Emerging Strategies for Cancer Immunoprevention

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REVIEW Emerging strategies for cancer immunoprevention

JC Roeser1, SD Leach2 and F McAllister3

The crucial role of the immune system in the formation and progression of tumors has been widely accepted. On one hand, the surveillance role of the immune system plays an important role in endogenous tumor prevention, but on the other hand, in some special circumstances such as in chronic inﬂammation, the immune system can actually contribute to the formation and progression of tumors. In recent years, there has been an explosion of novel targeted immunotherapies for advanced cancers. In the present manuscript, we explore known and potential various types of cancer prevention strategies and focus on nonvaccine- based cancer preventive strategies targeting the immune system at the early stages of tumorigenesis.

Oncogene (2015) 34, 6029–6039; doi:10.1038/onc.2015.98; published online 14 September 2015

INTRODUCTION Differences between chemoprevention and immunoprevention. As cancer is a collection of multiple complex individual diseases Whereas immunoprevention focuses on directly or indirectly that happen in different sites of the body and progress in different targeting the immune system, chemopreventive strategies utilize ways, a large amount of information is required to understand drugs or natural compounds to prevent cancer development.3 how to prevent each cancer type. Existing interventions for cancer Less than a dozen chemopreventive agents have been FDA prevention focus on the use of chemopreventive agents or approved: tamoxifen and raloxifene for breast cancer fi prevention,4,5 celecoxib for colorectal polyps prevention in vaccines designed and tested for prevention of speci c cancer 6,7 8,9 types. Cancer vaccines that aim to manipulate the patient’s own FAP, BCG and valrubicin for bladder cancer prevention and several topical agents for prevention of skin cancer in patients immune system to destroy existing cancer cells are called fl ‘therapeutic vaccines’.1 Cancer vaccines that target the immune with actinic keratosis: uorouracil, diclofenac, masoprocol and ingenol mebutate.10 system to prevent the development of cancer are called ‘prophylactic vaccines’ and could potentially be used for cancer immunoprevention. However, there are many other strategies to Differences between immunoprevention and immunotherapy prevent cancer aside from vaccines. In the present manuscript, we The term ‘immunoprevention’ refers to the manipulation of the will review existing and potential cancer prevention strategies and immune system for disease prophylaxis, while ‘immunotherapy’ is focus on nonvaccine approaches for the prevention of cancer reserved for the treatment of existing disease by targeting the targeting the immune system. immune system. ‘Immunoprevention’ or ‘immunoprophylaxis’ represent one of the most successful types of interventions ever developed. Preventive vaccines have practically eliminated infectious diseases such as polio. In contrast, immunotherapies Types of cancer interventions against chronic infectious diseases have not shown enough There are two global interventions to target cancer: preventive efficacy due to the mechanisms developed by infectious and therapeutic strategies (Table 1). Cancer prevention includes all pathogens to avoid recognition and elimination by the immune approaches that aim to reduce the risk and incidence of cancer. system. Until very recently, the same was believed about cancer There are two types of cancer-preventive strategies: primary and immunotherapies, with most of strategies failing due to the secondary. Primary cancer prevention focuses on health counsel- tolerance and escape of detection by the immune system.11 ing, education and environmental control to decrease exposure to However, in the past decade, huge advances have been made in risk factors.2 More than 30% of cancer deaths could be prevented the immunotherapy field, most of them based on a better by avoiding key risk factors, including tobacco use, an unhealthy understanding of the molecular events leading to tumor 12 diet, alcohol abuse, sexually transmitted HPV infections and so on. tolerance. Tobacco is the single most important risk factor for cancer causing 22% of global cancer deaths. Secondary cancer prevention Immunoprevention of tumors caused by infectious agents encompasses interventions leading to the diagnosis and control Two different types of tumors can be targeted by immunopreven- of cancer and precancerous lesions at the earliest stage possible, tion, tumors caused by infectious agents or the ones caused by including screening and active interventions. These active noninfectious agents. Effective vaccines are available for viruses prevention strategies can be further divided in two types: associated with cancer development. The vaccine against the chemoprevention and immunoprevention. hepatitis B virus initially developed for hepatitis prevention has

1Department of Oncology. Johns Hopkins University. Maryland, MD, USA; 2The David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA and 3Department of Clinical Cancer Prevention. The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Correspondence: Dr F McAllister, Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, 1515 Holcombe, Unit 1360, Houston, TX 77030, USA. E-mail: [email protected] Received 8 December 2015; revised 25 February 2015; accepted 27 February 2015; published online 14 September 2015 Cancer immunoprevention JC Roeser et al 6030

