Chemokine Receptor Signaling and the Hallmarks of Cancer

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Chemokine Receptor Signaling and the Hallmarks of Cancer R.A. Lacalle1, R. Blanco1, L. Carmona-Rodríguez1, A. Martín-Leal1, E. Mira1, S. Mañes2 Centro Nacional de Biotecnologı´a/CSIC, Madrid, Spain 2 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. The Chemokine Superfamily 2.1 Chemokines, the Ligands 2.2 Signal Transduction by Chemokine Receptors 3. Chemokine Activity on Neoplastic Cells 3.1 Chemokine Signaling Sustains Tumor Cell Proliferation 3.2 Chemokines Interact With Tumor Suppressor Genes 3.3 Chemokines as Regulators of Apoptosis in Cancer Cells 3.4 Chemokines as Drivers and Cues for Tumor Invasion and Metastasis 3.5 Chemokines and Cancer Stem Cell Plasticity 4. Chemokine Activities on Stromal Cells 4.1 Chemokines as Positive and Negative Regulators of Tumor Vascularization 4.2 Chemokines as Shapers of the Tumor Inflammatory Milieu 5. Concluding Remarks Acknowledgments References

2 3 3 7 13 13 16 17 21 24 27 28 33 41 43 43

Abstract The chemokines are a family of chemotactic cytokines that mediate their activity by acting on seven-transmembrane-spanning G protein-coupled receptors. Both the ability of the chemokines and their receptors to form homo- and heterodimers and the promiscuity of the chemokine–chemokine receptor interaction endow this protein family with enormous signaling plasticity and complexity that are not fully understood at present. Chemokines were initially identified as essential regulators of homeostatic and inflammatory trafficking of innate and adaptive leucocytes from lymphoid organs to tissues. Chemokines also mediate the host response to cancer. Nevertheless, chemokine function in this response is not limited to regulating leucocyte infiltration into the tumor 1

These authors contributed equally to this work.

International Review of Cell and Molecular Biology ISSN 1937-6448 http://dx.doi.org/10.1016/bs.ircmb.2016.09.011

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2016 Elsevier Inc. All rights reserved.

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microenvironment. It is now known that chemokines and their receptors influence most—if not all—hallmark processes of cancer; they act on both neoplastic and untransformed cells in the tumor microenvironment, including fibroblasts, endothelial cells (blood and lymphatic), bone marrow-derived stem cells, and, obviously, infiltrating leucocytes. This review begins with an overview of chemokine and chemokine receptor structure, to better define how chemokines affect the proliferation, survival, stemness, and metastatic potential of neoplastic cells. We also examine the main mechanisms by which chemokines regulate tumor angiogenesis and immune cell infiltration, emphasizing the pro- and antitumorigenic activity of this protein superfamily in these interrelated processes.

1. INTRODUCTION The chemokines are a family of low molecular weight, structurally related proteins that bind to cell membrane-anchored, seventransmembrane receptors linked to heterotrimeric G proteins. The word chemokine, derived from “chemotactic cytokine,” denotes the chemoattractant function and cytokine nature of these molecules. Indeed, the chemokine CXCL8 retains its former interleukin (cytokine) nomenclature (IL-8) in many scientific papers. Since the description of the first chemokine, platelet factor 4 (CXCL4) (Deutsch et al., 1955), 60 years ago, the family of chemokines and their receptors has expanded notably, with approximately 50 chemokines and 20 receptors now known in humans. The chemokine system is recognized as the major group of proteins that orchestrate leucocyte trafficking into/from lymphoid organs and tissues in homeostatic and in pathological conditions (Viola et al., 2006). Temporal and spatial regulation of leucocyte movement is essential for an efficient response to foreign antigens and other damaging agents. An example is the CCL19–21/ CCR7 chemokine/receptor pair, which regulates naı¨ve T cell encounter with mature antigen-presenting cells (APC) in lymph nodes (LN), the first step in the initiation of T cell-mediated responses. The so-called inflammatory chemokines steer activated leucocytes of the innate and adaptive immune systems to peripheral tissues for pathogen elimination or tissue repair. Inappropriate regulation of chemokine or chemokine receptor expression is also a major underlying factor in chronic inflammation and many autoimmune diseases (Comerford et al., 2014). Chemokines and their receptors are also involved in oncogenesis. Tumors are recognized as “organs” composed of different cell types that cohabit and communicate with one another. Evolution to clinically relevant cancers relies on the interactions between neoplastic cells and stromal cells,

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which include endothelial and immune cells. Chemokines are an important component of the cross-communication between the various cell types in the tumor microenvironment; they not only regulate leucocyte trafficking into the tumor but also act on neoplastic cells and on other stromal cell components to promote or inhibit tumor development. The chemokines and their receptors have been implicated in most hallmark processes of cancer (Hanahan and Weinberg, 2011). Here we will review distinct activities of chemokines in oncogenesis and discuss potential therapeutic opportunities based on these molecules.

2. THE CHEMOKINE SUPERFAMILY 2.1 Chemokines, the Ligands Chemokines are 8–12 kDa proteins that have from one to three disulfide bridges. The number and position of the N-terminal cysteines that form these bridges have been used to classify the chemokines in four families: the CXC or α-chemokines, in which a noncysteine amino acid is positioned between the first and second cysteines, the CC or β-chemokines, in which the two cysteines are adjacent, the XC or γ-chemokines that lack the first cysteine, and the CX3C or δ-chemokines, with three residues between the two cysteines (Bacon et al., 2002; Murphy et al., 2000). CXC and CC chemokines are the most numerous, whereas the XC and CX3C families are composed of two and one member, respectively. Chemokines were also grouped based on functional criteria: the inflammatory chemokines, produced by circulating leucocytes and other cells after activation, and the constitutively expressed homeostatic chemokines (Mantovani, 1999; Moser and Willimann, 2004). This nomenclature has nonetheless fallen into disuse, since some chemokines can belong to either category, depending on the physiological or pathological situation. Most chemokines have received a profusion of names based on their specific cellular activities. Table 1 shows the correspondence of these names with the cysteine-based nomenclature, which we will follow throughout this review. Although sequence identity among chemokines varies greatly (20–90%), nuclear magnetic resonance (NMR) and X-ray crystallography show that their tertiary structure is well conserved and is stabilized by the disulfide bonds (Allen et al., 2007). Most chemokines are secreted proteins, with two exceptions—CX3CL1 and CXCL16—anchored to the cell membrane through a
110 amino acid mucin-like stalk, a transmembrane domain, and a cytoplasmic tail. These membrane-tethered chemokines have a dual function. When membrane anchored, they can induce firm cell–cell adhesion by

Chemokine

Alternative name(s)

Receptor(s)a

CXCL1

GROα/MSGAα

CXCR2

CCL1

I-309

CCR8

CXCL2

GROβ/MSGAβ

CXCR2

CCL2

MCP1/MCAF/TDCF

CCR2

CXCL3

GROγ

CXCR2

CCL3

MIP-1α/LD78α

CCR1, CCR5

CXCL4

PF4

CXCR3B

CCL4

MIP-1β

CCR5

CXCL4V1

PF4V1

CXCR3B

CCL5

RANTES

CCR1, CCR3, CCR5

CXCL5

ENA-78

CXCR1, CXCR2

CCL7

MCP-3

CCR1, CCR2, CCR3

CXCL6

GCP-2

CXCR1, CXCR2

CCL8

MCP-2

CCR1, CCR2, CCR3, CCR5

CXCL7

NAP-2

CXCR2

CCL11

Eotaxin

CCR3

CXCL8

IL-8

CXCR1, CXCR2

CCL13

MCP-4

CCR1, CCR2, CCR3

CXCL9

MIG

CXCR3A, CXCR3B

CCL14

HCC-1

CCR1

CXCL10

IP-10

CXCR3A, CXCR3B

CCL15

HCC-2/LKN1/MIP-1γ

CCR1, CCR3

CXCL11

I-TAC

CXCR3A, CXCR3B

CCL16

HCC-4/LEC/LCC-1

CCR1, CCR2, CCR5

CXCL12

SDF-1 α/β

CXCR4

CCL17

TARC

CCR4

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Table 1 The Chemokine Superfamily Alternative Chemokine name(s) Receptor(s)a

CXCL13

BCL/BCA-1

CXCR5

CCL18

DCCK1/PARC/AMAC-1

CCR8

CXCL14

BRAK/bolckine

Unknown

CCL19

MIP-3β/ELC/exodus-3

CCR7

CXCL15

–

Unknown

CCL20

MIP-3α/LARC/exodus-1

CCR6

CXCL16

–

CXCR6

CCL21

SLC/6Ckine/exodus-2

CCR7

CCL22

MDC/STCP-1

CCR4

b

DMC

CXCR8 (GPR35)

–

–

–

CCL23

MPIF/CKβ8/CKβ8-1

CCR1

XCL1

Lymphotactin/ ATAC/

XCR1

CCL24

Eotaxin-2/MPIF-2

CCR3

–

SCM-1α

–

CCL25

TECK

CCR9

XCL2

SCM-1β

XCR1

CCL26

Eotaxin-3

CCR3

–

–

–

CCL27

CTAK/ILC

CCR10

CX3CL1

Fractalkine

CX3CR1

CCL28

MEC

CCR3, CCR10

a

Only signaling-competent receptors are considered in this table. For chemokine binding to atypical receptors, see Table 2. The orphan receptor GPR35 has been identified as a potential receptor for CXCL17 and renamed CXCR8 (Maravillas-Montero et al., 2015).

b

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CXCL17

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engagement with their receptors, in an integrin-independent signaling process (Borst et al., 2012; Haskell et al., 1999; Mionnet et al., 2010). They can also be shed sequentially by α- and γ-secretases and by the disintegrin-like metalloproteases ADAM-10 and ADAM-17 in a constitutive or inducible manner, which releases the chemokine moiety to the extracellular space (Hundhausen et al., 2007; Schulte et al., 2007). Neither cleaved nor secreted chemokines are found as soluble entities in the milieu, but are anchored to the extracellular matrix (ECM) or to the cell membrane through interaction with glycosaminoglycans (GAGs), carbohydrate structures found on the surface of virtually all mammalian cells and tissues. There is evidence of some selectivity between chemokines and GAG, and these GAGs can discriminate among chemokines (Allen et al., 2007; Witt and Lander, 1994). Interaction with GAGs enables chemokine retention and formation of the gradients needed for chemokine-induced cell attraction in vivo. Laboratory-generated chemokine mutants with impaired GAG binding are not functional as chemoattractants for leucocyte recruitment to the peritoneum, although the mutated chemokines are as effective as their nonmutant counterparts in Boyden chamber chemotaxis assays (Proudfoot et al., 2003); this indicates that mutation did not alter their receptor-binding and signaling capacities. Another striking characteristic of many chemokines is their ability to form dimers or higher-order oligomers. Heterodimerization is not only limited to chemokines of the same family (CCL or CXCL), but can also take place between chemokines of different families, as is the case for CCL21/ CXCL13 or CXCL4/CCL5 (Paoletti et al., 2005; von Hundelshausen et al., 2005). Homo- and heterodimers can form in solution (Clore et al., 1990), but evidence suggests that GAG might promote dimerization. For example, CCL2 forms tetramers and CCL8 forms dimers when incubated with octasaccharide heparin (Lau et al., 2004); this GAG also promotes CCL2/CCL8 heterodimerization (Crown et al., 2006). In contrast, other chemokines (CCL1, CCL7, CCL11, CCL13, or CX3CL1) are essentially monomeric, even in the presence of heparin (Kim et al., 1996; Salanga et al., 2014; Yu et al., 2005). The functional effects of oligomerization are debated, as it can potentiate or modulate specific chemokine functions. A case in point is the CXCL13/ CCL21 heterodimer, whose effects are more potent in vivo than those of CCL21 alone (Paoletti et al., 2005); similarly, CXCL4 enhances CCL5mediated monocyte arrest on endothelial cells (von Hundelshausen et al., 2005). Conversely, CXCL4 binding to CXCL8 attenuates the signal of

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the latter in CD34+ hematopoietic progenitor cells (Dudek et al., 2003), even though in endothelial cells, the CXCL4/CXCL8 dimer shows enhanced antiproliferative activity compared to CXCL4 alone (Nesmelova et al., 2005). It has not yet been resolved whether these functional differences between monomers and oligomers are the result of oligomerization or are due simply to the presence of two ligands. The current model assumes that chemokines bind as monomers to their receptors (Allen et al., 2007). For many chemokines, mutagenesis studies identified N-terminal residues as major determinants for receptor binding (Gong and Clark-Lewis, 1995; Jarnagin et al., 1999; Mizoue et al., 2001; Simmons et al., 1997); indeed, chemokines whose N-terminus has been truncated artificially act as potent antagonists in vitro and in vivo (Allen et al., 2007). In many chemokines, metalloproteinase (MMP) and dipeptidyl-peptidase IV cleavage of the N-terminus usually generate potent antagonists of the nontruncated forms (McQuibban et al., 2002; Proost et al., 1998; Van Damme et al., 2004). Structural studies showed that dimerization involves the N-terminus in part and buries specific chemokine amino acids involved in interaction with the receptor (Jarnagin et al., 1999; Laurence et al., 2000). This suggests that oligomerization prevents chemokine binding to the receptor, thus blocking receptor-induced signals. Other scenarios are nonetheless possible. As commented above, oligomerization could increase chemokine interaction with GAG, thus increasing local chemokine concentration on the cell surface or the ECM. Homo- or heterooligomers might bind directly to given receptor combinations, which can also homo- and heterooligomerize (see below). Much work is still needed to establish the structure–function relationships of the chemokine family.

