The expression and role of CXC chemokines in colorectal cancer

Cytokine & Growth Factor Reviews 22 (2011) 345–358

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The expression and role of CXC chemokines in colorectal cancer Hannelien Verbeke a, Sofie Struyf a, Genevie`ve Laureys b, Jo Van Damme a,* a b

Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven (K.U. Leuven), Minderbroedersstraat 10, B-3000 Leuven, Belgium Department of Pediatric Hemato-Oncology and Stem Cell Transplantation, Kinderziekenhuis Prinses Elisabeth, Ghent University Hospital, Ghent, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Available online 14 October 2011

Cancer is a life-threatening disease world-wide and colorectal cancer is the second common cause of cancer mortality. The interaction between tumor cells and stromal cells plays a crucial role in tumor initiation and progression and is partially mediated by chemokines. Chemokines predominantly participate in the chemoattraction of leukocytes to inflammatory sites. Nowadays, it is clear that CXC chemokines and their receptors (CXCR) may also modulate tumor behavior by several important mechanisms: regulation of angiogenesis, activation of a tumor-specific immune response by attracting leukocytes, stimulation of tumor cell proliferation and metastasis. Here, we review the expression and complex roles of CXC chemokines (CXCL1 to CXCL16) and their receptors (CXCR1 to CXCR6) in colorectal cancer. Overall, increased expression levels of CXC chemokines correlate with poor prognosis. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Angiogenesis Chemokine Colorectal cancer Leukocyte

Contents 1. 2. 3.

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The development of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemokines: classification and functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemokines in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of chemokines in tumor-related angiogenesis. . . . . . . . . . . . . . . . . . Link between chemokines, inflammation and cancer . . . . . . . . . . . . . . . . 3.2. Small and large intestinal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of CXC chemokines in colorectal cancer. . . . . . . . . . . . . . . . . . . . . . . . . ELR+ CXC chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. CXCR2 ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. 5.1.2. IL-8/CXCL8, CXCR1 and CXCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . ELR CXC chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. The CXCR3 ligands PF-4/CXCL4, Mig/CXCL9, IP-10/CXCL10 and 5.2.1. SDF-1/CXCL12 and its receptors CXCR4 and CXCR7. . . . . . . . . . 5.2.2. BCA-1/CXCL13 and CXCR5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. SR-PSOX/CXCL16 and CXCR6. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. The development of cancer Cancer is a world-wide disease of which the incidence is increasing every year. Tumorigenesis is a multistep process involving changes in the genome and disruption of cellular metabolic processes that drive

* Corresponding author. Tel.: +32 16337348; fax: +32 16337340. E-mail address: [email protected] (J. Van Damme). 1359-6101/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2011.09.002

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the progressive transformation of normal cells into highly malignant derivatives characterized by uncontrolled cell growth [1]. Cancer is a heterogeneous and complex disease presenting as familial/hereditary types and (acquired) sporadic types. The pathogenesis of the basic cellular abnormalities is not precisely known although the effects of different carcinogens including radioactive substances, chemical compounds and infectious agents have been clearly demonstrated. Alterations in three types of genes are responsible for carcinogenesis: stability genes, tumor-suppressor genes and oncogenes [2].

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Stability genes or caretakers include genes required for repairing mistakes made during normal DNA replication. Tumor-suppressor genes encode proteins that control cell processes such as cell cycle arrest and apoptosis (programmed cell death). Genetic alterations in stability genes and tumor-suppressor genes can reduce the activities of the gene products (loss of function). Such inactivations arise from missense mutations, from deletions or insertions of various sizes or from epigenetic silencing. Mutations in both the maternal and paternal allele are required to fully inactivate stability genes and tumor-suppressor genes. In contrast, alterations in oncogenes caused by chromosomal translocations, gene amplifications or intragenic mutations may render these constitutively active. Tumor cells with such ‘gain of function’ mutations are equipped with constitutive activation of anti-apoptotic and proliferative pathways. An activating somatic mutation in one allele is sufficient to activate an oncogene [2]. Besides disruption of the genomic structure, carcinogens and changes in the tumor environment (e.g. hypoxia, inflammation) during tumor progression also alter physiological and/or abnormal cell functions by enhancing or decreasing gene transcription [2]. Mutations in the three classes of genes can occur in the germline. Interestingly, the most common forms of hereditary cancer predisposition, leading to colon cancers, are caused by inherited mutations in stability genes rather than tumor-suppressor genes or oncogenes [2]. Six essential alterations in cell physiology contributing to malignant growth are well established: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis (cell migration) [1]. Recently, a seventh hallmark of cancer has been added: cancer-related inflammation [3]. Most of these cancer hallmarks are provided not only by the neoplastic cells itself but also by various stromal components [4]. Indeed, the formation of a tumor requires support from the surrounding stromal microenvironment of which the main constituents are stromal cells and extracellular matrix (ECM) composed of glycosaminoglycans (GAGs), adhesion glycoproteins such as fibronectin and laminin and fibers such as collagen and elastin [4,5]. Stromal cells comprise (myo)fibroblasts, cells of blood and lymphatic vessels (endothelial cells, pericytes and smooth muscle cells) and inflammatory leukocytes [5]. In the tumor microenvironment, fibroblasts, which are involved in the production of ECM, get activated and show an altered expression profile compared with normal fibroblasts [5,6]. Indeed, these cancer- or tumor-associated fibroblasts (CAFs/TAFs) are characterized by an increased secretion of ECM components and tumor promoting factors such as the CXC chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 [5,6]. Besides originating from normal fibroblast, CAFs might also differentiate from recruited bone marrow-derived mesenchymal stem cells [7]. In addition to fibroblasts, endothelial cells and infiltrated leukocytes may also participate in tumor progression by modulating their receptor expression profile and by releasing factors that in turn modulate leukocyte infiltration, angiogenesis and tumor growth [4,5]. Thus, the composition of a tumor is very complex and the different cell types might co-operate to promote tumor development. 2. Chemokines: classification and functions Chemotactic cytokines, designated chemokines, have been discovered as essential mediators of directional migration of leukocytes, also called chemotaxis. These small proteins are secreted by a variety of cell types such as leukocytes, epithelial cells, fibroblasts, endothelial cells and tumor cells, either constitutively or after induction by inflammatory stimuli (e.g.

infectious organisms, tissue injury) [8,9]. Chemokines play a role during physiological processes by mediating lymphoid tissue organogenesis, lymphocyte homing and hematopoiesis [8,9]. In addition, chemokines are also involved in pathological processes including microbial infections, auto-immune diseases, e.g. rheumatoid arthritis (RA) and inflammatory bowel diseases (IBD), as well as tumor growth and metastasis [8–10]. Indeed, besides their role in chemoattraction, chemokines possess other functions related to cell survival and proliferation and to stimulation or inhibition of angiogenesis. Chemokines have been subdivided in four families based on the relative position of their cysteine residues located in the Nterminal region: CXC, CC, C and CX3C chemokines, in which the X represents any amino acid [8,9,11]. CXC chemokines can be further subdivided depending on the presence or absence of an ELR (Glu, Leu, Arg)-motif, located in front of the first conserved cysteine residue [11–13]. ELR+ CXC chemokines have been described to possess neutrophil chemotactic and angiogenic properties, whereas most ELR CXC chemokines are angiostatic and attract lymphocytes and natural killer (NK) cells [12,13]. CC chemokines induce the migration of monocytes, dendritic cells (DC), lymphocytes, NK cells, eosinophils and basophils. The C and CX3C chemokine families comprise lymphotactin/XCL1 and fraktalkine/CX3CL1, respectively [8,14]. Lymphotactin attracts lymphocytes and NK cells, whereas fraktalkine acts on lymphocytes, NK cells and monocytes [8,11]. Chemokines mediate their functions through binding to seven transmembrane G-protein-coupled receptors defined as CXCR, CCR, CR or CX3CR [9,15]. Furthermore, there is a high degree of redundancy since some chemokines bind to multiple receptors and some receptors recognize more than one chemokine [9,11]. Besides binding to the specific chemokine receptors, chemokines can interact with the non-signaling DARC (Duffy antigen receptor for chemokines), D6 and CCX-CKR (ChemoCentryx chemokine receptor) [9,11,16–18]. In addition to signaling and non-signaling chemokine receptors, chemokines also bind to GAGs, present in the ECM and found on leukocytes, epithelial and endothelial cells [9,11,19]. By capturing chemokines, GAGs establish a local chemokine concentration gradient and assist in the attraction of cells expressing chemokine receptors [9,19]. In addition, GAGs may protect chemokines from clearance and degradation and may promote chemokine oligomerization [19]. 3. Chemokines in cancer The mutual interaction between tumor cells and stromal cells contributes to tumor progression and is mediated by multiple factors including growth factors, matrix-degrading enzymes and chemokines. Chemokines modulate tumor behavior by three important mechanisms: regulation of angiogenesis, activation of a tumor-specific immune response and direct stimulation of tumor proliferation in an autocrine or paracrine fashion [5]. 3.1. Role of chemokines in tumor-related angiogenesis Angiogenesis implies neovascular outgrowth of pre-existing blood vessels [20]. It is an important biological mechanism that plays a role in physiologic and pathologic processes such as tissue remodeling, wound repair, chronic inflammation and tumor development [5,20]. The regulation of angiogenesis depends on the balance between angiogenic and angiostatic factors. Angiogenesis occurs when the balance shifts in favor of endothelial growth factors, such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), and angiogenic chemokines [20]. In physiological circumstances, angiogenesis is a transient process leading to structured, hierarchically organized and well

