Context‐dependent role of IL‐18 in cancer biology and counter‐regulation by IL‐18BP

IL‐18 is a proinflammatory and immune regulatory cytokine, member of the IL‐1 family. IL‐18 was initially identified as an IFN‐γ‐inducing factor in T and NK cells, involved in Th1 responses. IL‐18 is produced as an inactive precursor (pro‐IL‐18) that is enzymatically processed into a mature form by Casp1. Different cells, such as macrophages, DCs, microglial cells, synovial fibroblasts, and epithelial cells, express pro‐IL‐18, and the production of bioactive IL‐18 is mainly regulated at the processing level. PAMP or DAMP molecules activate inflammasomes, which trigger Casp1 activation and IL‐18 conversion. The natural inhibitor IL‐18BP, whose production is enhanced by IFN‐γ and IL‐27, further regulates IL‐18 activity in the extracellular environment. Inflammasomes and IL‐18 represent double‐edged swords in cancer, as their activation may promote tumor development and progression or oppositely, enhance anti‐tumor immunity and limit tumor growth. IL‐18 has shown anti‐tumor activity in different preclinical models of cancer immunotherapy through the activation of NK and/or T cell responses and has been tested in clinical studies in cancer patients. However, the dual role of IL‐18 in different experimental tumor models and human cancers raises critical issues on its therapeutic use in cancer. This review will summarize the biology of the IL‐18/IL‐18R/IL‐18BP system and will address the role of IL‐18 and its inhibitor, IL‐18BP, in cancer biology and immunotherapy.


IL-18 BIOLOGY
IL-18 is a proinflammatory and immune regulatory cytokine, which belongs to the IL-1 family [1]. The IL-18 gene was cloned on the basis of the ability of the encoded protein to promote IFN-g production by T and NK cells and Th1 differentiation and was initially defined a IFN-g-inducing factor [2]. IL-18 is produced as an inactive precursor protein of 192 aa (pro-IL-18) that is converted into a mature form of 157 aa by Casp1-mediated cleavage of an N-terminal fragment (Fig. 1A). IL-18 is constitutively expressed by several cell types, including macrophages, DCs, microglial cells, synovial fibroblasts, and epithelial cells. Therefore, different from IL-1b, which is tightly controlled also at the transcription level, the IL-18 biologic activity is mainly regulated by its enzymatic processing [3]. PAMPs (e.g., muramyl dipeptide, bacterial RNA, and viral dsRNA) or DAMPs (e.g., extracellular ATP and uric acid crystals) activate protein complexes, known as inflammasomes, which mediate Casp1 activation and IL-18 processing [4,5]. The most studied inflammasome, Nlrp3, activates Casp1 through the adapter protein ASC, upon triggering by different PAMPs, DAMPs, or some adjuvants. Several studies indicate that inflammasomes represent a double-edged sword in cancer, as their activation may have tumor-promoting effects or on the other hand, immuneenhancing and anti-tumor activity [4].
Once released from cells, mature IL-18 binds to a heterodimer receptor complex, consisting of IL-18Ra and b-chains. These chains are members of the IL-1R family, characterized by a TIR domain in their intracellular portion [6]. IL-18 binding to the IL-18Ra recruits the IL-18Rb chain and initiates signal transduction through the adaptor protein MyD88, which binds to the TIR domain. IRAK is then phosphorylated and recruits TRAF-6, which initiates the signaling pathways leading to the activation of the transcription factors NF-kB and AP-1 (Fig. 1A). However, different from IL-1, IL-18 signal transduction primarily involves the MAPK p38 pathway, rather than NF-kB, in human epithelial cells [7]. Therefore, IL-18 does not induce COX-2 and fever, which are typical NF-kB-dependent, IL-1-mediated effects.