2.2 Signal Transduction by Chemokine Receptors Chemokines elicit intracellular signals after binding to receptors that cross the cell membrane seven times and are coupled to heterotrimeric G proteins (G protein-coupled receptors, GPCRs). Chemokine receptors belong to the class A rhodopsin-like group and share a common organization, with the N-terminus and three loops on the extracellular side of the membrane, all involved in ligand binding; three other loops and the C-terminus face the cytoplasm and participate in intracellular signaling. Like the chemokines, the receptors are grouped into four major subfamilies: CXCR, CCR, XCR, and CX3CR. There are currently seven known receptors for CXCL chemokines, 10 for CCL, and single receptor for

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XCL1/2 and for CX3CL1 (Bachelerie et al., 2015). There is correspondence between receptor and chemokine subfamilies (CXCR only bind to CXCL and so on), but despite this selectivity for binding to chemokines of the same group, most chemokine receptors show great promiscuity in binding to ligands within the same subfamily (Table 1). The functional consequences of this redundancy are not entirely known, although it could help to fine-tune chemoattraction of specific immune cell subpopulations (Devalaraja and Richmond, 1999; Mantovani, 1999). Another characteristic of chemokine receptors is their ability to form homo- and heterooligomers. GPCRs are found in the membrane as dimers or high-order oligomers (White et al., 1998). Despite initial skepticism (Thelen and Baggiolini, 2001), it is now well established that many chemokine receptors homo- and heterodimerize (Scholten et al., 2012). Heterodimerization usually takes place between receptors of the same subfamily, although it can also cross the CCR and CXCR subclasses (Contento et al., 2008; Rodriguez-Frade et al., 2004). Dimerization is a ligandindependent process thought to occur shortly after receptor synthesis (Hernanz-Falcon et al., 2004; Trettel et al., 2003). Chemokine receptor mutants engineered to be retained in the endoplasmatic reticulum have been used to sequester wild-type receptors in the cell interior (Mira et al., 2001). Other reports suggest that the agonist promotes receptor oligomerization (Rodriguez-Frade et al., 2001). There are also conflicting data regarding the number of ligands that interact with a receptor dimer. Experiments using crosslinking agents suggest that chemokine dimers bind to oligomeric receptors (Mellado et al., 2001b). Nonetheless, studies that measured binding– dissociation reactions at equilibrium showed negative cooperativity between the subunits of a receptor dimer during chemokine binding, which supports the idea of high affinity binding of only a single chemokine molecule per receptor dimer (Springael et al., 2005). The relevance of receptor oligomerization is not fully defined, although some studies show that blocking dimerization inhibits chemokine receptor function (Hernanz-Falcon et al., 2004). Receptor homo- and heterodimerization can activate distinct signaling pathways in response to a given ligand (Mellado et al., 2001b), which adds complexity to the redundancy of the chemokine network. The chemokine receptors described up to this point are able to transduce intracellular signals after interaction with their ligand. In addition to these “classical” receptors, there is another four-member family referred to as “atypical” chemokine receptors (Table 2), characterized by an alleged inability to induce signaling (Graham et al., 2012). These atypical receptors

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Chemokines and Cancer

Table 2 Atypical Chemokine Receptors Name Other Names Ligands

Expression

ACKR1 DARC, CCL1, 2, 5, 7, 8, 11, 13, 14, Erythrocytes, vascular CD234, 16, 17, 18 endothelial, Purkinje cells Duffy antigen CXCL1, 2, 3, 4, 5, 6, 8, 10, 11, 13 ACKR2 Ccbp2, D6, CMKBR9

CCL2, 3, 3L1, 4, 5, 6, 7, 8, Lymphatic endothelial 11, 12, 13, 14, 17, 22, 23, cells, B1 B cells, 24, 26 keratinocytes, trophoblasts

ACKR3 RDC1, CXCR7, CMKOR1

CXCL11, 12, opioid peptides, adrenomedullin

Hematopoietic cells, neurons, vascular endothelial cells

ACKR4 CCXCKR, CCRL1, CCR11

CCL19, 21, 25

Lymphatic endothelial cells, germinal center B cells, thymocytes

CX3CL1

cannot trigger G protein-mediated signaling (Steen et al., 2014), which suggests that they act as chemokine scavengers for fine regulation and/or resolution of chemokine-induced inflammatory responses (Bachelerie et al., 2015; Massara et al., 2016). Some are nonetheless able to transduce signals through the β-arrestin pathway; for instance, CXCL12, which normally induces G protein signaling through CXCR4, preferentially triggers β-arrestin-mediated signaling through ACKR3 (Rajagopal et al., 2010). Pathogens encode another family of unconventional receptors for chemokines. Some of these molecules are soluble chemokine-binding proteins that interfere with chemokine binding to its physiological receptor, whereas others are chemokine receptor mimics that induce specific signals in response to host ligands (Heidarieh et al., 2015; Murphy, 2015). We will not discuss this family of molecules here, although they might be relevant in hijacking the chemokine network in some virus-induced cancers. Chemokines are thought to bind to their cognate receptors in two steps. The first interaction yields high affinity chemokine binding to the receptor, and the second triggers conformational changes in the receptor that culminate in its coupling to GTP-loaded heterotrimeric G proteins (Allen et al., 2007; Monteclaro and Charo, 1996); the GTP-loaded Gα subunit and the Gβ/γ heterodimer then dissociate from the receptor and activate downstream effectors. Most of these responses are inhibited by pertussis toxin, which implicates the Gαi family in the initiation of signal transduction

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downstream of chemokine receptors (Murphy, 1994). Direct Gαi interaction has been observed with the second intracellular loop of most receptors (Steen et al., 2014). Nevertheless, chemokine receptors can be also coupled to Gαq or Gα11 in a cell- and stimulus-dependent manner (Molon et al., 2005; Shi et al., 2007). Gαi limits protein kinase A activation, leading to reduced intracellular cyclic AMP levels, whereas the Gβ/γ heterodimer is responsible for phospholipase C (PLC)-β activation, which generates the inositol trisphosphate necessary for intracellular Ca2+ release (Steen et al., 2014). This G protein-signaling module is important for cell chemotaxis and for activation of phosphoinositide-3-kinase (PI3K)-mediated pathways (Arai et al., 1997; Hirsch et al., 2000). Simultaneous with G protein activation, chemokines trigger a signaling pathway that leads to receptor desensitization and internalization. This signaling module is initiated by G protein receptor kinase (GRK)-mediated phosphorylation of key serine and threonine residues at the C-terminus of the agonist-occupied receptors (Ribas et al., 2007). This phosphorylation enables β-arrestin association to the C-terminal receptor, which promotes G protein uncoupling and receptor desensitization, and initiates receptor endocytosis by engaging the adaptor protein 2 (AP-2) and clathrin (Cheng et al., 2000; Marchese and Trejo, 2013). In addition to mediating the shutdown of signaling, β-arrestin acts as scaffolds for the activation of other signaling pathways such as those of the p38 mitogen-activated protein kinase (MAPK) and the p44/p42 extracellular-regulated kinases (ERK1/2) (Vroon et al., 2006). These G protein-independent, β-arrestin-mediated pathways have been studied intensively in CXCR4 (Sun et al., 2002). In humans, mutations in the final 10–19 C-terminal amino acids of CXCR4 result in hyperactivated receptors, whose inheritance is associated to a rare congenital immunodeficiency termed WHIM syndrome (warts, hypogammaglobulinaemia, infections, and myelokathexis) (Bachelerie, 2010; Hernandez et al., 2003; Liu et al., 2012). It is notable that these WHIM mutant receptors are able to interact with β-arrestin but are not internalized after agonist stimulation. These mutant receptors have been instrumental in identifying an additional site of β-arrestin binding to the N-terminal part of the CXCR4 third intracellular loop (ICL3); interaction at this site enhances CXCR4-mediated ERK activation and chemotaxis (Lagane et al., 2008). β-arrestin coupling to ICL3 is facilitated in a p160 Rho kinase-dependent manner by the actin-binding protein filamin A (Go´mez-Mouto´n et al., 2015). Filamin A also has two interaction CXCR4 sites, one in the C-terminus (Jimenez-Baranda et al., 2007) and

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other at the C-terminal end of the ICL3 (Go´mez-Mouto´n et al., 2015). Elimination of filamin A binding to ICL3 reduces the duration of CXCR4-induced ERK activation and, more importantly, enables agonist-induced internalization of mutant WHIM receptors (Go´mezMouto´n et al., 2015). Filamin A thus prolongs β-arrestin-induced signaling, probably by slowing CXCR4 internalization. Filamin A also regulates CCR2b internalization although here it appears to accelerate receptor endocytosis (Minsaas et al., 2010). Whether the β-arrestin/filamin A module has different effects in CC and CXC receptors requires research. A third main signaling module downstream of chemokine receptors is activation of the tyrosine kinase pathway. Chemokine stimulation leads to rapid tyrosine phosphorylation of several chemokine receptors, including CXCR4, CCR2, and CCR5 (Vila-Coro et al., 1999; Wong et al., 2001; Zhang et al., 2001), even though they are GPCR. This Gαi-independent signaling event depends on the Janus kinases (JAK), a family of cytosolic kinases typically activated by cytokines. JAKs control gene expression by phosphorylating STAT proteins (signal transducers and activators of transcription) (Gadina et al., 2001). JAK/STAT activation is associated with receptor homo- or heterodimerization (Vila-Coro et al., 1999). Which JAK and STAT isoforms are activated after chemokine stimulation appears to depend on cell type and/or other context-dependent factors, rather than the agonist or the receptor stimulated. Activation of the JAK/STAT module triggers a number of signaling pathways. CCL5-mediated activation of c-Fos in T cells requires STAT protein induction (Wong and Fish, 1998). Chemokine receptor-mediated triggering of STAT4 is also linked to the CD4+ T cell helper (Th)1-polarizing ability of dendritic cells (DC) (Zou et al., 2000), although a role for chemokines in T cell fate has not been clearly established. Pharmacological inhibition of the JAK/STAT pathway reduces phosphorylation of several focal adhesion proteins downstream of CCR5, including focal adhesion kinase (FAK) (Zhang et al., 2001). These findings explain the role of chemokines as boosters of integrin-mediated adhesion and, given FAK participation in other signaling pathways, also link chemokine receptors to these routes (Bacon et al., 1996). Chemokine receptor activation is precisely regulated in time and space; they concentrate at specific cell locations during migration and T cell activation (Man˜es and Viola, 2006). The spatiotemporal regulation of chemokine signaling is a result of chemokine receptor partitioning in specialized plasma membrane domains termed lipid rafts, which are membrane regions in a liquid-ordered conformation due to enrichment in glycosphingolipids

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and cholesterol (Rajendran and Simons, 2005). The lateral mobility of lipid rafts is fundamental to the organization of membrane-anchored proteins; these membrane domains act as efficient platforms to compartimentalize signaling (Gomez-Mouton and Man˜es, 2007; Gomez-Mouton et al., 2004). Several chemokine receptors of the CC and CXC subfamilies associate to lipid rafts, fostered by agonist binding (Gomez-Mouton et al., 2001; Man˜es et al., 1999, 2001; Nguyen and Taub, 2002; Wysoczynski et al., 2005). Lipid raft formation is highly dependent on membrane cholesterol levels. Cyclodextrin extraction of cholesterol eliminates chemokine receptor signaling (Man˜es et al., 1999; Nguyen and Taub, 2002), which suggests that receptor partitioning in these microdomains is essential for function. Fig. 1 summarizes the main signaling pathways downstream of chemokine receptors. In subsequent sections, we will analyze how chemokine-elicited signals affect the oncogenic process. For the sake of simplicity, we will separate their effects into those exerted directly on neoplastic cells and those that affect the stromal component of the tumor. It should nonetheless be stressed

Lipid rafts PKA

Gαi

I3P

Gβ/γ

PLCβ

JAK P P

P

P

FlnA

PKC cAMP

β−Arr

STAT

GRK

PI3K Nucleus

Ca2+ RhoA AKT

cdc42

p38

Rac ROCK

FAK

ERK1/2

c-fos

Cofilin

Survival

Migration

Actin reorganisation

Proliferation

Adhesion

Fig. 1 Chemokine receptor signaling. Summary of the main signaling modules activated downstream of chemokine receptors. Chemokine receptors, some of which bind to their agonists in lipid rafts, transduce intracellular signals through three main modules: (i) the heterotrimeric Gi protein hub, whose activation leads to release of the Gαi subunit, which inhibits PKA activity, and of the Gβ/γ heterodimer, which mobilizes Ca2+ from intracellular stores and triggers PI3K activation; (ii) the JAK/STAT hub, which regulates gene expression and transduces cell adhesion and migration signals; and (iii) the GRK/β-arrestin hub, whose activation initiates positive signals such as the MAPK cascade and the machinery for actin cytoskeleton remodeling, as well as shutdown of signaling through receptor internalization. β-Arr, β-arrestin; FlnA, filamin A.

