Biological Functions of CXCL12-CXCR4 in Human Malignant Tumors

WANG Sen, LUO Ji-Xian*, Istvan Boldogh

(1)School of Life Sciences, Shanxi University, Taiyuan 030006, China；2)Department of Microbiology and Immunology,University of Texas Medical Branch at Galveston, Galveston TX 77555， USA)

Abstract Metastasis and cell infiltration are the difficulties in the treatment of solid and lymphatic carcinoma and the main causes of disease recurrence and death. The migration of cancer cells is a prerequisite for tumor metastasis and invasion. The CXCL12-CXCR4 pathway plays an important role in the pathogenesis of solid tumors and leukemia. The interaction between CXCL12 and its receptor CXCR4 can activate multiple signaling pathways and regulate different physiological and pathophysiological processes. Thus, blocking CXCL12-CXCR4 binding and/or downstream pathways has clinical benefits in treating a variety of diseases and cancers. Although some CXCL12 and CXCR4 antagonists have been identified and have shown encouraging results in terms of antitumor activity, these drugs have not been widely used in clinical patients due to their serious toxic and side effects. There is an urgent need to develop novel CXCL12-CXCR4 axis antagonists for the treatment of tumors. Herein, we review the recent research progress of CXCR4 pathway in solid tumors and leukemia, and discuss the therapeutic value and future research direction of CXCR4 pathways in solid tumors and leukemia.

Key words C-X-C chemokine receptor 4 (CXCR4); C-X-C motif ligand 12 (CXCL12); cancer; leukemia; pathways

C-X-C motif ligand 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), is constitutively expressed in various tissues of the brain, lung, liver, kidney, heart, skeletal muscle, lymph nodes, bone marrow and skin[1,2]. TheCXCL12 gene is located at 10q11.1, and in humans it encodes six spliced variants that are distributed in a tissue-specific manner, where their expression/activity is controlled at multiple levels. They have key function(s) in both physiological and patho-physiological processes, including innate and adaptive inflammatory responses where it induces/activates migration of hematopoietic progenitor cells, stem and endothelial cells as well as leukocytes. CXCL12 has essential roles in embryogenesis, angiogenesis and hematopoiesis[3]. Recent studies have demonstrated that CXCL12 and its receptor contribute to tumor development, angiogenesis, and metastasis of several human cancers in an autocrine manner[4]. C-X-C chemokine receptor 4 (CXCR4), a G protein-coupled receptor, is expressed by numerous types of cells, including hematopoietic stem cells, embryonic stem cells, stromal fibroblast satellite progenitor cells, mesenchymal cells, lymphocytes, endothelial cells, epithelial cells, and germ cells[5, 6]. TheCXCR4 gene is located at 2q21 and has two exons with 103 nucleotides (nt) and 1 564 nt. The amino-terminal domain of CXCL12 binds the second extracellular loop of CXCR4 and activates downstream signaling pathways[7]. The third intracellular loop of CXCR4 is necessary for Gαi-dependent signal transduction, while the intracellular loop 2 and 3 of CXCR4 and the C-terminal of CXCR4 are necessary for chemotaxis[8,9]. The binding of CXCL12 with CXCR4 can trigger a variety of signal transduction pathways, thereby regulating cell chemotaxis, transcription and cell survival[6].