Table 1. Modalities of cancer prevention and therapy

Cancer

Prevention Therapy

Primary Secondary Chemo Targeted Immuno therapy therapy therapy

Active Targeted Screening Therapeutic prevention immuno- vaccines activators

Chemo Immuno prevention prevention

Infectious Non infectious

Prophylactic Immunomodulators vaccines

Targeted Targeted Anti-inflammatories immuno-activators

also been shown to reduce the incidence and mortality of liver antibodies to interferon (IFN)-γ in tumor-bearing mice or mice carcinoma.13,14 In 2006, the FDA approved two vaccines lacking the IFN-γ receptor showed accelerated tumor growth developed primarily for the prevention of cervical cancer compared with control mice.18,19 Studies using perforin-deficient associated with HPV infections: Gardasil (Merck) and Cervarix mice have shown increased susceptibility to both chemically (GlaxoSmithKline, Rixensart, Belgium). Gardasil is a quadrivalent induced and spontaneous cancer models.20 Mice lacking the vaccine that protects against four HPV types: HPVs 6, 11, 16 and recombinase-activating gene (RAG)-2 cannot somatically rear- 18. Cervarix is a bivalent vaccine against HPVs 16 and 18. These range lymphocyte antigen receptors and cannot produce 15 vaccines can reduce the incidence of cervical cancer by 70%. peripheral αβ T cells, B cells, NKT cells (NKTs) or γδ T. As RAG-2 is selectively expressed in the lymphoid system, RAG-2− / − mice Immunoprevention of tumors caused by noninfectious agents provided an appropriate model to exclusively study the effects of There are two kinds of approaches to noninfectious tumor host lymphocyte deficiency on tumor development. Following − / − immunoprevention: one that targets specific antigens (vaccines) subcutaneous injection of a chemical carcinogen RAG-2 mice or another that targets nonspecific immune components. The developed sarcomas at the injection site faster and with greater 21,22 drugs developed for the latter approach will be called ‘immune- frequency than strain-matched controls. Data about the role modulators’ in the present manuscript. At this point, immunopre- of immunosurveillance in cancer prevention are not only restricted 23 ventive approaches for tumors of non-infectious cause are at the to preclinical mouse models, but also exists in humans. As an preclinical level of development. Multiple transgenic mouse example of this, approximately a fourfold increase in the incidence models harboring activated oncogenes have been produced in of de novo malignant melanoma after organ transplantation has 24 the past decade and they can be used to test prevention been reported. In spite of that, tumors do develop in the strategies by targeting the immune system.16 presence of a functional immune system. The protection provided by the immune system may not be sufficient and immune- The immunosurveillance theory and implications for preventive strategies aiming to promote antitumor immune immunoprevention defenses may bring a proportional decrease in tumor incidence. In the 1950s, Thomas and Burnet developed the theory of tumor immunosurveillance in which they proposed that the immune Theory of immunoediting and implications for immunoprevention system protects against developing tumors by attacking abnormal The immunoediting theory later replaced the immunosurveillance cells before they progress to invasive malignancy. However, when theory to reflect the increasingly apparent dual role of immunity in this hypothesis was tested in CBA/H nude mice, the most not only preventing but also in sculpting the tumoral process. congenitally immunodeficient mice available in 1970s, mice were Dunn et al.25 proposed that the process of immunoediting was found to develop spontaneous tumors and methylcholanthrene- comprised by three phases and named them ‘three Es of cancer induced sarcomas in a similar rate to wild-type mice.17 The immunoediting’: elimination, equilibrium and escape. The first findings brought skepticism about the immunosurveillance phase, elimination, represents the original concept of immuno- theory. However, two issues in this experiment were found surveillance and when successful, the whole process of immu- decades later. First, nude mice are not entirely immunodeficient as noediting is completed. In the second phase, equilibrium, the host they still maintain natural killer (NK) cells and low numbers of immune system and the tumor cells that survived the elimination T cells. Second, the CBA/H strain of mice expresses a highly active step enter into a state in which the immune system imposes isoform of the enzyme that metabolizes methylcholanthrene to its selective pressure on the tumor cells that is enough to contain, carcinogenic form. Subsequent studies using neutralizing but not completely remove the tumor, resulting in a tumor