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that these activities are usually coordinated between tumor and stromal compartments. For example, CCL3 stimulation delays in vitro proliferation of hepatocarcinoma cells (HCC) through CCR1-mediated signals; however, CCL3 and CCR1 deficiency reduces HCC size and focus number in vivo due to impaired intratumor migration of Kupffer cells, which boost angiogenesis in hepatic cancers. Given the magnitude of the bibliography for this field, rather than a systematic review of the function of each chemokine/receptor in the oncogenic process, we have selected only mechanistic examples by which chemokines elicit each of the activities analyzed. The reader will be referred to more extensive reviews when appropriate.

3. CHEMOKINE ACTIVITY ON NEOPLASTIC CELLS 3.1 Chemokine Signaling Sustains Tumor Cell Proliferation One of the major differences between untransformed and transformed cells is their control of the production and release of growth-promoting signals that foster cell division (Hanahan and Weinberg, 2011). These proliferative cues are usually triggered by growth factors that transduce signals through cell receptors with intracellular kinase activity, the so-called tyrosine kinase receptors (RTKs). Although chemokines are less potent mitogens than growth factors, an enormous body of evidence indicates that the chemokine system elicits proliferation of epithelial and hematological cancers. Most, if not all, tumor cells produce chemokines and express chemokine receptors that can sustain proliferation in an autocrine or paracrine manner (for an extensive review, see Sarvaiya et al., 2013). Production of these chemokines/receptors is associated in many cases with mutation of protooncogenes that drive cell transformation. These activated oncogenes can directly up- or downregulate a specific set of chemokines and their receptors as part of the oncogenic programs. RET/PTC1, a transforming oncogene in human papillary thyroid carcinoma, induces expression of CXCL12 and its receptor CXCR4, thus triggering autocrine proliferation of thyroid carcinoma cells (Borrello et al., 2005). In other cases, oncogenes transactivate chemokine/receptor genes indirectly by regulating transcription factors involved in inflammation. For instance, H-ras-induced tumorigenesis in the skin requires NF-κB (Jo et al., 2000), which upregulates a large number of proinflammatory chemokines (Gupta et al., 2010).

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Activation of chemokine receptors in tumor cells can initiate a number of intracellular signals that lead to proliferation (Fig. 1). Most chemokine receptors trigger ERK1/2 activation through the PI3K or the β-catenin pathways. Gβ/γ dimers can also switch on the Ras/ERK pathway through Ca2+-dependent activation of the tyrosine kinases Pyk2 and Src (Bonacchi et al., 2001). CCR5 and CCR7 increase expression of the protooncogene c-Fos in leukemia cells through the JAK/STAT pathway (Wong and Fish, 1998). CCL20 and CXCL8 trigger cell cycle progression by upregulating cyclins E and D, which are involved in G1-to-S phase transition, and by downregulating cyclin D inhibitors such as p27 (Marsigliante et al., 2013; Shao et al., 2013). In addition to direct regulation of proliferative signaling, chemokine receptors can boost tumor growth indirectly through transactivation of the epidermal growth factor (EGF) receptor (EGFR). This can occur through physical interaction of the two receptors, as described for the atypical chemokine receptor ACKR3 (CXCR7) and EGFR in breast and prostate cancer cells (Salazar et al., 2014; Singh and Lokeshwar, 2011). ACKR3 was initially considered a “decoy” receptor that targets CXCL12 and CXCL11 for degradation (Massara et al., 2016); however, ACKR3 also elicits β-arrestin-biased signals in neoplastic and stromal cells (Bachelerie et al., 2015). In prostate and breast carcinoma cells, ACKR3 binds physically to EGFR through a β-arrestin-dependent, agonist-independent mechanism; this interaction enhances EGFR phosphorylation and proliferation (Salazar et al., 2014; Singh and Lokeshwar, 2011). The growth-promoting activity of ACKR3 has been extended to other cancers, and its expression is correlated with poor prognosis in renal and lung carcinoma patients (Ierano et al., 2014; Wu et al., 2016). Other atypical receptors have growth-inhibitory activity on cancer cells. Growth inhibition appears to be independent of the ability of these receptors to interact with β-arrestin; expression of ACKR1, ACKR2, and ACKR4 correlates with improved prognosis in cervical squamous and gastric cancer (Massara et al., 2016), although ACKR2 and ACKR4 interact with β-arrestins after ligand binding (Bachelerie et al., 2015). A second mechanism of chemokine-mediated EGFR transactivation involves the release of membrane-bound EGFR agonists. Stimulation of ovarian and breast cancer cells with CXCL1 or CX3CL1, respectively, induces ERK1/2-mediated cancer cell proliferation in a pertussis toxininsensitive, EGFR-dependent manner (Bolitho et al., 2010; Tardaguila and Man˜es, 2013; Tardaguila et al., 2013). The mechanism proposed involves MMP-driven shedding of EGF precursors anchored to the cancer

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cell membrane; this proteolysis releases the active form of the ligand, which can then activate the EGFR (Fig. 2, left). The in vivo relevance of this chemokine/EGFR crosstalk was demonstrated for CX3CL1 in a mouse model of spontaneous breast cancer, in which the neu protooncogene (an EGFR-related receptor) is expressed constitutively in the mammary epithelium (MMTV-neu); this mouse model mimics human HER-2+ breast tumors. MMTV-neu mice develop multifocal breast tumors with full penetrance (100% of nulliparous females have tumors by 14 months; t50 ¼ 31.7 weeks). Genetic ablation of CX3CL1 delays tumor onset to t50 ¼ 54 weeks, and
30% of the CX3CL1-deficient females do not develop tumors by 15 months of age (Tardaguila et al., 2013). CX3CL1 deficiency also reduces tumor multiplicity, suggesting that it acts as a promoter of incipient neoplastic lesions rather than as an initiator of the oncogenic process. Lack of CX3CL1 nonetheless does not affect tumor onset or multiplicity in MMTV-PyMT mice, which develop spontaneous breast tumors by overexpressing the polyoma virus middle-T antigen in the mammary epithelium (Tardaguila et al., 2013). These findings suggest that CX3CL1 promotes breast tumorigenesis through a “triple membrane passing” signaling pathway that bolsters EGFR signals at early states of malignancy (Fig. 2, left). It is not yet known whether this transactivation model applies to other chemokines and RTK.

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Fig. 2 Bidirectional cross-communication between RTK and GPCR. GPCR and RTK can transactivate each other via a similar triple membrane-passing signaling mechanism. (Left) The proposed model by which the CX3CL1/CX3CR1 axis boosts breast cancer cell proliferation by triggering transactivation of the EGFR signaling pathway. (Right) Scheme showing the process by which the IGF–IR triggers tumor cell invasiveness by activating the CCL5/CCR5 axis in breast cancer cells. “proEGF” indicates a generic membrane-anchored EGF precursor.

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3.2 Chemokines Interact With Tumor Suppressor Genes Chemokine activity does not always sustain growth-stimulatory signals on tumor cells. There is evidence that, in some cases, chemokines form part of negative regulatory programs that limit tumor cell proliferation and growth. Many of these programs depend on the activity of specific proteins termed tumor suppressors or antioncogenes. Tumor suppressors protect cells from becoming neoplastic by repressing the cell cycle or by inducing apoptosis, or both (Hanahan and Weinberg, 2011). The anticarcinogenic activity of tumor suppressors is not limited to their cell autonomous functions, but also seems to involve noncell autonomous regulatory programs in which specific chemokines and receptors are pivotal. This is epitomized by the prototypic tumor suppressor TP53, which represses CXCR4 (Mehta et al., 2007) and enhances CX3CL1 expression in cancer cells (Shiraishi et al., 2000). TP53-mediated upregulation of CX3CL1 could be a noncell autonomous, immune-based tumor suppression mechanism (Xin et al., 2007; Yu et al., 2007), whereas CXCR4 repression would restrict the growthpromoting and metastatic signals provided by this receptor in breast cancer cells (Mehta et al., 2007). In renal carcinoma cells, CXCR4 expression is also regulated by the von Hippel–Lindau tumor suppressor protein (pVHL), which in normoxic conditions targets the hypoxia-inducible factor (HIF)-1α for degradation. Renal cancers that harbor pVHL-inactivating mutations have high CXCR4 levels and increased mitotic score, both associated with hypoxia and poor prognosis (Staller et al., 2003). CXCR4 downmodulation could thus be a branch of the cell autonomous repressor activities shared by various tumor suppressor genes. Chemokines are not only targets, but can also modulate the activity of tumor suppressor genes. This was observed initially in CCR5-expressing breast cancer cell lines with nonmutated TP53. Stimulation of these cells with CCL5, a CCR5 agonist, activates TP53 through a Gαi-, JAK2-, and p38 MAPK-mediated mechanism. As a consequence, CCL5 stimulation leads to CCR5-dependent upregulation of TP53 transcriptional targets such as CDKN1A, which codes for the cyclin-dependent kinase inhibitor p21WAF and reduces breast cancer cell proliferation in vitro and in mouse models (Man˜es et al., 2003). In humans, the ccr5Δ32 polymorphism renders this receptor nonfunctional. Analysis of ccr5Δ32 allelic frequency in a cohort of 547 patients with primary nonmetastatic breast cancer suggested that the CCR5/TP53 connection is important for tumor progression; tumors from ccr5Δ32 patients were larger and tended to have a lower TP53 mutation rate

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than those harboring the ccr5 wild-type (ccr5WT) allele. Furthermore, diseasefree survival (DFS) was shorter in ccr5Δ32-bearing individuals whose tumors expressed nonmutated p53; conversely, DFS was comparable between ccr5Δ32 and ccr5WT patients whose tumors showed mutated p53 (Man˜es et al., 2003). The ccr5Δ32 polymorphism is also associated with susceptibility to gallbladder cancer, possibly through a similar mechanism (Srivastava et al., 2008). The observations that CCL5 induces stabilization of TP53 in neuronal and astrocytic nuclei (Jordan-Sciutto et al., 2001) and that CCL2 induces endothelial cell apoptosis through a p53-dependent pathway (Zhang et al., 2011) suggest that chemokine activation of TP53 is a fairly general phenomenon with physiological implications. Crosstalk between the chemokine network and tumor suppressors has also been associated with senescence, a process that inhibits cell reentry into the cell cycle, thus causing irreversible growth arrest (Hanahan and Weinberg, 2011). Senescence can occur due to excess oncogenic signaling (so-called oncogene-induced senescence, OIS) in preneoplastic lesions, but is not observed in established tumors. This led to the proposal that senescence acts as an additional barrier that must be bypassed for malignancy to be established. Senescence is linked to the DNA damage response, which activates TP53 and causes cell arrest by upregulating p21WAF or p16INK4a. Senescent cells are nonetheless metabolically active, and produce and secrete a complex cocktail of proteases, growth factors, cytokines, and chemokines (Acosta and Gil, 2009). These chemokines are proposed to reinforce the senescence program by TP53 activation through a CXCR2-dependent mechanism (Acosta and Gil, 2009; Ruan et al., 2012). TP53 increases cellular CXCR2 levels in response to DNA damage (Guo et al., 2013), which suggests a positive feedback loop in which CXCR2 ligands would trigger TP53 transcriptional activity, in turn transactivating the CXCR2 promoter to reinforce cell senescence. Since CCR5 can also elicit TP53 activation (Man˜es et al., 2003), it is tempting to propose a role for CCR5 in strengthening the senescence program, although this hypothesis has not been demonstrated formally.