Mature IL-18 is produced by different cell types in response to pathogens and activates host defense mechanisms [8][9][10][11]. Indeed, IL-18 augments antimicrobial properties of phagocytes through the up-regulation of reactive oxygen intermediate synthesis, NO production, and cytokine release. IL-18 potentiates the clearance of intracellular bacteria, fungi, and protozoa, and Il18 2/2 mice show increased susceptibility to several infections [12]. The clearance of different viruses is impaired in IL-18 2/2 Abbreviations: 2/2 = deficient mouse, ADAM-33 = a disintegrin and metalloproteinase domain-containing protein 33, ADCC = antibodydependent cellular cytotoxicity, ASC = apoptosis-associated speck-like protein containing a caspase recruitment domain, Casp1 = caspase -1, COX-2 = cyclooxygenase-2, DAMP = damage-associated molecular pattern, DC = dendritic cell, FasL = Fas ligand, HPV = human papillomavirus, (continued on next page) mice. Indeed, IL-18 mediates antiviral functions through the induction of NK [13] or CTL responses [14]. Repeated administrations of IL-18 restore the impaired immune response in mice with severe burn injury or splenectomy and improve survival after bacterial infections [11]. IL-18 acts in concert with other cytokines to modulate immune system functions. Optimal induction of IFN-g production, Th1 responses, and NK cell activation in response to pathogen products requires IL-18 and IL-12, which cooperate in these activities, as indicated by the study of Il18 2/2 , Il12 2/2 , and double-KO mice [15]. IL-18 and IL-12 cooperate for the induction of IFN-g, also in human NK cells. In addition, production of TNF-a, GM-CSF, and the chemokines CCL3 and CCL4 by human NK cells requires the cooperation of IL-18 with IL-15 or IL-12, which induce expression of the IL-18Rb chain, necessary for IL-18 signal transduction in target cells [16].
Importantly, IL-18-primed human NK cells develop a distinct helper differentiation phenotype (CD83 + CCR7 + CD25 + ) and acquire the ability to migrate to secondary lymphoid tissues in response to chemokines, such as CCL21 [17] (Fig. 1B). Helper NK cells show reduced cytotoxic functions but efficiently release IFN-g in response to DCs or Th-deriving signals, such as IFN-a, IL-12, IL-15, or IL-2. They may represent a link between innate and adaptive immunity, as they stimulate DCs to release IL-12, which induces Th1 responses, and chemokines, such as CXCL9, CXCL10, and CCL5 attracting CD8 + T cells [18]. Indeed, helper NK cells have been shown to prime tumor-specific Th1 and CTL responses via IFN-g and TNF-a DC activation [19].
In addition, a recent study showed that a subset of M-CSFactivated human macrophages expresses a membrane form of processed IL-18, which is released in a soluble form upon LPS stimulation. This soluble IL-18 induces the expression of CCR7 and the production of IFN-g in resting NK cells [20]. A similar membrane-bound form of IL-18 is also present in tumorassociated macrophages [21].
Further studies indicate a role of IL-18 also in Th2 and Th17 responses, in relationship to the different cytokine milieu. In the absence of IL-15 or IL-12, IL-18 induces naive T cells to polarize into Th2 cells and mediates IL-13 and/or IL-4 production also by NK cells, mast cells, and basophils [22][23][24]. In mouse basophils, IL-18-mediated Th2 cytokine production is dependent on MyD88 and MAPK-p38a signaling [24]. Therefore, IL-18 may play a role in allergic diseases, including asthma [25]. The combination of IL-18 or IL-1 with IL-23 stimulates IL-17 production by CD4 + or TCRab + T cells in the absence of TCR engagement, suggesting a role, not only for IL-1 but also for IL-18 in Th17 responses [26].
IL-18 may also have pathogenic effects in autoimmune disorders. For example, processing of IL-18 and IL-1 and the consequent IL-17 production is essential in experimental autoimmune encephalomyelitis induced by DCs primed with Mycobacterium tuberculosis and myelin antigen [27]. In fact, the administration of a Casp1 inhibitor reduces disease severity in this model. Several other studies indicate a role for IL-18 in human autoimmune disorders, such as lupus erythematosus [28,29], arthritis [30], and Crohn's disease [31]. Altogether, these data suggest that IL-18blocking agents represent novel, potential therapeutic approaches in inflammatory disorders or autoimmunity.
