Introduction

Cancer is characterized by unregulated proliferation of host cells, arising from alterations in cell physiology, such as: sufficiency in relation to growth factors, insensitivity to growth inhibitors, evasion of apoptosis and the immune system, dysregulation of energy metabolism, genomic instability, phenotypic plasticity, epigenetic reprogramming, tissue invasion and development of metastases [1, 2]. Although the tumor cell represents the main focus in the development of a neoplasm, it is important to consider that the tumor mass is not composed only of neoplastic cells, but of a set of tumor cells, mesenchymal cells and components of the vascular and immune system, which contribute substantially to carcinogenesis, tumor progression and metastasis of transformed cells [3].

Evidence indicates that interactions between tumor cells and stromal cells contribute to tumor initiation and progression. In this process, the role of chemokines and their receptors is highlighted, which can serve as a bridge for tumor-stromal cell communication, creating a permissive microenvironment for tumor growth and metastasis [4], sometimes facilitating tumor dissemination in each of the events of the process, including adherence of tumor cells to the endothelium, extravasation into blood vessels, metastatic colonization, angiogenesis, proliferation and protection against host response through activation of cell survival pathways [5, 6].

Bowel cancer encompasses tumors that start in the part of the large intestine: colon, rectum and anus. It is also known as colon and rectum or colorectal cancer (CRC). This cancer is the third most common cancer type worldwide; in 2020, almost 2 million cases were diagnosed. It is the second most common cause of cancer death, leading to almost 1 million deaths per year [7]. Although environmental and genetic factors play an important role in the pathogenesis of CRC, research suggests the involvement of chemokines and their receptors in the development of colon cancer [8, 9].

Binding of the C-X-C motif chemokine 12 (CXCL12) with its C-X-C motif chemokine receptor 4 (CXCR4) has been associated with several types of malignant neoplasms [10,11,12,13]. Studies have demonstrated increased expression of CXCR4 during CRC malignancy [14, 15]. However, despite contributing to tumor development and maintenance, the signaling mechanism through CXCR4 may be clinically relevant for CRC patients and represents a potential target for disease-directed therapy [16, 17].

Chemokines and their receptors

Chemokines are a large family of structurally homologous cytokines responsible for inducing chemotaxis, through binding to a receptor. They are divided into subfamilies according to the number and position of cysteine residues in the N-terminal portion: C, CC, CXC, and CX3C, where C represents the cysteine molecule and X or X3, refer to the number of variable amino acid residues separating these cysteines [18].

Produced by different cell types, both constitutively and after induction stimuli, chemokines exert their effects in an autocrine or paracrine manner, acting via G protein-coupled receptors [19]. In addition to its chemotactic activity, stimulating the migration of immune, endothelial, mesenchymal stem and malignant epithelial cells [20], chemokines have other functions, such as participating in embryonic development, acting in hematopoiesis, angiogenesis, lymphocyte development, cell maturation and directing the movement of mononuclear cells through the blood [21].

Chemokines are also gaining scientific prominence because of their crucial roles in diseases development and pathological processes, such as inflammation, autoimmune and infectious diseases, like in human immunodeficiency virus (HIV) infection and in cancer [5, 6] They are considered key mediators for tumor growth, angiogenesis, metastases and cellular recruitment for the composition of the tumor microenvironment [20].

Chemokines mediate their functions in target cells through their receptors, which are composed of transmembrane G protein-coupled receptors. In general, they are 320 to 380 amino acids in length and show significant sequence homology. In addition, receptors have an N-terminal portion on the outside of the cell surface, 3 intracellular loops, 3 extracellular loops and a C-terminal portion in the cytoplasm [22]. The binding of chemokines to their ligands triggers conformational alterations in the receptor that culminate in the activation of the G protein. In this way, the subunits of this protein dissociate, leading to the activation of several signal transduction pathways [19, 21].

Receptors are classified as CC, CXC, CX3C, or XC according to the subfamily of their ligands, followed by the letter R (receptor) and a number that reflects the order of their discovery [22]. Furthermore, the receptors are divided into two groups: G-protein-coupled chemokine receptors, which act in several signal transduction pathways, and atypical chemokine receptors, that appear to remove chemokines and contribute to the control of inflammation in a G protein-independent manner [16].

The CXCL12/CXCR4 axis

Among the chemokines, we can highlight the role of CXCL12 in tumor progression and metastasis. Its gene, CXCL12, is located at position 10q11.1 and was first cloned into a bone marrow-derived cell line and later identified as pre-B cell growth stimulating factor (PBSF) [23]. Also called stromal cell-derived factor 1 (SDF-1), this chemokine is constitutively produced in the bone marrow by immature osteoblasts and endothelial cells, as well as by epithelial cells in various organs, such as the lung, liver, adrenal glands and lymph nodes [24].