3.3 Chemokines as Regulators of Apoptosis in Cancer Cells The acquisition of molecular cues that permit neoplastic cells to evade programmed cell death by apoptosis is central for tumor progression to high-grade therapy-resistant malignancy (Kelly and Strasser, 2011). During the course of tumorigenesis, cancer cells must circumvent numerous forms

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of intra- and extracellular stresses able to activate intrinsic and extrinsic apoptotic pathways, respectively. Intracellular stresses that lead to cell death include aberrant oncogene-mediated signaling, DNA damage associated to hyperproliferation, and genomic instability. Extracellular signals that trigger apoptosis include engagement of “death receptors” (such as FAS) by cognate ligands of the tumor necrosis factor (TNF) family. The effectors of these death signals are a family of proteases termed caspases, which initiate a proteolytic cascade that activates a number of latent proteases responsible for the execution phase of apoptosis (Adams and Cory, 2007). Numerous regulatory proteins control communication between regulators and effectors. Bcl-2 and its relatives (Bcl-xl, Bcl-w, Mcl-1, and A1) act as antiapoptotic proteins by regulating the activity of the proapoptotic family members Bax and Bak (Adams and Cory, 2007). In contrast, proteins of the BH3-only family (Bim, Bid, Puma, Bad, Noxa, Bik/Blk, Bmf, Hrk/ DP5, and Beclin) suppress the antiapoptotic activity of Bcl-2 family members, thus promoting cell death (Shamas-Din et al., 2011). Many reports state that chemokines produced by tumor or by stromal cells exert antiapoptotic activity in the former, although the mechanism by which this occurs has not been documented. Chemokine receptors mediate activation of the PI3K/AKT/NF-κB pathway (Massara et al., 2016), which upregulates Bcl-2 and its relatives and downregulates Bax and Bak; these changes protect cells from apoptosis by altering the balance between pro- and antiapoptotic proteins. For instance, CCR5-mediated activation of NF-κB is a major factor for tumor development in several mouse models (Lee et al., 2012; Song et al., 2012). CCR5-deficient tumors have a higher frequency of apoptotic cells, which correlates with increased levels of cleaved caspases-3, -9, PARP, and Bax, and reduced expression of Bcl-2 and the inhibitor of apoptosis protein (IAP). These effects are associated to NF-κB inactivation in CCR5-deficient tumors, which in turn caused downmodulation of NF-κB target genes such as CCL2. Blockade of CCL2 using a specific inhibitor delays lung cancer progression in these mice (Lee et al., 2012), which suggests that the CCR5/NF-κB/CCL2 connection is important for providing survival signals and potentiating tumor progression. ERK pathway activation also participates in the prosurvival activity of many chemokine receptors. CXCR4-mediated ERK activation protects chronic lymphocytic leukemia (CLL) B cells from apoptosis by direct phosphorylation and inactivation of the proapoptotic protein Bad, and indirectly

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by activation of CREB (cAMP response element-binding protein), a transcription factor that upregulates the antiapoptotic bcl2 gene (Burger et al., 2000). ERK activation is also implicated in CCR3, CCR8, and CCR7 prosurvival activity in lymphoma and nonsmall lung cancer cells, by inducing phosphorylation and inactivation of proapoptotic proteins and activating survivin (Miyagaki et al., 2011; Mo et al., 2015; Spinetti et al., 2003). Chemokine-mediated upregulation of antiapoptotic proteins also confers cancer cell resistance to chemotherapeutic drugs and to the cytotoxic activity of immune cells. The former is exemplified by CCL25, which enhances resistance to TNF-α-induced apoptosis in leukemia cells through JNK1 (c-Jun N-terminal kinase)-mediated upregulation of livin, an IAP family member (Qiuping et al., 2004). CCR10 activation in melanoma cells by locally produced CCL27 has a role in resistance to FAS-mediated cell death induced by tumor antigen-specific CD8+ T cells; the mechanism proposed involves a CCR10-mediated, PI3K-dependent pathway (Murakami et al., 2003). In contrast, shutdown of chemokine receptor signaling in neoplastic cells increases the effectiveness of chemotherapeutic drugs. For instance, blockade of CXCR1/2 with repertaxin or the genetic knockdown of CXCR4 increases Bax expression and renders gastric and triple-negative breast cancer cells more susceptible to 5-fluorouracil (5-FU) and cisplatin, respectively (Liang et al., 2015; Wang et al., 2016). Blockade of CCR5 with maraviroc directly upregulates FAS and other proapoptotic proteins, as well as increasing levels of cleaved caspases, causing apoptosis in colorectal cancer cells (Pervaiz et al., 2015). This suggests that chemokine antagonists could be used as chemotherapeutic agents for some tumors. The evidence discussed above indicates that cancer cells use chemokines to evade apoptosis by inducing upregulation of antiapoptotic proteins, repressing proapoptotic protein expression, or by short-circuiting the extrinsic apoptotic pathway. Chemokines also protect cancer cells from apoptosis by regulating autophagy. Autophagy is a physiological cell response induced in specific cell stress situations (Rabinowitz and White, 2010). Activation of the autophagy program causes sequestration of proteins and cell organelles into cytosolic vesicles termed autophagosomes, which fuse with lysosomes wherein degradation occurs. This generates new metabolites that sustain the survival of stressed cells, particularly in nutrient-limited environments similar to those of tumors. According to this interpretation, radiotherapy or certain chemotherapeutic drugs could induce autophagy, which is cytoprotective for cancer cells, and thus limits their own therapeutic

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effectiveness (Kondo et al., 2005). Genetic deletion of the autophagy effector Beclin-1 or other of the autophagy machinery components nonetheless increases cancer susceptibility, suggesting that autophagy acts as a barrier for neoplastic transformation (Galluzzi et al., 2015). Beclin-1, the mammalian orthologue of the autophagy-related gene (atg)-6 in yeast, is also a member of the BH3-only group of apoptotic regulatory proteins, and thus links the autophagic and apoptotic machineries. The balance between Beclin-1induced apoptosis or autophagy depends on its interaction with Bcl-2/ Bcl-xl, which is regulated in turn by BH3 proteins such as Bid or Puma (Hanahan and Weinberg, 2011). Furthermore, the autophagy program is precisely regulated through the PI3K and the mammalian target of rapamycin (mTOR) pathways, which also participate in the apoptotic regulatory program. There are thus numerous interconnections between the regulatory circuits that govern autophagy and apoptosis, two critical processes in cell homeostasis. Although the literature is not extensive, some reports have implicated chemokines in the regulation of autophagy. This is not surprising, since many chemokine receptors activate the mTOR pathway. This is the case of CXCR4, which contributes to the development of peritoneal carcinomatosis of gastric cancer by activating the PI3K–mTOR pathway. mTOR blockade not only inhibits migration of these gastric cancer cells but also activates autophagic cell death (Hashimoto et al., 2008), which suggests that CXCR4 negatively regulates autophagy through PI3K/AKT/mTOR pathway activation. Given the dual outcome of autophagy in the oncogenic process, its repression might also contribute to tumor growth inhibition. CXCR3 has two splice variants, CXCR3A and CXCR3B, which in breast cancer cells transduce growth-promoting and growth-inhibitory signals, respectively (Datta et al., 2006). Some of the CXCR3B-elicited inhibitory signals include downmodulation of Beclin-1 and LC3B (a ubiquitin-like modifier) (Balan and Pal, 2014), which is the mammalian orthologue of the yeast atg-8 gene. Concomitant with the CXCR3B-induced inhibition of autophagy, there is an increase in apoptotic regulators, which supports the reverse regulation of apoptosis and autophagy in some cancer cells. Although most evidence pinpoints chemokines as prosurvival signals, chemokine receptor signaling can also transduce death signals in some cancer cells. CXCR4 is needed for acute myeloid leukemia (AML) cells to migrate to the bone marrow; nonetheless, CXCR4 engagement also induces AML cell apoptosis by reducing expression of Bcl-xl, upregulating Bak and increasing the stability of Noxa, a Bak activator (Kremer et al., 2013).

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Chemokine receptor signaling can also induce apoptosis in tumorinfiltrating T cells, which will be discussed later as an immune evasion mechanism. These results highlight the concept that survival or apoptotic signals provided by chemokines could be cell type specific and/or the result of a cell context-dependent cross-communication with yet unknown signals.

3.4 Chemokines as Drivers and Cues for Tumor Invasion and Metastasis A hallmark of high-grade malignancies is the dissemination of cancer cells to distant tissues, a process termed metastasis. Metastasis is a multistep process that involves local invasion of the tissue adjacent to the primary tumor, intravasation of cancer cells to nearby blood and lymphatic vessels, resistance of the intravasated cells to anoikis during their transit through the hematic and lymphatic systems, extravasation at distant tissues, the formation of micrometastatic nodules of cancer cells and, finally, colonization of the affected organ due to outgrowth of the micrometastases. Hematopoietic- and epithelial-derived cancers coopt the signals provided by chemokines to influence most, if not all, steps in this cascade. Since chemokines are well-recognized chemoattractants for cells of hematopoietic origin, we will focus mainly on the role of these molecules in the metastasis of epithelialderived tumors. The progression from localized to metastatic carcinomas is associated with remarkable changes that enable the conversion of an essentially static cell into a cell able to move and invade surrounding tissue. These changes are usually associated with a process termed epithelial-to-mesenchymal transition (EMT, which will be addressed in the next section) and with extracellular cues, such as chemokines, that are able to trigger migration of cancer cells (Sarvaiya et al., 2013). Activation of chemokine receptors not only directly transduces migratory signals in many cancer cells but also seems to be a requisite for growth factor-induced cancer cell invasiveness. This was described initially in the low-invasive, HER2-negative MCF7 breast cancer cell line. Insulin-like growth factor (IGF)-I-induced invasion of MCF7 cells is switched on and off by modification of CCR5 levels at the cell membrane (Mira et al., 2001); high CCR5 levels provide a “permissive” signal that endows the tumor cell with the ability to invade. The mechanism involves IGF-I-induced upregulation of CCL5, a CCR5 ligand, which is secreted to activate the receptor in an autocrine or paracrine manner. These data establish a curious parallel between GPCR–RTK, which is involved in proliferation, and RTK–GPCR transactivation,

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involved in invasion, through extracellular cross-communication between chemokines and growth factors (Fig. 2). The potential of CCR5 to foster cancer cell invasiveness has been validated in mouse models of breast cancer metastasis (Datar et al., 2015; Velasco-Velazquez and Pestell, 2013), as well as in observational studies in humans; indeed, ccr5Δ32 postmenopausal breast cancer patients showed longer metastasis-free survival than those with the wild-type allele (Span et al., 2015). Growth factor-induced chemokine crosstalk appears to be a general requirement for cancer cell invasion, although the partners and mechanisms involved might depend on tumor cell characteristics. For instance, CXCR4 is critical for HER2-enhanced invasiveness of HER2expressing breast tumors (Li et al., 2004); in this case, HER2 increases CXCR4 levels in two ways, by triggering CXCR4 transcription and by impairing agonist-induced CXCR4 degradation. Since many invasive cancer cells constitutively secrete growth factors and chemokines, it is tempting to speculate that RTK/chemokine receptor cross-communication endows these tumor cells with built-in invasive capacity. Although it is counterintuitive, growth factor-stimulated autocrine production of chemokines can guide tumor cells through a process referred to as “autologous chemotaxis” (Shields et al., 2007). Due to the interstitial flow of the lymphatic system, CCL19 and CCL21 secreted by CCR7expressing tumor cells form a gradient that guides these cells toward lymphatic endothelial cells (LEC). The invasive phenotype might not arise in a strictly cell autonomous manner, however, but could involve heterotypic interactions between cancer and nonmalignant cells of the tumor microenvironment. Chemokines produced by cancer-associated fibroblasts (CAF) form gradients that guide tumor cells to blood or lymphatic vessels (reviewed extensively in Raman et al., 2007). Growth factors produced by breast carcinoma cells stimulate CCL5 production in bone marrow-derived mesenchymal stem cells (MSC); CCL5 then drives breast cancer metastasis in a CCR5-dependent manner (Karnoub et al., 2007). CCL5 produced by MSC also increases prostate cancer cell invasion, in this case by altering androgen receptor signals through a HIF-2α pathway (Luo et al., 2015). CXCL12 is also involved in recruitment of MSC and inflammatory monocytes into the tumor stroma to promote metastasis. Indeed, miR-126/miR126* (a microRNA pair derived from a single precursor) represses lung metastasis of breast cancer xenografts by inhibiting CXCL12 expression and MSC and monocyte recruitment (Zhang et al., 2013). It is interesting that miR-126/miR-126* levels are usually low in cancer cells, due to