Oncogene (2015) 6029 – 6039 © 2015 Macmillan Publishers Limited Cancer immunoprevention JC Roeser et al 6031 composed of many genetically unstable and mutating tumoral IL-6-gp130-STAT3 axis was fundamentally required for the cells. In the third phase, escape, tumor cells selected by the orchestration of the inflammatory process.34 equilibrium phase now grow and become clinically detectable. IL-6 and its downstream targets are involved in the regulation of Some of the escape mechanisms include: elaboration of cell proliferation, survival and metabolism, explaining the close immunosuppressive cytokines (TGF-β), growth factors (VEGF), association between IL-6 signaling and tumorigenesis.34 IL-6 recruitment of regulatory T cells and changes in tumor cells that regulates normal and tumor stem cell self-renewal through affect their recognition by immune effector cells (loss of MHC, Notch-3 activation and upregulation of the hypoxia response development of IFN-γ insensitivity, expression of anti-apoptotic protein carbonic anhydrase IX (CA-IX), which creates a permissive signals and so on).26 The accumulation of metabolic enzymes, environment for breast cancer stem cells survival in hypoxic 35 36 indoleamine 2,3-dioxygenase and arginase, that suppress T-cell conditions. Gao et al. have shown that activated EGFR induces proliferation and activation as well as the high expression of epithelial expression of IL-6, which induces STAT3 (signal tolerance-inducing molecules (PD-1 and CTLA-4) are additional transducers and activators of transcription 3) activation in lung mechanisms that contribute to tumor escape.27 Further under- carcinoma. Lesina et al. have demonstrated that pancreatic standing of the mechanisms responsible for tumor equilibrium expression of oncogenic Kras(G12D) causes an IL-6 transsignal- and escape will be invaluable to generate better strategies for ing-dependent activation of Stat3/Socs3 required to promote 37 cancer immunoprevention. pancreatic tumorigenesis. In a model of colitis-associated cancer, IL-6 produced by hematopoietic cells enhances adenoma growth38 and its ablation reduces the number and size of colonic Advantages of Immunoprevention without vaccines adenomas.39 Toclizumab, a humanized anti–IL-6 receptor mono- A key issue in developing effective cancer preventive vaccines is clonal antibody, globally blocks IL-6 activities since it competes for the large variability of tumor antigens (even within the same both the membrane-bound and soluble types of IL-6 receptors.40 patient tumor). Even in the event of a very common antigen This antibody was approved by the FDA in 2010 for the treatment 28 selected as the target, results have been less than optimal. of patients with rheumatoid arthritis that have failed at least one Immunopreventive strategies that target the immune system anti-TNF therapy. When targeting IL-6 with antibodies, the impact directly would have to deal with less variability and therefore, of blocking classical membrane-bound signaling and IL-6 trans- potentially could result in more effective outcomes as evidenced signaling will have to be considered. A recent Phase I clinical trial by the recently FDA-approved nonvaccine cancer immunotherapies.12 of a chimeric IL-6 monoclonal antibody, siltuximab, has reported good tolerance in cancer patients and phase II therapeutic trials 41 Risk profile of immune-preventive agents are currently ongoing. Clinical preventive strategies targeting fl Given the fact that preventive strategies are directed to healthy IL-6 would be highly promising, particularly in in ammation- patients who, even in the case of pertaining to high-risk groups, associated cancers. A proof of principle for anti-IL-6 immune prevention of pancreatic cancer in preclinical model with few may actually never develop cancer, safety is a major concern. 42 As immune-preventive agents continue to being developed, long-term side effects has been recently reported. major attention should be focused on dissecting their risk profile. As some protumoral targets may also have a role in host defense Interleukin-8 against pathogens, strategies that aim to suppress protumoral Interleukin-8 (IL-8), also known as CXCL8, is a proinflammatory targets may actually result in an increased risk for infections.29 On CXC chemokine that binds to CXCR1 and CXCR2, two cell-surface the other hand, strategies that focus on inducing antitumoral G-protein-coupled receptors.43,44 The receptors CXCR1 and CXCR2 targets may actually result in the development of autoimmune have also been found overexpressed on cancer cells and multiple conditions.30 reports have described an autocrine IL-8 signaling loop associated with cell proliferation, invasion and angiogenesis as mechanisms 45–47 Shotgun vs targeted immune-preventive approaches to induce tumorigenesis in various models. Humanized monoclonal antibodies against IL-8 have been used in murine Broad anti-inflammatory agents such as celecoxib and aspirin fi models and have shown to decrease bladder cancer and have proven bene cious as preventive agents in high-risk melanoma growth in xenografts models.45,46 A liposomal encap- populations but even these drugs have several toxicities, including sulated small interfering RNA that supressed IL-8 expression in a cardiovascular toxicities and increased of bleeding risk, when used 31 model of ovarian cancer xenografts resulted in decreased chronically. It would be expected that better molecular 48 fi microvessel density and growth retardation. Targeting IL-8 characterization of the speci c immune events surrounding expression for cancer prevention would fail to account for the fi preneoplastic lesions may theoretically result in high ef cacy with signaling effect of other CXC-chemokines. In this regard, several few side effects through the use of targeted immune-preventive CXCR1/2 receptors49–55 inhibitors are now being developed and strategies. they may also have a potential utility for immunoprevention.