A natural inhibitor of IL-18 biologic activity is IL-18BP, which has high affinity for mature IL-18 and blocks its interaction with  (Pro-IL-18), which is processed by Casp1 in the mature form (Mat-IL-18). DAMPs or PAMPs activate the inflammasome, which triggers Casp1 activation. Mature IL-18 binds to IL-18Ra, which recruits the b chain to the complex. The recruitment of MyD88 and IRAK initiates signal transduction through TRAF6. (B) IL-18 primes human NK (hNK) cells to acquire CCR7 expression and migratory capacity to secondary lymphoid organs. In this environment, cytokines produced by DCs or Th cells activate "helper" functions of IL-18-primed NK cells, consisting of high IFN-g and TNF-a production, which increases DC activation. In turn, DCs express higher IL-12 and release chemokines attracting T cells. IFN-g, produced by NK cells and DC-released IL-12, promotes Th1 polarization and CTL responses. the IL-18Ra, thus preventing receptor dimerization. Therefore IL-18BP is considered a therapeutic tool to limit IL-18-based inflammation [32]. IL-18BP is a member of the Ig superfamily [33]. Alternative splicing of the IL18BP transcript leads to the generation of different isoforms, among which, IL-18BPa is the most widely expressed [34]. High levels of IL-18BPa in IL18BPtransgenic mice limit IL-18-mediated tissue injuries in response to different inflammatory stimuli, such as bacterial endotoxin or Con A [35]. IL-18BP accumulation may inhibit IL-18-mediated responses also in humans, as in the case of renal failure, where high levels of IL-18BP contribute to the defective immune response [36]. IL-18BP is constitutively produced by monocytes and macrophages and is present in the systemic circulation of healthy donors in molar excess relative to IL-18 [37]. However, IFN-g up-regulates IL-18BP expression in myeloid cells [38] and induces de novo expression in nonleukocytic cells, such as normal keratinocytes and mesangial cells [39]. Therefore, IL-18BP secretion is part of a negative-feedback loop, which proceeds from IL-18-triggered IFN-g production to IFN-ginduced IL18BP gene expression to prevent exaggerated Th1 responses. STAT1 signaling induced by IFN-g and STAT1 sites in the IL18BP promoter region are crucial for IL18BP expression [40] (Fig. 2). Recent data showed that another STAT1-activating cytokine, IL-27, induces IL-18BPa expression in human keratinocytes in vitro [41]. This finding suggests a possible antiinflammatory role of IL-27-induced IL-18BP in skin diseases, such as psoriasis, where keratinocyte-produced IL-18 may play a pathogenic role.
In view of its Th1-enhancing properties, IL-18 has been considered as a molecule with potential anticancer activity.
However, as a result of the complexity of IL-18 biologic functions, which are dependent on the context (e.g., cytokine milieu, different tissues, and counter-regulation by IL-18BP), IL-18 may play divergent roles in cancer [42,43]. We will review the anticancer and the tumor-promoting activities of IL-18 in experimental tumor models and human tumors.

ANTI-CANCER EFFECTS OF IL-18
Protective effects of IL-18 have been reported in different murine models of carcinogenesis (see Table 1). PAMP-mediated activation of the inflammasomes may be relevant during carcinogenesis in pathogen-associated cancers, such as gastric cancer. Indeed, Casp1 is activated, and IL-1b and IL-18 are processed as a consequence of Helicobacter pylori infection. Studies in Il1R 2/2 and Il18 2/2 mouse strains indicate that IL-18 counteracts the proinflammatory effects of IL-1b and limits IL-1-mediated gastritis [44]. Indeed, Il1R 2/2 mice infected with Helicobacter show a limited control of the colonization level but develop less gastritis and gastric pre-neoplastic lesions as a result of reduced IFN-g-and Th17-mediated inflammatory responses. Oppositely, Il18 2/2 mice show reduced Helicobacter colonization, a modest defect in IFN-g but strongly enhanced mucosal production of IL-17, and rapidly progressive gastric immunopathology. Therefore, activation of Casp1 by Helicobacter triggers IL-1-mediated Th17 responses that limit bacterial growth but induce gastritis. These effects are counterbalanced by IL-18 that limits Th17-mediated gastric immunopathology and prevents the onset of gastric cancer.
Another study showed that upon in vitro or in vivo exposure to Helicobacter, mouse DCs produce IL-18 and mediate the conversion of naive CD4 + T cells in CD4 + CD25 + Forkhead box P3 + regulatory T cells, endowed with immune-suppressive properties. IL-18, produced by DCs, takes part in this process, which limits T cell-driven gastric immunopathology. Indeed, depletion of DCs in newborn mice infected with H. pylori limits the infection but also worsens T cell-driven gastric immunopathology [45]. Similar to mouse models, also in humans, increased levels of IL-18 expression are found in gastric mucosa during H. pylori infection, where IL-18 may contribute to Th1 responses [46].