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methylation of their host gene egfl7. There is thus solid evidence that chemokines, directly or indirectly, provide carcinoma cells with the ability for invasive growth and to initiate the metastastic process. The role of chemokines in tumor dissemination is not restricted to the acquisition of a motile phenotype. Experimental and clinical evidence indicate that metastasis is not random, but occurs in an organ-specific manner. Chemokine guidance appears to be one of the molecular mechanisms that control the organ specificity of metastatic cells. One hypothesis is that cancer cells hijack the network of homeostatic chemokines, which signal the tissuespecific homing of hematopoietic precursors in physiological conditions, to metastasize in certain organs (Zlotnik et al., 2011). Evidence from animal models and retrospective studies in patients supports this idea. For instance, CXCR4 is strongly expressed in many cancer cell types and appears to capacitate metastatic cell homing to tissues that express high CXCL12 levels such as bone marrow, lung, liver, and brain (Muller et al., 2001; Murakami et al., 2002; Romain et al., 2014; Smith et al., 2004). Metastasis of hematopoietic (Lopez-Giral et al., 2004) and nonhematopoietic tumors (Muller et al., 2001; Shields et al., 2007; Takeuchi et al., 2004) to LN is associated with tumor cell expression of CCR7, which in physiological conditions enables recruitment of mature dendritic cells (mDC) and naı¨ve T lymphocytes to LN. Furthermore, forced CCR7 expression in CCR7 breast cancer cells shifts metastasis from lung to the LN (Cunningham et al., 2010) and indicates that a single chemokine receptor could be sufficient to determine the organ specificity of metastatic cancer cells. Another ligand/receptor pair involved in organ-specific metastasis is CCL25/CCR9, which participates in physiological seeding of T cell progenitors to the thymus as well as in homing of a CCR9+ T cell subset to the small intestine (Johansson-Lindbom and Agace, 2007). High CCL25 levels in the small intestine submucosa appears to be a central signal for targeting CCR9-expressing cutaneous melanoma, ovarian, and breast cancer cells to this infrequently metastasized site (Amersi et al., 2008; JohnsonHoliday et al., 2011). CCR9 levels in cancer cells correlate negatively with invasiveness and metastasis of colorectal cancers with high CCL25 expression in the gastrointestinal microenvironment (Chen et al., 2012). This observation highlights the direct relationship between chemokines and their receptors as a rate-limiting step for organ-specific metastases. The role of chemokines in the formation of distant metastases is not restricted to neoplastic cell homing to a given tissue. Chemokines might also

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modify the target tissue to create a hospitable environment for the growth of engrafted cells. LEC expression of CCL5 is considered an important factor for conditioning premetastatic niches in experimental breast cancer models (Lee et al., 2014). CCL5 is not expressed by LEC in physiological conditions, but its transcription is induced in these cells by tumor cell-secreted IL-6, through STAT3 phosphorylation. Inflammatory chemokines might also contribute to formation of metastatic niches by recruiting hematopoietic cells (Hiratsuka et al., 2006). In a syngeneic mouse model, tumor cell-produced VEGF-A (vascular endothelial growth factor), TGF-β (transforming growth factor), and TNF-α-induced production of chemokines and S-100 chemoattractant cytokines that attract Mac-1+ -myeloid cells to the lung parenchyma (Hiratsuka et al., 2006). Inhibition of Mac-1+ cell infiltration led to an
90% reduction in tumor cell colonization of the lung, which suggests the importance of these myeloid cells for preconditioning the niche before tumor cell arrival. In another study, the formation of these niches depended on tumor-induced mobilization of CD45+CD13+ mesenchymal cells (also termed fibrocytes), which infiltrate the lung in a CCR5-dependent manner (van Deventer et al., 2008). Fibrocyte infiltration is a critical factor for cancer cell engraftment in the lung through production of MMP9, a metalloproteinase that facilitates tumor cell invasion and mediates neoangiogenesis at the metastatic niche (Mira et al., 2004). Although evidence is limited, chemokines are important for survival and growth of micrometastases. Blockade of CXCR4 is thought to reduce lung metastasis of colon cancer cells, not by impairing invasion or extravasation to lungs, but by inhibiting micrometastasis outgrowth (Smith et al., 2004; Zeelenberg et al., 2003). A similar role is proposed for CCR6/CCL20 in colon cancer metastasis to the liver (Ghadjar et al., 2009). By acting at different levels in the metastatic cascade, including stimulation of cancer cell invasiveness, organ-specific homing of circulating tumor cells, and micrometastasis outgrowth during organ colonization, chemokines, and their receptors appear to be central to cancer cell dissemination to distant organs.

3.5 Chemokines and Cancer Stem Cell Plasticity Cancer cells within tumors have traditionally been viewed as relatively homogeneous cell populations, particularly at early disease stages. The use of single-cell genomic analysis has nonetheless demonstrated enormous heterogeneity in the neoplastic cells that form a tumor from very early stages. It

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is now accepted that clonal heterogeneity within tumors affects disease progression as well as resistance to therapies. Although classically considered a late-stage event in tumor progression, cancer cell subclones with metastasisprone genetic signatures can be detected very early in the tumorigenic process (Weng et al., 2012). Clonal heterogeneity within tumors has been explained using two models, the stochastic and the hierarchical. The stochastic model predicts that subclonal variation occurs through evolution over the course of the malignancy, as hyperproliferation, genetic instability, and environmental signals give rise to mutations in the original clone (Nowell, 1976). According to this model, all subclones from a tumor will have similar tumorigenic potential. In contrast, the hierarchical model predicts that cells in a tumor are organized in a pyramid; cells with stem-like, self-renewal, and differentiation properties (cancer stem cells, CSCs) are at the apex, and terminally differentiated cancer cells are at the base (Bonnet and Dick, 1997). This model considers that CSCs are not only the tumor-initiating cells, but that they are also the only cells with the potential to give rise to a tumor. The two models are not mutually exclusive, however, since cancer cells are able to shift from a CSC-like condition to a non-CSC state and vice versa (Cabrera et al., 2015). The combination of clonal evolution and CSC plasticity might explain the extraordinary heterogeneity found in solid tumors, as well as the inherent resistance of poorly differentiated tumors to chemoand radiotherapy. Chemokines have been implicated in promotion and/or maintenance of the CSC phenotype, and hence thought to contribute to tumor heterogeneity. CXCL8 increases the sphere-forming activity (stem cell specific) and self-renewal of CXCR1-expressing breast CSC (Ginestier et al., 2010), and CXCL8 blockade is associated with greater effectiveness of anti-HER2based treatments (Singh et al., 2013). CCR7 is also linked to CSC regulation in murine and human breast tumors (Boyle et al., 2016). The CXCR4/ CXCL12 pair helps to promote tumor-initiating cells in renal (Gassenmaier et al., 2013) and lung carcinomas (Bertolini et al., 2015), glioblastoma (Gatti et al., 2013), and T cell leukemia (Passaro et al., 2015); in some cases, this is associated with resistance to chemotherapy-induced cell death, tumor recurrence, or metastasis. Chemokines form part of the cytokine loops that are established between stem and stromal cells and that contribute to maintenance of the stem-like phenotype in the former. As an example, breast cancer cells induce fibroblast production of CCL2, which in turn enhances the sphere-forming phenotype and self-renewal of the CSC-like population (Tsuyada et al., 2012).

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CXCL7 also participates in the MSC/CSC crosstalk in breast xenografts, the formation of MSC/CSC niches, and the acceleration of tumor growth (Liu et al., 2011b). CSC plasticity resembles that of embryonic stem cells (ESC) during development. Some gene expression signatures are shared between ESC and poorly differentiated solid tumors (Ben-Porath et al., 2008), which suggests that cancer cells coopt normal developmental programs to achieve a selective advantage. An example is EMT, a reversible embryonic program involved in gastrulation that is recapitulated during tumor progression (Larue and Bellacosa, 2005). Induction of EMT in cancer cells converts an essentially static epithelial cell into a mesenchymal-like cell able to move and invade surrounding tissue. These changes not only alter cancer cell morphology and cell-to-cell and cell-to-ECM adhesive properties, but are also important for maintenance of stem cell properties. EMT induction in nontumorigenic epithelial cells leads to expression of CSC markers and acquisition of stem-like properties (Mani et al., 2008). The chemokine CCL5 is proposed to be a key molecule in the transformation of nonCSC to CSC-like ovary carcinoma cells through EMT induction (Long et al., 2012). EMT is induced by the integration of numerous signals in the tumor microenvironment that include soluble factors such as members of the TGF-β family, and intercellular communication systems such as Notch and its ligands (Bolos et al., 2013; Thiery, 2002; Zavadil et al., 2004). These stimuli activate important transcription factors (Snail and Slug, among others) that regulate expression of EMT effector genes, including chemokines and their receptors. In breast cancer cells undergoing EMT, for instance, TGF-β upregulates CCL21 through a p38 MAPK-dependent mechanism (Pang et al., 2016). CXCL12/CXCR4 and CXCL8/CXCR1 are also linked to the EMT program induced by TGF-β and Snail in several cancers (Bertran et al., 2009; Fernando et al., 2011; Hwang et al., 2011). Chemokines appear to act not only as intermediates but also as direct drivers of the EMT. CXCL12/CXCR4 induces EMT in colorectal and pancreatic cancer cells by activating the Wnt/β-catenin and the noncanonical Hedgehog pathways, respectively (Hu et al., 2014; Li et al., 2012). CCL21/CCR7 and CXCL5/CXCR2 trigger EMT in hepatocellular carcinoma cells through PI3K-mediated induction of glycogen synthase kinase (GSK)-3β and Snail (Zhang et al., 2015; Zhou et al., 2015). CCR7 is reported to mediate TGF-β-induced EMT of gastric cancer cells via crosstalk with the NF-κB signaling pathway (Ma et al., 2015). In some cases, tumor cells

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produce chemokines that induce EMT, but in others chemokines are produced by bone marrow-derived cells, which are attracted by other chemokines to the proximity of tumor cells. Melanoma-produced CXCL5 attracts myeloid-derived suppressor cells (MDSC) to the tumor site, which reciprocally produce TGF-β and other factors to induce EMT in the melanoma cells (Toh et al., 2011). By inducing the formation of functionally distinct subpopulations within a tumor, CSC plasticity might also enable the acquisition of stromal support for tumor progression. A growing number of studies report that, in addition to the EMT, transdifferentiation of CSC subpopulations into functional endothelial-like cells contributes to the formation of tumor-associated neovasculature in glioblastoma and breast cancer (Ricci-Vitiani et al., 2010; Tang et al., 2014). This transdifferentiation was also documented for ovarian CSC, a process in which CCL5 is a central molecule (Tang et al., 2016). Ovarian CSC (characterized by CD133 expression) secretes CCL5, which acts in an autocrine manner to stimulate migration and invasion of non-CSC cells through an NF-κB-mediated mechanism (Long et al., 2012). Tang et al. found that CCL5 also activates STAT3, and that this activation is critical for ovarian CSC transdifferentiation into endothelial cells (Tang et al., 2016). CCL5 blockade inhibits but does not completely block formation of vessel-like structures in vitro, which suggests the involvement of additional factors in this transdifferentiation process. Grafting of CCL5-silenced human ovarian CSC into immunodeficient mice does not affect tumor growth or the number of mouse-derived CD31+ vessels compared to nonsilenced cells, but produces a notable reduction in the number of human CD31+ structures observed in these xenografts, which implicates CCL5 in in vivo CSC transdifferentiation into endothelial cells (Tang et al., 2016). In summary, chemokines influence the stem-like properties of cancer cells, which have important implications for the invasiveness, metastasis, and therapeutic resistance of tumors.

4. CHEMOKINE ACTIVITIES ON STROMAL CELLS Chemokines can act as an efficient intercellular communication system between neoplastic cells and other cellular elements that constitute the tumor stroma. Of these elements, the tissue-resident fibroblasts usually produce chemokines rather than serving as their targets (Raman et al., 2007). In some tumors, fibroblasts associated to neoplastic cells are derived from MSC that originate in the bone marrow (Guo et al., 2008); in these cases,

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chemokines could have a major role in guiding bone marrow cells to the tumor stroma, as indicated in previous sections. Here we will focus on chemokine activity in vascular and immune cells, and how these molecules strike a balance between the host defensive response and the growthpromoting activity of stromal cells in tumorigenesis.