1 The signal transduction mechanism of CXCL12-CXCR4

CXCR4 is a pertusis toxin-sensitive GTP-binding protein of Gitype. After binding with CXCL12, the heterotrimer G protein is activated by the exchange of GDP and GTP, and is then separated into the GTP-bound α and βγ subunits[10]. The dissociated βγ subunits activate phosphatidylinositol specific phospholipase C-β (PLC-β) and phosphatidylinositol-3-OH kinase (PI3K)[11]. The PLC-β cleaves phosphatidylinositol (4, 5)-bisphosphate into two secondary messengers, inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG). IP3 can induce Ca2+release by binding to endoplasmic reticulum (ER) specific receptors. DAG and Ca2+act to activate Mitogen-activated protein kinase (MAPK) and promote cell migration[12,13]. Ca2+binding to calmodulin (CaM) stimulates NOS to produce NO. Ca2+can also stimulate the production of ROS by Nicotinamide adenine dinucleotide phosphate (NADPH; a reduced form of NADP+) oxidase, of which activity regulated by p40phox, p47phox, p67phox and Rac1 or Rac2. ROS-driven signaling can promote cell proliferation; however, supra physiological levels of ROS can promote cell apoptosis[14]. PI3K is activated by Gβγor Gαsubunits, which rapidly generate phosphatidylinositol (3,4,5)-triphosphate and activate the Protein kinase B (AKT) pathway[15]. Activated AKT plays a key role in tumor cell survival by inactivating phosphorylation of the B-cell lymphoma 2 (BCL2)-associated agonist of cell death (BAD). AKT-mediated CXCR4 signal transduction enables β-catenin to move to the nucleus, activates gene transcription and promotes proliferation[16]. Gαregulates gene expression through PI3K-AKT-MEK1/2, ERK1/2, and NF-κB axes. PI3K can also activate protein-rich tyrosine kinase 2 (Pyk2) and thereby regulate cell migration[17]. As the Gαsubunit is activated, the Ras pathway activates ERK, which can phosphorylate and regulate other cell proteins as well as be imported to the nucleus where it can activate transcription factors, leading to changes in gene expression and cell cycle progression[18]. In addition, Gαmay also activate the Rac/Rho pathway, leading to phosphorylation of the p38 protein, which then promotes cell proliferation and survival[18]. There are three processes to regulate CXCR4: desensitization (homologous and heterologous), internalization, and degradation[17]. When CXCR4 combines with CXCL12, it is rapidly phosphorylated, and in the process of phosphorylation, CXCR4 recruits arrestin to make the receptor internalized[19-21], after which CXCR4 can be recycled back to the plasma membrane or sorted to the lysosome for degradation[22](Fig.1).

Fig.1 Signaling pathways and regulation of CXCR4 PLC (phospholipase C), PIP2 (phosphatidylinosital biphosphate), IP3(inositol triphosphate), CaM (calmodulin), NOS(nitric oxide synthase), NO (nitric oxide), p47phox(NADPH oxidase p47phox), ROS(reactive oxygen species), DAG(diacylglycerol), PKC (protein kinase C), MAPK (mitogen-acticated protein kinase), PI3K (phosphatidylinositol 3-kinase), NF-kB (nuclear factor-kB), Pyk2 (protein-rich tyrosine kinase 2), FAK (focal adhesion kinase), Crk (CT10 regulator of kinase), MEK1/2(Mitogen-activated protein kinase kinase 1/2), JAK(Janus kinase), ERK1/2(extracellular signal-regulated kinase1/2), STAT(signal transducer and activator of transcription)

2 The role of CXCR4 in cancer

The cancer genome atlas (TCGA) showed that CXCR4 was upregulated in breast adenocarcinoma, kidney renal clear cell carcinoma, kidney renal papillary carcinoma, esophageal carcinoma, glioblastoma multiforme, cholang iocarcinoma and stomach adenocarcinoma compared with normal controls. However, only patients with stomach adenocarcinoma showed that the high and low expression of CXCR4 had a significant impact on patient survival.

Although CXCR4 expression was not significantly altered in brain low grade glioma, head and neck squamous cell carcinoma and acute myeloid leukemia compared to normal controls, differences in CXCR4 expression in these patients demonstrated a significant survival difference (Table 1). This suggests that the expression of CXCR4 may influence the survival of tumor patients. By analyzing the literature on CXCR4, we found that CXCR4 plays an important role in tumorigenesis and development, including angiogenesis, invasion and migration, proliferation and anti-apoptosis.