IL-18 has a protective role also in mouse models of inflammation-driven colon carcinogenesis induced by azoxymethane and dextran sulfate [47]. Myd88-KO mice, which have defects in IL-1b and IL-18 systems, show increased colorectal tumor development [48]. Il1R 2/2 mice show no increase in colorectal tumorigenesis, whereas Il18 2/2 and Il18R 2/2 mice are highly susceptible to colitis and colorectal cancer development. These findings suggest a protective role of IL-18 but not of IL-1 in colorectal cancer development [48]. Another report shows that Casp1 2/2 , Asc 2/2 , and Nlrp3 2/2 mice are more susceptible to experimental colitis and colitis-related carcinogenesis. At early stages of tumorigenesis, their colon tissue shows reduced IL-18 production, increased macrophage infiltration, and COX-2 expression and higher numbers of proliferating epithelial cells in the dysplastic regions. In addition, Nlrp3 2/2 and Casp1 2/2 mice show reduced IFN-g mRNA and protein expression and STAT1 signaling in their colon tissues during early phases of tumorigenesis, suggesting a role of IFN-g and STAT1 as mediators of IL-18 anti-tumor effects. Indeed, IL-18 administration to Casp1 2/2 mice reduces the signs of colitis and epithelial cell dysplasia and restores pSTAT1 [49]. Altogether, these data support the concept that IL-18 has a protective role at early stages of the colorectal carcinogenesis.
In view of its immune-enhancing properties, IL-18 has been investigated for anti-tumor activity in preclinical models ( Table 1). Administration of rIL-18 mediated melanoma or sarcoma regression in syngeneic mice through the activation of CD4 + T and/or NK cell-mediated responses [50,51]. Several other reports indicate that administration of rIL-18 or Il18 gene transfer has anti-tumor effects in different experimental models (reviewed in ref. [61]). In some of these studies, the IL-18 anti-tumor effects required IFN-g and involved antiangiogenic mechanisms [52,53]. The inhibition of angiogenesis is important for the anti-tumor effect of IL-18 and is mediated by IFN-g-dependent induction of the antiangiogenic chemokines CXCL9 and CXCL10 and downregulation of angiogenin expression [53].
The combination of IL-18 with other cytokines, such as IL-12 [54] or costimulatory molecules (e.g., CD80) [38], increases the IL-18-mediated anti-tumor effects. For example, Il18 and Il12A/B or CD80 genes have been integrated successfully in the genome of oncolytic viruses, with the aim to trigger synergistically T cellmediated anti-tumor immune responses [55,62]. IL-2/IL-18 fusion proteins also display enhanced anti-tumor properties relative to either cytokine alone and low toxicity in preclinical models [63].
IL-18 also demonstrated potent adjuvant activity in combination with a variety of cell-based or molecularly defined anticancer vaccines. For instance, a lung cancer cell vaccine, genetically modified to coexpress GM-CSF and IL-18, inhibits tumor growth and increases survival of mice bearing LL/2 tumors [56]. Administration of DNA plasmid vaccines encoding the human tumor antigen MUC1 and mouse IL-18 is an effective treatment for pulmonary metastases in MUC1-transgenic mice, whereas MUC1-or IL-18-encoding plasmids alone have no effect. CD8 + T cells and IFN-g mediate the anti-tumor immunity in this model [57]. An attenuated strain of Salmonella typhimurium, engineered to express IL-18, inhibits the growth of s.c. tumors or pulmonary metastases in syngeneic mice by systemic administration without toxic effects. This treatment induces accumulation of T and NK cells and granulocytes in tumors and intratumor production of cytokines [58].
IL-18 may be an important factor for NK-based cell therapies. Mouse NK cells, preactivated in vitro with a combination of IL-12, IL-15, and IL-18, persist with sustained effector function in vivo when transferred into syngeneic mice. These cytokine-stimulated NK cells display "memory-like" features, as they produce more IFN-g than naïve NK cells when restimulated in vitro with cytokines or antibodies engaging NKRs [64]. A subsequent study shows that the adoptive transfer of IL-12/15/18-activated NK cells, combined with irradiation, inhibits the growth of established mouse tumors through mechanisms requiring host CD4 + T cells [59].
The finding that human IL-12/15/18-preactivated NK cells also display memory-like features and secrete more IFN-g in response to cytokines or to K562 cells in vitro suggests possible applications in human cancer therapy [65]. Human IL-12/15/ 18-preactivated NK cells display increased IL-2Ra (CD25) expression, which allows the formation of high-affinity trimeric IL-2Rs. Therefore, IL-12/15/18-preactivated NK cells respond to picomolar concentrations of IL-2 with increased proliferation, survival, and cytotoxicity. This finding provides a rationale for IL-12/15/18 NK-adoptive cell-therapy strategies combined with low-dose IL-2 [66]. Another report proposes the use of IL-18containing combinatorial cytokine adjuvants to induce the intranodal helper NK functions, which activate DCs to recruit and activate T cells [67].