4.1 Chemokines as Positive and Negative Regulators of Tumor Vascularization Tumor growth, progression, and metastasis rely on the ability of neoplastic cells to induce formation of new blood vessels that infiltrate the tumor parenchyma. This neovascularization occurs through two main processes: vasculogenesis, that is, the assembly of newly generated endothelial cells, and angiogenesis, the sprouting of new blood vessels from existing ones (Ahn and Brown, 2009). Generation of tumor-associated neovasculature follows the same principles as those that govern blood vessel formation during embryogenesis or wound healing in adulthood. Normal vasculature nonetheless returns to quiescence when the physiological stimulus disappears, whereas progressing tumors undergo an “angiogenic switch” that enables constitutive formation of new vascular structures (Hanahan and Weinberg, 2011). The angiogenic switch arises due to an imbalance in pro- and antiangiogenic factors in the tumor milieu (Hanahan and Weinberg, 2011). Many cancer and/or stromal cells secrete proangiogenic inducers such as VEGF, basic fibroblast growth factor (bFGF), or prostaglandin E2 (PGE2), whereas they silence production of antiangiogenic factors such as thrombospondin-1 or angiostatin. Chemokines in the tumor microenvironment also contribute to regulation of the angiogenic switch, and thus influence both vasculogenic and angiogenic responses. Chemokines modulate the mobilization and plasticity of endothelial progenitor cells, which are responsible for tumor-associated vasculogenesis. CXCL12 and CCL2 are the main chemokines involved in mobilization of these progenitors from bone marrow (Nolan et al., 2007; Reddy et al., 2008; Smadja et al., 2005). Furthermore, CXCL12 produced in hypoxic areas of tumors is linked to the recruitment of these progenitors (Ceradini and Gurtner, 2005) and participates in formation of vascular structures by increasing integrin-mediated progenitor adhesion to fibronectin and collagen I (De Falco et al., 2004). CCL20 and CCL5 assist in vasculogenesis by inducing transdifferentiation of hematopoietic cells or CSC to endothelial-like cells able to form new vessels (Shih et al., 2012; Tang et al., 2016). CXC

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chemokines can exert direct pro- or antiangiogenic effects on vascular cells depending on the presence or absence of the glutamic acid–leucine–arginine motif (ELR, in single-letter amino acid code) at their N-terminus (Strieter et al., 2005). The ERL+ CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) act as angiogenesis promoters, whereas the ERL CXC chemokines (CXCL4, CXCL4L1, CXCL9, CXCL10, CXCL11, and CXCL14) function as angiostatics (Strieter et al., 1995). The exceptions are CXCL12 and CXCL17, two ERL chemokines with angiogenic activity (Lee et al., 2013; Strieter et al., 2005). Some CCL chemokines are also proangiogenic when overexpressed in the tumor environment. Although most of these CCL chemokines trigger angiogenesis through indirect and/or paracrine mechanisms (discussed later), those that act through CCR1 can have direct angiogenic activity on the endothelium. CCL23, CCL16, and CCL15 induce endothelial cell migration and proliferation (Hwang et al., 2004, 2005; Strasly et al., 2004). This activity appears to be CCR1 specific, since the angiogenic activity of CCL16 is inhibited only by CCR1 antagonists but not by blockade of CCR8 or CCR2 (Strasly et al., 2004), the other two CCL16 receptors. The atypical chemokine receptors nonetheless have a dual role in angiogenesis. ACKR1 expression in erythrocytes and in endothelial cells limits tumor angiogenesis by sequestering proangiogenic chemokines. Prostate tumors implanted in ACKR1-deficient mice have greater tumor vessel density and growth than those grafted in wild-type animals, and this correlates with reduced ERL+ CXC chemokines (Shen et al., 2006). In contrast, ACKR3, whose expression is upregulated by VEGF (Yamada et al., 2015), mediates the angiogenic effects of CXCL12 through an ERK1/2 signaling pathway (Yamada et al., 2015). ACKR3 blockade by genetic interference or the compound CCX771 inhibits migration and tube formation in vitro and reduces tumor growth, metastasis, and angiogenesis in vivo (Yamada et al., 2015). In many cases, the angiogenic activity of chemokines takes place through more or less complex paracrine interactions. This is epitomized by CXCL17, which induces chemoattraction of monocytes to the tumor microenvironment and stimulates VEGF production in the attracted cells (Lee et al., 2013). Other proinflammatory chemokines such as CCL2 and CCL3 induce angiogenesis by attracting neutrophils and macrophages to the tumor site and simultaneously trigger expression of the angiogenic protease MMP-9 (Pahler et al., 2008; Wu et al., 2008) or of VEGF through a HIF-1α-dependent pathway (Varney et al., 2005). CCL2 is also involved in

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the TGF-β angiogenic effect through recruitment of MSC and vascular smooth muscle cells to endothelial cells in the tumor parenchyma (Ma et al., 2007). CCL chemokines might even be mediators of wellestablished angiogenic factors such as bFGF; bFGF interaction with its receptor on endothelial cells triggers CX3CL1, CCL2, and CXCL1 secretion, which attracts inflammatory cells that foster tumor neovascularization (Andres et al., 2009). From the examples mentioned to this point, a close relationship is evident between chemokines and other angiogenic factors in the tumor milieu. In glioblastoma, CXCL8 and VEGF are coregulated at the posttranscriptional level through the microRNA miR-93; this regulatory mechanism is clinically relevant for predicting the progression of these brain tumors (Fabbri et al., 2015). In melanoma and ovarian cancers, constitutive expression of CXCL1, CXCL2, CXCL3, and CXCL8 is associated to an enhanceosome-like complex formed by the NF-κB, HMG(I)Y, and SP1 elements activated by VEGF stimulation (Duckworth et al., 2016; Luan et al., 1997). In colorectal cancer, however, CXCL1 appears to be regulated mostly through a PGE2-induced signaling pathway (Wang et al., 2006). PGE2 regulates CXCR4 on endothelial cells downstream of classical angiogenic factors such as bFGF or VEGF (Salcedo et al., 2003), suggesting that CXCL12/CXCR4 is integral to tumor neovascularization induced by these factors. CXCL12/CXCR4 stimulates VEGF expression in several tumor types, including prostate (Darash-Yahana et al., 2004), breast (Liang et al., 2007), and glioblastoma stem cells (Ping et al., 2011), through a PI3Kmediated pathway. Together these results indicate potential positive feedback loops between angiogenic chemokines and growth factors that could boost neoangiogenesis in tumors. Treatment of tumor-bearing mice with the CXCR4 antagonist ADM3100 not only reduced VEGF expression, but also inhibited angiogenesis of the tumor xenografts (Darash-Yahana et al., 2004; Ping et al., 2011), which indicates the in vivo relevance of these feedback loops. In osteosarcoma cells, CCR5 increases VEGF levels by downregulating miR-374b via the PKCδ/HIF-1α (Wang et al., 2015) and the JNK/ERK/ p38 MAPK-signaling pathways (Liao et al., 2016). In chondrosarcoma, CCR5 activation also increases VEGF levels by downregulating various microRNA. Liu and coworkers found positive correlation between CCL5 and VEGF levels in chondrosarcoma patients, as well as negative association between CCL5 and miR-200b expression (Liu et al., 2014). In vitro CCR5 activation leads to reduction of miR-200b levels through a

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PI3K-dependent pathway; miR-200b binds to the 30 UTR (untranslated region) of the VEGF mRNA, with a direct regulatory role in VEGF protein levels (Liu et al., 2014). Another study in chondrosarcoma found positive association between CCR5 activation and increased VEGF expression, although in this case through miR-199a downregulation (Liu et al., 2015a). It is not clear whether miR-199a directly targets the 30 UTR of VEGF mRNA, but the miR-199 family is known to regulate endothelial cell differentiation via silencing of JAG1 (Jagged-1), a Notch ligand that enhances VEGF expression and angiogenesis (Chen et al., 2015). These examples establish a strong link between CCR5 and miR in the control of the angiogenic switch, although the means and the mechanisms through which this regulation occurs appear to be cell context- and/or tumor type-dependent. The crosstalk between angiogenic chemokines and growth factors may be also indirect. CXCL12 controls the angiogenic switch in prostate cancer by regulating the expression and secretion of the enzyme phosphoglycerate kinase (PGK)-1 (Wang et al., 2007). PGK-1 is a cytosolic enzyme involved in ATP generation through the glycolytic pathway and is hence part of the metabolic reprograming from oxidative phosphorylation to glycolysis in tumors, the so-called Warburg effect (Hanahan and Weinberg, 2011). Nevertheless, PGK-1 is also secreted to the extracellular space, where it acts as a disulfide reductase, facilitating plasminogen cleavage to generate angiostatin (Lay et al., 2000). High PGK-1 levels in the extracellular space also downregulate production of proangiogenic factors such as CXCL8 and VEGF (Wang et al., 2007), thus inhibiting tumor neovascularization. CXCR4 activation in prostate cancer cells inhibits PGK-1 expression and secretion (Wang et al., 2007), which releases the PGK-1-imposed brake on tumor angiogenesis. The activity of angiostatic and angiogenic chemokines can be regulated posttranscriptionally by limited proteolysis at their N-terminus. There is evidence that the ERL motif determines the functional outcome of CXC chemokines after proteolytic cleavage. Proteolysis by cysteine cathepsins (cathepsin K, L, and S) generates more potent forms of ERL+ chemokines (CXCL1, CXCL3, CXCL5, and CXCL8), whereas it inactivates and in some cases degrades ERL chemokines (CXCL9, CXCL10, or CXCL12) (Repnik et al., 2015). The ERL sequence also protects chemokines from proteolysis by aminopeptidase N (CD13), a protease that cleaves and inactivates the ERL chemokine CXCL11 (Proost et al., 2007). Cleavage of ERL chemokines by CD26 (dipeptidyl-peptidase IV) generates potent antagonists (Proost et al., 2001), which indicates that proteolysis might be

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a general mechanism to shift the functional balance of pro- and antiangiogenic chemokines in the tumor milieu. The angiogenic switch has been explained as an adaptive mechanism to satisfy the oxygen and nutrient demands of exponentially growing cancer cells, which is paradoxical, as the blood vessels generated within tumors are generally abnormal and their function is poor (Carmeliet and Jain, 2011). Tumor-associated blood vessels are tortuous and enlarged, poorly perfused, with erratic blood flow, microhemorrhage, and leakiness. Blood vessel leakiness increases tumor interstitial pressure, which complicates interchange of gasses and substances, including therapeutic compounds, between the bloodstream and the tumor parenchyma (Goel et al., 2011). Many factors contribute to the abnormalization of tumor blood vessels, including dysregulation of the chemokine network. High CCL2 production by colon carcinoma cells activates JAK2/STAT5- and p38 MAPKdependent signaling pathways in endothelial cells to enhance vascular permeability and tumor cell extravasation (Wolf et al., 2012). In an apparent contradiction, CCL2 also recruits pericytes and other mural cells to forming blood vessels (Aplin et al., 2010), a process associated with vascular maturation (Carmeliet and Jain, 2011). Pericytes are major producers of angiostatic chemokines such as CXCL11 (Suyama et al., 2005), which links blood vessel maturation with reduced angiogenic potential. CXCL8 triggers endothelial permeability through a signaling cascade that activates the Rac1/PAK-1 (p21-activated kinase) pathway; PAK-1 then phosphorylates VE-cadherin, a critical protein in endothelial adherens junctions, which causes its endocytosis and thus, vessel permeability (Gavard et al., 2009). Since high levels of specific chemokines can lead to anomalous vasculature, drugs that normalize vasculature would be predicted to regulate chemokine expression. For instance, treatment of tumor-bearing mice with lovastatin, a mevalonate pathway inhibitor, improves vascular function, and downregulates the chemokines CCL22 and CCL17 (Mira et al., 2013). This downmodulation is associated with a reduction in the number of proangiogenic M2-like macrophages in the tumor environment, improved vascular function and delivery of chemotherapeutics, and increased T cell infiltration. Production of CCL5, CXCL9, and CXCL10 is also linked to eosinophil-induced normalization of tumor vasculature. Coinjection of eosinophils with tumor-specific T cells led to normalization of blood vessels in experimental melanoma tumors (Carretero et al., 2015). IFNγ and TNF-α produced by these tumor-specific T cells induce eosinophils to upregulate CCL5, CXCL9, and CXCL10 secretion; this attracts

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more CD8+ T cells that eradicate the tumor cells and collaborate in the angiostatic response (Carretero et al., 2015). Although chemokine dysregulation is integral to aberrant angiogenesis and vascular abnormalization, these two studies also show that targeting specific chemokines might be a therapeutic strategy for use against tumors.

