(2008) 27, 218–224 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc REVIEW TLR signaling by tumor and immune cells: a double-edged sword

B Huang1, J Zhao2, JC Unkeless3, ZH Feng1 and H Xiong3

1Department of Biochemistry and Molecular Biology, Tongji Medical College, Wuhan, China; 2Department of Gynecology and Obstetrics, Tongji Medical College, Wuhan, China and 3Immunology Institute, Mount Sinai School of Medicine, New York, NY, USA

The tumor pathways that trigger the between tumor cells and the host’s has uncontrolled proliferation, resistance to , meta- led to the realization that tumor cells have devised multiple stasis and escape from immune surveillance are partially strategies to evade immune attack. For example, tumors understood.Toll-like receptors (TLRs), which recognize a secrete transforming growth factor-b and vascular endothe- variety of pathogen-associated molecular patterns, are lial growth factor, which inhibit dendritic cell activation and centrally involved in the initiation of the innate and impair tumor-specific T-cell immunity (Vicari et al., 2002). adaptive immune responses.However, recent evidence To escape attack from natural killer cells and cytotoxic T shows that functional TLRs are also expressed on a wide lymphocytes (CTL), tumor cells upregulate certain surface variety of tumors suggesting that TLRs may play molecules (B7-H1 and HLA-G), downregulate others important roles in tumor biology.Activation of tumor cell (MHC class I and Fas), and shed some surface molecules TLRs not only promotes tumor cell proliferation and in soluble form such as MIC, the ligand for NKG2D resistance to apoptosis, but also enhances tumor cell (Gratas et al., 1998; Dong et al., 2002; Rouas-Freiss et al., invasion and metastasis by regulating metalloproteinases 2003; Wu et al., 2004). However, the signaling pathways and .Moreover, the activation of TLR signaling in controlling expression by tumor cells of factors/molecules tumor cells induces the synthesis of proinflammatory that are important for tumor cell immune evasion are not factors and immunosuppressive molecules, which enhance well characterized. Toll-like (TLR) signaling, the resistance of tumor cells to cytotoxic lymphocyte attack critical for innate and adaptive immune responses, has and lead to immune evasion.Thus, the neoplastic process been thought to be restricted to immune cells (Iwasaki and may usurp TLR signaling pathways to advance Medzhitov, 2004). However, recent studies suggest that progression, which suggests that targeting tumor TLR tumor cells bear TLRs and that TLR signaling promotes signaling pathways may open novel therapeutic avenues. tumor growth and immune evasion (Huang et al., 2005, Oncogene (2008) 27, 218–224; doi:10.1038/sj.onc.1210904 2007; Kelly et al., 2006).

