TLR7 and TLR8 As Targets in Cancer Therapy

MP Scho¨ n and M Scho¨ n

Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine and Department of Dermatology, Julius-Maximilians- University, Wu¨rzburg, Germany

Small-molecule agonists at Toll-like receptor 7 (TLR7) expressed at the cell surface, both TLR7 and TLR8 are and TLR8 have sparked a vivid interest in cancer research localized intracellularly to endosomal membranes, similar owing to their profound antitumoral activity. The lead to TLR3 and TLR9 (Lee et al., 2003; Nishiya and compound of the imidazoquinoline family, imiquimod, is DeFranco, 2004; Miggin and O’Neill, 2006). These four marketed as a topical formulation. It is efficacious against intracellular receptors represent a group of so-called many primary skin tumors and cutaneous metastases. antiviral TLRs. However, the molecular mechanisms Using different imidazoquinoline species, distinct functions guiding endosomal localization of TLR3 and TLR7 of TLR7 and TLR8 have been discovered. The predomi- appear to be distinct inasmuch as TLR3 is targeted nant antitumoral mode of action of these agents is TLR7/ intracellularly by a sequence of 23 amino acids within the 8-mediated activation of the central transcription factor linker region between the TIR domain and the trans- nuclear factor-jB, which leads to induction of proinflam- membrane domain, while TLR7 localizes intracellularly matory cytokines and other mediators. Cutaneous den- via its transmembrane domain (Nishiya et al., 2005). Both dritic cells are the primary responsive cell type and initiate TLR7 and TLR8 have been identified as natural a strong Th1-weighted antitumoral cellular immune receptors for single-stranded RNA, and they are thought response. Recent research has shown that dendritic cells to act as potent activators of innate immune responses themselves acquire direct antitumoral activity upon upon viral infections. Sequence specificities of TLR7 and stimulation by imiquimod. In addition, there are a number TLR8 have not been conclusively defined yet, but ssRNA of secondary effects on the molecular and cellular level sequences containing GU-rich or poly-U sequences can that can be explained through the activation of TLR7/8. stimulateboth receptors (Diebold et al., 2004; Heil et al., The proinflammatory activity of imiquimod, but not 2004; Lund et al., 2004). In addition, siRNA sequences resiquimod, appears to be augmented by suppression of a containing an internal 50-UGUGU-30 motif appear to regulatory mechanism, which normally limits inflamma- bind to TLR7/8 (Judge et al., 2005). Certain siRNA tory responses. This is achieved independently of TLR7/8 sequences may even stimulate TLR7/8 independently of through interference with adenosine receptor signaling their GU content suggesting specific sequences recognized pathways. Finally, at higher concentrations imiquimod by TLR7/8 (Hornung et al., 2005). The level of nucleoside exerts Bcl-2- and caspase-dependent proapoptotic activity modification appears to be a discriminating feature for against tumor cells. TLR7 and TLR8 to distinguish between viral and Oncogene (2008) 27, 190–199; doi:10.1038/sj.onc.1210913 mammalian ssRNA (Kariko et al., 2005). The scientific and clinical interest in TLR7 and TLR8 Keywords: TLR7; TLR8; skin cancer; imiquimod; for cancer biology has originated from the antitumoral imidazoquinolines; cancer immunity activity of somesmall-moleculecompounds (Sidky et al., 1992) which have later been shown to act as agonists at one or both receptors (Hemmi et al., 2002; Jurk et al., 2002). Indeed, together with the study of genetically engineered mice (MyD88 or TLR deficient) TLR7, TLR8 and small-molecule agonists (Gibson et al., 2002; Hemmi et al., 2002) many functions of TLR7 and TLR8 have been unraveled primarily TLR7 and TLR8 functions through studies of the mode of action of such small Toll-like receptor 7 (TLR7) and TLR8 are phylogeneti- molecule compounds, imidazoquinolines in particular cally and structurally closely related members of the TLR (Gorden et al., 2005; Jurk et al., 2006; Gorden et al., family; togetherwith TLR9 theyconstituteoneof thesix 2006b; Ma et al., 2007). Theintracellular localization of major TLR clades (Roach et al., 2005). With the TLR7 and TLR8 usually necessitates cellular uptake of exception of a small proportion of TLR8 which is drugs targeting these receptors.