4.2 Chemokines as Shapers of the Tumor Inflammatory Milieu Immune cells infiltrate most, if not all, solid tumors, although the precise role of these cells in tumorigenesis is not completely defined. In addition to the immune system surveillance function in eliminating pathogens that initiate and/or promote certain tumor types, accumulated evidence mainly in experimental models indicates dual activity of immune cells at different stages of malignancy. It is now clear that immune cells of both the innate and the adaptive immune systems can specifically identify cancer cells based on nonspecific “alarm” signals (innate cells) and on the specific expression of tumor antigens (adaptive cells), both of which are generated as a result of the genomic instability and somatic mutations intrinsic to neoplastic cells. Recognition of these signals can lead to elimination of transformed cells through a process referred to by Burnet and Thomas as cancer immunosurveillance (Burnet, 1970). The main adaptive and innate immune cells involved in tumor elimination are effector CD4+ (Th1) and CD8+ T lymphocytes, γδ T cells, natural killer (NK), and NKT cells, CD8+ DC, M1-skewed macrophages, and eosinophils (Vesely et al., 2011). The basic idea of immunosurveillance has now been extended by the theory of immunoediting (Dunn et al., 2002), in which the immune system not only protects the host against tumor onset, but can also select for tumor cells able to escape immune eradication. Tumor cell variants that resist the elimination phase are then able to evade the antineoplastic activity of the immune system and to hijack immune cells that boost tumor progression. The microenvironment of these “evasive” tumors is dominated by immunosuppressive cells [regulatory T lymphocytes (Treg), immature, and plasmacytoid DC (pDC), MDSC, and tumor-associated M2 macrophages (TAM)], which either suppress effector cell function directly or skew cells toward a Th2 phenotype that eradicates cancer cells less effectively (Vesely et al., 2011). Given the role of chemokines in homeostatic and inflammatory leucocyte trafficking into and between tissues and organs, it is not surprising that these proteins are central in shaping the tumor immune infiltrate (Bindea et al., 2014; Ma et al., 2014). The expression of specific chemokines

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is associated with the transforming and suppressive activities of oncogenes and tumor suppressor genes, respectively. The leukemogenic activity of the BCR/ABL oncogene is linked to CCL9 repression, with concomitant inhibition of CD3+ cell recruitment to neoplastic lesions (Iotti et al., 2007). TP53-mediated upregulation of CX3CL1 might suppress emergent tumors by recruiting NK cells and cytotoxic T lymphocytes (Xin et al., 2007; Yu et al., 2007). TP53 is also a negative regulator of NF-κB, a key transcription factor that fosters immunosuppressive environments. In ovarian (Son et al., 2012) and breast cancer cells (Mitkin et al., 2015), TP53 mediates upregulation of the E3 ubiquitin ligase Mdm2 (mouse double minute 2 homolog), which increases stability of IκB, an NF-κB inhibitor. Mutation of TP53 during oncogenesis hampers negative NF-κB regulation, and restoration of TP53 function in ovarian carcinoma cells harboring mutated TP53 negatively inhibits NF-κB-mediated expression of CXCL1, 2, 3, and 8, and infiltration of immunosuppressive CXCR1- and CXCR2expressing granulocytes and mastocytes. Moreover, oncogenic mutations of TP53 can generate gain-of-function transcription factors that increase expression of protumorigenic chemokines such as CXCL5 and CXCL8 (Yeudall et al., 2012). These data imply that mutation of oncogenes and tumor suppressor genes alters the chemokine expression pattern and reshapes the tumor environment from effective immune surveillance to an inflammatory context that aids cancer progression. The immune infiltrate is not only important at the initial stages of carcinogenesis, but also determines progression of the malignancy. Tumor infiltration by specific cell subtypes (CD8+ T lymphocytes, NK, and NKT cells) is associated with better prognosis, whereas massive infiltration by cells of myeloid origin (macrophages, MDSC) or Treg cells correlates with advanced stages of various tumor types (Draghiciu et al., 2015; Liu et al., 2015c; Sato et al., 2005). It is tempting to speculate that chemokines that predominate in the tumor environment establish the balance between anti- and protumor activities of the inflammatory infiltrate. There is nonetheless considerable overlap between the chemokine receptors expressed on effector and immunosuppressive cells (Zhou et al., 2014), which could explain the dual activity observed for most chemokines in modulating tumor immunity (Fig. 3). This is the case for CCR7 and its ligands CCL19 and CCL21. This chemokine trio is crucial for efficient induction of immune reactions by controlling entry of immune cells into secondary lymphoid organs and their positioning within defined functional compartments (Forster et al., 2008). CCR7 regulates entry of naı¨ve T cells, central memory T cells, Treg cells,

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Tumor progression CX3CL1, CXCL12 CXCL8, CCL2, CCL20, CCL25, CCL27

CXCL7, CXCL8 CXCL5, CXCL12 CCL2, CCL19/21

Tumor regression

Apoptosis

Survival

senescence

proliferation

CXCL12 CCL5, CXCL8

Tumor cells

Selfrenewal Immune surveillance

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M2 macrophage

CTL NK/NKT

MDSC

Metastasis CCL2, CCL5 CCL19/21 CCL22 CX3CL1

Immune evasion

CXCL12, CCL25 CCL19/21, CCL5

CXCL9/10/11 CCL2, CCL5 CCL17, CCL19/21 CX3CL1

M1 macrophage mDC CD4+ Th1 cell

Treg cell MSC

CXCL1/2/3/5/6/7/8 CXCL17, CXCL12 CCL2, CCL15/16 CCL17, CCL23

CCL3/4/5 CXCL12

CXCL9/10/11 CXCL4, CXCL14 CCL2, CCL5

iDC

CD4+/CD8+ T cell priming

Blood vessel

Angiogenesis

Vasculogenesis

Angiostasis

Fig. 3 The multifaceted and opposite activities of chemokines in carcinogenesis. The figure highlights direct and indirect chemokine effects on tumor and infiltrating cells, and their influence on various cancer processes. Some of the chemokines implicated in each process are also indicated, to highlight their dual activity. Chemokines can be produced initially by tumor cells, but tumor-infiltrating leukocytes and mesenchymal stem cells (MSC) supply an additional chemokine wave that can amplify or control their own pro- or antitumorigenic activities. CSC, cancer stem cells; MDSC, myeloid-derived suppressor cells; iDC, immature dendritic cell; mDC, mature dendritic cell; CTL, cytotoxic T lymphocyte; NK, natural killer cells; NKT, NK T cells.

and mDC into the LN; DC maturation involves CCR7 upregulation (as well as of MHC class II and CD80/86 costimulatory receptors) and repression of other chemokine receptors such as CCR1, CCR5, or CCR6. These genetic changes make mDC sensitive only to CCL19/CCL21 gradients, which enable correct DC and T cell positioning in the LN T cell zone in a CCR7-dependent manner (Forster et al., 2008). Capture of CCL21 and CCL19 on the DC surface also increases the basal mobility of T cells, which allows them to scan more DC, thus increasing the probability of engagement with the DC that present their cognate antigen (AspertiBoursin et al., 2007). CCL21 and CCL19 can stabilize contacts between

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APC and T cells during immune synapse formation, thus increasing T cell priming and costimulation (Friedman et al., 2006; Gollmer et al., 2009). CCR7 expression not only promotes T cell encounter with appropriate APC, but also extend APC longevity in LN by inhibiting apoptosis (Sanchez-Sanchez et al., 2004). The antiapopotic activity of CCR7 is also observed in tumor-specific T cells (Kim et al., 2005), and loss of CCR7 expression in fully differentiated effector CD8+ T cells is associated with reduced antitumor activity in adoptive cell transfer (ACT) protocols (Gattinoni et al., 2005). The evidence outlined above indicates that CCR7 and its ligands must be central to tumor immunity. In some experimental settings, CCL21 overexpression by tumor cells recruits tumor-specific T cells and improves immune-mediated elimination of cancer cells (Phan-Lai et al., 2014). In other systems, however, CCR7 activation is linked to immunosuppression. In mouse melanoma, tumors with the lowest CCL21 levels were smaller than those that expressed high or normal CCL21 levels, which showed increased Treg cell infiltration and formation of tertiary lymphoid structures (Shields et al., 2010). This indicates an inverse association between CCL21 levels and the antitumor activity of the immune system. CCR7 and its ligands are essential for the immunosuppressive function of Treg cells and the induction of peripheral tolerance (Forster et al., 2008), both implicated in the suppression of tumor immunity. In support of this conclusion, upregulation of CCL21 production combined with administration of an anti-CD25 antibody to deplete Treg cells was sufficient to generate potent antitumor responses (high IL-12 and IFNγ, low IL-10 and TGF-β) to mouse hepatocellular carcinoma cells, with increased cytotoxic cell recruitment to the tumor mass and prolonged mouse survival (Zhou et al., 2013). The chemokines CXCL9, CXCL10, and CXCL11 have also been associated with effective tumor immunity. Their receptor, CXCR3, is expressed in most effector cell types, such as Th1 CD4+ T cells, CD8+ cytotoxic lymphocytes (CTL), and NK cells, which are attracted to the tumor parenchyma by these chemokines (Hensbergen et al., 2005; Wendel et al., 2008). CXCR3 also cooperates with CCR5 (see below) to enhance tumor infiltration by cytotoxic T lymphocytes and IFNγ-producing CD4+ T cells, a phenomenon associated with reduced metastasis in colon and oesophageal cancers (Liu et al., 2015b; Zumwalt et al., 2015). Indirect evidence that CXCR3 deficiency leads to macrophage differentiation to the protumorigenic M2 phenotype (Oghumu et al., 2014) suggests that CXCR3-transduced signals can also polarize macrophages toward the

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tumoricidal M1 subtype. Since CXCL9-11 are inducible by type I and type II IFN (Viola et al., 2006), high levels of these chemokines could trigger infiltration of IFN-producing effector cells into a tumor and establish a positive feedback loop that would skew macrophages to the M1 phenotype. With the angiostatic properties of CXCL9-11 (see above), this explains the potent antitumor responses associated with their upregulation in distinct tumorigenesis models (reviewed in Ma et al., 2014). Upregulation of CXCL10 production by tumor-infiltrating macrophages is proposed as a necessary event for success of radiotherapy (Lim et al., 2014). Many tumors develop mechanisms to suppress expression of CXCR3 and/or its ligands, which indicates the importance of this chemokine hub in restraining tumor progression. Peng et al. showed epigenetic mediated silencing of CXCL9 and CXCL10 to be an effective immune evasion strategy (Peng et al., 2015). CXCL9/10 transcription is repressed through trimethylation of histone H3K27, mediated by the enhancer of zeste homologue (EZH)2, and through promoter methylation, mediated by DNA methyl transferase (DNMT)1. Inhibition of these epigenetic modulators increases effector cell infiltration into tumors and improves the effectiveness of immunotherapeutic regimes (Peng et al., 2015). An inverse correlation was also observed between EZH2 and DNMT1 levels and tumorinfiltrating CD8+ T cells in patients with renal carcinoma and was associated with patient outcome. Posttranslational cleavage of CXCL10 by dipeptidyl-peptidase 4 (DPP4/ CD26) is another mechanism used by tumors to reduce CXCR3-mediated trafficking of effector immune cells into the parenchyma (Barreira da Silva et al., 2015). Genetic ablation or pharmacological inhibition of DPP4 improves the effectiveness of a number of immunotherapeutic strategies, including checkpoint blockade, ACT, and adjuvant therapy, by increasing CD8+ T lymphocyte and NK cell infiltration (Barreira da Silva et al., 2015). The CXCR3 axis appears to have a dual role in tumor immunity, and some reports suggest that these chemokines might also foster an immunosuppressive tumor environment. CXCL10 and CXCL11 production by neuroendocrine cells in the gut triggers macrophage infiltration into colorectal tumors, which correlates with enhanced metastasis and poor prognosis (Zeng et al., 2016). CXCR3 is also found in a subset of CD4+ Treg cells that coexpress the transcription factors T-bet (specific to Th1 responses) and FoxP3 (Treg cell specific); in vivo, this unique Treg cell subset mediates suppression of Th1 responses (Koch et al., 2009). These CXCR3+ Treg cells