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functioning vascular networks. In contrast to physiological conditions, a permanent angiogenic imbalance during tumor development leads to the establishment of new capillaries, which are characterized by a fenestrated, disorganized and chaotic endothelial barrier. Such blood vessels, with many branches, dilated lumen and irregular diameter function poorly, but favor metastasis by facilitating tumor cell entry into the vascularization [20]. Besides angiogenesis, vasculogenesis can also occur during tumor development. Vasculogenesis is the formation of vascular structures from bone marrow-derived endothelial progenitor cells, which proliferate and differentiate into de novo endothelial cells [20]. Chemokines have been implicated in both processes of vascular growth (angiogenesis and vasculogenesis) and may exert their regulatory activity directly or indirectly as a consequence of leukocyte infiltration. Angiogenic ELR+ CXC chemokines include growth-regulated oncogene-a (GRO-a)/CXCL1, GRO-b/CXCL2, GRO-g/CXCL3, epithelial cell-derived neutrophil activating peptide-78 (ENA-78)/CXCL5, granulocyte chemotactic protein-2 (GCP2)/CXCL6, interleukin-8 (IL-8)/CXCL8 and neutrophil-activating peptide-2 (NAP-2)/CXCL7 [10,12,13]. SDF-1 lacks the ELR-motif, but nevertheless has angiogenic properties [10]. In addition, SDF-1 mediates the process of vasculogenesis by attraction of endothelial progenitor cells [21]. CXCR3 binding ELR CXC chemokines have angiostatic activities and include platelet factor-4 (PF-4)/CXCL4, platelet factor-4 variant (PF-4var)/CXCL4L1, monokine induced by interferon-g (Mig)/CXCL9, interferon-g inducible protein-10 (IP10)/CXCL10 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11 [10,12]. Also B cell-attracting chemokine-1 (BCA1)/CXCL13, which binds CXCR5, is an angiostatic ELR CXC chemokine [22]. 3.2. Link between chemokines, inflammation and cancer The first evidence that non-neoplastic cells might influence tumor formation and growth came from the field of inflammation. A link between inflammation and cancer was discovered already in 1863 by Rudolf Virchow who reported the presence of leukocytes within the tumor environment [4]. Based on this observation, he suggested that cancer may originate at sites of inflammation. Later on, the presence of leukocytes in the tumor was interpreted as an aborted attempt of the immune system to reject the tumor [4]. During early stages of carcinogenesis, innate responses, in which granulocytes, macrophages and NK cells initiate tumor rejection, are beneficial and likely involved in the activation of effective immune surveillance requiring action of adaptive immune cells such as DC and lymphocytes [23]. If immune surveillance would be effective, the immune system would protect against nascent cancer by destroying malignant cells before these develop into tumors. However, immune surveillance is not always successful as some tumor cells might escape the immune response resulting in progressive tumor growth [23,24]. The process of immune escape is not simply due to the absence of anti-tumor immunity, but because of pro-tumoral immunity which blocks anti-tumoral adaptive and innate responses [23,24]. Indeed, several pro-tumoral immune cells such as neutrophils, M2 macrophages and Th2 lymphocytes have been shown to inhibit the differentiation and proliferation of anti-tumoral M1 macrophages and cytotoxic Th1 lymphocytes. The chemoattraction of either pro-tumoral or anti-tumoral leukocytes depends on the secretion of ELR+ and ELR CXC chemokines, respectively. Indeed, it has been shown that ELR+ CXC chemokines, which bind to CXCR1 and/or CXCR2 (e.g. GRO, ENA-78, IL-8, GCP-2), mediate the infiltration of pro-tumoral neutrophils, whereas CXCR3 binding ELR CXC chemokines (e.g. Mig, IP-10, I-TAC, PF-4) attract antitumoral DC, T lymphocytes and NK cells. In general, tumor

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progression or rejection caused by tumor immunity depends on the balance between the infiltrated pro-tumoral and anti-tumoral leukocytes (Fig. 1). Monocytes attracted by chemokines, especially CC chemokines, produced by tumor cells and stromal cells may be the first line of defense in tumors as they colonize rapidly and secrete cytokines and chemokines, which attract and activate immature DC and NK cells [4,25]. When exposed to bacterial products such as lipopolysaccharide (LPS) or to the cytokine interferon (IFN)-g, which is released by infiltrating NK cells and T cells, monocytes differentiate into type I (M1) macrophages [24,25]. M1 macrophages are potent effector cells as they function as antigenpresenting cells (APC) that are capable of killing tumor cells directly and produce factors which favor the activation of NK cells and the differentiation of naı¨ve T cells into Th1 effector cells and Th17 cells [24–26]. Killing of tumor cells by M1 macrophages and NK cells stimulates the release of tumor antigens which are captured and processed by DC and M1 macrophages. In the draining lymph nodes, these APC present the tumor antigens to naı¨ve T cells [5,23,25]. CD4+ and CD8+ T cells are the principal helper and effector cells of the adaptive cellular immunity, respectively [23,24]. Type I CD4+ T cells (Th1), producing IFN-g, facilitate tumor rejection by providing help to cytotoxic CD8+ T cells (CTL), whereas type II CD4+ T cells (Th2), which secrete IL-4, IL-10 and IL-13, facilitate antibody production by B cells [24]. Tumor-reactive monoclonal antibodies are believed to have antitumor efficacy [4,24]. Besides differentiating into Th1, Th2 and CTL, naı¨ve T lymphocytes can also differentiate into CD4+ T regulatory cells (Treg) and CD4+ Th17 cells [24]. In particular, Treg cells allow tumor development by blocking the activation of CTL and the killing capacity of NK cells [24,27]. On the contrary, it is not yet clear whether Th17 cells promote or inhibit tumor progression [28]. On one hand, it has been described that cytokines (e.g. IL-17, IL-1 and tumor necrosis factor-a (TNF-a)) released by Th17 cells may favor angiogenesis either directly or indirectly by the induced secretion of angiogenic factors [24,28]. On the other hand, Th17 cells may contribute to protective human tumor immunity by facilitating DC recruitment into the tumor and promoting the activation of tumor-specific CTL [28]. Even though an adaptive immune response can be provoked during tumor progression by antigen-specific T lymphocytes, such anti-tumor immune response generates immune selection pressure and only tumor cell variants adapted to escape the immune

Fig. 1. Inflammatory cells mediating tumor development. Tumor immunity influences tumor development and is dependent on the balance between the infiltration of leukocytes promoting tumor progression and the infiltration of leukocytes inducing tumor regression. M2 or tumor-associated macrophages (TAM), T helper 2 (Th2) cells, regulatory T (Treg) cells, B cells, N2 neutrophils and probably mast cells promote tumor progression, whereas M1 macrophages, natural killer (NK) cells, dendritic cells (DC), eosinophils, T helper 1 (Th1) cells, cytotoxic T cells (CTL), N1 neutrophils and probably Th17 cells are responsible for tumor rejection.