A recent study examined the effects of IL-18 on NK cell function mediated through FcgRs. IL-18 augments IFN-g production and ADCC of NK cells mediated by anti-CD20 antibody (Rituximab) against Burkitt lymphoma cells [60]. Moreover, IL-18 and Rituximab cooperate in mediating regression of human lymphoma xenografts in immune-deficient mice. Altogether, these studies indicate that IL-18 activates NK or T cell-mediated antitumor immune responses in different preclinical tumor models.
It has been proposed that endogenous IL-18 may have antitumor properties in some human cancers. For example, the normal colon and ovarian epithelia are capable to process and release mature IL-18, which is involved in local immunity to pathogens. However, during neoplastic transformation, colon or ovarian cancer cells may develop mechanisms that limit the potential Th1/CTL-mediated anti-tumor immunity triggered by IL-18. Indeed, colon and ovarian cancer cells are unable to process IL-18 and express only pro-IL-18 as a result of defective Casp1 expression or activation [68][69][70]. Moreover, pro-IL-18, present in some ovarian cancer cells, is partially resistant to in vitro digestion with Casp1 as a result of the presence of a Casp1-resistant splice variant of pro-IL-18 [71]. Immune-reactive IL-18 is present at very high levels in ovarian cancer ascites and is also elevated in the patient sera. However, biochemical analyses of ascites show the predominance of the 23-kDa pro-IL-18, whereas the 18-kDa mature form is undetectable. In agreement with this finding, IFN-g is not increased in patients' sera and is undetectable in most ascites [72]. Altogether, these data suggest that the loss of the capacity to process IL-18 during neoplastic transformation may represent a potential mechanism of escape from Th1/IFN-gmediated responses, at least in the colon and ovary.
Another potential mechanism limiting IL-18 activity in some cancers is related to the increased expression of its natural inhibitor IL-18BP. In prostatic cancer, urinary and serum IL-18BP levels are increased, and serum IL-18BP levels correlate with the Gleason score. IFN-g up-regulates IL-18BP production by prostate cancer cells in vitro, and costimulation with other cytokines, such as TNF-a or IFN-a, increases IL-18BP production further [73]. Furthermore, in ovarian cancer, IL-18BP is increased in sera and even more in the ascites. This is likely related to local production, as tumor-associated myeloid cells and cancer cells show IL-18BP expression in vivo by immunohistochemistry [74]. Several findings suggest that different factors present in the microenvironment mediate IL-18BP expression in the tumor cells. Indeed, ovarian cancer cell lines do not express IL-18BP in vitro, unless treated with IFN-g or IL-27, and IL-27 is a heterodimer cytokine-member of the IL-12 family-that mediates pSTAT1 in ovarian cancer cells. A potential role of IL-27 in vivo is suggested by the expression of the 2 IL-27 chains (IL-27A and EBV-induced 3) in ovarian cancer-associated leukocytes [74]. Prostate or ovarian cancer-derived IL-18BP is functional and inhibits IL-18 biologic activity in vitro. Altogether, these findings suggest that IL-18BP, produced in the microenvironment of ovarian and prostate tumors, may limit the activity of therapeutic IL-18 and represent a potential mechanism of tumor escape from Th1 responses by inhibiting endogenous IL-18, eventually present (Fig. 2). IL-18BP may then contribute to other known mechanisms of immune regulation at the tumor site, such as the induction of PD-L1 or of the immune-suppressive enzyme IDO in tumor cells, which can be driven by IFN-g [75], or alternatively, by IL-27 in ovarian cancer [unpublished results].
Based on the efficacy of IL-18 in preclinical studies of cancer immunotherapy, clinical trials of IL-18 have been conducted ( Table 2). Phase I clinical studies of IL-18 in advanced solid tumors and lymphomas showed limited toxicity and evidence of immune modulatory activity and identified a bioactive dose range [76,77]. IL-18 administration had biologic effects on the immune system, as indicated by the increase in plasma concentrations of the proinflammatory cytokines IFN-g and GM-CSF, soluble FasL, and IL-18BP. In addition, IL-18 induced a transient lymphopenia, reaching a maximum at 2 h from infusion. The reduction in lymphocyte counts was more evident for NK cells than for CD8 + and CD4 + T cells, and the remaining NK and T cells showed increased expression of FasL and the activation marker CD69. In addition, these studies showed some clinical activity and disease stabilization in a fraction of patients.