Keywords: TLR signaling; tumor; endogenous ligand

TLRs are essential for immune defense against microbes and viruses

Toll-like receptors, expressed on sentinel cells of the Introduction immune system, including and dendritic cells (DCs), are the key sensors of pathogen invasion Dynamic interaction between tumors and the immune (Miyake, 2007). Currently, 13 mammalian-TLR analogs system is essential for tumor survival, growth and metastasis have been identified. TLRs 1, 2, 4, 5 and 6 are expressed (Yu et al., 2007). Tumors are infiltrated with large number on the cell surface; TLRs 3, 7, 8 and 9 are found almost of immune cells that constitute a major cell population in exclusively within endosomes. Different TLRs exhibit the tumor microenvironment. Tumor cells depend on their specificity for pathogen-derived ligands; TLRs 2, 3, 4, 5, microenvironment to provide signals for growth, antiapop- 7 and 9 recognize bacterial lipoproteins, double-stranded tosis, and metastasis (Hanahan and Weinberg, RNA/poly (I:C), lipopolysaccharides, flagellin, single- 2000). However, tumor cells are also under the surveillance stranded RNA and CpG-containing DNA, respectively due to their recognition by immune cells as foreign. (Poltorak et al., 1998; Aliprantis et al., 1999; Hemmi et al., Therefore, tumors have to overcome such immune 2000; Alexopoulou et al., 2001; Hayashi et al., 2001; Heil surveillance to progress. Analysis of the interactions et al., 2004). The ligands for TLRs 10, 12 and 13 remain unidentified. TLR10 is expressed in humans but not in Correspondence:Dr H Xiong, Immunology Institute, Mount Sinai mice, TLR8 is not functional in mice and TLRs 11, 12 and School of Medicine, New York, NY, USA. 13 are expressed in mice but not in humans. E-mail:[email protected] or Dr B Huang, Department of Biochemistry and Molecular Biology, The signaling pathways utilized by TLRs differ, which Tongji Medical College, Wuhan, China. result in varied cellular responses. For example, TLR3, E-mail:[email protected] the receptor for double-stranded RNA couples to the TLR signaling by tumor and immune cells B Huang et al 219 adaptor . This pathway culminates in solid tumor types and are currently in clinical trials. In activation of TRAF3 and IRF3, resulting in the secretion addition to the positive regulation by TLR signaling of of IFN-b, which is required for an effective antiviral the , TLR signaling may also decrease response. In contrast, other TLRs couple to the adapter or relieve the suppression of regulatory T cells on DCs or myeloid differentiation primary response 88 (MyD88) CD8 þ -T cells (Pasare and Medzhitov, 2003; Yang et al., (Takeda and Akira, 2004; O’Neill, 2006). The MyD88- 2004). adapter protein recruits IRAKs and TRAF6. The Although there are intriguing data about the effects of TRAF6 in turn activates TAK1 that phosphorylates TLR agonists on tumors, some concerns remain and activates the IKK complex culminating in the release unresolved at the moment. The immune stimulation of and translocation of NF-kB to the nucleus. TAK1 also TLR agonists may cause subpopulations of splenic DCs activates stress-activated protein kinase (SAPK) path- to make indoleamine 2,3-dioxygenase, which degrades ways and activates c-Jun-NH2-kinases (JNK) and p38. required by effector T cells, and thus may The MyD88-coupled TLRs induce the synthesis of cyto- downregulate antitumor immunity and even promote kines such as TNF-a, IL-6 and IL-1, key mediators of the tumor growth (Mellor et al., 2005; Wingender et al., inflammatory response (Kawai and Akira, 2005; Akira 2006). Regulatory T cells can impair antitumor immune et al., 2006). TLR4, the receptor for lipopolysaccharide, is responses (Wang and Wang, 2007). Several groups have unique in that it activates both MyD88-dependent and recently reported that TLR agonists stimulate the TRIF-dependent pathways. TLR signaling also activates proliferation and suppressor function of regulatory T DCs and macrophages to secrete IL-12, a that cells, thus attenuating the antitumor effects discussed directs the adaptive immune response toward a Th1 earlier (Liu et al., 2006; Sutmuller et al., 2006; Kabelitz, phenotype. TLRs play a major role as the initiator of the 2007). So far, there have been no definitive reports of innate immune responses to defend against bacteria, induction of systemic autoimmune disease from the viruses and other pathogens and also required for an administration of TLR agonists. However, studies with effective secondary immune response. TLR agonists as vaccine adjuvants have showed that they provide a remarkable acceleration and amplifica- tion of both T- and B-cell responses. Thus, there exists a possibility for the activated T or B cells to attack self- TLR signaling and components (Deane and Bolland, 2006; Ehlers and Ravetch, 2007). It remains unclear to what extent TLR- Cancer immunotherapy based on the induction and activation will be able to overcome the multiple activation of tumor-specific CTLs in principle can strategies developed by tumors to evade vaccine-induced eradicate even metastatic tumor cells. CTL cytotoxicity antitumor immunity. can be mediated by different mechanisms:(1) indirect killing through the release of IFN-g and TNF- a; (2) induction of apoptosis by FasL expressed on the CTL interaction with Fas on the target cell and (3) direct Expression of TLRs by tumor cells killing by secretion of perforin and granzymes into the intercellular space (Andersen et al., 2006). Regrettably, The importance of TLR for tumor immunity is evident antitumor , which impairs the ther- in an increasing body of evidence that TLR-variants are apeutic effect of antitumor vaccines, is usually induced by associated with cancer risk. Zheng et al. (2004) first tumor-derived factors or tumor-associated suppressor reported and Chen et al. (2005) and Cheng et al. (2007) cells (Khazaie and von Boehmer, 2006; Li et al., 2006; confirmed that sequence variants of TLR4 are associated Lizee et al., 2006; Serafini et al., 2006; Munn and Mellor, with prostate cancer risk. Subsequently, variants of 2007). Microbial products have been utilized as adjuvants TLR1, TLR6 and TLR10 were also confirmed to to potentiate antitumor immune responses, because of the increase prostate cancer risk (Sun et al., 2005). Sequence pioneering work of William Coley with Coley’s toxin. We variants of TLR4 and TLR10 are associated with now understand that such adjuvants work by stimulating nasopharyngeal risk (Song et al., 2006; Zhou TLR signaling and activating both innate and adaptive et al., 2006). The risk of gastric carcinoma is increased by immune responses to enhance tumor immunotherapy. a functional polymorphism of TLR4 (Ohara et al., 2006; Several TLR agonists have been demonstrated to Hold et al., 2007). The etiology of some produce antitumor effects. Okamoto et al. (2006) used subtypes is connected to single-nucleotide polymorph- Streptococcal agent OK-432 to activate TLR4 signaling, isms in TLR1, TLR2, TLR4, TLR5 and TLR9 (Nieters whichresultedinanIFN-g-mediated antitumor immune et al., 2006). response. Whitmore et al. (2001, 2004) reported that Toll-like receptors are expressed on cells of the immune double-stranded RNA and CpG DNA can synergistically system but we have also found that TLRs are also expressed activate innate immunity leading to enhanced antitumor on tumor cells, where they may influence tumor growth activity. Current efforts to use TLR agonists to enhance and host immune responses (Huang et al., 2005). We tumor immunity are largely focused on TLR7 and TLR9 screened murine tumor cell lines of different tissue-origin (Lee et al., 2003; Craft et al., 2005; Pashenkov et al., 2006; including MC26 (colon), 4T1 (breast), RM1 (prostate), B16 Krieg, 2007). TLR7 and TLR9 agonists, nucleic acid () and LLC1 (lung) for the expression of TLRs analogues improve antitumor immunity in or (including TLR1-6 and -9) by reverse transcriptase–PCR