CXCL12 has six isoforms (CXCL12 α to φ) derived from alternative splicing, with CXCL12α and CXCL12β being the most widely studied subtypes. The α isoform is increased in tissue damage but is rapidly degraded in the blood. In addition, it is the main form secreted by bone marrow cells and endothelial cells, found in almost all organs. The β isoform is more resistant to degradation, stimulates angiogenesis and is present in highly vascularized organs such as liver, spleen and kidney [16].

Literature data suggest that CXCL12 acts as a modulator of cell growth and survival by binding to its receptor, CXCR4, playing a key role in the homing of stem and hematopoietic progenitor cells. The gene encoding CXCR4 is located on chromosome 2q2 and is expressed in dendritic cells, naive T cells, natural killer cells and monocytes [25]. In addition, the production of this chemokine in the initial stage of the disease aids in the process of angiogenesis and in the growth of tumor cells, while in more advanced stages, production decreases in order to avoid the recruitment of cytotoxic lymphocytes and increases the metastatic potential of tumor cells [26], being implicated in the spread of malignant tumors from the primary site, transendothelial migration of tumor cellsand homing of precursor tumor cells [27]. The expression of CXCR4 in malignant cells indicates that the CXCL12/CXCR4 axis can influence tumor biology and play a key role in directing metastasis of CXCR4 + tumor cells towards organs that express CXCL12 [28].

The CXCL12/CXCR4 interaction was believed to be unique, until the description of another receptor, CXCR7, with a strong affinity for CXCL12 [29]. CXCR7 is part of the G protein-coupled cell surface receptor family and is also associated with tumor cell survival, migration, adhesion, angiogenesis and metastasis [25]. CXCR7-mediated activation of intracellular signals remains controversial since, unlike typical chemokine receptors, CXCR7 does not activate heterotrimeric G proteins [16]. The CXCR7 sequence presents a small modification and when bound to CXCL12, there is no increase in intracellular Ca2+ release, an essential step in the recruitment and activation of G proteins [30]. The CXCR7 receptor is reported to be highly expressed in many tumors and tumor-associated blood vessels, including cancers of the liver, colon, pancreas, prostate and lungs [16, 31,32,33]. In CRC, its role in tumorigenesis probably occurs by means of histone demethylation, through the formation of heterodimers with CXCR4 [34]. Furthermore, overexpression of CXCR7 is significantly correlated with the presence of distant metastasis, advanced TNM stage, reduced overall survival and disease-free survival in patients with CRC [35].

Actuation of the CXCL12/CXCR4 axis in the CRC

The CXCL12 chemokine acts in cancer biology by two main mechanisms. The first is related to direct autocrine effects, through the activation of signaling pathways that promote tumor cell growth, metastasis and angiogenesis. The second occurs through indirect effects, where high levels of CXCL12 in tumors attract CXCR4+ inflammatory, vascular and stromal cells to the tumor mass. This favorable microenvironment supports tumor growth through the secretion of growth factors, cytokines, chemokines and pro-angiogenic factors, so an indirect effect is that CXCR4+ tumor cells can be recruited to CXCL12-rich mesenchymal stromal niches to initiate metastasis [4], which can be seen in CRC [28]. Overall, a high CXCR4 expression is clearly associated with an advanced stage of the tumor, an increased risk of recurrence and distant metastases and poor overall survival of CRC [13], with an increased risk of death and progression in patients (Figure 1) [17].

The CXCL12/CXCR4 axis can be regulated by epigenetic, transcriptional and post-transcriptional factors. Regulation of CXCL12/CXCR4 expression by promoter hypermethylation is common in cancer and studies show that the CXCL12 gene modulates metastatic potential, where it controls its own regulation in an autocrine loop [4]. CRC tumor cells can also undergo DNA hypermethylation at the CXCL12 promoter, resulting in an imbalance in CXCL12/CXCR4 expression. In this way, tumor cells that lack CXCL12 expression but maintain overexpression of CXCR4 can selectively spread to target organs in which CXCL12 is highly secreted [36, 37].

CXCL12 significantly increases several genes associated with angiogenesis in tumor cells, such as the IL-6 gene [10]. In CRC, IL-6 activates the janus kinase/signal transducer and activator of transcription (JAK/STAT3) to promote tumor initiation and tumor growth. Thus, the IL-6/STAT3 pathway activates target genes to protect tumor cells from apoptosis, promote angiogenesis and drive tumor cell proliferation, cell cycle progression, invasion and metastasis [38].