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response are able to persist [23]. Furthermore, tumor-derived soluble factors such as cytokines and chemokines (e.g. ELR+ CXC and CC chemokines) may facilitate the escape from immune attack by altering the immune response in favor of pro-tumoral leukocytes. Indeed, the differentiation of monocytes into type II (M2) macrophages in response to IL-4, transforming growth factorb (TGF-b), IL-10 or IL-13 stimulates tumor growth by the release of cytokines (e.g. VEGF), chemokines (e.g. IL-8, SDF-1) and matrixdegrading enzymes which are responsible for tuning inflammatory responses, stimulating tumor cell proliferation and promoting angiogenesis [3,24,25]. In addition, M2 macrophages stimulate CD4+ Th2 cell and CD4+ Treg differentiation, which in turn favor M2 development by releasing IL-4, IL-10 and IL-13 [26]. Furthermore, M2 macrophages are less capable of presenting tumor antigens (lower levels of major histocompatibility complex (MHC) II) and have a reduced tumoricidal activity. Recent information supports the view that tumor-associated macrophages (TAM) are type II polarized [24–26]. In addition to macrophages, mast cells and neutrophilic granulocytes can also support tumor progression. Neutrophils, which are attracted by ELR+ CXC chemokines such as IL-8, ENA-78 and GCP-2, exhibit a dual role in tumor development [29]. At one hand, neutrophils can contribute to immune surveillance as they have potent cytotoxic ability and interact with the adaptive immune system. On the other hand, neutrophils can favor malignancy by releasing growth stimulating signals, matrix-degrading proteases (e.g. matrix-metalloprotease-9/gelatinase B), as well as mediators of angiogenesis such as IL-8 and VEGF [29]. Recently, Fridlender et al. have demonstrated that tumor-associated neutrophils possess anti-tumoral (N1) or protumoral (N2) activities depending on their phenotype, which is regulated by TGF-b [30]. Tumors lacking the expression of TGF-b favor the accumulation of N1 neutrophils, which possess enhanced expression of immunoactivating cytokines and chemokines and show increased capacity to kill tumor cells. However, many tumors are characterized by the over-expression of TGF-b mediating the differentiation of neutrophils toward the N2 pro-tumorigenic phenotype [30]. In contrast to the pro-tumoral neutrophilic granulocytes, eosinophilic granulocytes are rather detrimental for tumor development whereas the role of basophilic granulocytes in tumor progression is rather uncertain [31]. Similar to neutrophils, also the mast cell phenotype is variable and profoundly altered according to the tumor environment [32]. As another means of immune escape, the presence of various cytokines in the tumor environment may block the maturation of DC impairing their ability to stimulate T cells [23,25]. Indeed, many tumors are characterized by the presence of a high number of tumor-associated immature DC [23,25]. Another feature in tumor development is the presence of tumoral self-antigens which are not recognized by the immune system [33]. The majority of tumor antigens is not unique to tumor cells but is also present on normal cells. These tumor-associated antigens may be proteins usually expressed only on fetal cells and not on normal adult cells, or they may be proteins expressed at low levels by normal cells but at much higher levels by tumor cells. Alternatively, tumor antigens may be tumor specific, especially when tumors are induced by viruses, causing activation of the immune system [33]. At a later phase, immune escape occurs as a result of altered gene transcription and adapted tumor cells. Indeed, the expression of class I MHC molecules is decreased in a number of tumors, thereby limiting their destruction by specific CTL [4,33]. Additionally, malignant transformation is also often associated with a reduced expression of tumorspecific antigens [33]. In general, the adaptive immune response against tumor cells is often very weak and largely inefficient, since tumors develop strategies, to evade the effects of this immune response [33]. The altered gene transcription profile in which the expression of

pro-tumoral chemokines, e.g. ELR+ CXC chemokines attracting neutrophils, is up-regulated and the expression of anti-tumoral chemokines such as the ELR CXC chemokines attracting lymphocytes is down-regulated, evokes a shift to the innate type II immune response promoting angiogenesis, tumor growth and metastasis. 4. Small and large intestinal cancer The main risk factors for adenocarcinoma of the small and large intestine include cigarette smoking, excessive alcohol consumption, obesity, nutrition, microbial infections and inflammatory bowel disease, which is characterized by chronic mucosal inflammation [34,35]. In IBD the cancer precursor lesion is called ‘‘dysplasia’’. The development of malignant intestinal adenocarcinoma begins with the formation of an adenoma, which is considered to be the precursor lesion [34]. Several underlying genetic pathways may contribute to the progression of an adenoma. An adenoma is characterized by extension of highly replicating cells from the base of the crypts to the mucosal surface, replacing the normal goblet and absorptive cells. The proliferating adenomatous cells may extend laterally into adjacent crypts, replacing normal epithelial cells while maintaining the basic mucosal architecture. These benign tumors most often demonstrate a polypoid configuration (adenomatous polyps or polypoid adenoma) mostly pedunculated with a stalk, but some produce sessile lesions (sessile adenomas) [34]. An adenoma is considered to be malignant when there is evidence that neoplastic cells pass the muscularis mucosa and infiltrate the submucosa; in this case the definition of adenocarcinoma is appropriate [34]. Adenocarcinoma cells form irregular tubular structures, harboring pluristratification, multiple lumens and contain less stroma. Depending on the presence of glandular structure, cellular pleomorphism and mucosecretion, adenocarcinomas may present three degrees of differentiation: well, moderately and poorly differentiated. Neoplasms of the small intestine are rare throughout the world. Indeed, only 2% of the total cancer incidence of the digestive system occurs in the small intestine [35]. Adenocarcinomas are most common in the duodenum and proximal jejunum, whereas neuroendocrine tumors and lymphomas predominate in the distal jejunum and ileum [35]. Neuroendocrine tumors and adenocarcinomas account for 42% and 30–40% of the total of registered cancers in the small intestine, respectively [35]. Colorectal cancer is the second most common cause of cancer mortality and is typically a disease of the Western lifestyle [36]. The most common colon cancer type is adenocarcinoma arising from the columnar surface epithelium, which accounts for 95% of the cases. Other, rarer types of colorectal cancer include lymphoma, neuroendocrine carcinoma and squamous cell carcinoma, the latter usually originates from the stratified squamous epithelium of the anal region [34]. 5. The role of CXC chemokines in colorectal cancer 5.1. ELR+ CXC chemokines 5.1.1. CXCR2 ligands Growth-regulated oncogene (GRO) has been first purified from human malignant melanoma cells and has been characterized as an autocrine growth factor [37]. Three different genetic variants GRO-a, GRO-b and GRO-g have been described of which GRO-a binds CXCR2 with a higher affinity [38]. As mentioned above, CXCR2 ligands including GRO-a, GRO-b, GRO-g, ENA-78, GCP-2, NAP-2 and IL-8, possess pro-tumoral capacities since they chemoattract pro-tumoral neutrophils and stimulate angiogenesis [12,37,39–42]. Besides IL-8, GCP-2 is the only chemokine which is

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Table 1 Cell types expressing CXC chemokines and chemokine receptors in colorectal cancer. Chemokines and receptors

Cellular expression

Reference

GRO-a,b,g/CXCL1,2,3 ENA-78/CXCL5 GCP-2/CXCL6 IL-8/CXCL8 CXCR2 CXCR1

tu, tu EC tu, tu, tu,

[48,53] [57] [59] [65,66] [53,66,74] [65,66]

CXCR3

ND tu ND tu, MF tu tu, T, EC

[96] [105] [105–107] [105,108] [110]

CXCR4 CXCR7

tu, EC, fibro tu, EC, ly, fibro tu, EC

[122,123,126,145] [123,125,126,128,133,140,142,145] [145]

CXCR5 CXCR6

tu tu, MF

[149] [150]

PF-4/CXCL4 PF-4var/CXCL4L1 Mig/CXCL9 IP-10/CXCL10 I-TAC/CXCL11

SDF-1/CXCL12

BCA-1/CXCL13 SR-PSOX/CXCL16

leu

neu, MF, ly, EC, fibro EC EC

Abbreviations: EC: endothelial cells, fibro: fibroblasts, leu: leukocytes, ly: lymphocytes (no distinction made between T or B lymphocytes), MF: macrophages, ND: not determined, neu: neutrophils, T: T lymphocytes, tu: tumor cells.