Oncogene TLR signaling by tumor and immune cells B Huang et al 220 (RT–PCR). All the tumor cell lines expressed multiple tumor growth and chemoresistance in ovarian cancer TLRs (Huang et al., 2005). We also screened five human cells. (Yuan et al., 2003; Dan et al., 2004a, b) report that tumor cell lines including HCT15 and SW620 (colon), activation of TLR4 in ovarian cancer cells results in a MCF7 (breast), UACC-62 (melanoma) and MDA-MB435 significant increase of X-linked inhibitor of apoptosis and (breast). The human tumor cell lines also expressed multiple phosphorylated Akt. The inhibition of tumor-cell apop- TLRs. TLR9 was most strongly expressed on human tumor tosis by TLR-signaling is also observed in lymphoma lines rather than TLR4, as in murine tumors (unpublished cells and lung cancer cells (Jego et al., 2006; He et al., data). Similar results were reported by others. Human lung 2007). The highly invasive MDA-MB-231 breast cancer cancer cells express functionally active TLR9 (Droemann cell line expresses TLR9, which when activated promotes et al., 2005); human gastric carcinoma cells express TLR4, MDA-MB-231 cell invasion by increasing the activity of TLR5 and TLR9 (Schmausser et al., 2005); human 13 (MMP13), but not MMP8 laryngeal carcinoma cells express TLR2, TLR3 and TLR4 (Merrell et al., 2006). Moreover, two earlier studies (Szczepanski et al., 2007); human cervical tumor cells demonstrated that lipopolysaccharides may also promote express TLR9 (Lee et al., 2007); human NB-1 neuroblas- tumor invasion through the lipopolysaccharides-acti- toma cells express an intracellular form of TLR4 (Hassan vated NF-kB pathway resulting in upregulation of iNOS et al., 2006); human prostate-cancer cells express TLR9 and MMP2 and the b1 subunit (Harmey et al., (Ilvesaro et al., 2007) and human melanoma cell lines 2002; Wang et al., 2003). Thus, activation of TLR4 express TLR4 (Molteni et al., 2006). In addition, many expressed by tumor cells results in adherence to extra- reports indicate that TLRs are expressed on leukemia cells cellular matrix and endothelial cells promoting invasion (Bohnhorst et al., 2006; Jego et al., 2006), which is expected and metastasis. In addition, a new in vitro study indicates since leukemic cells are the immune cells derived from bone that TLR9 agonists can stimulate prostate cancer marrow. The general expression of TLRs by tumor cells invasion by increasing MMP13 activity (Ilvesaro et al., suggests that TLR-signaling may play an important role in 2007). tumor development.