Targets for cancer Immunoprevention TH17 and Interleukin-17 IL-17A, a proinflammatory cytokine produced mainly by CD4+ T cells known as TH17 (T-helper 17) cells, has a key role in chronic inflammation-related human diseases and autoimmunity.56,57 In PROTUMORAL 58 addition to TH17 cells, IL-17A can also be produced by NKTs, Interleukin-6 gamma–delta T cells59 and CD8+T cells.60 IL-17A binds the The complex of IL-6 and IL-6R does not lead to signaling but heteromeric IL-17R complex consisting of at IL17RA and instead associates with the protein gp130 inducing its dimeriza- IL17RC.61 TGF-β and IL-6 are the most important cytokines 62 tion and starting intracellular signaling via the JAK/STAT and the required for TH17 differentiation, while IL-23 is crucial for its – – 32 63 Ras Raf MAPK signaling pathways. IL-6 is the only cytokine that phenotype maintenance. The differentiation of TH17 cells also in vivo utilizes both classical membrane-bound receptor signaling requires the expression of retinoic acid receptor-related orphan and trans-signaling through its soluble receptor.33 Using mice in nuclear receptor gamma (RORγt), which is a STAT-3-dependent 64 which the portions of gp130 have been genetically modified in transcription factor. TH17 cells are increased in the tumor models of inflammatory diseases led to the conclusion that the microenvironment during tumor development65,66 and several

© 2015 Macmillan Publishers Limited Oncogene (2015) 6029 – 6039 Cancer immunoprevention JC Roeser et al 6032 manuscripts have reported the role of IL-17 in promoting constitutive STAT3 phosphorylation,95 and the ablation of STAT3 tumorigenesis.65,67–72 The mechanisms for the protumorigenic results in impairment of malignant transformation by tyrosine effects include the induction of angiogenic and anti-apoptotic kinase oncoproteins.96–98 STAT3 signaling within the tumor factors, migration and invasion and recruitment of myeloid microenvironment induces a procarcinogenic cytokine, IL-23, derived suppresors cells. Conversely, TH17 cells have been also while inhibiting a central anticarcinogenic cytokine, IL-12, thereby reported to mediate antitumor effects by promoting CTLs shifting the balance of tumor immunity toward carcinogenesis.99 activities, MHC antigen expression and production of IFNγ. The Small molecule inhibitors of STAT3 have been developed and antitumor effects reported were mostly described in the setting of used in the preclinical setting, but they mostly target upstream established cancer73 and some of them relied on IFNγ, rather than receptor and non-receptor tyrosine kinases, resulting in low on IL-17A secretion. The FDA has approved the use of specificity of action. Sorafenib, a multikinase inhibitor, decreased ustekinumab, an anti-IL-23 antibody that would decrease TH17 STAT3 phosphorylation in hepatocellular carcinoma by inhibiting responses, for moderate-to-severe psoriatic arthritis.74 Ixekizumab phosphoinositide 3-kinase/Akt and MEK/ERK pathways.100 WP1-66, and secukinumab, IL-17A-specific antibodies and brodalumab, a JAK2 inhibitor, has also been used to inhibit STAT3 activity in a monoclonal antibody directed against IL-17RA, have shown safety model of renal cell carcinoma.101 RNA interference approach has and efficacy in early phase trials for autoimmune and chronic also been done to knock down STAT3 and in preclinical models it inflammatory conditions75–77 and are currently being tested in supressed tumorigenesis.102 Finally, a chemically modified double- phase III trials for autoimmune diseases. RORγt small molecule stranded STAT3 oligonucleotide decoy that inhibited tumorigen- inhibitors are currently being developed and under preclinical esis in murine models has also been clinically tested in a phase 0 testing. In any case, the potential idea of targeting IL-17 producing setting and proved to be safe and effective to selectively suppress cells as a strategy for cancer immunoprevention should consider STAT3 target genes in head and neck tumors.103 Given the key their impacts in autoimmunity as well as the potential protumoral role of STAT3 in promoting prostate, colon and pancreatic effects.78–80 Potential preventive strategies targeting IL-17 would be cancer,104–106 STAT3 inhibitors could be useful for cancer ofparticularinterestforinflammation-induced cancer. prevention in patients with high risk for these diseases.