A Phase II study of IL-18 was performed in untreated Stage IV melanoma [78]. Patients were randomized to different groups receiving different dose levels of i.v. IL-18 for 5 days, repeated every 28 days. Among 63 evaluable patients, a partial response and 4 disease stabilizations, lasting for 6 months or more, were observed. It was concluded that rIL-18 was well tolerated but had limited activity in metastatic melanoma as a single agent.
The in vitro and in vivo synergy of IL-18 and anti-CD20 mAb in a preclinical study [60] led to the design of a Phase I study of IL-18 and Rituximab in patients with B cell lymphoma. IL-18 administration was followed by an increase in plasma proinflammatory cytokines, CXC chemokines (CXCL9 and CXCL10), and the CC chemokine, CCL2. The IL-18-induced lymphopenia, recorded also in other studies [77] is possibly a result of lymphocyte activation and their subsequent extravasation into tissues. Indeed, an increased tumor infiltration of CD69 +activated lymphocytes was recorded in a patient with mantle cell lymphoma [79]. Objective tumor responses were seen in 5 patients out of 18 treated, including 2 complete and 3 partial responses. It was concluded that further studies of human rIL-18 and anti-CD20 mAb in B cell malignancies are warranted [79].
A Phase I dose-escalation study of i.v. IL-18, in combination with pegylated liposomal doxorubicin (Doxil) in recurrent epithelial ovarian cancer, has been concluded recently (NCT00659178). rIL-18, in combination with Doxil, is safe and mediates biologic responses. One out of 10 evaluable patients showed a partial response and approximately one-third had stable disease. The authors concluded that this combination therapy is feasible and deserves further evaluation in a Phase II trial [80].
Collectively, clinical trials show that IL-18 has low toxicity in humans but limited therapeutic effects as a single agent. Nonetheless, IL-18 may be incorporated as an immuneenhancing molecule in combinational therapies with other agents (e.g., mAb, cytotoxic drugs, or vaccines).
In this respect, a Phase I study of IL-18, in combination with the anti-CD20 antibody ofatumumab, after autologous peripheral blood stem cell transplantation for lymphoma, is ongoing (NCT01768338). Moreover, a Phase I study of cyclophosphamide/ fludarabine lymphodepletion, followed by adoptive transfer of vaccine-primed peripheral blood autologous T cells and IL-18 treatment, will be conducted in patients with recurrent ovarian, fallopian-tube, or primary peritoneal cancer, who were vaccinated previously with a whole-tumor vaccine (NCT02277392).

TUMOR-PROMOTING ACTIVITIES OF IL-18
Although the preclinical studies and some clinical trials suggest that IL-18 has anti-tumor activities, other studies indicate that IL-18 has a dual role in tumors, as it may exert proinvasive, To assess the safety and biologic activity in patients with recurrent cancer, who previously underwent vaccination with whole tumor vaccine proangiogenic, and immune-regulatory activities in different tumor models (Table 3) [42,43]. In spite of its protective effect in initial stages of H. pylori infection, IL-18 may support tumor progression in advanced gastric cancer. Indeed, it stimulates the production of the proangiogenic factor, thrombospondin-1, in IL-18R-expressing gastric cancer cells through the activation of the c-Jun N-terminal kinase [81]. In another study, VEGF stimulates IL-18 production and processing in gastric cancer cells, and IL-18, in turn, promotes cell migration through tensin down-regulation and actin polymerization. Therefore, IL-18 may be part of a loop that amplifies gastric cancer cell migration, angiogenesis, and progression [82]. In addition, VEGF induces ADAM-33 expression, which up-regulates IL-18 secretion, resulting in increased gastric cancer cell migration and proliferation [83]. Besides its activity on tumor invasiveness and angiogenesis, IL-18 induced expression of a granzyme B inhibitor, protease inhibitor 9, in gastric cancer cells and decreased their susceptibility to lymphocytemediated cytotoxicity [84].
A recent report showed that IL-18, produced by stromal cells, is a growth factor for T-ALL cells [85]. MEK inhibitors enhance the growth of human T-ALL cells cocultured with stromal cells through the transcriptional up-regulation of IL18 in stromal cells. In addition, rIL-18 promotes T-ALL growth in vitro through activation of the NF-kB pathway, whereas silencing of IL-18R in T-ALL cells inhibits their proliferation in vitro and in vivo.