Correspondence: Professor MP Scho¨ n, Rudolf Virchow Center, DFG Small-molecule agonists at TLR7 and TLR8 Research Center for Experimental Biomedicine and Department of Dermatology, Bayerische Julius-Maximilians-University, Versbacher Imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4, Str. 9, Wu¨ rzburg 97078, Germany. 5-c]quinolin-4-amine), is a nucleoside analogue with a E-mail: [email protected] molecular mass of Mr ¼ 240.3. It is theleadcompound TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 191 of the imidazoquinoline family and has been studied receptors. Indeed, these recent studies have been instru- most extensively. Most of the findings concerning the mental for dissecting distinct cytokine induction profiles antitumoral modeof action of TLR7/8 agonists, clinical and other functions of both receptors (Gorden et al., efficacy and safety reviewed here have been obtained 2005, 2006a, b; Gorski et al., 2006; Jurk et al., 2006). with imiquimod (Scho¨ n and Scho¨ n, 2007). Imiquimod In thefirst study of that kind it was found that the activates preferentially TLR7; its agonistic activity at specific TLR7 agonist (3M-001, (N-[4-(4-amino-2-ethyl- TLR8 appears to be much weaker (Lee et al., 2003). 1H-imidazo[4,5-c]quinolin-1-yl)butyl-]methanesulfonamide) Resiquimod (R848; 4-amino-2-(ethoxymethyl)-a,a-di- primarily activated plasmacytoid dendritic cells, B cells methyl-1H-imidazo[4,5-c]quinoline-1-ethanol), Mr ¼ 314.4) and monocytes, while a TLR8 agonist (3M-002, CL075; is a selectiveligand for TLR7 in themurineand for TLR7 2-propylthiazolo[4,5-c]quinolin-4-amine) induced cytokine and TLR8 in the human system. Resiquimod induces more production by myeloid dendritic cells, monocytes and pronounced cytokine secretion, macrophage activation and monocyte-derived dendritic cells (Gorden et al., 2005). enhancement of cellular immunity as compared to Accordingly, the TLR7 agonist preferentially induced imiquimod (Wagner et al., 1997, 1999; Imbertson et al., secretion of IFNa and IFN-regulated chemokines, such 1998; Bernstein et al., 2000; Burns et al., 2000). as I-TAC (CXCL11) and IP-10 (CXCL10), whilethe Gardiquimod (1-(4-amino-2-ethylaminomethylimidazo- TLR8 agonist predominantly induced proinflammatory [4,5-c]quinolin-1-yl)-2-methylpropan-2-ol; Mr ¼ 313.4) is cytokines and chemokines including TNFa, IL-12 and another imidazoquinoline derivative that, similar to MIP-1a (CCL3) (Gorden et al., 2005). These observations imiquimod, induces activation of nuclear factor (NF)-kB are consistent with the known activities of imiquimod, in cells expressing human or murine TLR7. However, which also acts predominantly as activator of TLR7 basedondataderivedfromHEK293cellsexpressing(Gibson et al., 2002), and indicated differential target human or mouse TLR7 gardiquimod is approximately ten cell selectivity of TLR7 and TLR8 agonists as well as times more potent as compared to imiquimod. At high distinct cytokine induction profiles of the two receptors concentrations (that is, X3 mgmlÀ1), gardiquimod also (Gorden et al., 2005). Theactivity of imidazoquinoline activates TLR8. agonists at TLR7 and TLR8, respectively, can be Another commercially available compound with the modulated by modified oligodeoxynucleotides. Interest- thiozoloquinolonebackbonestructureis CL075 (3M-002), ingly, such treatments inhibit TLR7 while they enhance a compound of Mr ¼ 243.3 that preferentially stimulates TLR8 signaling induced by the respective agonists (Jurk TLR8 in human PBMC. It has been demonstrated recently et al., 2006; Gorden et al., 2006b). In addition, the that CL075 induces secretion of TNFa and IL-12 by functionality of murine TLR8 has first been demonstrated human PBMC through agonistic activity at TLR8. In by using a small-molecule TLR8 agonist (3M-002) together addition, although to a markedly lesser extent, the with polyT oligodeoxynucleotides (Gorden et al., 2006a). compound activates TLR7. Based upon studies using Of particular relevance for antitumoral immune responses is transfected HEK293 cells, concentrations of CL075 the differential triggering of NK cell activity by imidazo- required for activation of TLR7 are approximately 10-fold quinolines inasmuch as TLR8 agonists showed more higher than those needed for TLR8 stimulation. In contrast pronounced indirect NK cell activation as compared to to CL075, CL097 (Mr ¼ 242.3) predominantly activates TLR7 agonists (Gorski et al., 2006). However, direct TLR7. Again using transfected HEK293 cells it was found interaction of any of the compounds with TLRs has not that CL097 induces TLR7-mediated activation of NF-kB been demonstrated (yet). at concentrations of 0.1 mgmlÀ1,while1.0mgmlÀ1 are required to induce NF-kB activation in a TLR8-dependent fashion (Gorden et al., 2005; Gorski et al., 2006; Levy et al., 2006; Qin et al., 2006; Wille-Reece et al., 2006). Cellular events induced by small-molecule agonists at The L-nucleoside loxoribine (Mr ¼ 339.3) is a guano- TLR7/8 sine analog derivatized at positions C8 and N7. This compound does not share the basic imidazoquinoline Modulation of inflammatory mediators by imiquimod and structure with the aforementioned agents. The activa- related compounds tion of theinnateimmunesystemthrough loxoribineis, Already thefirst scientific investigations into themodeof similarly to imiquimod, largely restricted to TLR7 (Pope action of imiquimod and numerous studies since have et al., 1995; Heil et al., 2003; Lee et al., 2003). demonstrated that the compound stimulates the produc- The delineated small-molecule agonists at TLR7 and tion and secretion of proinflammatory cytokines, which TLR8 areschematically summarizedin Figure1. then induce a profound tumor-directed cellular immune response (Reiter et al., 1994; Stanley, 2002). Given that dendritic cells produce inflammatory cytokines in re- Dissecting functions of TLR7 and TLR8 by sponse to markedly lower concentrations of imiquimod small-molecule agonists as compared to other cells, they are the primary Following thediscoverythat imiquimod and resiquimod responsive cell type (Reiter et al., 1994; Gibson et al., somewhat differentially activate TLR7 and TLR8 1995, 2002; Megyeri et al., 1995). In most experimental signaling, respectively (Hemmi et al., 2002; Jurk et al., studies in vitro, imiquimod concentrations of up to 2002), several other compounds of the imidazoquinoline 5 mgmlÀ1 elicited a robust proinflammatory response by family have been studied regarding their activity at both dendritic cells. A number of additional imidazoquinolines

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 192

Figure 1 Agonistic activity of small-molecule compounds at Toll-like receptor 7 (TLR7) and/or TLR8. Thickness of arrows corresponds to preferential activity. Imiquimod activated TLR7 in the murine and TLR7 and TLR8 in the human system.

(some have been mentioned above) have been tested results in the activation of central transcription factors, in vitro for their capacity to inducecytokineand NF-kB (nuclear factor-kappa B) in particular. Under chemokine production, and for some species effective resting conditions, the (hetero)-dimeric NF-kBiskept concentrations of as low as 0.05 mgmlÀ1 havebeen inactivewithin thecytoplasm through binding to the described (Gerster et al., 2005). In addition, it has been inhibitory factor, IkB. Following receptor-mediated sti- shown in vivo that topically applied imiquimod induces mulation, IkB is phosphorylated through the IKK functional maturation of epidermal Langerhans cells and complex. This phosphorylation results in release, activa- stimulates migration of these antigen-presenting cells to tion and nuclear translocation of NF-kB, where it induces regional lymph nodes where they presumably promote a transcription of a multitudeof genes,including thoseof speciﬁc T-cell response (Stanley, 2002). cytokines, chemokines, adhesion molecules and apoptosis- Stimulation of TLR7- or TLR8-mediated signaling related proteins (Karin, 2006). As demonstrated in large pathways through imiquimod or other imidazoquinolines gene expression array analyses, topical treatment of