Interleukin-1 PD-1/ PD-L1 The IL-1 family encompasses three proteins: IL-1α, IL-1β and the Programmed cell death-1 (PD-1), a CD28 family receptor, has a key IL-1 receptor antagonist (IL-1ra).81,82 There are two IL-1 receptors, role in tumor tolerance.107 The interaction between PD-1/PD-L1 the biologically active IL-1 receptor type I (IL-1RI), agonistic results in the inhibition of T-lymphocyte effector functions, with receptor ubiquitously expressed with preferential binding to IL-1a subsequent downregulation of antitumor immune responses.108 and the IL-1 receptor type II (IL-1RII or decoy IL-1R), antagonist PD-1 has two ligands, PD-L1 and PD-L2 with a different pattern of receptor expressed on B cells, neutrophils and monocytes with expression and regulation. PD-L1 is expressed in most hemato- preferential binding to IL-1β.83,84 The mechanisms responsible for poietic cells and in multiple tumors including melanoma, the IL-1- tumor growth promotion are: induction of prometastatic pancreatic, breast and lung tumors109–113 but PD-L2 expression genes (for example, matrix metalloproteinases) and stimulation of is restricted to macrophages, mast cells and dendritic cells (DCs). adjacent cells to produce angiogenic proteins and growth factors High expression of tumoral PD-L1 has been associated with poor (VEGF, IL-8, IL-6, TNFα and TGFβ).82,83,85 Cancer cells are able to prognosis in multiple studies.111,114 Preclinical studies have produce IL-1 or can induce its production from cells within the suggested that PD-1/PD-L1 signaling favors tumor immune tumor microenvironment.86 IL-1 has autocrine behavior by evasion and that blockade of the PD-1 pathway can restore inducing proliferation and invasion of the tumor cells and is also antitumor immune responses. Tumor cells that express PD-L1 a paracrine effector on the stromal cells. From a clinical have increased resistance to T-cell-mediated lysis with increased perspective, elevated IL-1 levels within the serum have been tumorigenesis and invasiveness, when compared with tumor cells associated with more aggressive tumors.87 that do not express PD-L1 and the administration of anti-PD-L1 The IL-1ra is an inhibitor of IL-1 and has been shown to antibodies reversed these effects.115 Also, PD-1-deficient mice decrease tumor growth, angiogenesis and metastases in murine presented inhibition of tumor growth, suggesting a strong xenograft models.84 IL-1ra is an approved treatment for patients antitumor immune response in the absence of PD-1/PD-L1 with rheumatoid arthritis, has shown some promising results in signaling.116 Finally, in mice with established tumors, the indolent multiple myeloma treatment88 and is now being tested administration of anti-PD-1 or anti-PD-L1 antibodies led to on other cancers. IL-1ra could represent effective novel immune- reduction in tumor size and increased survival.117 preventive agents but further studies are needed.81 In recent years, early phase studies reported clinical activity of PD-L1118 and PD-1119,120 monoclonal antibodies in patients with STAT3 advanced solid tumors. As larger therapeutic trials get conducted for efficacy validation, the utilization of these antibodies for tumor STATs are a family of transcription factors discovered in the early immunoprevention in high-risk populations may become a 1990s that regulate cell growth and differentiation through potential effective strategy, particularly if PD-1/PD-L1 biomarkers phosphorylation by receptor-associated kinases.89 STAT3 was are well defined in preneoplastic lesions and side effects are found to mediate the acute phase response observed with manageable.121 Given the success of these agents in melanoma inflammation and stress that is often mediated by interleukin-6 and non-small cell lung cancer, immunoprevention targeting (IL-6) and other proinflammatory cytokines.90 Activated STAT3 is PD-1/PD-L1 might be of high relevance in patients with phosphorylated on tyrosine and forms a dimer through phospho- preneoplastic lesions associated with either disease. tyrosine/src homology 2 (SH2) domain interaction. The dimer enters the nucleus via interaction with importins and binds genes with critical role in cellular proliferation, survival, pluripotency, CTLA-4 invasion and angiogenesis, explaining its association with cancer CTLA-4 is a cell-surface protein constitutively expressed on T cells, development. Fibroblasts engineered to express an activated but upregulated by the interaction of the T-cell receptors and STAT3 formed tumors in nude mice, thus establishing STAT3 as an CD28.122 A splice variant of CTLA-4 may also be secreted by T cells oncogene.91 Increased STAT3 activity is present in 470% of in a soluble form that binds to T-cell receptors. CTLA-4 interacts solid tumors,92,93 and the inhibition of STAT3 can mediate with CD80 (B7-1) and CD86 (B7-2) and competitively competes tumor regression.94 Tyrosine kinase oncoproteins can target with CD28 for binding with these receptors, therefore, acting as an