able to activate both CXCR1 and CXCR2 [41,43]. As a consequence, the neutrophil chemotactic activity of GCP-2 and IL-8 are mediated by both receptors. However, possibly only CXCR2 is responsible for the angiogenic effect of ELR+ CXC chemokines [44]. Colorectal adenocarcinoma cells have been described to constitutively express GRO-a and ENA-78. The presence of chronic inflammation (mediated by pro-inflammatory cytokines TNF-a and IL-1) or bacterial infection with e.g. Salmonella Dublin, Bacteroides fragilis or Escherichia coli further up-regulated the expression of GRO-a, ENA-78 and also GRO-g [45–48]. Kinetic studies performed on cytokine-stimulated colon adenocarcinoma cells showed a delayed but more prolonged production of ENA-78 in comparison with IL-8. This can be attributed to differences in their upstream promoter regions and/or the activation of their relevant transcription factors [47,48]. IL-8 is a more potent neutrophil chemoattractant, more rapidly up-regulated in tumor cells and produced in larger amounts than ENA-78 [41,47]. Elevated expression of GRO-a, GRO-b and GRO-g has been evidenced in colon adenocarcinomas and adenomas, which was (except for GRO-b) significantly higher than in normal colon epithelium (Table 1) [49–51]. It has been demonstrated that the non-metastatic and less metastatic colon adenocarcinoma cell lines (Caco-2 and HT-29, respectively) express lower levels of GRO-a compared with the highly metastatic colon cancer cell line LS147T [52]. A higher constitutive expression of CXCR2 has also been found in highly metastatic variants, whereas GRO-b and GRO-g expression did not correlate with metastatic potential in colorectal cancer cell lines [52]. GRO-a is produced by many cell types and may have multiple roles in the progression of colorectal cancer. Indeed, immunohistochemical staining on colon cancer biopsies provided intense staining of GRO-a in epithelial and stromal cells (Table 1). Strong CXCR2 immunostaining was seen mainly in stromal endothelial cells but not in epithelial cells, which contrasts with the detection of CXCR2 on colon adenocarcinoma cell lines in vitro [52,53]. The up-regulation of both GRO-a and CXCR2 in human colon adenocarcinoma cell lines may stimulate tumor progression as GRO-a acts as an autocrine growth factor and increases the invasive potential (Table 2) [52]. Moreover, tumors over-expressing GRO-a showed reduced expression of fibulin-1, a component known to participate in the organization of the basement

membrane and extracellular matrix [49]. Abrogation of fibulin-1 expression may facilitate the ability of neoplastic cells to permeate the basement membrane and consequently promote invasion and metastasis [49]. However, Wang et al. did not find any expression of CXCR2 in colon cancer epithelial cells in situ and mentioned that GRO-a promotes colon cancer through stimulation of angiogenesis [53]. A schematic representation of the expression and role of GRO in colorectal cancer is provided in Table 2 and in Fig. 2. In contrast to the above mentioned results demonstrating the pro-tumoral role of GRO-a, Chiu and colleagues evidenced a protective effect of GRO-a in preventing the progression of colon cancer [54]. Apparently, GRO-a was significantly up-regulated in patients

TNF-α, IL-1β, bacterial infection leu

tu GRO

ENA-78

tu proliferation

EC angiogenesis

GRO ENA-78 GCP-2 GRO ENA-78 GCP-2

EC GCP-2

T

neu

chemoattraction

fibulin tumor progression metastasis

tumor regression

Fig. 2. Expression and roles of GRO, ENA-78 and GCP-2 in colorectal cancer. Bacterial infection and the pro-inflammatory cytokines IL-1b and TNF-a increase the expression of GRO-a, GRO-g and ENA-78 in colorectal cancer cells (tu). GRO-a may act as an autocrine growth factor and may increase the invasive potential of the tumor cells by reducing the expression of fibulin-1. In contrast, ENA-78 does not induce colorectal tumor growth proliferation. GRO-a may also promote colon cancer through stimulation of angiogenesis. However, the effect of ENA-78 on angiogenesis and neutrophil (neu) infiltration in colorectal cancer is less evidenced. ENA-78 may also chemoattract CD8+ T cells (T), which results in tumor regression. GCP-2 can be detected in endothelial cells (EC) in colorectal adenocarcinoma and may stimulate tumor growth by chemoattracting neutrophils and by inducing angiogenesis.

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350

Table 2 Presence and pro- or anti-tumoral functions of ELR+ CXC chemokines in colorectal cancer. Chemokine

GRO

Test Systema In vitro (cell lines)

In vivo (mice)

Cell proliferation assay, Induction/neutralization, GRO transfection/siRNA. GRO transfection/siRNA, Western blot, qRT-PCR, Invasion assay. Transwell system,

Tumor xenografts

Pro/anti-tumoralb

Reference

Enhanced tumor cell survival and proliferation

[49,52,56]

Reduced fibulin-1 expression, Enhanced tumor cell migration and invasion

[52,59]

Angiogenesis

[53]

Microarray qRT-PCR

Protective effect: Tumor less invasive Tumor growth arrest

[54,55]

IHC

Infiltration anti-tumoral CD8+ T cells

[58]

IHC

Angiogenesis Neutrophil infiltration

[59] [59]

Cell proliferation by up-regulation of EGFR and by proteolytic processing of EGFR ligands

[74–76,78]

Tumor cell migration via protease-mediated EGFR activation Tumor cell invasion

[76–78,80,81]

Angiogenesis

[71,75,76]

Neutrophil infiltration and adhesion to tumor cells

[70,75,87]

Chemotherapeutic resistance

[76]

Tumor cell adhesion to matrix via CD44 variant

[82]

Ex vivo (human)

Microarray qRT-PCRc IHCc IHC on tumor xenografts

Matrigel (tube formation).

ENA-78 GCP-2

IL-8

Tumor allografts Cell proliferation assay, Boyden chemotaxis assay. Induction/neutralization, IL-8 transfection/siRNA, Cell proliferation assay, Western/Northern blot, Flow cytometry. Wounding assay, Boyden and transwell chemotaxis assays. Invasion assay, Cell adhesion assay. CAMd, Tumor xenografts, qRT-PCR, IHC. Boyden chemotaxis assay, Flow cytometry, Northern blot, Transendothelial migration, Cell adhesion assay. Clonogenicity assay, Growth inhibition assay. Induction/neutralization, qRT-PCR, flow cytometry, Cell adhesion assay.

a b c d

[74,76,78]

This table gives an overview of the different techniques used to unravel either the pro- or anti-tumoral effects of ELR+ CXC chemokines in colorectal cancer. The mechanisms by which CXC chemokines exhibit either pro-tumoral or anti-tumoral effects on tumors. qRT-PCR: quantitative reverse transcriptase-polymerase chain reaction, IHC: immunohistochemistry. Chorioallantoic membrane.

younger than 65 years old, in which the immune response is generally stronger than in elderly, and was frequently overexpressed in less invasive tumors [54]. Also the discovery that CXCR2 ligands may reinforce tumor growth arrest can explain these contradictory results [54,55]. In comparison with GRO-a expression, a much more pronounced over-expression of ENA-78 was noticed in colorectal adenocarcinoma [50]. Despite the extensive surface expression of CXCR2 on colorectal tumor cells, ENA-78 did not induce tumor cell proliferation [56]. Furthermore, no correlation was found between the concentrations of ENA-78 in the circulation and in tissues and reports on the expression of ENA-78 in normal versus colon adenoma and adenocarcinoma are conflicting [50,57]. Dimberg et al. have observed lower plasma concentrations of ENA-78 in patients with colorectal cancer than in healthy control patients indicating that patients with colorectal cancer have an immunologic imbalance resulting in impaired production of ENA-78 from leukocytes and/or in local secretion of ENA-78 from epithelial cells in the colon and rectum [57]. Furthermore, Speetjens et al. have demonstrated that disrupted expression of ENA-78 in colorectal cancer cells resulted in reduced infiltration of CXCR2 expressing cytotoxic CD8+ T lymphocytes and was associated with poor prognosis and decreased survival [58]. Taken together, ENA-78

may have both pro-tumoral (e.g. angiogenesis, neutrophil infiltration) and anti-tumoral (e.g. cytotoxic T cell chemoattraction) capacities (Fig. 2). However, it seems that in colorectal cancer, the chemoattraction of anti-tumoral immune cells by ENA-78 is dominant [58]. Nevertheless, more extensive in vivo studies with immune-competent animals instead of immune-deficient mice and ex vivo studies on human tumor biopsy material are needed to confirm the results obtained by Speetjens and colleagues. Gijsbers et al. detected GCP-2 in endothelial cells in colorectal adenocarcinomas, but the chemokine was not expressed by endothelial cells in normal tissue (Table 1) [59]. In addition, a positive correlation was found between the presence and localization of leukocytes and the expression of GCP-2 within the tumor [59]. The production of GCP-2 by endothelial cells within the tumor would contribute to tumor development, invasion and metastasis through neovascularization and chemoattraction of neutrophils. The latter are loaded with matrix-degrading enzymes which enable tumor cells to migrate through the extracellular matrix, to enter the vasculature (Fig. 2). NAP-2 is generated by cleavage of the inactive precursors, platelet basic protein (PBP) and its derivatives connective-tissueactivating peptide-III (CTAP-III) and b-thromboglobulin (b-TG), which are stored in the a-granules of blood platelets and released