The finding that high serum levels of IL-18 in some patients with autoimmune diseases correlate with impaired NK cell survival [98] suggested immune-regulatory activities of IL-18 on NK cells, other than the "helper NK" cell priming. This hypothesis was addressed in syngeneic tumor models. Intriguingly, whereas daily administration of IL-18 had anti-tumor effects in a syngeneic melanoma model, similar to previous reports [50], a twice/wk schedule of IL-18 showed protumor Human gastric cancer Gastric cancer cell lines in vitro IL-18 mediates production of the proangiogenic factor thrombospondin-1. [81] Human gastric cancer cells SNU-601 cell line in vitro VEGF-induced IL-18 promotes cell migration and proliferation through ADAM-33 induction. [82,83] Human gastric cancer cells Gastric cancer cell lines in vitro IL-18 induces resistance to lymphocyte-mediated cytotoxicity via granzyme B inhibitor. [84] Human T-ALL In vitro assays on primary T-ALL cells and T-ALL xenografts activity [86]. These data suggest that the administration schedule of IL-18 may be crucial for biologic effects on tumors. IL-18 upregulates PD-1 expression by activated, mature NK cells in lymphoid organs and reduces NK cell antimetastatic activity in a PD-1-dependent mode [86]. Further studies indicated that IL-18 converts a subset of Kit 2 NK cells into Kit + NK cells, which overexpress PD-L1 and mediate immune-ablative functions, in mouse models. Indeed, the silencing of IL-18 in tumors or its blockade by IL-18BP restores NK cell-dependent immune surveillance [87].
A recent report indicates that IL-18 induces the differentiation of MDSC expressing iNOS and arginase-1 from murine bone marrow precursors. These cells efficiently suppress T cell responses in vitro. Treatment of mice with IL-18, twice/wk, increased the accumulation of MDSC in s.c. B16 melanoma tumors [88].
Although IL-18-induced IFN-g plays a role in the defense against infections and cancer, IFN-g may also have immunesuppressive effects in some models of disease, as in a mouse model of HPV-associated epidermal hyperplasia, driven by transgenic expression of the HPV16 E7 oncoprotein. Production of IFN-g requires IL-18 but not IL-12 in this model. These findings indicate that IL-18 contributes to the generation of an immunosuppressive environment during viral oncogene-driven epidermal hyperplasia [89].
Moreover, IL-18 showed proangiogenic and prometastatic activities in mouse models of melanoma hepatic metastases. IL-18 mediates VCAM1 expression in endothelial cells, favoring the adhesion of melanoma cells in vitro [90]. In the B16 melanoma model, the administration of IL-18BP, which blocks endogenous IL-18, inhibits the development of hepatic metastases through inhibition of VCAM1 expression on hepatic sinusoid endothelium [91]. More recent data indicate that resveratrol [92] or the NLRP3 inflammasome inhibitor thymoquinone [93] suppresses metastases of murine melanoma cells through inhibition of IL-18mediated VCAM1 expression and/or IL-18 secretion.
A subset of human melanomas expressing IL-18R shows enhanced prometastatic activity in nude mice relative to IL-18Rnegative melanomas. The increased metastatic potential is related to a cascade of IL-18-mediated inflammatory factors leading to expression of VLA-4 integrin [94]. VLA-4, a ligand for VCAM1, mediates adhesion of circulating tumor cells to the vessel endothelium, the initial step in tissue transmigration and metastasis formation. The study of human metastatic melanomas, with or without IL-18-dependent gene-expression signatures, indicates the involvement of an IL-18-induced inflammatory phenotype in some metastasizing melanomas [95].
Altogether, these studies suggested that IL-18BP or inflammasome/ Casp1 inhibitors may be regarded as a therapeutic option in cancers where IL-18 acts as a tumor-promoting agent. Indeed, IL-18BP-Fc treatment was effective in inhibiting the lung metastasis progression by blocking endogenous, tumor-released IL-18 [99]. In addition, IL-18BP may represent a useful tool for the detection of IL-18-expressing tumors. Indeed, (64)Cu-DOTA-IL-18BP-Fc shows specific accumulation by positron emission tomography in a syngeneic lung metastasis model [99].