Studies demonstrate that transcription factors, such as NF-κB, Sp1 and C/EBP-β can be activated through mitogen-activated protein kinase (MAPK) pathways in CRC cells [39]. Tung et al. [40] proposed a relationship between this signal transduction pathway, the chemokine CXCL12 and the expression of intercellular adhesion molecule-1 (ICAM-1). According to the authors, CXCL12 induces extracellular signal-regulated kinase (ERK) phosphorylation, c-Jun N-terminal kinase (JNK) and p38, which consequently activates NF-κB, Sp1 and C/EBP-β, leading to their binding to the respective sites on the ICAM-1 promoter, thus resulting in the transcriptional activation of ICAM-1, facilitating the adhesion of cancer cells to the vascular endothelium and later the promotion of metastases.

The increase in NF-κB mediated by the activation of the MAPK pathway can also occur by stimulation of an external factor, such as visfatin, an adipokine produced by adipose tissue. Huang et al. [41] suggest that visfatin induces the expression of CXCL12 through the activation of β1 integrin, the ERK and p38 intracellular signaling cascades, and the NF-κB transcription factors and activator protein 1 (AP-1), thus contributing to the progression of CRC. In addition, lipopolysaccharides (LPS), normally produced by the microbiota, also use NF-κB signaling to induce CXCR4 expression in tumor cells, promoting epithelial-mesenchymal transition (EMT) and metastasis in the CRC [39, 42].

A study by Hu et al. [43] demonstrated a cross-relationship between the CXCL12/CXCR4 and Wnt/β-catenin axis in the CRC, where the CXCR4 receptor could aid in disease progression, invasion and the EMT through the activation of this signaling pathway. Constitutive activation of the Wnt/β-catenin pathway in CRC can occur through aberrant secretion of Wnt factors or a mutation in the adenomatous polyposis coli (APC) gene [44].

Activation of CXCL12 indirectly exerts anti-apoptotic effects on tumor cells. The CXCL12/CXCR4 axis activates serine-threonine kinase (AKT) and ERK, leading to the accumulation of NF-κB. Accumulation of this transcription factor can suppress apoptotic signaling. In addition, CXCL12 can suppress tumor cell apoptosis by inducing the MAPK-ERK and PI3K pathways, which inactivate the pro-apoptotic protein Bcl-2-associated cell death agonist (BAD), leading to upregulation of the anti-apoptotic gene Bcl-2 [25, 45].

A hypoxic tumor microenvironment also favors tumor progression during carcinogenesis through upregulation of CXCR4 expression in tumor cells by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that responds to tissue oxygen concentrations. Thus, in hypoxic regions of expanding tumors, CXCR4 levels can be increased to facilitate tumor survival and invasion [14, 46].

Figure 2 presents the main mechanisms of action of the CXCL12/CXCR4 axis in the CRC.

Fig. 1
figure 1

The tumor microenvironment in CRC. The tumor microenvironment includes both the cellular componentssurrounding the tumor mass such as immune cells, fibroblasts and epithelial cells and the acecellular components including the extracellular matrix and blood vessels. Cancer cells release molecules (CXCL12) that modulate the TME and contribute to cancer growth through immune evasion, metastatic niche formation, neoangiogenesisamong other functions that contribute to the hallmarks of cancer. “Created with BioRender.com.”

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Fig. 2
figure 2

CXCL12/CXCR4 signaling pathway. Bcl-2– anti-apoptotic gene; EMT – Epithelial-to-mesenchymal transition; MAPK – mitogen-activated protein kinase; ERK – extracellular signal-regulated kinase; PI3K – phosphatidylinositol-3-kinase; AKT - serine-threonine kinase; JAK/STAT – Janus kinase /signal transducer and activator of transcription; IL-6 – interleukin 6; ICAM-1 – Intercellular Adhesion Molecule 1; NF-κB – nuclear factor kappa B; LPS – Lipopolysaccharide; HIF-1 – hypoxia-inducible factor 1

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Therapeutic strategies targeting the CXCL12/CXCR4 axis in CRC

This axis is directly involved in the progression of cancer, which, therefore, have an important role in the signaling network, attracting great interest on the part of researchers in exploring therapeutic measures for this target. Thus, studies are seeking inhibitory or antagonist molecules of CXCR4 and CXCL12 that can be used in the treatment of various types of tumors, including CRC [16, 47].