Table 1 Expression of CXCR4 and survival rate of cancer patients

2.1 CXCR4 induces angiogenesis

Angiogenesis is a hallmark of cancer, which provides additional access to nutrients for cancers. CXCL12 is one of the most potent angiogenic chemokines. Activation of the CXCL12-CXCR4 axis stimulated angiogenesis in endothelial cells[23]. Endothelial cells exposed to CXCL12 showed high level expression of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP-2).CXCR4-knockdown cells failed to express these mediators, suggesting important roles of CXCL12-CXCR4 in angiogenesis[23]. CXCL12-CXCR4 may regulate tumor angiogenesis through the following three mechanisms: (1) CXCL12 signaling increases VEGF expression by activating PI3K/AKT pathways or by decreasing the expression of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1)[11]. The increase in VEGF levels promote the proliferation of vascular endothelial cells, leading to the formation of new blood vessels[24]. (2) CXCL12 may also enhance angiogenesis via interleukin (IL)-6. CXCL12 mediates the transcriptional regulation of IL-6 by phosphorylation of ERK1/2 and activation of NF-κB[25]. In turn, IL-6 indirectly induces angiogenesis by increasing the expression of VEGF, fibroblast growth factor and cyclooxygenase-2[26,27]. (3) CXCL12 secreted by fibroblasts recruits endothelial progenitor cells near neovascularization, thus promoting tumor angiogenesis.

In addition to classical pathways, mammalian target of rapamycin complex 2 (mTORC2) is a downstream target of CXCL12-CXCR4 signaling, which is involved in the regulation of angiogenesis[28]. In 2018, studies showed that the activation of the CXCL12-CXCR4 axis in vascular endothelial cells stimulates angiogenesis by upregulating MAPK/ERK, PI3K/AKT and wingless-type MMTV integration site family protein (Wnt)/beta (β)-catenin pathways[22]. At the same time, a large number of experimental data show that the inhibition of CXCL12-CXCR4invitroorinvivoby CXCR4 blocker can decrease the angiogenesis of non-small cell lung cancer and increase the sensitivity of cancer cells to chemotherapy[29,30]. Although there is no definite conclusion about the mechanism of angiogenesis induced by CXCR4, CXCR4 does play a role in angiogenesis.

2.2 CXCR4 promotes metastasis of cancer cells

Metastasis refers to the spreading of malignant cells from a primary location, causing their progressive extension to distant organs that eventually kills most cancer patients. Epithelial mesenchymal transition (EMT) is an important process related to tumor metastasis. In this process epithelial cells lose their polarity and acquire the phenotype of migration and invasion[31]. EMT is characterized by the loss of E-cadherin and the upregulation of vimentin[32]. It has been shown that the CXCL12-CXCR4 signal promotes EMT by activating MEK/ERK, PI3K/AKT or Wnt/β-catenin pathways[33,34]. CXCL12-CXCR4 also plays an important role in the invasion and metastasis of cancer cells[35].

Studies have shown that inhibition of CXCR4 with AMD3100 and knockout ofCXCR4 can decrease the invasion and migration of cancer cells[34,36]. This suggests that CXCR4 does play a role in tumor invasion and migration, probably via the PI3K/AKT, ERK and NF-κB pathways. Activation of the ERK1/2 signaling pathway can increase the expression of MMP-2 and MMP-9, thus promoting the metastasis of cancer cells[37,38]. The signaling pathway of CXCL12-CXCR4 in tumor metastasis remains to be further studied.