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 193 cutaneous tumors or virally induced skin lesions with indication for a direct melanocyte-directed immune imiquimod results in markedly elevated expression of reaction, localized vitiligo (depigmentation due to an numerous gene products involved in the regulation of autoimmune reaction against melanocytes) was ob- innateimmunefunctions (Buatesand Matlashewski,2001; served. Another TLR-dependent immunomodulatory Dummer et al., 2003a). To namebut a few,imiquimod effect of imiquimod is the induction of 2050-oligoadenyl- induces expression of proinflammatory cytokines includ- synthase, which is associated with stimulation of NK ing IFNa,TNFa, IL-2, -6, -8, -12, G-CSF and GM-CSF, (natural killer) cells, immunocytes that are pivotally as well as chemokines such as CCL3 (MIP-1a), CCL4 involved in antiviral and antitumoral immune responses (MIP-1b) and CCL2 (MCP-1) (Reiter et al., 1994; Weeks (Navi and Huntley, 2004). However, compounds activat- and Gibson, 1994; Gibson et al., 1995; Megyeri et al., ing preferentially TLR8 may even prove superior with 1995; Wagner et al., 1997). In addition to theNF- kB- regard to NK cell activation (Gorski et al., 2006). Given mediated transcription of proinflammatory mediators, it that imiquimod has been shown to induce perforin in appears that TLR-7- (and TLR-8)-agonistic activities of cytotoxic T cells under experimental conditions in vitro imiquimod inducesomeproinflammatory cytokines,such (Ambach et al., 2004) it is conceivable that imiquimod as IFNg,inaNF-kB-independent fashion. The known exerts even broader (pleiotropic) stimulatory effects on T functions of these mediators explain, at least in part, many cells and other immune cells. cellular responses to imiquimod including activation and Based upon the activation of dendritic cells through application-site-directed migration of dendritic cells and imiquimod, it has been hypothesized that the compound their precursors as well as attraction and stimulation of elicits an indirect vaccination effect, thus leading to cytotoxic T-lymphocytes and other immune cells. Along prolonged Th1-weighted immune responses against this line, it has been demonstrated that plasmacytoid tumors and virally infected cells (Harrison et al., 1994; dendritic cells accumulate in subcutaneous melanoma Bernstein et al., 1995; Burns et al., 2000; Suzuki et al., metastases in a mouse model (Palamara et al., 2004) and 2000). However, untreated tumors in the vicinity of that plasmacytoid predendritic cells migrate into the skin tumors that responded well to imiquimod did not show (Urosevic et al., 2005) following application of imiquimod. signs of inflammation or regression, and experimental The net result of the imiquimod-induced cytokine evidence to support this intriguing hypothesis has not production is a Th1-weighted cellular immune response, been published yet. whileTh2 signaling pathways appearto besuppressed. In addition to the indirect stimulation of lymphocytes The latter activity is exemplified by IFNg-dependent and NK cells via activation of dendritic cells, a recent downregulation of IgE-synthesis by resiquimod (Frotscher study has demonstrated elegantly that imiquimod- et al., 2002). Other indirect biological effects that can be treated dendritic cells themselves presumably acquire explained by the TLR-dependent activation of NF-kBby direct antitumoral functions in vivo (Stary et al., 2007). imiquimod and subsequent expression of IFNa,IL-1and In this study, peritumoral myeloid dendritic cells in the other mediators include enhanced expression of the opioid vicinity of imiquimod-treated basal cell carcinomas growth factor receptor on tumor cells and cells of the expressed perforin and granzyme B, and tumor-infiltrat- inflammatory infiltrate(Urosevic et al., 2004) as well as ing plasmacytoid dendritic cells expressed TRAIL. changes in the epidermal barrier function (Barland et al., In vitro, stimulation of myeloid dendritic cells by 2004). imiquimod led to lysis of MHC class Ilo tumor cells by these cells, and plasmacytoid dendritic cells acquired the ability to induceapoptosis in Jurkat cellsthrough Cellular immune reactions induced by imidazoquinolines TRAIL signaling (Stary et al., 2007). It is conceivable Strong tumor-directed cellular immune responses have that this novel dendritic cell-based antitumoral mechan- been observed in most skin tumors treated topically with ism contributes to the apoptosis of imiquimod-treated imiquimod (Sullivan et al., 2003). These results are tumors described earlier (Scho¨ n et al., 2003). consistent with and in support of observations that Overall, although some aspects of the TLR-dependent imiquimod strongly augmented the mounting of a activity of imiquimod and related compounds are not cellular immune response in cutaneous vaccinations with entirely clear yet, there is compelling evidence for the nontumoral antigens (Lore et al., 2003; Thomsen et al., notion that imiquimod—through stimulation of anti- 2004; Zuber et al., 2004), thus employing the same tumoral T cells following activation of antigen present- principleas otheradjuvants which utilizeTLR-depen- ing cells as well as through direct antitumoral activation dent signaling cascades in order to enhance activation of of dendritic cell subsets—exerts its activity primarily at cytotoxic T cells (Schwarz et al., 2003). Imiquimod- the interface between innate and acquired immunity enhanced activation and immigration of cytotoxic T cells (Hurwitz et al., 2003; Urosevic et al., 2003). has been observed in vaccination studies with melanoma antigens (Shackleton et al., 2004). Moreover, recent in vivo data have demonstrated that imiquimod strongly enhances antigen-specific activation of antitumoral T Antitumoral efficacy of TLR7/8 agonists in clinical trials cells (Rechtsteiner et al., 2005; Prins et al., 2006) and augments protective cellular antitumoral immunity Imidazoquinolines were originally developed in order to against melanoma cells following vaccination with generate antiviral agents. Although these substances did Listeria (Craft et al., 2005). As a conspicuous clinical not exert direct antiviral activities, some of them showed

