [18].
The G α subunit produces an inhibition of the adenylate cyclase leading to an increase in the intracellular calcium mediated by the decrease in the concentration of adenosine 3′,5′-cyclic monophosphate (cAMP). It also interacts directly with the Src family of tyrosine kinases and then activates the signaling pathway of MEK1/2-Erk1/2 [19]. The G βγ subunit activates phosphatidyl-inositol-3-OH kinase (PI3K) and consequently generates an increase in phosphatidylinositol triphosphate (PIP3), while the interaction with phospholipase C generates diacylglycerol (DAG) and inositol-(1,4,5)-triphosphate (IP3). IP3 increases intracellular calcium deposition after outflow from the endoplasmic reticulum (ER), while DAG interacts with protein kinase C and mitogen-activated protein kinase (MAPK) [20].
Representation of the signaling pathway in the activation of CXCR4 (left) and ACKR3 (right). Blue arrows mean activation while red arrows represent metabolic pathway inhibition. Note that G proteins and calcium do not participate as second messengers after binding CXCL12 to ACKR3. Acronyms: C-X-C motif chemokine ligand 12 (CXCL12), chemokine C-X-C motif receptor 4 (CXCR4), atypical cytokine receptor type 3 (ACKR3), protein kinase C (PKC), adenylate cyclase (AC), adenosine 3′,5′-cyclic monophosphate (cAMP), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK), diacylglycerol (DAG), inositol-(1,4,5)-triphosphate (IP3), phosphatidylinositol triphosphate (PIP3), phospholipase C (PLC), mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER).
When CXCL12 binds ACKR3/CXCR7, a different signaling pathway is developed because of the biochemical difference between classical and atypical chemokine receptors, which basically boils down to the fact that atypical cytokine receptors (ACKRs) lack G proteins and its effects are calcium-independent [21]. The signal pathway through β-arrestin proteins becomes the main way the ACKR3 activation leads to its tumorigenic properties. β-arrestins increase the MEK/ERK axis and the protein kinase B (also known as Akt) activity [22]. The binding of CXCR4 to its agonist ligand results in phosphorylation and internalization of the receptor [23,24]. However, once inside the cell, it can be recycled and transported back to the plasma membrane or it can be degraded in the cell lysosomes [25]. The first scenario occurs in a PKC-mediated phenomenon [26], whereas the second case takes place after interaction with E3 ubiquitin ligase [27].
Firstly, the involvement of CXCR4 as a co-receptor in HIV infection overshadowed its potential as a tumorigenesis-related agent and it was not until 1999 when Burger et al. noticed that this protein favored migration of B cells in chronic lymphocytic leukemia. Since then, the link between CXCR4 and tumoral disease has been reviewed and, for instance, the implication of CXCR4 in more than 23 cancers is well known [28,29,30]. Considering that CXCR4 functions involve the promotion of cell growth, proinflammatory cell recruitment, angiogenesis, and cell migration, it is not surprising that the pathological activation of this receptor favors the development of tumoral disease. To be more accurate, the hyperactivation of the CXCL12/CXCR4/AKR3 axis is associated with increased tumor size, lower degree of cell differentiation, higher probability of recurrence, worse response to chemotherapy, and decreased overall survival [31,32]. The role it plays in cell growth and its different effects on stromal tissue have placed this receptor in the spotlight of the scientific community. CXCR4 has been studied in practically all the different types of cancer because its expression is independently associated with decreased survival [33]. In fact, it is being investigated as to whether it could be a pan-cancer marker of the microenvironment status [34].
The presence of metastases drastically worsens cancer prognosis and CXCR4 is closely related to this phenomenon in various solid tumors. It is hypothesized that the upward adjustment of the CXCR4/CXCL12 axis occurs in organs on which metastases frequently settle such as the liver, lung, brain, or bone [5,35,36] and this fact can be ratified if it is taken into account that the blockade of this axis leads to metastatic dissemination in animal studies [37,38]. Regarding the possible underlying mechanisms, the influence on the epithelial–mesenchymal transition (EMT) is postulated. This is a process characterized by the disarticulation of tight junctions and loss of apicobasal polarity [39] that facilitates distant dissemination and invasion of different organs by the acquisition of a mesenchymal phenotype. This process involves interleukin 11 [40], the NF-kB receptor [41], and CXCR4 [42,43].
Lastly, CXCR4 is closely related not only to solid tumors but also to the hematopoietic system [44]. Such is the case that the CXCR4/CXCL12-knockout mice exhibit specific characteristics which consist of heart malformations, abnormal cerebellar development, and absence of myelopoiesis and B lymphopoiesis [45,46,47]. This phenomenon can be explained if we take into consideration that CXCL12 is one of the most relevant cytokines involved in the chemotactic response of hematopoietic stem cells (HSCs) [48]. Having this ligand-receptor axis intact results necessary not only for the homing of HSCs through the bone marrow but also in retaining them in the hematopoietic microenvironment [49,50]. This knowledge has led to the development of strategies that target this level, such as the CXCR4 antagonist plerixafor, which is used in bone marrow transplant in patients with multiple myeloma or non-Hodgkin lymphoma due to its ability to mobilize HSCs from the bone stroma to the peripheral blood [51].
NENs are a heterogeneous group of malignant tumors whose origin relies in the cells of the diffuse endocrine system, which are scattered throughout the human body, although the most frequent locations are in the gastrointestinal (GI) tract or in the lung. The incidence of NENs varies substantially according to the location of the primary tumor, being approximately 3.56 new cases per 100,000 in gastroenteropancreatic NENs (GEP-NENs), 1.49/100,000 in bronchopulmonary NENs (BP-NENs), and 0.84/100,000 in unknown primary NENs [52]. It is important to highlight the association of NENs with genetic syndromes such as multiple endocrine neoplasia syndrome type 1 [53]. NENs can be classified depending on whether they produce biologically active substances or not. Currently, it is considered that about 60% of NENs are non-functioning [54]. Carcinoid syndrome is the most common of the many syndromes that can develop due to hormone production [55] (such as insulinoma, glucagonoma, and gastrinoma).
The expression of somatostatin receptors (SSTR) on the cell membrane is a typical feature of NENs and it has diagnostic as well as therapeutic approaches [56]. In fact, the ability to diagnose NENs has improved substantially thanks to the incorporation of gallium-68(68Ga)-labeled DOTA tracers, such as DOTA-TOC, DOTA-TATE, and DOTA-NOC because of their sensitivity and specificity that reach 97% and 92%, respectively [57], compared to Indium-111 scintigraphy (sensitivity 72% and specificity 92%).
Somatostatin analogs (SSA) constitute the first line treatment in NENs due to an antisecretory as well as an antiproliferative effect. In fact, administration of both octreotide [58] or lanreotide [59] has demonstrated increases in progression-free survival (PFS) versus placebo (14.3 vs. 6.0 months, HR 0.34, and >27 vs. 18 months, HR 0.47, respectively) in GEP-NENs. Regarding BP-NENs, only lanreotide has shown benefits in PFS [60] (16.6 vs. 13.6 months, HR 0.90). There are five types of SSTRs, although the drugs currently available focus on SSTR2A and SSTR5 agonism [61]. Lanreotide and octreotide mainly stimulate SSTR2 while pasireotide exerts its action after binding to SSTR2 and SSTR5. In general, NENs are indolent and slow-growing tumors. The main prognostic factor for GEP-NENs is the tumor grade according to the latest WHO classification, which takes into account cytologic features, the number of mitoses per field, and the Ki-67 proliferation index [62]. BP-NENs are governed by a similar classification but this does not take into account the proliferation index but