2.3 CXCR4 promotes cancer cell survival

Unlimited proliferation is a significant feature of cancer. CXCL12-CXCR4 axis promotes the survival and proliferation of cancer cells via inhibition of apoptotic processes in cancer cells[39]. CXCL12-CXCR4 may inhibit apoptosis in two ways: (1) Activation of the anti-apoptotic program via the JAK/STAT signaling pathway[40]; (2) Inhibition of the PI3K/AKT/NF-κB signaling pathway against apoptosis[29]. The expression of CXCL12 can lead to the activation of NF-κB[41]. Activated NF-κB increases expression of Bcl-2 and apoptosis regulator Bcl-X (Bcl-XL), thereby inhibiting apoptosis. In addition, CXCL12 can upregulate the anti-apoptotic geneBcl-2 by directly inhibiting the proapoptotic protein BAD or indirectly activating the transcription factor cAMP response element binding protein, so as to inhibit the apoptosis of tumor cells[4]. Cancer associated fibroblasts (CAF) are an essential part of the tumor environment. They are continuously activated, which facilitates tumor cell growth/proliferation, angiogenesis, tumor-associated inflammation, and remodeling of the extracellular matrix[42]. When the diameter of the tumor reaches a certain size, hypoxic areas will form because neovascularization cannot provide sufficient nutrition and oxygen. Within the anoxic environment, the increase levels of CXCL12 in CAFs lead to CXCR4 expression on cancer cells, which promotes cell proliferation, invasion and migration[43,44]. CXCL12-CXCR4-mediated cancer cell proliferation is MAPK pathway dependent, which up-regulates the expression of CXCR4 by increasing DNA occupancy of multiple transcription factors promoting the survival of cancer cells[45,46]. Thus inhibition of the CXCL12-CXCR4 pathway inhibits cancer proliferation and has clinical relevance.

3 The relationship between CXCR4 and leukemia

Various forms of leukemia (acute and chronic myeloid [AML, CML], acute and chronic lymphocytic [ALL, CLL]) are a seriously threat to human health, whose pathogenetic mechanism is very complex. On one hand, the interaction between leukemic cells and bone marrow (BM) stroma will lead to migration of leukemic cells to the BM microenvironment nest to enhance the proliferation and viability of leukemic cells[47]. On the other hand, a large number of abnormal leukocytes infiltrate the extramedullary tissues[48]. Under physiological conditions, CXCL12 secreted by bone marrow stromal cells specifically bind to CXCR4 on the surface of bone marrow hematopoietic stem cells, which is crucial for hematopoietic stem cells homing, quiescence and long-term maintenance in niches[49,50]. However, leukemia cells compete with hematopoietic stem cells to enter the niche of hematopoietic stem cells, destroying the normal interaction between hematopoietic stem cells and their microenvironment, thus replacing hematopoietic stem cells and inhibiting hematopoietic function[51].

TCGA data show that perturbed expression of CXCR4 is primarily related to AML, B cell-ALL and CLL, while recent studies extend it to other types of leukemia. CXCL12 is produced by the BM microenvironment where it binds and activates its cognate receptor CXCR4 on leukemic cells, facilitates leukemia cell trafficking and homing in the BM microenvironment, and keeps leukemia cells in close contact with the stromal cells and extracellular matrix that constitutively generate growth-promoting and anti-apoptotic signals[52]. When CXCR4 binds to CXCL12, it can activate MAPK, AKT and ERK pathways, release Ca2+from the endoplasmic reticulum, and finally coordinate homing, proliferation and cell survival[53].

3.1 CXCR4 and ALL

Acute lymphocytic/lymphoblastic leukemia (ALL) is a malignant proliferation of lymphoid progenitor cells of B or T cell lineage. ALL is the most common malignancy in children, accounting for approximately 80% of leukemia in the pediatric group, and its etiology is unknown[54]. There are two subtypes of ALL: T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL). T-ALL arises from the clonal proliferation of T lymphocyte progenitor cells[55]. It accounts for 10-15% of ALL in children and 25% of ALL in adults[56]. Despite major advances, the treatment of T-ALL lags behind B cell ALL (B-ALL) and other leukemia subtypes in the availability of immunotherapy and molecular targeted therapies[57]. Relapsed T-ALL remains a major clinical problem. Bone marrow and central nervous system (CNS) relapse are important reasons for the poor prognosis of T-AL[58]. The exact etiology that caused T-ALL CNS relapse have not been determined. Some studies suggest that CXCR4 is involved in the infiltration of T-ALL cells in leukemia[59]. Furthermore, high CXCR4 expression was also found in B-ALL cells and correlated with a higher incidence of relapse[60]. The expression of CXCR4 was positively correlated with the migration ability of leukemia cells[61]. The Zeta chain of T cell receptor associated protein kinase 70 (Zap70) regulates the expression of CXCR4 in T-ALL cells through ERK1/2 and promotes migration to CXCL12 in an ERK1/2-dependent manner, which is also consistent with the central nerve infiltration in T-ALL[62, 63]. CXCR4 is also involved in the development of T-ALL disease[64-66]. However, the downstream signals of CXCR4 and mechanisms involved in infiltration still need to be further studied[67].