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 194 considerable indirect efficacy against virally induced exceptions which cannot be explained easily by TLR7- lesions through TLR7/8-mediated induction of cytokines or TLR8-dependent mechanisms and whose molecular (Chen et al., 1988; Harrison et al., 1988; Gerster et al., basisisnotentirelyclearyet. 2005). Following thedemonstrationof profound anti- Under experimental conditions, there is some evidence tumoral activities in preclinical models (Sidky et al., 1992), suggesting that imiquimod can enhance antibody pro- imidazoquinolines, the lead compound imiquimod in duction by B cells (Hengge and Ruzicka, 2004), a particular, havemovedinto thelimelight of clinical and function that is typically governed through a Th2- experimental oncology. The small size of imiquimod, its dominated microenvironment. It appears that imiqui- hydrophobicity and ability to penetrate the epidermal mod and related compounds exert a direct stimulatory barrieron theonehand, and, on theotherhand, effect on antibody production inasmuch as they mimic considerable unwanted side effects when the compound CD40-dependent signals, which are normally sent by T is applied systemically, led to the development of a topical cells (Bishop et al., 2001). In addition, imidazoquinolines formulation (Aldara 5% cream) for the treatment of viral can stimulate the proliferation of cultured B cells, even in lesions (HPV papillomas) and malignant tumors of the the absence of other immunocytes, although the increase skin. Imiquimod was first approved for the treatment of in B-cell proliferation was more pronounced when genital warts and is usually well tolerated (Gollnick et al., preactivated cultures were exposed to imiquimod (Tomai 2001). In addition, several controlled clinical trials as well et al., 2000). However, resiquimod suppressed IgE as many smaller series of cases and case reports have production in an IFNg-dependent fashion (Frotscher demonstrated that imiquimod is also effective against a et al., 2002), an activity that needs to be reconciled with variety of primary skin cancers as well as cutaneous theputativeCD40-mimicking activity in futurestudies. metastases of some malignancies. Cutaneous tumors that Recent research has demonstrated that imiquimod have responded well to topical treatment with imiquimod can activateNF- kB and increase expression of some include basal cell carcinomas (Sterry et al., 2002; Bath- proinflammatory cytokines (IL-6, IL-8, TNFa and IL- Hextall et al., 2004; Geisse et al., 2004; Gollnick et al., 1b) in TLR-7- and TLR-8-negative cell lines (Scho¨ n 2005; Schulze et al., 2005), keratoacanthomas (Dendorfer et al., 2006). In addition, imiquimod increased IL-1b et al., 2003; Peris et al., 2003), actinic keratoses (Stockfleth mRNA in mononuclear cells from mice deficient for et al., 2001, 2002; Lebwohl et al., 2004; Szeimies et al., MyD88 (myeloid differentiation factor 88, an adaptor 2004; Korman et al., 2005) and Bowen’s disease (the latter molecule essential for TLR-mediated signaling), albeit two entities represent epidermal carcinoma in situ)(Patel at lower levels than in the counterpart cells from wild- et al., 2006; Peris et al., 2006), cutaneous metastases of type mice. Competitive radioligand binding studies have melanoma (Steinmann et al., 2000; Bong et al., 2002; shown that imiquimod binds to adenosine receptors Ugurel et al., 2002; Wolf et al., 2003; Zeitouni et al., 2005), with highest affinity at the A2A receptor (Ki-value some cases of primary melanoma in situ (Fleming et al., 2.16 mM), followed by the A1 receptor (Ki-value 2004; Kamin et al., 2005; Ray et al., 2005; Wolf et al., 2.94 mM) where it presumably acts as a partial antago- 2005; Lonsdale-Eccles et al., 2006) and cutaneous T-cell nist. In addition, a moderate but consistent receptor- lymphomas (Suchin et al., 2002; Dummer et al., 2003b; independent inhibition on adenylyl cyclase activity was Chong et al., 2004; Deeths et al., 2005). Clinical responses observed. Activity of resiquimod at adenosine receptors of cutaneous neoplasias to topical treatment with imiqui- could not be detected, however. Given that adenosine mod have also been observed in difficult-to-treat patient receptor-mediated signaling pathways can suppress populations, such as organ transplant patients under transcription of proinflammatory cytokines (Odashima immunosuppressive therapy (Smith et al., 2001; Prinz et al., 2005), it is conceivable that imiquimod interferes et al., 2004; Brown et al., 2005) or Xeroderma pigmento- with a negative feedback mechanism that normally sum patients suffering from rapid development of multiple contains inflammatory reactions. The net result of this UV-induced cutaneous malignancies (Giannotti et al., additional activity of imiquimod would bean augmen- 2003; Roseeuw, 2003). A recent meta-analysis of five tation of inflammation synergistically to the TLR- randomized, double blind clinical trials showed that mediated proinflammatory activity (Scho¨ n et al., 2006). approximately 50% of imiquimod-treated patients with actinic keratoses were cleared of their lesions, as compared to only 5% in the vehicle-treated group (Hadley et al., Proapoptotic activity of imiquimod 2006). Other imidazoquinolines, resiquimod in particular, In addition to its predominant activation of NF-kB, have also been assessed in preclinical and early clinical imiquimod modulates STAT-1 (signal transducer and trials (Miller et al., 2002; Sauder et al., 2003). activator of transcription-1) signaling pathways (Dock- rell and Kinghorn, 2001). This interaction may contributeto thetranscription of proinflammatory cytokines (Bottrell et al., 1999), but also to theinduction Off-target effects of imiquimod of apoptosis in various cell types (Stephanou and Latchman, 2005). In addition, imiquimod led to Other activities influencing immunological effects increased expression of the death receptor CD95 (Ber- Although imiquimod induces primarily a Th1-dominated man et al., 2003) and to decreased expression of the anti- cellular immune response under most clinical and experi- apoptotic protein Bcl-2 (Vidal et al., 2004) in somebasal mentalcircumstances,this rulehas someremarkable cell carcinomas in vivo. Thus, it is conceivable that