What are the endogenous ligands for tumor cell TLRs? Tumor cell TLR-signaling and tumor progression We and others have shown by using microbial TLR Activation of TLR expressed on tumor cells may have ligands that tumors express a variety of functionally profound consequences for tumor growth. Tumor active TLRs but it is not clear what the ligands in the immune evasion may be facilitated by inhibitory tumor microenvironment are (Huang et al., 2005). We cytokines, inflammatory factors, proteinases, and other have demonstrated that L. monocytogenes directly affects small molecules such as nitric oxide. The sources of such tumor cells and promotes tumor cell growth through the tumor-promoting factors are still poorly understood. We TLR2-signaling pathway in vivo (Huang et al., 2007) but stimulated tumor cells with lipopolysaccharides, the the endogenous ligands (that is, TLR ligands apart from prototypical ligand for TLR4, and found that tumors those of microbial origin) for tumor cell TLRs have not produce proinflammatory factors including nitric oxide, been identified (Berg, 2002; Tsan and Gao, 2004; Rifkin IL-6 and IL-12 mimicking some characteristics of et al., 2005). Identification of endogenous ligands is of inflammatory cells. These factors result in resistance of obvious interest for tumor biology, but will also be tumor cells to CTL and attack and pertinent to dissect the role of TLRs in autoimmune evasion from immune surveillance (Huang et al., 2005). diseases such as systemic lupus erythematosus and These effects can be attributed to TLR4 expression since chronic sterile inflammatory disorders such as athero- blockade of the TLR4 pathway by either TLR4 siRNA sclerosis and arthritis (Xu, 2002; Liu-Bryan et al., 2005; or a cell-permeable TLR4 inhibitory peptide delays Marshak-Rothstein, 2006; Sioud, 2006). tumor growth and thus prolongs the survival of tumor- Heat-shock (HSP) such as HSP60, HSP70 and bearing mice. In another system, we show that Listeria HSP90 from a variety of sources induce the production of monocytogenes infection of a local tumor can promote proinflammatory cytokines such as TNF-a, IL-1, IL-6 tumor growth via the TLR2-signaling pathway, which and IL-12; the release of nitric oxide and C–C leads to the production of immunosuppressive molecules by monocytes, macrophages and DCs (Asea such as nitric oxide and IL-6 by tumor cells (Huang et al., 2000; Kol et al., 2000; Singh-Jasuja et al., 2000). et al., 2007). Thus, under some conditions, TLRs HSP also induce the maturation of DCs as demonstrated expressed by tumor cells do play a role in regulating by the upregulation of MHC class I and II molecules and tumor growth. co-stimulatory molecules such as CD80 and CD86. Using Recent evidence suggests that TLRs also contribute to TLR4 mutant mice and TLR2 KO mice, these functions tumor-cell resistance to apoptosis and increased invasive- of HSPs have been shown to proceed through TLR4- or ness. Kelly et al. (2006) find that activation of TLR4 TLR2-dependent pathways. Therefore, HSP60, HSP70 signaling promotes the growth and chemoresistance of and HSP90 are putative endogenous ligands for TLR4 epithelial ovarian cancer cells. The expression of X-linked and TLR2 (Tsan and Gao, 2004; Rifkin et al., 2005). In inhibitor of apoptosis, a major inhibitor of caspase-3 and addition, a recent study indicated that a small HSP -9 and the expression of Akt have been associated with (HSP22) may also act as an endogenous TLR4 ligand in