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by exocytosis upon platelet stimulation [42,60]. The processing of PBP and its derivatives b-TG and CTAP-III to yield NAP-2 is mediated by proteases released by monocytes and neutrophils [60]. Furthermore, NAP-2 attracts neutrophils and may also contribute to breakdown of the basement membrane and extracellular matrix during angiogenesis and metastasis [60,61]. As a consequence, platelets and the platelet-derived factors may play an important role in tumor growth and metastasis. Moreover, neoplastic cells of various tissues are capable of platelet activation [62]. b-TG blood levels have been demonstrated to be significantly increased in patients with gastric and colon cancer compared with healthy controls [62,63]. However, controversial results are obtained concerning the correlation between increased b-TG blood levels and the presence of lymph node and distant metastasis in colorectal cancer [62,63]. 5.1.2. IL-8/CXCL8, CXCR1 and CXCR2 The ELR+ CXC chemokine IL-8 is the most potent human neutrophil chemoattractant and activator in vitro and in vivo [64]. In addition, IL-8 is the first chemokine described to have angiogenic properties [12]. The migration of neutrophils is provoked by interaction with CXCR1 and CXCR2, whereas the angiogenic activity of IL-8 is mediated predominantly by binding to CXCR2 [44]. As a consequence, IL-8 might play a role in tumorigenesis. Several studies describe that IL-8 is highly up-regulated in colon cancer cells and surrounding stromal cells in comparison with its normal counterparts [65–67]. CXCR1 and CXCR2 have been shown to be expressed on both stromal and cancer cells, however no differences in immunohistochemical staining pattern and intensity between different tumor stages and control subjects were observed [66]. Amongst the stromal cells especially endothelial cells, fibroblasts and some infiltrating leukocytes (mainly neutrophils) within tumor sections showed a positive staining for IL-8, whereas CXCR2 was mainly detected in tumor-associated microvessels (Table 1) [65,66]. The expression of IL-8 by endothelial cells was induced after stimulation with IL-1a, released by colorectal cancer cells [68]. Additionally, it has been demonstrated that the IL-8 positive stromal cells were observed in the invading edges [66]. Furthermore, a significantly more pronounced over-expression of IL-8 was detected in colorectal adenocarcinoma specimens in comparison with colorectal adenoma, in which IL-8 was significantly more expressed than in normal mucosa [66,67]. On top, the increase of IL-8 expression levels was associated with an increase of dysplastic grades in the adenomas [66]. Interestingly, IL-8 upregulation was most enhanced in colorectal cancer metastasis in the liver, when compared with the corresponding primary colorectal cancer tissues [67]. In contrast, Doll and colleagues have not found significant differences in IL-8 gene expression levels, neither between adenoma and normal colonic mucosa neither between liver metastasis and their primary tumors [51]. Altogether, elevated levels of IL-8 in tumor tissue are significantly associated with tumor size, depth of infiltration, tumor stage and liver metastasis and high levels of IL-8 within the tumor are significantly associated with a shorter overall survival time [67,69]. Several components have been described to be responsible for the increased expression of IL-8 in colorectal adenocarcinoma cells including pro-inflammatory cytokines such as IL-1b and TNF-a, microorganisms and even hypoxia, a major characteristic occurring during tumor progression [46,47,70–72]. Therefore, pathogenic organisms and the concomitantly induced inflammatory reaction are considered to play prominent roles in the etiology and progression of gastrointestinal cancer. Indeed, Abdulamir et al. have shown that colorectal adenoma and adenocarcinoma are associated with higher levels of Streptococcus gallolyticus infection in comparison with control tissue and the tumor promoting role of S. gallolyticus is due to induction of inflammatory, anti-apoptotic and

351

angiogenic factors including NF-kB and IL-8 in colon tumor cells [73]. Obviously, the amount of IL-8 secretion by colon adenocarcinoma cells in response to pro-inflammatory cytokines varies from cell line to cell line, probably due to differences in receptor distribution and the magnitude of the generated signal [72]. IL-8 plays multiple roles in the progression of colorectal adenocarcinoma (Table 2, Fig. 3). First of all, IL-8 activates neutrophils by increasing the expression of adhesion molecules and thereby inducing neutrophil transendothelial migration [70]. In addition to pro-inflammatory cytokines, IL-8 is also able to increase the expression of intercellular adhesion molecule-1 (ICAM-1) on colon cancer cells causing increased leukocyte adhesion to these cells [70]. Upon activation and adhesion of neutrophils to tumor cells, angiogenic proteins and matrix-degrading proteases are released, promoting tumor progression [70]. Additionally, Li et al. have demonstrated the importance of IL-8 in mediating binding of colon carcinoma cells to endothelial cells, which might be critical for tumor cell invasion and metastasis [74]. IL-8 promotes the angiogenic outgrowth of vascular vessels providing oxygen and nutrients to the tumor, required for tumor growth and metastasis [12,13,75,76]. Furthermore, it has been reported that IL-8 enhances the expression of CXCR1 on tumor cells and acts as an autocrine growth factor for colon adenocarcinoma cells [74–77]. Besides its direct stimulation of proliferation, IL-8 may also indirectly promote cell proliferation by inducing cleavage of EGFR ligands via activated metalloproteases, [78]. Conflicting reports also exist concerning the association between the constitutive CXCR1 and CXCR2 expression and the aggressiveness of human colon carcinoma cells [66,74]. Sturm et al. suggest that intestinal cells lose the capacity to express CXCR2 upon differentiation as CXCR2 was not detectable on highly differentiated colorectal cancer cell lines e.g. Caco-2 and HT-29 cells [77]. Similarly, IL-8 production, especially after stimulation with IL1b, was impaired in colon cancer cells upon differentiation [75,79]. Indeed, Bates et al. have demonstrated that loss of cell-cell adhesion and consequently cell polarity may be responsible for the increased IL-8 production in colon carcinoma [80]. Next, it has been observed

Fig. 3. Expression and role of IL-8 in colorectal cancer. Several studies describe that IL-8 is highly up-regulated in colon cancer cells (tu) and surrounding stromal cells including endothelial cells (EC), fibroblasts (fibro), macrophages (MF) and neutrophils (neu). Several components may increase the expression of IL-8 in colorectal adenocarcinoma cells including IL-1b, TNF-a, microorganisms and hypoxia. IL-8 plays multiple roles in the progression of colorectal adenocarcinoma. First of all, IL-8 acts as an autocrine growth factor for colon adenocarcinoma cells. Furthermore, IL-8 stimulates the migration of colorectal cancer cells, chemoattracts neutrophils and promotes angiogenesis. In addition, it has been described recently that over-expression of IL-8 by colorectal cancer cells leads to significant resistance to the cytotoxic effects of chemotherapeutic drugs. On the contrary, IL-8 may increase the cell-to-matrix adhesion by up-regulation of CD44 variant proteins inhibiting tumor metastasis.

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that IL-8 has a putative role on the invasive potential of human colon carcinoma cells as it stimulates the migration of colorectal cancer cells with increasing mitogenic activity in higher metastatic tumor cells [74,76,81]. In addition, the chemotactic response of colorectal cancer cells to IL-8 has been shown to be at least partly dependent on CXCR1 and not CXCR2 [77,80]. On the contrary, it has been reported that IL-8 increases the cell-to-matrix adhesion by up-regulation of CD44 variant proteins (the receptors for extracellular matrix components) on colon cancer cells [82]. The variant isoforms of homing cell adhesion molecule (HCAM)/CD44 are characterized by enhanced binding capacity to extracellular matrix components [82]. These findings suggest that therapeutic strategies, targeted to the chemokine system, should be considered with caution as they may affect malignancy of tumors with low metastatic potential [82]. Furthermore, it has been described recently that over-expression of IL-8 by colorectal cancer cells leads to significant resistance to the cytotoxic effects of oxaliplatin, a platinum-based chemotherapeutic drug [76]. Taken together, these findings indicate that overexpression of IL-8 promotes tumor growth, angiogenesis, metastasis and chemoresistance, implying IL-8 to be an important therapeutic target in colorectal cancer (Fig. 3). Gene alterations such as single nucleotide polymorphisms (SNPs) may occur during tumor development. The IL-8 gene contains four exons, three introns and a proximal promoter region. Three well-characterized SNPs in the IL-8 gene have been noticed so far: in the proximal promoter at 251 T/A and in intron 1 at +396 T/G and +781 C/T [83]. The number of studies performed on patients with colorectal adenocarcinoma to examine whether a correlation exists between SNPs in the IL-8 gene and the risk for colorectal cancer, is rather sparse. Contradictory results are obtained about the correlation between the IL-8 -251 SNP and the risk of colorectal cancer [84,85]. This discrepancy may be ascribed to differences in etiology and environmental factors. Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared with microsatellite stable (MSS) cancers [86]. Microsatellites are very short repetitive nucleotide sequences, distributed throughout the human genome, that are prone to insertion of errors during DNA replication. The appearance of abnormally long or short microsatellites is referred to as microsatellite instability (MSI). It has been described that MSI cancers generate abnormal peptides, specifically tumor specific antigens, which can generate cytotoxic T cell responses. In spite of its pro-tumoral potential, IL-8 has been found to be elevated in MSI cancers compared with MSS cancers [86]. This is a quite surprising result as IL-8 mediates the infiltration of neutrophils and acts as a pro-angiogenic chemokine. However, in some cases it has been described that massive infiltration of neutrophils in response to high concentrations of IL8 within the tumor has anti-tumoral potentials by inducing necrosis and tumor regression. Indeed, as already mentioned, two subtypes of neutrophils have been described with either protumoral or anti-tumoral activities [30]. In particular, membrane interactions between neutrophils and target cells have been shown to activate the neutrophils, which then release cytotoxic factors including reactive-oxygen species, acids, proteases, defensins and perforins [87]. 5.2. ELR CXC chemokines 5.2.1. The CXCR3 ligands PF-4/CXCL4, Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 Platelet factor-4 (PF-4)/CXCL4 is an ELR CXC chemokine which is synthesized in megakaryocytes and stored in a-granules until it is released during platelet aggregation [88]. PF-4 exerts various biological functions such as inhibition of angiogenesis and chemoattraction of DC, T lymphocytes and NK cells via CXCR3