The first therapy targeted at CXCR4 was AMD3100 (Plerixafor), which acts as an antagonist by binding to the CXCR4 receptor, preventing the conformational change necessary to activate intracellular kinases [48]. In addition, its mechanism of action may be related to the mobilization of CD34+ hematopoietic progenitor cells (HPCs), an increase in circulating neutrophils, lymphocytes and monocytes, a reduction in myeloid-derived suppressor cells and increased infiltration of cytotoxic T cells into tumors [47]. Because of these functions, its use was cleared by the Food and Drug Administration (FDA) in 2008 to mobilize HPCs cells for autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma [49].

Recent studies have shown that AMD3100 can inhibit tumor growth and metastasis, in addition to acting as a potent immunomodulator to enhance antitumor immune responses and prevent the development of a multifaceted immunosuppressive intratumoral microenvironment [50]. Fearon, Janowitz [51] showed that infusion of AMD3100 in patients with CRC for only 1 week induced intratumoral immune responses that involved multiple mediators and cells of both innate and adaptive immune responses. According to the authors, inhibition of CXCR4 by AMD3100 in patients with CRC allowed their pre-existing anticancer immune responses to overcome intratumoral immunosuppression mediated by CXCL12, through increased expression of genes whose products mediate cytotoxicity by CD8+ T cells, that correlate with decreased expression of genes characteristic of tumor cells. Biasci et al. [52] also demonstrate that continuous administration of AMD3100 promotes an integrated immune response in metastatic lesions of patients with CRC.

Another drug under clinical study is LY2510924, which also acts as a potent and selective antagonist of CXCR4 [53]. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non-Hodgkin’s lymphoma, renal cell carcinoma, lung cancer cells and CRC expressing functional CXCR4. Furthermore, its mechanism of action involves the inhibition of the CXCL12/CXCR4 interaction, with consequent blocking of signaling [54]. In addition, several clinical studies show that the administration of LY2510924 was associated with a significant increase in CD34+ HSCs and leukocytes in circulation [55, 56].

The CXCL12 chemokine has also been studied as a possible therapeutic target in different types of tumors, including CRC. CXCL12 acts as a communication bridge between tumor cells and their environment and may confer resistance to checkpoint inhibitors by excluding T cells in preclinical models. Studies show that the use of molecules that interfere with the mechanisms of failure of cytotoxic T cells to physically reach tumor cells can support combined cancer immunotherapy treatments [57, 58].

Evidence indicates that inactivation of CXCL12 by Olaptesed Pegol (NOX-A12) induces alterations in the tumor microenvironment of patients with CRC and pancreatic cancer, making tumors more susceptible to immuno-oncological approaches, such as checkpoint inhibition by anti-PD-1. NOX-A12 is an L-configured aptamer (Spiegelmer) that binds to CXCL12 with high affinity and specificity, blocking the CXCL12/CXCR4 axis. Thus, inhibition of CXCL12 leads to an increase in the infiltration of T cells and natural killer (NK) cells into the tumor [58, 59].

A phase I/II clinical trial (NCT03168139) evaluated the effects of a combination treatment with NOX-A12 and anti-PD-1 in patients with later-line CRC and pancreatic ductal adenocarcinoma [60]. According to the authors, the new approach of specifically inhibiting CXCL12 through a spiegelmer showed activity alone and in combination with anti-PD-1 in these patients, where the mechanisms of NOX-A12-mediated transformation of the tumor immune microenvironment included the migration of T and NK cells into the tumor.

New molecules targeting the CXCL12/CXCR4 axis have been proposed as possible therapies for CRC. D’Alterio et al. [61] demonstrated in a preclinical trial that the newly developed CXCR4 antagonist, Peptide R (Pep R) is able to improve the efficacy of standard CRC therapy targeting cell growth and mesenchymal transition, through inactivation of CXCR4.

Therapeutic strategies have focused on the potential of CXCR4 antagonists to enhance the cytotoxic effect of chemotherapy and immunotherapy. Furthermore, future therapies may also involve genome editing technologies to remove harmful “driver mutations” and insert “preventive mutations” on the CXCL12/CXCR4 axis [16].

Conclusion

The CXCL12/CXCR4 plays an important role in the development of several types of malignant neoplasms. Activation of this axis results in the induction of signal transduction pathways, with effects on chemotaxis, cell migration and gene expression. In CRC, increased expression of this axis leads to tumor progression and the development of metastases, with unfavorable disease progression and poor patient survival.

Preclinical and clinical studies have been carried out in order to study several drugs that can inhibit CXCL12/CXCR4 and used in combination with chemotherapy or immunotherapy for the treatment of CRC. In addition, the development of new research involving genome editing technologies may also represent a future strategy, allowing a more effective therapeutic targeting against the disease.