Different studies reported the association of polymorphisms in the IL18 gene promoter (2607 C . A and 2137 G . C) and the development of different human cancers, suggesting a possible functional involvement of IL-18 in human carcinogenesis. Indeed, these polymorphisms alter binding sites of specific transcription factors and result in altered IL18 gene transcription [100]. Although these studies produced controversial results, in different cancers and populations, a recent meta-analysis suggested that the 2607 C . A polymorphism is associated with increased overall cancer risk, particularly in nasopharyngeal carcinoma and esophageal cancer in the Asian population [96]. Another meta-analysis indicated that the 2137 G . C polymorphism is associated with increased risk of nasopharyngeal carcinoma in the Asian population but not in Caucasians [97]. On the other hand, no association between the 2607 C . A polymorphisms and the risk of prostate, colorectal, breast, cervical, or other cancers was found. The divergent role of IL18 gene polymorphisms may account for differences in carcinogenetic mechanisms in various cancers.
High levels of IL-18 were found in tumor tissues or in the systemic circulation of human cancers, including esophageal [101], gastrointestinal [102], breast [103], ovarian [72,104], pancreatic [105], hepatocellular [106], lung [107], and renal cancer [108]; diffuse large B cell lymphoma [109]; and multiple myeloma [110]. In some instances, high levels of IL-18 were associated with advanced tumor stages and/or with a poor prognosis, suggesting that IL-18 promotes tumor progression. However, some IL-18 ELISAs show substantial cross-reactivity with the pro-IL-18 [72], leaving unresolved the question of whether the high levels of IL-18 found in tumors always correspond to an increase of the mature IL-18 form. Therefore, in most tumors, the impact of IL-18 processing and the role of endogenous bioactive IL-18 are still an open issue. For example, serum IL-18BPa and IL-18 levels were increased in pancreatic cancer patients, and calculated "free" IL-18 levels were correlated with disease severity and poor survival. Chemotherapy further increased free IL-18 levels, without affecting IL-18BPa. The authors concluded that caution in the use of IL-18 therapy should be used in pancreatic cancer in light of a potential role of IL-18 in tumor progression or angiogenesis [105]. Nonetheless, the possibility that pro-IL-18 may interfere with mature IL-18 detection and explain the apparent paradox of high, free IL-18 levels cannot be excluded, as pancreatic cancer cells secrete pro-IL-18 [111]. However, upon treatment with 5-fluorouracil, Casp1 is activated, leading to secretion of mature IL-18 by pancreatic cancer cells. This finding supports the concept that in cancer, the inflammasomes may be activated through DAMPs, produced as a consequence of spontaneous or therapy-related tumor cell death, and lead to IL-18 processing.

CONCLUDING REMARKS
In conclusion, the current literature uncovers a complex and sometimes divergent role of the IL-18/IL-18R/IL-18BP system in different neoplastic conditions. Anti-tumor effects of endogenous or exogenous IL-18 were reported in early stages of colon and gastric carcinogenesis and in several preclinical models of cancer immunotherapy, respectively. On the other hand, procancer effects of IL-18 were described in advanced gastric cancer, in a subset of melanomas, and in T-ALL. Along this line, high levels of IL-18 were found in different cancers, and IL18 gene polymorphisms were associated with some cancers. However, IL-18, released by ovarian and colon cancer cells, is the unprocessed, inactive form, which can cross-react with mature IL-18 in ELISA. These findings suggest that more studies on the biologic role of cancer-related IL-18 are warranted.
Altogether, the context-dependent effects of IL-18 in cancer pose the question of whether IL-18, or rather its antagonist IL-18BP, should be used for cancer therapy. Most likely, as for other biologic medicinal products, the choice should depend on the specific characteristics of a given tumor. In some tumor types, where tumor-promoting IL-18 effects prevail, IL-18BP could be helpful, whereas IL-18 therapy should be regarded with caution. This is the case of a subset of melanomas, gastric cancers, and T-ALL, which expresses IL-18R and may progress in response to IL-18. In other tumors, such as ovarian and prostate cancer, high endogenous IL-18BP levels may limit the activity of therapeutic IL-18, particularly at the tumor site. Therefore, the biologic impact of the IL-18/IL-18R/IL-18BP system in specific cancers should be considered in the design of clinical studies. AUTHORSHIP M.F., G.C., and S.F. wrote the manuscript together.

ACKNOWLEDGMENTS
This work was supported by a grant from Fondazione Compagnia di San Paolo, and a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC; IG 13518). Grazia Carbotti is recipient of a fellowship from FIRC, Fondazione Italiana per la Ricerca sul Cancro.

DISCLOSURES
The authors have no conflict of interest to declare.