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 195 imiquimod enhances susceptibility of tumor cells to 2004) treated with imiquimod, an increased number of apoptotic stimuli, for example, to CD95L, TRAIL or apoptotic cells was detected as compared to the other death ligands, which can be produced by respective controls. However, it appears likely that neighboring tumor cells, infiltrating lymphocytes or in vivo additional factors, for example, infiltrating dendritic cells (Stary et al., 2007). This proapoptotic immunecells(Stary et al., 2007), contributeto the activity of imiquimod could be mediated by TLR- induction of apoptosis in imiquimod-treated tumors. dependent regulation of the respective apoptosis-related Theexactcontribution of apoptosis induction to the proteins; it would thus be indirect. overall antitumoral activity of imiquimod (and possibly However, imiquimod exerts an additional direct other TLR7/8 agonists) is not entirely clear at this time. proapoptotic activity against tumor cells (Scho¨ n et al., The in vivo situation is certainly complex and comprises 2003), a surprising observation that was confirmed various levels of interaction between multiple players in vivo in tumors of different origin (Sidbury et al., 2003; that are affected directly or indirectly by imiquimod Sullivan et al., 2003; Scho¨ n et al., 2004; Vidal et al., (and presumably other TLR7/8 agonists). 2004) as well as on the cellular level in vitro (Meyer et al., Overall, the predominant antitumoral activity of 2003). Whiletheproapoptotic activity of imiquimod is imidazoquinolines is induction of proinflammatory independent of membrane-bound death receptors, shifts cytokines achieved through agonistic activity at TLR- of Bcl-2 proteins in favor of the proapoptotic Bax were 7- and TLR-8-mediated signaling cascades, and con- observed. Bcl-2 overexpression rendered tumor cells secutively, activation of NF-kB. This activity stimulates resistant against imiquimod-induced apoptosis (Scho¨ n theproduction of proinflammatory cytokinesand et al., 2003, 2004). In addition, imiquimod-induced chemokines resulting in a profound Th1-weighted direct apoptosis of tumor cells could be abrogated by antitumoral cellular immune response and direct anti- inhibition of caspaseactivation. Theproapoptotic tumoral capacities of dendritic cells. Several secondary activity of resiquimod was markedly less pronounced effects can also be explained through the TLR7/ as compared to imiquimod (Scho¨ n et al., 2003). 8-dependent activation of NF-kB. In addition, off- Imiquimod-induced apoptosis occurred (cell type-de- target activities of imiquimod (and presumably other pendent) at rather high concentrations of approximately compounds) include interference with adenosine recep- 25–50 mgmlÀ1, that is, five- to ten-fold higher than those tor signaling pathways and direct proapoptotic activity required for TLR-mediated cytokine induction in against tumor cells. dendritic cells. However, these concentrations are approximately 3 logs below the marketed formulation Acknowledgements (Aldara 5% cream). Thus, it is reasonable to assume that the proapoptotic activity contributes to the overall This work was supported by a research grant from the Dr antitumoral effect of imiquimod in vivo. Indeed, in both Mildred Scheel Stiftung/Deutsche Krebshilfe (10-2196 Scho¨ -2) basal cell carcinomas (Scho¨ n et al., 2003; Vidal et al., to MS and MPS, and by a Rudolf Virchow Award from the 2004) and cutaneous melanoma metastases (Scho¨ n et al., Deutsche Forschungsgemeinschaft to MPS.

References

Ambach A, Bonnekoh B, Nguyen M, Scho¨ n MP, Gollnick H. resiquimod mimics CD40-induced B cell activation. Cell (2004). Imiquimod, a toll-like receptor-7 agonist, induces Immunol 25: 9–17. perforin in cytotoxic T lymphocytes in vitro. Mol Immunol Bong AB, Bonnekoh B, Franke I, Scho¨ n MP, Ulrich J, 40: 1307–1314. Gollnick H. (2002). Imiquimod, a novel immune response Barland CO, Zettersten E, Brown BS, Ye J, Elias PM, modifier, in the treatment of cutaneous metastases of Ghadially R. (2004). Imiquimod-induced interleukin-1 malignant melanoma. Dermatology 205: 135–138. alpha stimulation improves barrier homeostasis in aged Bottrell RL, Yang YL, Levy DE, Tomai MA, Reis LF. murine epidermis. J Invest Dermatol 122: 330–336. (1999). The immune response modifier imiquimod requires Bath-Hextall F, Bong F, Perkins W, Williams H. (2004). STAT-1 for induction of interferon, interferon-stimulated Interventions for basal cell carcinoma of the skin: systematic genes, and interleukin-6. Antimicrob Agents Chemother 43: review. Br Med J 329: 705. 856–861. Berman B, Sullivan TP, De Araujo T, Nadji T. (2003). Brown VL, Atkins CL, Ghali L, Cerio R, Harwood CA, Proby Expression of Fas-receptor on basal cell carcinomas after CM. (2005). Safety and efficacy of 5% imiquimod cream for treatment with imiquimod 5% cream or vehicle. Br J the treatment of skin dysplasia in high-risk renal transplant Dermatol 149(Suppl. 66): 59–61. recipients: randomized, double-blind, placebo-controlled Bernstein DI, Harrison CJ, Tepe ER, Shahwan A, Miller RL. trial. Arch Dermatol 141: 985–993. (1995). Effect of imiquimod as an adjuvant for immunother- Buates S, Matlashewski G. (2001). Identification of genes apy of genital HSV in guinea pigs. Vaccine 13: 72–76. induced by a macrophage activator, S-28463, using gene Bernstein DI, Harrison CJ, Tomai MA, Miller RL. (2000). expression array analysis. Antimicrob Agents Chemother 45: Daily or weekly therapy with resiquimod (R848) reduces 1137–1142. genital recurrences in herpes simplex virus-infected guinea Burns R, Ferbel B, Tomai MA, Miller RL, Gaspari A. (2000). pigs during and after treatment. J Infect Dis 183: 844–849. Theimidazoquinolines,imiquimod and R-848, induce Bishop GA, Ramirez LM, Baccam M, Busch LK, Pederson functional, but not phenotypic, maturation of human LK, Tomai MA. (2001). The immune response modifier epidermal Langerhans cells. Clin Immunol 94: 13–23.