Oncogene TLR signaling by tumor and immune cells B Huang et al 221 pathways involved in inflammation. In tumors both apoptosis and necrosis occur releasing proteins such as high-mobility group box 1 protein and HSPs that are ligands for TLR2 and TLR4. However, in addition RNA– and DNA– nucleoprotein complexes could also be released, which could also constitute ligands for TLR activation. Chromatin and DNA immune complexes have been proposed as endogenous ligands for TLR9 (Boule et al., 2004; Barrat et al., 2005), and mRNA may be an endogenous ligand for TLR3 (Kariko et al., 2004). We propose that DNA– and RNA–protein complexes released from dying cells act as tumor-facilitating factors by paracrine activation of tumor cell TLR3, TLR7, TLR8 and TLR9. Identification of the endogenous ligands for tumor TLRs would further make us to Figure 1 Expression of TLRs in murine tumor cell lines. understand the mechanisms for tumor cell growth and Expression of TLRs (1-6, 9) in different mouse tumor cell lines was analyzed by RT–PCR. escape from the host defense system. However, exclud- ing microbial contamination such as lipopolysacchar- ides is a major concern in attempting to identify the pathogenesis of rheumatoid arthritis (Roelofs et al., endogenous TLR ligands (Tsan and Gao, 2004, 2007). 2006). Other molecules besides HSP have been identified as potential TLR ligands including fibrinogen (Smiley et al., 2001), surfactant protein-A (Guillot et al., 2002), domain A of fibronectin (Okamura et al., 2001), heparan Conclusion sulfate (Johnson et al., 2002), hyaluronan oligosaccharide (soluble hyaluronan) (Termeer et al., 2002), b-defensin 2 The activation of TLRs is required for host defense (Biragyn et al., 2002) and high-mobility group box 1 against invading microbes and TLR ligands may be protein (Park et al., 2004). These endogenous ligands utilized as effective immuno-adjuvants in tumor im- were characterized with murine cells and are ligands for munotherapy or combined tumor therapy. However, TLR2 and/or TLR4. These observations are significant TLR activation may be a two-edged sword, with both since:(1) murine-tumor cells highly express TLR4 antitumor and pro-tumor consequences. The general (Figure 1); (2) fibrinogen, fibronectin, heparan sulfate expression by tumor cells of functionally active TLRs and hyaluronan are critical components of the tumor and the broad distribution of putative endogenous and all-cause tumor progression ligands suggest tumor cell TLR signaling may be (Rybarczyk and Simpson-Haidaris, 2000; Sanderson continually activated and contribute to tumor progres- et al., 2004; Gotte and Yip, 2006; Liu et al., 2007); (3) sion. Understanding TLR function in tumor biology surfactant protein-A is expressed in lung cancer (Camilo may lead to discovery of new therapeutic targets in et al., 2006; Iino et al., 2007); (4) b-defensin 2 possesses a cancer therapy. strong bactericidal effect on Gram-negative bacteria (Ganz and Weiss, 1997) and is expressed by oral cancer cells (Abiko et al., 1999); (5) although HSPs and high- Acknowledgements mobility group box 1 protein are intracellular proteins, they can be passively released from dead cells (Ciocca and Dr Huabao Xiong was supported by NIH P01 DK72201, a Crohn’s and Colitis Foundation of America, a grant from the Calderwood, 2005; Ellerman et al., 2007) and therefore Eli and Edythe L Broad Foundation. Dr Zuo-Hua Feng was could be present in the tumor microenvironment. supported by National Development Program (973) For Key Apoptosis or programmed cell death, necessary for Basic Research (2002CB513100) of China. Dr Jay Unkeless tissue remodeling and removal of cells that fail to repair was supported by a Crohn’s and Colitis Foundation of a damage, is tightly regulated by many of the same America Grant.

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