Oncogene (2015) 6029 – 6039 © 2015 Macmillan Publishers Limited Cancer immunoprevention JC Roeser et al 6033 inhibitor of T-cell proliferation.123 The receptor CD28 is similar to Murine MDSCs are characterized by the coexpression of two CTLA4 and shares a homologous amino-acid sequence. CD28 myeloid differentiation antigens: Gr1 and CD11b.135 The normal binding causes costimulation and activation of T cells, whereas murine bone marrow can contain up to 30% cells with this CTLA-4 binding causes T-cell inhibition.122 As CTLA-4 provides a phenotype but the spleen contains only 2–4%. Human MDSCs regulatory signal that inhibits T-cell activation, it serves as an have a different phenotype, being defined by CD11b+/CD14 − immune checkpoint inhibitor to prevent over activation of expression.136 The levels of immune cells with these phenotypes T cells.124 Two antibodies have been tested against CTLA-4 have been shown to correlate with prognosis and overall – clinically: ipilimumab and tremelimumab. Ipilimumab has become survival.137 139 Multiple methods of inhibiting MDSCs are currently the standard of care for melanoma patients after a phase III trial under investigation. These methods involve (1) induction of showed survival benefit in patients with metastatic melanoma.125 MDSC maturation to nonsuppressive cells (all trans retinoic acid, Tremelimumab failed to show a survival advantage in a phase III vitamin D), (2) decrease MDSC levels (sunitinib, gemcitabine, 5-FU, clinical trial in melanoma126 and is currently being investigated in CDDO-Me) or (c) functional inhibition of MDSC (PDE-5 inhibitors, 140 combination with other agents. The recent success in targeting cyclooxygenase 2 inhibitors). These approaches may enhance CTLA-4, particularly in melanoma has led to increased interest in antitumor immunity through reduction in immune suppression understanding the role that CTLA4 has in immune surveillance and thus would be relevant in the immune-preventive scenario. and immunoprevention, particularly for patients with preneoplastic lesions. Tregs Regulatory T cells (Tregs) were initially called suppressive CD4+ Innate lymphoid cells T cells as they were found to negatively regulate tumor immunity 141 Innate lymphoid cells (ILCs) have been recently described as a leading to tumor growth in mice. The transcription factor Foxp3 group of innate immune cells that can regulate immunity, is crucial for the development and functionality of T regulatory fl cells and the IL-2 receptor α-chain, CD25, serves as the phenotypic in ammation and tissue repair in multiple anatomical compart- + ments. ILCs can regulate commensal bacterial communities, marker for CD4 T regulatory cells. Therefore, the classic regulatory contribute to resistance to bacterial pathogens, promote T cells are phenotyped as CD4+CD25+FOXP3+T cells (Tregs). inflammation and orchestrate tissue repair and wound Multiple possible suppressive mechanisms for regulatory T cells healing.127 RORγt+ ILCs that express and require the nuclear have been proposed: induction of B7-H4 expression by antigen- hormone receptor retinoic acid receptor related orphan receptor presenting cells, CTLA4-mediated suppression of T-cell activity and (RORγt), are involved in lymphoid tissue genesis and can produce direct inhibition of T-cell activation by the secreted cytokines IL-10 and TGF-b.142 The role of Tregs (CD4+CD25+Foxp3+) on cancer TH17-associated cytokines IL-17 and IL-22. On the basis of functional characteristics, three subpopulations of RORγt-depen- progression has been widely demonstrated in several preclinical models.143 Tumors trigger an increase of T-regulatory cells dent ILCs can be distinguished: (a) lymphoid tissue-inducer (LTi) (CD4+CD25+Foxp3+) numbers in lymph nodes and in the tumor cells, (b) ILCs dedicated to IL-22 production and (c) IL-17- microenvironment, leading to immunosuppression. Several producing ILCs. ILCs that produce both IL-17 and IL-22 have also reports have linked the abundance of Tregs in tumors with worse been found that may be cells derived from either IL-17- or IL-22- prognosis.144,145 producing cells.128 RORγt ILCs have recently been the target of Cyclophosphamide is an alkylating agent that causes DNA increasing attention due to their key role in multiple models of crosslinking and is utilized as a chemotherapeutic agent for microbe-related tumorigenesis. In a murine model of bacteria- several types of tumors. High dose cyclophosphamide can induce induced colon cancer, innate lymphoid cells have been shown to 129 effective antitumor activity. However, low dose cyclophosphamide sustain tumorigenesis through production of interleukin-22. In can also induce improved antitumor immune responses as a result a melanoma model, the chemokine CCL21 expressed by the of Tregs depletion. This was demonstrated by the evidence that tumor cells recruited not only myeloid-derived suppressor cells cyclophosphamide treatment caused an immune-mediated that can directly suppress antitumor immune responses but also regression of a cyclophosphamide-resistant lymphoma.142 Several γ recruited ROR t+ ILCs that mediated the enhancement of tumor preclinical studies have confirmed the effect of cyclophosphamide growth as shown by the absence of effect in tumor growth in on Tregs depletion and clinical studies have also been conducted γ fi 130 ROR t-de cient mice. Further understanding of these cells with similar results.146 Studies have also shown that anti-CD25 will be required for the development of immune strategies antibodies induce tumor rejection in mice147,148 and humans. targeting them. However, this strategy is not as effective in advanced tumors, highlighting the critical role that Tregs may have in the tumor Myeloid-derived supressor cells immune-preventive setting. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that are expanded in various Macrophages disease states with the capability of suppressing T-cell Macrophages are a major component of the inflammatory tumor 131–133 responses. In addition to their suppressive effect on stroma and macrophages that infiltrate tumors are called tumor- adaptive immune responses, MDSCs have also been reported to associated macrophages.149 Macrophages are classified in M1 regulate innate immune responses by modulating the cytokines (classically activated) and M2 (alternatively activated) phenotypes 134 secretion of macrophages. in analogy with the Th1 and Th2 classification.150,151 Macrophages Myeloid progenitor cells and immature myeloid cells (IMCs) have functional plasticity and can change their functional status in constitute the major types of MDSCs. In healthy patients, IMCs that response to changes in the microenvironment.152 When macro- are generated in the bone marrow quickly differentiate into phages are exposed to lipopolysaccharides and IFN-α, they mature granulocytes, macrophages or DCs. In contrast, during polarize to an M1 phenotype with antitumor properties and pathological states like cancer and infections, a block in the when they are exposed to Th2 cytokines (IL-4, IL-13, IL-10), they differentiation of IMCs into mature cells results in an increase of polarize to an immune suppressive M2 phenotype that promotes the immature populations. Another relevant event during these tumor growth.150,153 Tumor-associated macrophages are a major pathological states is the upregulation of arginase1 (ARG1) and source of COX-2 and the selective COX-2 inhibitor celecoxib has inducible nitric oxide on IMCs, conferring them immune been shown to change the tumor-associated macrophages suppresive properties. phenotype from M2 to M1, in parallel to a reduction in number