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 196 Chen M, Griffith BP, Lucia HL, Hsiung GD. (1988). Efficacy carcinoma following successful treatment with imiquimod of S-26308 against guinea pig cytomegalovirus infection. 5% cream: interim 2-year results from an ongoing 5-year Antimicrob Agents Chemother 32: 678–683. follow-up study in Europe. Eur J Dermatol 15: 374–381. Chong A, Loo WJ, Banney L, Grant JW, Norris PG. (2004). Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Imiquimod 5% cream in the treatment of mycosis Qiu X et al. (2005). Synthetic TLR agonists reveal functional fungoides—a pilot study. J Dermatolog Treat 15: 118–119. differences between human TLR7 and TLR8. J Immunol Craft N, Bruhn KW, Nguyen BD, Prins RM, Lin JW, Liau LM 174: 1259–1268. et al. (2005). The TLR7 agonist imiquimod enhances the anti- Gorden KB, Qiu X, Binsfeld CCA, Vasilakos JP, Alkan SS. melanoma effects of a recombinant Listeria monocytogenes 2006a. Cutting edge: Activation of murine TLR8 by a vaccine. JImmunol175: 1983–1990. combination of imidazoquinolineimmuneresponsemodi- Deeths MJ, Chapman JT, Dellavalle RP, Zeng C, Aeling JL. fiers and polyT oligodeoxynucleotides. J Immunol 177: (2005). Treatment of patch and plaque stage mycosis 6584–6587. fungoides with imiquimod 5% cream. J Am Acad Dermatol Gorden KB, Xiaohong Q, Battiste JJL, Wightman PPD, 52: 275–280. Vasilakos JP, Alkan SS. (2006b). Oligodeoxynucleotides Dendorfer M, Oppel T, Wollenberg A, Prinz JC. (2003). differentially modulate activation of TLR7 and TLR8 by Topical treatment with imiquimod may induce regression of imidazoquinolines. J Immunol 177: 8164–8170. facial keratoacanthoma. Eur J Dermatol 13: 80–82. Gorski KS, Waller EL, Bjornton-Severson J, Hanten JA, Riter CL, Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Kieper WC et al. (2006). Distinct indirect pathways govern (2004). Innate antiviral responses by means of single- human NK-cell activation by TLR-7 and TLR-8 agonists. Int stranded RNA. Science 303: 1529–1531. Immunol 18: 1115–1126. Dockrell DH, Kinghorn GR. (2001). Imiquimod and resiqui- Hadley G, Derry S, Moore RA. 2006. Imiquimod for actinic mod as novel immunomodulators. J Antimicrob Chemother keratosis: systematic review and meta-analysis. J Invest 48: 751–755. Dermatol 126: 1251–1255. Dummer R, Urosevic M, Kempf W, Hoek K, Hafner J, Burg G. Harrison CJ, Jenski L, Voychehovski T, Bernstein DI. (1988). (2003a). Imiquimod in basal cell carcinoma: how does it work? Modification of immunological responses and clinical Br J Dermatol 149(Suppl. 66): 57–58. disease during topical R-837 treatment of genital HSV-2 Dummer R, Urosevic M, Kempf W, Kazakov D, Burg G. infection. Antiviral Res 10: 209–224. (2003b). Imiquimod induces complete clearance of a PUVA- Harrison CJ, Miller RL, Bernstein DI. (1994). Posttherapy resistant plaque in mycosis fungoides. Dermatology 207: suppression of genital herpes simplex virus (HSV) recur- 116–118. rences and enhancement of HSV-specific T-cell memory by Fleming CJ, Bryden AM, Evans A, Dawe RS, Ibbotson SH. imiquimod in guinea pigs. Antimicrob Agents Chemother 38: (2004). A pilot study of treatment of lentigo maligna with 2059–2064. 5% imiquimod cream. Br J Dermatol 151: 485–488. Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, Frotscher B, Anton K, Worm M. (2002). Inhibition of Gellert T et al. (2003). TheToll-likereceptor7 (TLR7)-specific IgE production by theimidazoquinolineresiquimodin stimulus loxoribineuncoversa strong relationship within the nonallergic and allergic donors. J Invest Dermatol 119: TLR7,8and9subfamily.Eur J Immunol 33: 2987–2997. 1059–1064. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Geisse J, Caro I, Lindholm J, Golitz L, Stampone P, Owens M. Akira S et al. (2004). Species-specific recognition of single- (2004). Imiquimod 5% cream for the treatment of super- stranded RNA via Toll-like receptor 7 and 8. Science 303: ficial basal cell carcinoma: results from two phase III, 1526–1529. randomized, vehicle-controlled studies. J Am Acad Dermatol Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K 50: 722–733. et al. (2002). Small anti-viral compounds activateimmune Gerster JF, Lindstrom KJ, Miller RL, Tomai MA, Birmachu cells via the TLR7 MyD88-dependent signaling pathway. W, Bomersine SN et al. (2005). Synthesis and structure- Nat Immunol 3: 196–200. activity-relationships of 1H-imidazo[4,5-c]quinolines that Hengge UR, Ruzicka T. (2004). Topical immunomodulation induce interferon production. J Med Chem 48: 3481–3491. in dermatology: potential of toll-like receptor agonists. Giannotti B, Vanzi L, Difonzo EM, Pimpinelli N. (2003). The Dermatol Surg 30: 1101–1112. treatment of basal cell carcinomas in a patient with Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Xeroderma pigmentosum with a combination of imiquimod Schlee M, Uematsu S et al. (2005). Sequence-specific potent 5% cream and oral acitretin. Clin Exp Dermatol 28(Suppl. 1): induction of IFN-alpha by short interfering RNA in 33–35. plasmacytoid dendritic cells through TLR7. Nat Med 11: Gibson SJ, Imbertson LM, Wagner TL. (1995). Cellular 263–270. requirements for cytokine production in response to the Hurwitz DJ, Pincus L, Kupper TS. (2003). Imiquimod: a immunomodulators imiquimod and S-27609. J Interferon topically applied link between innate and acquired immu- Cytokine Res 15: 537–545. nity. Arch Dermatol 139: 1347–1350. Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Imbertson LM, Beaurline JM, Couture AM, Gibson SJ, Smith Fuller AE et al. (2002). Plasmacytoid dendritic cells produce RMA, Miller RL et al. (1998). Cytokineinduction in cytokines and mature in response to the TLR7 agonists, hairless mouse and rat skin after topical application of the imiquimod and resiquimod. Cell Immunol 218: 74–86. immune response modifiers imiquimod and S-28463. J Invest Gollnick H, Barasso R, JappeU, Ward K, Eul A, Carey-YardM Dermatol 110: 734–739. et al. (2001). Safety and efficacy of imiquimod 5% cream in the JudgeAD, Sood V, Shaw JR, Fang D, McClintock K, treatment of penile genital warts in uncircumcised men when MacLachlan I. (2005). Sequence-dependent stimulation of applied three times weekly or one per day. Int J STD AIDS 12: the mammalian innate immune response by synthetic 22–28. siRNA. Nat Biotechnol 23: 457–462. Gollnick H, Barona CG, Frank RG, Ruzicka T, Megahed M, Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H Tebbs V et al. (2005). Recurrence rate of superficial basal cell et al. (2002). Human TLR7 or TLR8 independently confer