[89–91]. The angiostatic functions of PF-4 are mediated by binding CXCR3 and/or heparan sulfate proteoglycans (HSPG), both present on endothelial cells [90]. The IFN-g-inducible CXCR3 ligands, Mig/ CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, are produced in vitro by a variety of cells, including endothelial cells, fibroblasts, mononuclear cells and tumor cells. These ELR CXC chemokines are characterized by their anti-tumoral activities as they act as angiostatic regulators and mediate the infiltration of anti-tumoral T lymphocytes and NK cells [11,12]. During metastasis, tumor cells that have detached from the primary tumor and have entered the surrounding vasculature or lymphatics must attach to capillary beds and other sites in distant organs before metastatic lesions can arise [92]. Angiogenin, which can be secreted by colorectal tumor cells and is also a normal constituent of plasma, binds to endothelial cells and the ECM to support the adhesion of tumor cells to stromal components [92]. It has been reported that PF-4 inhibits the binding of tumor cells to angiogenin by interfering with HSPG, the putative receptor of angiogenin. Thus, PF-4 has been shown to counteract the protumoral action of angiogenin, which is likely to be present at sites where tumor cell arrest and invasion occur [92]. Besides, PF-4 suppresses the angiogenic process by inhibition of endothelial cell proliferation and induction of apoptosis [89,93,94]. Indeed, it has been demonstrated that PF-4 inhibits the growth of human colorectal tumors in nude mice by inhibition of angiogenesis (Table 3) [94]. Recently, a variant of PF-4 (PF-4var/CXCL4L1) has been described which has stronger angiostatic and anti-tumoral activities than PF-4 [95]. In tumor biopsy specimens of patients with colorectal adenocarcinoma, a strong expression of PF-4var has been detected by immunohistochemistry, whereas the staining of PF-4var in esophageal adenocarcinoma and squamous cell carcinoma was rather weak [96]. Further studies are needed to establish a correlation between PF-4var expression in colorectal adenocarcinoma and decreased blood vessel density, metastasis and better prognosis. Human colon cancer cell lines were found to constitutively release Mig, IP-10 and I-TAC and expression can be further increased by IFN-g, IL-1b and TNF-a [47,97,98]. IP-10 and I-TAC, but not Mig, were up-regulated in response to TNF-a, whereas only IP-10 was up-regulated in response to IL-1 [47,97]. Infection with bacteria and stimulation with TNF-a and IL-1 strongly potentiated the IFN-g-induced cancer cell production of these CXCR3 ligands [97,98]. Generally, the response of colorectal cancer cells to proinflammatory stimuli varied from cell line to cell line and on the state of differentiation probably due to differences in receptor distribution and signaling pathways. The induction of a cytotoxic T cell response capable of eradicating disseminated tumors and the establishment of a persistent tumorprotective immunity remain major goals of cancer immunotherapy. Mig and IP-10 have been demonstrated to be amongst the most potent chemokines in reducing cancer progression. Indeed, Mig, especially in combination with IL-2 has been found to reduce tumor growth and lung metastasis of colon adenocarcinoma [99]. Combined treatment of IP-10 with cytokines contributing to activation of T and NK cells such as TNF-a or IL-12 enhanced the anti-tumor effects not only in treated primary tumors, but also in distant untreated metastatic tumors [100,101]. The anti-tumor effect obtained by Mig and IP-10 was mediated predominantly by MHC class I antigen-restricted CD8+ T cells with the help from MHC class II antigen-restricted CD4+ T lymphocytes, by NK cells and by inhibition of angiogenesis (Table 3) [99,101,102]. Moreover, it has been described that Mig and IP-10 not only chemoattract T lymphocytes but also augment their functional expansion. Indeed, tumor infiltrating lymphocytes from IP-10-treated tumors had a greater proliferating potential, had more potent cytolytic activity and produced higher levels of IFN-g, which in turn up-regulates the

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353

Table 3 Presence and pro- or anti-tumoral role of ELR CXC chemokines in colorectal cancer. Chemokine

Test Systema In vitro

PF-4

Mig

IP-10

T cell cytotoxicity assay

Boyden and transwell chemotaxis assay, T cell cytotoxicity assay.

Pro-tumoralb

Anti-tumoralb

Reference

Chorioallantoic membrane, Tumor xenografts.

Angiostasis

[94]

Tumor allografts, IHCc Depletion of T cells Microarray, qRT-PCRc and IHC (human). Tumor allografts, IHC, Matrigel plug angiogenesis assay.

T cell chemoattraction

[99,104]

Angiostasis

[99]

Tumor allografts, T and NK cell depletion, IHC.

NK and T cell infiltration

[101,102]

Tumor allografts, IHC.

Angiostasis Enhanced adhesion to laminin

[102] [102]

In vivo

Cell adhesion assay Boyden chemotaxis assay, Western blot.

I-TAC

T cell migration in organotypic colon carcinoma culture, T cell cytotoxicity assay.

Tumor allografts, IHC.

SDF-1

Cell proliferation assay

Tumor allografts, IHC.

Boyden and transwell chemotaxis assay, Wounding assay. Cell adhesion assay to fibronectin and collagen, Flow cytometry, Invasion assay. qRT-PCR, Western blot, ELISA, Gelatin zymography. Boyden chemotaxis assay, T cell cytotoxicity assay.

Injection of SDF-1 transfected tumor cells, flow cytometry and qRT-PCR.

BCA-1

SR-PSOX a b c

Cell proliferation assay, Migration assay.

[102]

Tumor cell migration and invasion by up-regulated MMP-9 T cell infiltration

[108]

Inhibition of apoptosis Tumor cell proliferation Tumor cell migration and metastasis

[117,120,121,128]

Increased ICAM expression, tumor cell adhesion and invasion

[117,121,128,131]

Tumor allografts, IHC, microcirculation analysis. Tumor allografts and IHC.

Increased production of VEGF and MMPs, angiogenesis

[117,120,121,133]

Injection of BCA-1 transfected tumor cells

Tumor cell proliferation and migration

IHC (human)

[117,128–130]

DC and CD8+ T cell recruitment

[118]

Angiostasis

[22,149]

T cell infiltration

[150]

This table gives an overview of the different techniques used to unravel either the pro- or anti-tumoral effects of ELR CXC chemokines in colorectal cancer. The mechanisms by which CXC chemokines exhibit either pro-tumoral or anti-tumoral effects on tumors. IHC: immunohistochemistry, qRT-PCR: quantitative reverse transcriptase-polymerase chain reaction.