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were recently detected in human pancreatic tumors, and their presence was associated with poor patient survival (Lunardi et al., 2014, 2015). Another chemokine axis with dual antitumor activity is that of CCR4 and its ligands CCL17 and CCL22. CCR4 is a well-established marker of natural Treg cells; high CCL22 levels are thus associated with massive Treg cell infiltration and immunosuppression in human ovarian cancers and T cell leukemias (Curiel et al., 2004; Ishida et al., 2003). Hepatitis B virus downregulates miR-34a, which increases CCL22 levels in virus-associated hepatocellular carcinomas and promotes metastasis by recruiting Treg cells (Yang et al., 2012). CCR4-mediated Treg cell infiltration can also induce apoptosis of NK and other effector cells in the lung, thus favoring outgrowth of metastatic lesions (Olkhanud et al., 2009). IFNγ inhibition of CCL22 production and CCR4 blockade with specific antibodies enhance antitumor immunity by depleting Treg cells from the tumor (Anz et al., 2015; Chang et al., 2016). Transduction of melanoma, lung, and ovarian carcinoma cells with CCL22 can nonetheless induce tumor regression in an IL-4-, CD4+-, and CD8+ T cell-dependent manner (Guo et al., 2002; Okada et al., 2004). In mice in which these tumors regressed, CCL22 also induced long-term specific protection against the parental cell line (Okada et al., 2004), which concurs with CCR4 expression in memory T cells (Andrew et al., 2001). CCL17 is also involved in the alternative, NKT-mediated licensing of DC to activate CCR4+ cytotoxic T cells (Semmling et al., 2010). Crosspriming allows DC to induce cytotoxic CD8+ T cells, a process classically mediated by CD4+ helper cells (see below). Semmling and coworkers observed that interaction of activated NKT cells with spleen CD8α+ DC induces autocrine CCL17 production by DC; CCL17 then attracts CD8+ T cells and stabilizes their interaction with antigen-loaded DC, thus increasing CD8+ T cell priming (Semmling et al., 2010). This alternative crosspriming by CD1d-restricted NKT cells has been exploited in vaccines with tumor antigens to induce robust antitumor immune responses (Shimizu et al., 2013). CCL2 was initially isolated as a monocyte chemotactic and activating factor with tumor cytostatic activity (Matsushima et al., 1989). Consistent with an antitumor function, overexpression of CCL2 suppresses tumor formation in animal models by increasing tumor-infiltrating effector cells (Lanca et al., 2013; Li et al., 2013; Rollins and Sunday, 1991); high CCL2 levels in soft tissue sarcomas and endometrial cancers are also associated with good prognosis in humans (Kehlen et al., 2014; Pen˜a et al., 2015).

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Repression of CCL2 by amplification of the MYCN oncogene is also proposed as an immune escape strategy in glioblastoma, in which it reduces NKT infiltration to the tumor vicinity (Song et al., 2007). High CCL2 levels are nevertheless associated with poor prognosis in various cancers (Izumi et al., 2013; Li et al., 2013), and this chemokine is directly linked to the immunosuppressive program elicited by MDSC and TAM (Schmall et al., 2015; Stewart et al., 2009). In these cases, CCL2 or CCR2 blockade results in reduced infiltration of monocyte-derived cells, reversal of immunosuppressive status, and activation of tumoricidal effector cells (Chun et al., 2015; Li et al., 2013; Loberg et al., 2007). This preclinical evidence prompted the initiation of clinical trials with a monoclonal antibody (carlumab, CNTO 888) that targets CCL2; although this antibody can delay tumor progression in some patients, disease rebound increases metastasis (De Sanctis et al., 2014). Nitrosylation of CCL2 in some tumor conditions might reconcile the dual activity of this chemokine, since nitrated CCL2 no longer acts as a chemoattractant for CD8+ T cells, but remains able to induce MDSC infiltration into tumors (Molon et al., 2011). CCR5 and its ligands (CCL3, CCL4, and CCL5) merit special mention, since this axis probably constitutes the prime example of dual chemokine activity in tumor immunity. CCR5 is expressed by various effector cell subsets, including CD4+ Th1 cells, cytotoxic (CD8+) T cells, effector memory T cells, NK and NKT cells, and by immunosuppressive cells such as Treg cells, monocytes, macrophages, and immature DC (Gonzalez-Martin et al., 2012a). Indeed, the ability of CCR5 ligands to recruit suppressive or effector cells into tumors has been linked to pro- or antitumorigenic activities. The CCR5 antagonist met-RANTES (methylated CCL5) inhibits mouse breast tumor growth by reducing the number of TAM, which implicates CCR5 in monocyte infiltration and tumor progression (Robinson et al., 2003). The CCR5 inhibitor TAK-779 also reduces Treg cell infiltration and growth of pancreatic tumors (Tan et al., 2009), although a link between CCR5 and Treg cell infiltration has not been observed in other cases (Ward et al., 2015). In contrast to these reports, dozens of carcinogenesis models in mice have associated CCR5 and its ligands to host protective responses through recruitment of innate and adaptive effector cells (Gonzalez-Martin et al., 2012b; Lavergne et al., 2004; Mule et al., 1996; Nesbeth et al., 2010; Uekusa et al., 2002). Epidemiological studies in breast and colon cancer also indicate a tendency to reduced numbers of tumor-infiltrating lymphocytes (TIL) in patients with low CCR5 expression (Man˜es et al., 2003; Zimmermann et al., 2010). Finally, CCR5 not only

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recruits TIL into the tumor but retains them at the tumor site via a CD103 (integrin αEβ7)-mediated mechanism (Franciszkiewicz et al., 2009). CCR5 is also a central regulator of T cell priming and activation. CCR5 acts as a direct costimulatory receptor in CD4+ T cells by providing a stop signal that enhances their antigen-dependent interaction with APC (Molon et al., 2005). CCR5 accumulates at the synapse formed between the T cell and the APC in a Gαi-independent but Gαq/11-dependent manner. As a result of this costimulatory activity, CCR5 in primed T cells increases expression of IL-2, CD25 (the IL-2 receptor subunit), and transactivation of the transcription factors NFAT (nuclear factor of activated T cells) and STAT5 (Camargo et al., 2009). CCR5 costimulatory activity on CD4+ T cells requires its ability to form heterodimers with CXCR4; indeed, CXCR4 and CCR5 homodimers do not costimulate CD4+ T cells, although they are entirely able to transduce chemotactic responses (Contento et al., 2008). In addition, CXCR4 interacts with the TCR CD3ξ and CD3ε subunits (Kumar et al., 2006) and stabilizes T cell-APC contacts through integrins and specific APs (Cascio et al., 2015; Smith et al., 2013). These results indicate that the costimulatory activity of the CCR5/CXCR4 heterodimer is a result of direct and indirect interactions with the TCR signaling machinery during activation. CCR5 activity is also important for optimal CD8+ T cell priming. Effective CTL responses to tumor antigens entail CD4+ T cell cooperation (Melief, 2008). This CD4+/CD8+ cooperation involves recognition of antigens copresented by the same APC, although the time frame in which these CD4+/APC/CD8+ interactions occur is debated. The “three cell model” proposes that CD4+ and CD8+ T cells interact simultaneously with the same APC, whereas the “kinetic model” suggests that the initial CD4+ T cell interaction with APC “licenses” the latter for subsequent activation of the CD8+ T cell (Hugues et al., 2007). CCR5 has been associated with both of these models. Nesbeth et al. showed that CCL5 produced by tumorinfiltrating CD4+ T cells steers CCR5+ DC for in situ CD40-induced licensing (Nesbeth et al., 2010). In contrast, an intravital microscopy study showed that CCL3 and CCL4 produced by CD4+/APC complexes guide naı¨ve CD8+ T cells to these complexes to form trimeric CD4+/APC/CD8+ cell clusters to activate CD8+ T cells (Castellino et al., 2006). These interactions influence the generation of protective immunity against tumors, since maximal antitumor responses are achieved only when both CD4+ and CD8+ T cells express CCR5 (Gonzalez-Martin et al., 2011, 2012b). CCR5 activation in CD4+ T cells increases levels of CD40 ligand

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(CD40L), which augments expression of costimulatory receptors on the APC and maximizes CD8+ T cell activation. CCR5 also recruits naı¨ve CD8+ T cells to complexes composed of antigen-specific CD8+ T cells and mature APC; this helps to prime unrelated polyclonal CD8+ T cells in settings of low antigen frequency (Hugues et al., 2007). Whereas CCR5-mediated signaling primes antigen-specific T cells for activation, it can also transduce apoptotic signals to the activated cells. Melanoma cell-secreted CXCL12 induces CCL5 production by tumorexperienced TIL; this CCL5 causes CCR5+-dependent CD8+ T cell death via a pathway involving cytochrome c release and activation of caspases 9 and 3 (Mellado et al., 2001a). The ability of CCL5 to induce direct T cell apoptosis depends on its oligomerization after binding to GAG on the cell surface (Murooka et al., 2006). CCR5-mediated CD8+ T cell apoptosis can also be the result of upregulated TGF-β production by tumor-infiltrating Treg cells (Chang et al., 2012). CCR5 and its ligands can eliminate cytotoxic T cells by direct and indirect mechanisms, and thus contribute to local tumor immunoprivilege.

5. CONCLUDING REMARKS The evidence presented in these pages indicates a multifactorial role for chemokines and their receptors in carcinogenesis. The chemokines not only shape the immune infiltrate of tumors, but might also act directly on neoplastic, fibroblast, or endothelial cells to regulate critical processes in tumor development such as cancer cell proliferation, apoptosis, tumor suppressor function, CSC stemness and transdifferentiation, metastasis, and angiogenesis. Given the metabolic changes observed in immune cells after chemokine receptor activation, we can anticipate that studies in the near future will implicate chemokines in the metabolic reprograming of cancer cells. Despite the multifunctional nature of these molecules, the use of chemokines/receptors as targets for cancer therapy is hampered by their dual roles in carcinogenesis. As we have seen throughout this review, the same chemokine can have context-dependent tumor-promoting and tumorsuppressive abilities. This Jekyll and Hyde behavior probably indicates that chemokines are just one part of the complex inter- and intracellular signal communication networks that operate in the tumor milieu. Further studies are therefore warranted to define the still-unexplored areas of chemokine interaction with other mediators, although a humanized anti-CCR4 antibody (mogamulizumab) is in phase II/III clinical trials for several T cell

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malignancies (Vela et al., 2015), and maraviroc (a CCR5 antagonist approved for treatment of HIV-1-infected patients) as well as plerixafor (AMD-3100, a CXCR4 antagonist approved for mobilization of hematopoietic progenitors from bone marrow) are in clinical trials for advanced colon, ovarian, or pancreatic cancers (Halama et al., 2016; https:// clinicaltrials.gov/ct2/show/NCT02179970). Another characteristic of the chemokine network that hinders clinical application is its intrinsic redundancy, which is found at several levels. For instance, tumor infiltration by Treg cells can be driven directly at least by CCR4, CCR5, CCR6, CCR7, and CXCR3 (Curiel et al., 2004; Liu et al., 2011a; Lunardi et al., 2015; Shields et al., 2010; Tan et al., 2009), which would preclude inhibition of this process through blockade of a single receptor. Nonetheless, redundancy is not only functional, but is also observed at the molecular level, as different ligands bind to the same receptor and different receptors bind the same ligand. This is further complicated by the ability of chemokines and receptors to homo- and heterooligomerize, which can modify the receptor/ligand-binding pattern as well as the signaling pathways initiated by these oligomers. Indeed, we have begun to recognize that chemokine receptors do not simply switch between on and off states, but also can adopt a number of intermediate conformations. Recent evidence discussed here indicates the association of distinct receptor conformations with the activation of specific signaling pathways. The molecular factors that endow chemokine receptors with this functional selectivity that allows them to activate certain signaling pathways over others are not known. The next few years should bring new information on the molecular cues that create bias in chemokine signaling, such as structural data on the interaction of a given receptor with its distinct agonists, the effect of receptor/ligand oligomerization in signaling selectivity, and the influence of membrane composition on binding kinetics or receptor conformation. These analyses must be performed in relevant cells, since coexpressed receptors can modulate the response of a given chemokine receptor in a specific biological process. To provide meaningful information, however, these analyses must be carried out in functionally specific cells, since other cellular receptors that are also coexpressed can modulate the responses of given individual chemokine receptors in a specific biological process. Understanding signaling bias in the chemokine network could provide a conceptual framework for the design of new inhibitors or agonists that can selectively target chemokine activities in cancer.

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ACKNOWLEDGMENTS We thank J. Ogando and J. Santos for discussions and C. Mark for editorial assistance. Given the size of the field and the depth of its literature, we must apologize to those authors whose work could not be cited in this review. L.C.-.R and A.M.-L. are recipients of the Formacio´n del Personal Universitario predoctoral fellowships from the Spanish Ministry of Education. This work was supported in part by the Spanish Ministry of Economy and Competitiveness (SAF2014-54475-R), the Comunidad de Madrid (INMUNOTHERCAN, S2010/BMD2326), and the Domingo Martı´nez Foundation.

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