© 2015 Macmillan Publishers Limited Oncogene (2015) 6029 – 6039 Cancer immunoprevention JC Roeser et al 6034 of colon polyps in a murine model of colon cancer.154 A large solid malignancies.174 Peptide based vaccines with adjuvants clinical trial has shown that celecoxib reduces the incidence of containing cytokines, chemokines or costimulatory molecules adenomas, but increased the risk of cardiovascular events.155 have been developed to amplify and direct the immune response Other selective COX2 inhibitors, with potentially less side effects, but are beyond the scope of the present review. are currently being tested and could represent potential colorectal cancer chemopreventive strategies. In a different manner, a recent CD4+ T cells (TH1) murine and human study has shown that CD40 agonists can cause TH1 CD4+ T cells are driven by IL-12 activation of Stat4 and T-bet macrophage activation with rapid stromal infiltration and 175,176 156 transcription factors on naïve T cells that induces and up- tumoricidal effect in pancreatic adenocarcinoma. This led us regulates IFNγ.177 Ectopic expression of T-bet in T 2orT 17 cells to speculate that similar agents could be used for prevention in H H results in their conversion into IFNγ-producing TH1-like cells. The patients with pancreatic preneoplastic lesions. central role of IFNγ- producing CD4+ T cells in antitumor immunity has been known for a long time.178 CD4+ T cells have an Mast cells indispensable role in the generation of vaccine-related therapeutic Tumor-infiltrating mast cells remodel the tumor microenviron- primary immune responses for the generation of long-term ment and promote tumor growth. Mast-cell infiltration and immune memory.179 In multiple human tumors, TH1 CD4+ T-cell activation in tumors is mainly mediated by tumor-derived stem infiltrates have been found associated with a better prognosis.180 cell factor and its receptor c-Kit.157 Mast cells accumulate in the Specific effector immune cells proliferate within the tumor tumor microenvironment and exacerbate immunosuppression by microenvironment but the contribution to anticancer immunity inducing MDSC and Treg infiltration in an IL-17-dependent is unclear, given the immunosuppressive factors within the tumor. manner.158 The presence of mast cells in many precancerous Vaccines targeting specific antigens should be planned in lesions and tumors is associated with a poor prognosis, suggesting combination with agents that aim to decrease the immunosup- that mast cells may promote an immunosuppressive tumor pressive environment. microenvironment and impede the development of protective Cancers with high mutational loads have a high number of antitumor immunity.159–161 tumor-specific neoantigens. Recently, Linnemann et al.181 devel- Targeting mast cells can be achieved indirectly via targeting oped a screening platform to evaluate CD4+ T-cell responses to mast cells mediators with agents like anti-TNF monoclonal tumor neoepitopes in melanoma patients and the results antibodies (infliximab) or directly through the use of mast-cell- indicated that CD4+ T-cell reactivity toward patient-specific stabilizing agents like sodium cromoglycate. Preclinical work with neoantigens is a common feature of melanoma and suggest that + infliximab in DSS-induced colitis has decreased development of therapeutic and potentially preventive strategies to boost CD4 colorectal tumors in association with decreased mast cell T-cell responses may be effective in melanoma and other cancers numbers.162 Stabilization of mast cells through sodium cromogly- with similar mutational loads. cate has also shown efficacy in preclinical studies,163 but the specificity of this compound has been questioned.164 Interven- NKT cells tions aimed at inhibiting mast cells function hold promise as NKTs are a subset of T cells that recognize self- and microbial- potential effective cancer preventive strategy, particularly because derived glycolipids such as α-galactosylceramide (α-GalCer) of the low side effects of these agents. presented by an HLA class I-like molecule, CD1d. NKTs are capable of producing key cytokines of both Th1 and Th2 T cells that ANTITUMORAL provide