expression of IP-10 by colorectal cancer cells to recruit more T cells [103]. Hence, high expression levels of Mig and IP-10 in colorectal tumors correlated with enhanced infiltration of memory CD8+ T cells, CD4+ T cells and macrophages as well as with better prognosis and prolonged disease-free survival [104–106]. Moreover, the expression levels of IP-10 have been shown to be lower in colorectal cancer patients with recurrence than in patients without recurrence [105,106]. Elevated IP-10 expression in tumor cells of colorectal adenocarcinoma, but not in the adjacent normal tissues, was observed in approximately 50% of the cases [107]. Additionally, the expression of IP-10 in colorectal adenocarcinoma has been detected in both cancer cells and macrophages, especially along the invasive margin where also the inflammatory T cell infiltrates were abundant (Table 1) [105]. In contrast, in the majority of the cases, T cell infiltration was rather poor within the stroma of the central area of the tumor [105]. Immunohistochemical staining of Mig and I-TAC in colorectal cancer was not observed within the tumor [105]. However, Berencsi et al. have demonstrated that I-TAC was expressed by organotypic colorectal cancer cell cultures, which caused chemoattraction of cytotoxic T cells [108]. In addition to the expression of CXCR3 by T lymphocytes, it has been reported that colorectal cancer cells can also express CXCR3, which may be up-regulated after stimulation with IFN-g (Table 1) [109,110]. Expression of CXCR3 by colon tumor cells was

demonstrated to promote colon cancer metastasis to lymph nodes, whereas metastasis to liver and lung was rare and unaffected by CXCR3 expression [109]. In contrast, Cambien and colleagues showed the implication of CXCR3 in metastasis of colorectal cancer cells toward the lungs and the liver [111]. Indeed, expression of IP10 has been shown to occur in all three colorectal cancer sites: liver, lungs and lymph nodes [110]. In approximately one third of the analyzed clinical colon cancer biopsies, the expression of CXCR3 was found in tumor cells, most of these cases had lymph node metastasis, whereas normal epithelial cells of the colon did not express CXCR3 [109]. Hence, patients with CXCR3 positive tumor cells showed significantly poorer prognosis than those without CXCR3 [109]. Furthermore, stimulation of colorectal cancer cells in vitro with IP-10 promoted the expression of MMP-9 facilitating colorectal cancer cell migration. However, tumor cell growth was not affected (Table 3) [109,110]. Taken together, CXCR3 ligands have been shown to be expressed constitutively in colorectal carcinomas and primary metastatic sites exerting anti-tumor activity by recruiting CXCR3 positive T lymphocytes and NK cells and by inhibiting angiogenesis (Fig. 4). However, at the same time CXCR3 ligands can promote tumor cell migration and metastatic adhesion. So, care should be taken considering CXCR3 ligands as an anti-tumoral therapy option (Fig. 4).

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Fig. 4. Expression and role of Mig and IP-10 in colorectal cancer. Human colon cancer cell lines (tu) have been found to express Mig, IP-10 and I-TAC constitutively, however chemokine levels can be increased by IFN-g, IL-1b and TNF-a. Additionally, IP-10 has also been detected in macrophages (MF). The antitumor effects exerted by Mig and IP-10 are mediated predominantly by CD8+ T cells (T) with help of CD4+ T cells, by NK cells and by inhibition of angiogenesis. Nevertheless, stimulation of colorectal cancer cells with IP-10 may promote the expression of MMP-9 and induce migration of CXCR3 expressing colorectal cancer cells, which favors metastasis.

5.2.2. SDF-1/CXCL12 and its receptors CXCR4 and CXCR7 Stromal cell-derived factor-1/CXCL12, an ELR CXC chemokine, also plays prominent roles in tumorigenesis, but in contrast to the ELR CXCR3 agonists it is an angiogenic chemokine. SDF-1 modulates the angiogenic process directly by binding to its receptor CXCR4 expressed on endothelial cells and indirectly by the induced secretion of matrix-metalloproteases or angiogenic factors (e.g. IL-8, VEGF) [112]. In addition, SDF-1 also mediates neovascularization by the attraction of endothelial progenitor cells. Furthermore, SDF-1 positively regulates the proliferation of cancer cells and affects their survival [112]. Moreover, the SDF-1/ CXCR4 axis is involved in tumor metastasis toward tissues which are characterized by high production of SDF-1 such as lymph nodes, liver, lung and bone marrow [112,113]. Several studies have reported that SDF-1 expression is reduced in colorectal adenoma and adenocarcinoma in comparison with normal colon mucosa [114–116]. Immunohistochemical staining on normal colon biopsies provided intense staining of SDF-1 in normal intestinal epithelial cells lining the upper parts of the crypts [117]. It has been postulated that colonic epithelial expression of SDF-1 upon transformation into cancer cells is silenced by DNA hypermethylation of the promoter region [116]. Additionally, it has been demonstrated that re-expression of functional, endogenous SDF-1 in colorectal adenocarcinoma cells dramatically reduced metastatic tumor formation in mice and increased apoptosis in colorectal cancer cells [116]. Moreover, Fushimi et al. have demonstrated that over-expression of SDF-1 by colorectal cancer cells resulted in accumulation of DC and CD8+ T cells, thereby suppressing tumor growth (Table 3) [118]. However, the role of SDF-1 in apoptotic cell death is still controversial, with studies describing SDF-1 either as an inducer or a repressor of apoptosis [116,117,119–121]. Furthermore, it has been proposed that tumor cells which do not express endogenous SDF-1, respond better to exogenous SDF-1 produced by distal organs, resulting in metastasis [116]. In contrast to the above results, several studies have demonstrated that SDF-1 is increased in colorectal cancer cells, particularly at the invasive front, compared with normal colorectal epithelium

[122,123]. In addition, SDF-1 expression has been shown to be higher in colorectal adenocarcinomas in comparison with colorectal adenomas, which in turn was significantly higher than in normal colon mucosa [124]. Besides tumor cells, also vascular endothelial cells and fibroblasts in the tumor stroma showed strong SDF-1 immunoreactivity (Table 1) [122–125]. Furthermore, the increased expression of SDF-1 in colorectal tumors has been described to be significantly associated with tumor stage, lymphatic invasion, venous invasion, lymph node metastasis, distant metastasis and decreased survival [122,123,125]. On the contrary, in one report not a single correlation was found between high SDF-1 expression levels in colon cancer cells and clinicopathological parameters [126]. Altogether, to reconcile these apparently contradictory findings, we can speculate that, during early phases, SDF-1 production might sustain angiogenesis and tumor growth, whereas in later stages, a lower expression might favor tumor development avoiding recruitment of cytotoxic lymphocytes and enhancing the metastatic ability of colorectal cancer cells to tissues expressing high levels of SDF-1. CXCR4 was consistently found to be expressed in both normal colonic epithelium and colonic carcinoma cells [114,116,127]. In normal colon mucosa CXCR4 has been detected at both the surface of the mucosa and in the crypts, however the expression of CXCR4 was strongest in the epithelial cells at the base of the crypts [114,117]. It has been proposed that CXCR4 plays a role in maintenance and renewal of colonic epithelium since differentiation of colorectal cancer cells resulted in a down-regulation of CXCR4 [114,117]. Additionally, it has been described that stimulation of CXCR4 with SDF-1 promotes the migration of colon cancer cells to distant organs, which highly express SDF-1 (Table 3) [117,128–130]. Activation of CXCR4 with SDF-1 stimulated the upregulation of ICAM-1 in tumor cells and thereby enhanced their adhesion to endothelial cells and ECM, which is crucial for invasion of the cancer cells into distant organs (Table 3) [114,128,129]. Once tumor cells invaded distant organs, the local expression of SDF-1 promotes the outgrowth of tumor cells in the metastatic site by initiating tumor cell proliferation and survival, stimulating angiogenesis and inducing MMP-9 secretion (Table 3) [117,120,121,128,129,131]. MMP-9 facilitates migration of tumor cells by the breakdown of ECM and regulates angiogenesis [117,132]. SDF-1 may directly and indirectly promote the outgrowth of new blood vessels. On one hand, SDF-1 directly stimulates angiogenesis by binding CXCR4 on endothelial cells [133]. On the other hand, SDF-1 may induce the secretion of proangiogenic cytokines and chemokines including, VEGF, IL-8 and GRO by CXCR4+ leukocytes, endothelial and tumor cells [117,120,127]. In turn, VEGF not only induces CXCR4 expression, but also enhances SDF-1 production by endothelial cells and tumor cells, establishing a positive feedback loop [126,134,135]. Further, colorectal cancer cells have been shown to activate normal fibroblasts to undergo transformation into CAFs producing increased levels of SDF-1, which in turn promotes invasion of more tumor cells and tumoral outgrowth [131,136]. Also tumorinfiltrated macrophages may promote tumor cell progression by a positive feedback loop [137]. Indeed, stimulation of mononuclear phagocytes with SDF-1 induced the shedding of EGF, which subsequently activated EGFR on the cell surface of colorectal cancer cells, resulting in tumor cell proliferation. Upon activation of EGFR, colorectal tumor cells released granulocyte macrophage colony-stimulating factor (GM-CSF), which in turn activated mononuclear phagocytes to release more soluble EGF [137]. In contrast to Jordan and colleagues, who did not find differences in CXCR4 expression between normal colon mucosa and colorectal cancer cells, Ottaiano et al. have shown that CXCR4 is over-expressed in both colorectal adenocarcinomas and hepatic metastases in comparison with normal colon mucosa [114,128]. Besides tumor cells, CXCR4 expression was also found in vascular