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 197 responsiveness to the antiviral compound R-848. Nat Nishiya T, Kajita E, Miwa S, DeFranco AL. (2005). TLR3 and Immunol 3: 499. TLR7 are targeted to the same intracellular compartments Jurk M, Kritzler A, Schulte B, Tluk S, Schetter C, Krieg AM by distinct regulatory elements. J Biol Chem 280: et al. (2006). Modulating responsiveness of human 37107–37117. TLR7 and 8 to small molecule ligands with T-rich Odashima M, Bamias G, Rivera-Nieves J, Linden J, Nast CC, phosphorothiate oligodeoxynucleotides. Eur J Immunol 36: Moskaluk CA et al. (2005). Activation of A2A adenosine 1815–1826. receptor attenuates intestinal inflammation in animal Kamin A, Eigentler TK, Radny P, Bauer J, Weide B, Garbe C. models of inflammatory bowel disease. Gastroenterology (2005). Imiquimod in the treatment of extensive recurrent 129: 26–33. lentigo maligna. J Am Acad Dermatol 52(Suppl. 1): 51–52. Palamara F, Meindl S, Holcmann M, Luhrs P, Stingl G, Kariko K, Buckstein M, Ni H, Weissman D. (2005). Sibilia M. (2004). Identification and characterization of Suppression of RNA recognition by Toll-like receptors: pDC-like cells in normal mouse skin and melanomas treated the impact of nucleoside modification and the evolutionary with imiquimod. J Immunol 173: 3051–3061. origin of RNA. Immunity 23: 165–175. Patel K, Goodwin R, Chawla M, Laidler P, Price PE, Finlay Karin M. (2006). Nuclear factor-kappaB in cancer develop- AY et al. (2006). Imiquimod 5% cream monotherapy for ment and progression. Nature 441: 431–436. cutaneous squamous cell carcinoma in situ (Bowen’s Korman N, Moy R, Ling M, Matheson R, Smith S, McKane S disease): a randomized, double-blind, placebo-controlled et al. (2005). Dosing with 5% imiquimod cream 3 times per trial. J Am Acad Dermatol 54: 1025–1032. week for the treatment of actinic keratosis: results of two Peris K, Micantonio T, Fargnoli MC. (2003). Successful phase 3, randomized, double-blind, parallel-group, vehicle- treatment of keratoacanthoma and actinic keratoses with controlled trials. Arch Dermatol 141: 467–473. imiquimod 5% cream. Eur J Dermatol 13: 413–414. Lebwohl M, Dinehart S, Whiting D, Lee PK, Tawfik N, Peris K, Micantonio T, Fargnoli MC, Lozzi GP, Chimenti S. Jorizzo J et al. (2004). Imiquimod 5% cream for the (2006). Imiquimod 5% cream in the treatment of Bowen’s treatment of actinic keratosis: results from two phase III, disease and invasive squamous cell carcinoma. J Am Acad randomized, double-blind, parallel group, vehicle-controlled Dermatol 55: 324–327. trials. J Am Acad Dermatol 50: 714–721. PopeBL, MacIntyreJP, Kimball E, LeeS,Zhou L, Taylor GR Lee J, Chuang TH, Redecke V, She L, Pitha PM, Carson DA et al. (1995). Theimmunostimulatory compound 7-allyl-8- et al. (2003). Molecular basis for immunostimulatory oxoguanosine(loxoribine)inducesa distinct subsetof murine activity of guaninenucleosideanalogs: activation of Toll- cytokines. Cell Immunol 162: 333–339. like receptor 7. Proc Natl Acad Sci USA 100: 6646–6651. Prins RM, Craft N, Bruhn KW, Khan-Farooqi H, Koya RC, Levy O, Suter EE, Miller RL, Wessels MR. (2006). Unique Stripecke R et al. (2006). TheTLR-7 agonist, imiquimod, efficacy of Toll-like receptor 8 agonists in activating human enhances dendritic cell survival and promotes tumor neonatal antigen-presenting cells. Blood 108: 1284–1289. antigen-specific T cell priming: relation to central nervous Lonsdale-Eccles AA, Morgan JM, Nagarajan S, Cruickshank DJ. system antitumor immunity. J Immunol 176: 157–164. (2006). Successful treatment of vulval melanoma in situ with Prinz BM, Hafner J, Dummer R, Burg G, Bruswanger U, topical 5% imiquimod cream. Br J Dermatol 155: 215–217. Kempf W. (2004). Treatment of Bowen’s disease with Lore K, Betts MR, Brenchley JM, Kuruppu J, Khojasteh S, imiquimod 5% cream in transplant recipients. Transplanta- Perfetto S et al. (2003). Toll-like receptor ligands modulate tion 77: 790–791. dendritic cells to augment cytomegalovirus- and HIV-1- Qin J, Yao J, Cui G, Xiao H, Kim TW, Fraczek J et al. (2006). specific T cell responses. J Immunol 171: 4320–4328. TLR8-mediated NF-kappaB and JNK activation are Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, TAK1-independent and MEKK3-dependent. J Biol Chem GaleNW et al. (2004). Recognition of single-stranded RNA 281: 21013–21021. viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 101: Ray CM, Kluk M, Grin CM, Grant-Kels JM. (2005). 5598–5603. Successful treatment of malignant melanoma in situ Ma Y, Pawar S, Sanchez-Schmitz G, Poisson L, Byers A, with topical 5% imiquimod cream. Int J Dermatol 44: Shanen BC et al. (2007). In vitro vaccination site: a novel test 428–434. bed for immunopotentiators. J Immunol 178: 36.30. Rechtsteiner G, Warger T, Osterloh P, Schild H, Radsak MP. Megyeri K, Au W-C, Rosztoczy I. (1995). Stimulation of (2005). Cutting edge: priming of CTL by transcutaneous interferon and cytokine gene expression by imiquimod and peptide immunization with imiquimod. J Immunol 174: stimulation by sendai virus utilize similar signal transduc- 2476–2480. tion pathways. Mol Cell Biol 15: 2207–2218. Reiter MJ, Testerman TL, Miller RL, Weeks CE, Tomai MA. Meyer T, Nindl I, Schmook T, Ulrich C, Sterry W, Stockfleth (1994). Cytokineinduction in miceby theimmunomodu- E. (2003). Induction of apoptosis by toll-like receptor-7 lator imiquimod. J Leukoc Biol 55: 234–240. agonist in tissuecultures. Br J Dermatol 149: 9–14. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith Miggin SM, O’Neill LAJ. (2006). New insights into the KD et al. (2005). The evolution of vertebrate Toll-like regulation of TLR signaling. J Leukoc Biol 80: 220–226. receptors. Proc Natl Acad Sci USA 102: 9577–9578. Miller RL, Tomai MA, Harrison CJ, Bernstein DI. (2002). Roseeuw D. (2003). The treatment of basal skin carcinomas in Immunomodulation as a treatment strategy for genital two sisters with Xeroderma pigmentosum. Clin Exp herpes: review of the evidence. Int Immunopharmacol 2: Dermatol 28(Suppl. 1): 30–32. 443–451. Sauder DN, Smith MH, Senta-McMillian T, Soria I, Meng TC. Navi D, Huntley A. (2004). Imiquimod 5% cream and the (2003). Randomized, single-blind, placebo-controlled study treatment of cutaneous malignancy. Dermatol Online J 10. of topical application of theimmuneresponsemodulator Nishiya T, DeFranco AL. (2004). Ligand-regulated chimeric resiquimod in healthy adults. Antimicrob Agents Chemother receptor approach reveals distinctive subcellular localization 47: 3846–3852. and signaling properties of the Toll-like receptors. J Biol Scho¨ n M, Bong AB, Drewniok C, Herz J, Geilen CC, Chem 279: 19008–19017. Reifenberger J et al. (2003). Tumor-selective induction of