3.2 CXCR4 and AML

Acute myeloid leukemia (AML) is a heterogeneous malignancy, which is characterized by infiltration of the bone marrow, blood, and other tissues[68-70]. In AML, malignant cells arising from immature myeloid progenitors or stem cells increasingly occupy the BM space as the disease progresses, leading to rapidly fatal complications without treatment[71]. Primary refractory disease and relapse after a complete remission are the greatest challenges in treating AML[71]. In AML patients, the surface of leukemia cells expresses high levels of CXCR4[5]. In recent years, it was found that chemotherapeutic agents upregulated CXCR4 in AML cell lines, suggesting CXCR4 is one of the mechanistic reasons for cellular resistance to chemotherapy[52, 72]. CXCL12-CXCR4 signaling has a role in the development of human AML stem cells and has been found to regulate theirinvivomotility[73]. Accordingly, CXCR4 overexpression correlates with poor prognosis in AML patients[74]. AMD3100, the CXCR4 inhibitor has been shown to decrease in CXCL12-induced migration of AML blasts and AML xenotransplants in NOD/SCID mice[75]. Therefore, targeting the CXCL12-CXCR4 axis may have therapeutic potential for AML.

3.3 CXCR4 and CLL

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia[76]. Combination of CXCL12 and CXCR4 promotes B-cell progenitors and CLL cell survival through CXCR4 signaling[77-79]. The CXCL12-CXCR4 axis promotes cell survival and proliferation, which may contribute to the orientation of leukemia cells to the lymphoid tissue and bone marrow[80]. Thus, activation of the CXCL12-CXCR4 axis plays an important role in stromal cell-dependent resistance to therapy in CLL patients[81]. In the last decade, a number of inhibitors targeting CXCR4 have been developed, and they have been shown to inhibit the CXCL12-CXCR4 signaling in CLL cells.

3.4 CXCR4 and CML

Chronic myeloid leukemia is characterized by myeloproliferative disorder, and represents 20% of all adult leukemia. In contrast to CLL, around 16% of CML patients develop extramedullary blast crisis, which usually involves lymph nodes, BM and the CNS[82]. A proposed mechanism for molecular relapse in CML patients involves leukemic stem cell homing to the bone marrow, mediated through the CXCL12-CXCR4 axis[83]. CXCR4 directly delivered growth-promoting and/or anti-apoptotic signals to CML cells[84]. CXCR4 activated its downstream PI3K/AKT signal pathway and promoted the activation and nuclear translocation of NF-κB in CML cells, resulting in a decreased expression of apoptosis-related molecules[85]. Furthermore, CXCL12 increased the expression of ATP-binding cassette transporters, and their enhanced expression might substantially contribute to resistance to chemotherapeutic agents by exporting chemotherapeutic drugs outside cells[84, 86].

4 CXCL12-CXCR4 axis and immunity

The immune system is the best tool for human beings to fight against various diseases. The immune system can not only remove the pathogens that infect the human body, but also clear the cancerous cells in the body. Anti-PD ligand-1, anti-programmed death receptor 1 (anti-PD-1) and checkpoint antagonists such as anti-CTLA-4 have achieved good therapeutic effects in some cancers[87]. However, immunotherapy for more solid tumor patients such as ovarian cancer, colon cancer has not achieved good results, which may be related to the CXCL12-CXCR4 axis[45]. Studies have shown that blocking the interaction between CXCR4-expressing T cells and CXCL12-secreting cells in microenvironment can regulate the immunotherapy anti-CTLA-4 or anti-PD-1[7]. In pancreatic ductal adenocarcinoma (PDA) model, CXCL12 released by CAF may regulate T cell entry into tumors by binding with PDA cancer cells[7]. Plerixafor inhibits the expression of CXCR4, increases the accumulation of T cells among cancer cells, inhibits tumor growth, and increases the sensitivity of tumor to anti PD-L1[7, 45]. CXCR4-dependent immunoregulation was confirmed in the hepatocellular carcinoma (HCC) model. In HCC model, anti PD-1 combined with sorafenib and AMD3100 could reduce the growth of HCC[34]. Therefore, the CXCL12-CXCR4 axis can cooperate with immunotherapy in cancer patients.