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endothelial cells, fibroblasts and tumor-infiltrated leukocytes (Table 1) [125,126]. Additionally, several studies have shown that CXCR4 expression was even higher in liver metastases compared with primary colorectal tumors [131,138]. These results indicate that especially colorectal cancer cells expressing high levels of CXCR4 metastasize to distant organs. Several factors are responsible for the increased expression of CXCR4 in colorectal cancer cells including hypoxia and VEGF [134,135,139]. Furthermore, it has been demonstrated by several studies that expression of CXCR4 by colorectal cancer cells correlated with increased risk for local recurrence, lymph node metastasis, distant metastasis, T stage and consequently with decreased disease-free and overall survival [138,140]. Additionally, it has recently been described that especially nuclear and not cytoplasmic expression of CXCR4 significantly correlated with lymph node metastasis and poor survival [123,141,142]. Particularly, SDF-1 ligand binding to CXCR4 induces translocation of CXCR4 to the cytoplasm and to the nucleus where the SDF-1/CXCR4 complex functions as a transcription factor and thereby regulates biological processes [141,142]. In contrast, several studies have not detected nuclear localization of CXCR4 in colorectal cancer cells indicating that more studies are needed to confirm the nuclear staining pattern of CXCR4 in colorectal cancer cells and to explore the role of different CXCR4 staining patterns in colorectal tumor progression [126,128,138,143]. Recently, a new SDF-1 receptor named CXCR7 has been discovered [144]. Few studies have investigated the expression of CXCR7 in colorectal cancer. In particular, CXCR7 expression has been noticed in colorectal cancer cells and endothelial cells (Table 1) [145]. Until now, only one study demonstrated that activation of CXCR7 by SDF-1 may induce angiogenesis [120]. Taken together, silencing of SDF-1 in primary colorectal cancer may stimulate the migration of CXCR4 expressing tumor cells to distant organs expressing high levels of SDF-1 including lymph nodes, bone marrow, lungs and liver. Once invaded in the distant organ, a positive feedback loop between cancer cells and host stromal cells promotes tumor cell outgrowth via SDF-1 by increasing cell survival, proliferation and angiogenesis. 5.2.3. BCA-1/CXCL13 and CXCR5 The ELR CXC chemokine, B cell-attracting chemokine (BCA)-1/ CXCL13, which is mainly found in secondary lymphoid organs, has originally been considered to play a role in the chemoattraction of human B lymphocytes [146]. The receptor for BCA-1, Burkitt’s lymphoma receptor (BLR)-1/CXCR5 is not found on immature, but only on mature B cells. This is in contrast to SDF-1 which was originally described as a growth factor for progenitor B cells and chemoattractant for human pre- and pro-B cells, but does not attract mature B cells [146]. Besides mature B cells, CXCR5 is also expressed on skin-derived DC, but not on bone marrow-derived DC [147]. The chemoattraction of DC to secondary lymphoid organs, especially into the B cell zone, may mediate the differentiation of B cells and consequently alter Ag-specific immune responses [147]. CXCR5 is not specific for hematopoietic cells, since it is also expressed in several human colon carcinomas as detected by immunohistochemistry [148,149]. Moreover, the expression of CXCR5 on human colorectal adenocarcinoma cells may drive the migration of tumor cells to organs highly expressing BCA-1, such as spleen and liver (Table 3) [149]. In addition, BCA-1 directly enhances the proliferation of adenocarcinoma cells, thereby promoting tumor cell outgrowth [149]. 5.2.4. SR-PSOX/CXCL16 and CXCR6 CXCL16, also called scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), is a unique ELR CXC chemokine that exists both in a transmembrane form and a soluble form [150]. SR-PSOX has been shown to possess multiple biological

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activities. Soluble SR-PSOX is chemotactic for cells expressing its receptor CXCR6 including CD8+ and CD4+ T cells. The cell surfaceanchored form SR-PSOX rather functions as a cell adhesion molecule for CXCR6 expressing cells. The expression of SR-PSOX has previously been observed in macrophages, DC, fibroblasts and endothelial cells [150]. It has been shown that SR-PSOX is up-regulated in colorectal cancer cells in comparison with normal colon mucosa [150]. Besides its expression in tumor cells, SR-PSOX is also expressed in macrophages (Table 1). In addition, colorectal tumors expressing high levels of SR-PSOX contained higher levels of CD4+ and CD8+ T lymphocytes than those with weak expression of SR-PSOX (Table 3) [150]. Hence, patients with strong expression of SR-PSOX in colorectal adenocarcinoma had a significantly better prognosis [150]. 6. Conclusion Chemokines have originally been identified as chemotactic cytokines, mainly involved in the migration of leukocytes. It has become clear that expression of chemokines and their receptors within colon tumors participates in the different steps of tumorigenesis including survival, tumor growth, attraction of leukocytes, angiogenesis and finally metastasis. Furthermore, the interaction between colon tumor cells and normal stromal cells influences the chemokine expression pattern in both cell types, which provides a favorable environment for tumor growth and spread. ELR+ CXC chemokines are widely expressed in gastrointestinal cancers and are correlated with poor prognosis. The ELR+ CXCR2 ligands favor tumor progression by inducing tumor cell proliferation and migration, attracting neutrophils and promoting angiogenesis and metastasis. The ELR CXC chemokine SDF-1 is widely accepted to promote metastasis of CXCR4 positive tumor cells to distant sites where it stimulates the outgrowth of metastasized tumor cells by increasing tumor cell survival and proliferation and by stimulation of the angiogenic process. Expression of the ELR CXCR3 ligands rather protects against tumor outgrowth as these mediate the infiltration of cytotoxic T lymphocytes and inhibit angiogenesis. However, recent reports have described that CXCR3 positive tumor cells migrate to distant organs highly expressing CXCR3 ligands. Taken together, the role of different chemokines in tumor development is very complex. More extensive studies are required to unravel the intricate chemokine network in tumor development in order to provide additional insights, which may lead to therapeutical applications in patients with cancer. Acknowledgements This work was supported by the Concerted Research Actions (G.O.A.) of the Regional Government of Flanders, the Fund for Scientific Research of Flanders (F.W.O.-Vlaanderen) and the Interuniversity Attraction Poles Program (I.A.P.)-Belgian Science Policy. The authors thank Prof. Dr. Ghislain Opdenakker (Rega Institute, Leuven, Belgium), Prof. Patrick Matthys (Rega Institute, Leuven, Belgium), Prof. Gert De Hertogh (University Hospitals, Leuven, Belgium), Prof. Christiane Dinsart (Deutsches Krebsforschungszentrum, Heidelberg, Germany) and Prof. Daniel Desmecht (Universite´ de Lie`ge, Lie`ge, Belgium) for critically evaluating this manuscript. References [1] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70. [2] Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004;10:789–99. [3] Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 2009;30:1073–81.

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Hannelien Verbeke graduated as master in biomedical sciences in 2007 and obtained a PhD in biomedical sciences in 2011, under the promotership of Prof Dr Sofie Struyf and Prof Dr Jo Van Damme at the Rega Institute, University of Leuven, Belgium. Her research interests are primarily focused on the role of chemokines in inflammation and cancer, in particular gastrointestinal cancer.

Sofie Struyf graduated as bio-engineer in 1996 at the University of Leuven, Belgium. She obtained her PhD degree (2002) in applied biological sciences at the Rega Institute (Laboratory of Molecular Immunology, University of Leuven, Belgium) on post-translational modifications of chemokines. She is currently holding a position of associate professor at the Rega Institute. Her research is currently focused on the role of chemokines in angiogenesis and cancer.

Genevie`ve Laureys (MD, University of Antwerp; Specialist in Pediatrics and Pediatric Hematology and Oncology, University Hospital Ghent; Postdoctoral Fellow at Yale University, USA, Laboratory of Prof Uta Francke, 1986–1987; PhD, University of Ghent, Belgium). She is mainly interested in research on genetic aberrations in cancer, e.g. in neuroblastoma, and the role of chemokines in the development of cancer. Presently, she is Chief Clinical Staff in the Department of Pediatrics, Hematology and Oncology and Stem Cell Transplantation, Associate Professor in the Faculty of Medicine at the University of Ghent, and Member-elect of the Royal Academy of Medicine of Belgium.

Jo Van Damme (1950, bio-engineer, PhD, University of Ghent, Belgium) is professor at the Medical Faculty of the University of Leuven, Belgium, and Head of the Laboratory of Molecular Immunology, Department of Microbiology and Immunology, at the Rega Institute for Medical Research. He was president of the European Cytokine Society (2001–2007). He has done pioneer work in cytokine research and was involved in the identification of several human interleukins (IL-1, IL6, IL-8) and chemokines (CXCL6, CXCL8, CCL7, CCL8). His current research is dealing with the role of chemokines and posttranslationally modified forms thereof in infection, inflammation and cancer. He has published about 400 scientific papers in peer-reviewed journals.