signals for other immune cells to start innate and adaptive responses.182 Low levels of circulating NKTs can predict poor α IFN- clinical outcome in certain human tumors183 and tumor infiltration Interferons alpha (IFNs-α) are pleiotropic cytokines from the type I by NKT is a predictor of favorable outcomes.184 Treatment of mice IFN family, initially described for their antiviral activity achieved by with α-GalCer has been shown to suppress tumor metastasis185 directly inhibiting viral replication in infected cells.165 IFN-α can yet clinical trials have not shown efficacy.186 Novel α-GalCer induce anti-tumoral effects through induction/promotion of analogs have been designed to favor Th1-biased immunity, with apoptosis and inhibition of cell growth.166 Multiple studies have greater preclinical anticancer potential and are currently being shown that IFN-α can induce the rapid differentiation of tested.187 monocytes into activated DCs that are involved in effective anti- 167 α tumor T-cell immunity. Moreover, IFN- can induce polarization Dendritic cells of T-helper (TH) cells to TH1 cells and activation of cytotoxic 168 DCs have a critical role in linking innate and adaptive immunity. T cells. Finally, IFN-α can promote B cell differentiation, 169 They have a key role in role in capturing and presenting antigens antibodies production and Ig class switching. In sum, IFN-α in the form of MHC II receptors to T cells stimulating them to has a broad effect on immune system activation and prolonged develop into effector T cells. In this role, DCs have a significant role activation can lead to autoimmune reactions. IFN-a is used for the in the tumor microenvironment, surrounding tumors and provid- treatment of multiple malignancies including melanoma and renal ing signals to other immune cells as to stimulate or suppress T-cell cell carcinoma,170,171 but its utilization for cancer prevention has activation.188 Tumor tolerant DCs are one reason that budding not been explored yet. tumors can escape immune surveillance. IL-10 secreted from tumors can inhibit the maturation of DCs, and promote DCs + CD8 T cells apoptosis, thereby promoting tumor tolerance.188 Potent DCs are It is well known that CD8+ T cells are important for antitumoral required for a strong antitumor immune response after che- responses. The expansion of tumor-specific CD8+ T cells can motherapy or radiation. There is a great degree of variability in the trigger effective antitumoral immunity,172 and this constitutes the location and number of DCs in tumors.189 However, most basis for multiple vaccine trials. Recent reports indicate that CD8+ evidence shows that although there are some levels of DCs T cells may also be capable of protumoral activity depending on present in tumors, they are suppressed by a variety of the antigen specificity.173 There is large evidence about the value immunosuppressing cytokines and other factors from the tumor of high numbers of T cells at the tumor site with CD8 memory microenvironment. DCs can also have prognostic value in treating T cells as a prognostic factor for overall survival in patients with patients. Studies have shown that tumors with DCs expressing low

IMMUNO-MODULATORS EFFECTIVE IMMUNO-PREVENTION ANTI-INFLAMMATORIES IMMUNO-ACTIVATORS

IMMUNE RESPONSE

Pro-tumoral Anti-tumoral

CYTOKINES CELL TYPES CYTOKINES CELL TYPES AND SIGNALS Tregs AND SIGNALS CD8+T cells IL-6 Stat3 TH17 CD4+ (TH1) γ IL-8 PD-1 ROR t+ ILC IFN-a NKT IL-17 CTLA-4 MDSC DC IL-1B M0. (M2) Mast cells

Figure 1. Immune response equilibrium. Activation of protumoral signals and cytokines as well as recruitment of specific immune cells populations with protumoral activity (Red circle) are counteracted by activation of signals and cytokines with antitumoral effects as well as tumor infiltration by immune cells populations with antitumoral effect (Green circle). Effective immunopreventive strategies should focus on either opposing the protumoral signals and immune cells by the use of anti-inflammatories or by increasing the activity of the anti-tumoral signals and immune cells through the use of immune-activators.

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