As a co-receptor of HIV (human immunodeficiency virus), CXCR4 is necessary for the entry of human immunodeficiency virus type HIV-Ⅰ into target cells[88]. After infection with HIV, CD4+T cells will be destroyed, and CD4+T cells play an important role in the human immune system[89]. A large amount of destruction of these cells will cause the loss of human immune function. Without the protection of the immune system, patients can develop multiple infections early on and malignant tumors later on. CXCL12 can inhibit CXCR4-mediated virus entry, but direct use of CXCL12 as an inhibitor may have serious consequences[90]. The CXCL12 mutant SDF-1/54 can maintain its ability to bind to CXCR4 while showing resistance to HIV. Therefore, based on the CXCL12-CXCR4 axis, it is possible to find new drugs to treat Acquired Immune Deficiency Syndrome (AIDS).

5 Prospects

As mentioned above, the CXCL12-CXCR4 axis promotes the invasion and metastasis of cancer cells through a variety of signaling pathways, thus promoting the occurrence and development of cancer. Therefore, the CXCL12-CXCR4 axis is considered as a new target for tumor therapy. So far, many inhibitors of CXCL12-CXCR4 axis have been reported, which can inhibit the growth of cancer cellsinvivoandinvitro, and show good anticancer activity in a variety of tumor cells. These inhibitors mainly include four categories: (1) peptides, including BKT140, LY2510924, T140 and so on; (2) small molecules, AMD11070, AMD3100, MSX-122，POL6326(balixafortide),etc; (3) antibodies, such as MDX-1338/BMS 93656; (4) modifying agonists and antagonists of CXCL12, such as CTCE-9908(Table 2).

Table 2 Some antagonists of CXCL12-CXCR4 in clinical research

At present, the most studied inhibitor is AMD3100, which can inhibit the binding of CXCL12 and CXCR4. In 2008, FDA approved the combination of AMD3100 and granulocyte colony-stimulating factor to mobilize hematopoietic stem cells into peripheral blood for autologous transplantation in patients with non Hodgkin’s lymphoma. Now AMD3100 has been proved to prevent ALL, AML recurrence, and can reduce the growth and metastasis of primary small cell lung cancer[106,107]. A study of breast cancer patients showed that AMD3100 has a certain therapeutic effect on breast cancer[108]. However, the long-term use of AMD3100 is limited due to its cardiotoxicity. In addition, AMD3100 lacks the specificity of CXCR4 because it can also bind to CXCR7 as an allosteric agonist. Therefore, there is still room for drug development of the CXCL12-CXCR4 axis.

The CXCL12-CXCR4 axis can not only be used as a single therapy target, but also can be used in combination with immunotherapy in cancer patients. A large number of preclinical data have shown that CXCL12-CXCR4 axis antagonists have good antitumor activities in a variety of tumor cells, including (1) affecting CXCR4 expressing cancer cells; (2) regulating immune response; (3) cooperating with other targeted therapies. Although some gratifying results have been achieved, the clinical results so far are not satisfactory. It is necessary to further study the CXCL12-CXCR4 axis inhibitors that have been found so far. At the same time, it is urgent to develop new CXCL12-CXCR4 axis antagonists to produce enough anticancer activity, so as to produce good therapeutic effects in a complex tumor microenvironment.

AcknowledgementsWe thank Dr. Roy Ambli Dalmo, UiT-The Arctic University of Norway for suggestions and editing of the manuscript.For editing this manuscript,we also thank Sherry L. Haller, PhD, English Editor, Research Development Specialist, The University of Texas Medical Branch, Galveston.