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 198 apoptosis and the small-molecule immune response modifier application of the immune response modifier imiquimod. imiquimod. J Natl Cancer Inst 95: 1138–1149. Arch Dermatol 138: 1137–1139. Scho¨ n M, Scho¨ n MP. (2007). Theantitumoral modeof action Sullivan TP, Dearaujo T, Vincek V, Berman B. (2003). of imiquimod and other imidazoquinolines. Curr Med Chem Evaluation of superficial basal cell carcinomas after 14: 681–687. treatment with imiquimod 5% cream or vehicle for Scho¨ n MP, Scho¨ n M, Klotz KN. (2006). Thesmall anti- apoptosis and lymphocytephenotyping. Dermatol Surg 29: tumoral immune response modifier imiquimod interacts 1181–1186. with adenosine receptor signaling in a TLR7- and TLR8- Suzuki H, Wang B, Shivji GM, Toto P, Amerio P, Tomai MA independent fashion. J Invest Dermatol 126: 1338–1347. et al. (2000). Imiquimod, a topical immuneresponse Scho¨ n MP, Wienrich BG, Drewniok C, Bong AB, Eberle J, modifier, induces migration of Langerhans cells. J Invest Geilen CC et al. (2004). Death receptor-independent Dermatol 114: 135–141. apoptosis in malignant melanoma induced by the small- Szeimies RM, Gerritsen MJ, Gupta G, Ortonne JP, Serresi S, molecule immune response modifier imiquimod. J Invest Bichel J et al. (2004). Imiquimod 5% cream for the Dermatol 122: 1266–1276. treatment of actinic keratosis: results from a phase III, Schulze HJ, Cribier B, Requena L, Reifenberger J, Ferrandiz C, randomized, double-blind, vehicle-controlled, clinical trial Garcia Diez A et al. (2005). Imiquimod 5% cream for the with histology. J Am Acad Dermatol 51: 547–555. treatment of superficial basal cell carcinoma: results from a Thomsen LL, Topley P, Daly MG, Brett SJ, Tite JP. (2004). randomized vehicle-controlled phase III study in Europe. Imiquimod and resiquimod in a mouse model: adjuvants for Br J Dermatol 152: 939–947. DNA vaccination by particle-mediated immunotherapeutic Schwarz K, Storni T, Manolova V, Didierlaurent A, Sirard JC, delivery. Vaccine 22: 1799–1809. Rothlisberger P et al. (2003). Role of toll-like receptors in Tomai MA, Imbertson LM, Stanczak TL, Tygrett LT, costimulating cytotoxic T cell responses. Eur J Immunol 33: Waldschmidt TJ. (2000). The immune response modifiers 1465–1470. imiquimod and R-848 arepotentactivators of B lympho- Shackleton M, Davis ID, Hopkins W, Jackson H, Dimopoulos N, cytes. Cell Immunol 203: 55–65. Tai T et al. (2004). Theimpact of imiquimod, a toll-like Ugurel S, Wagner A, Pfo¨ hler C, Tilgen W, Reinhold U. (2002). receptor-7 ligand (TLR7L), on the immunogenicity of melano- Topical imiquimod eradicates skin metastases of malignant ma peptide vaccination with adjuvant Flt3 ligand. Cancer melanoma but fails to prevent rapid lymphogenous meta- Immun 23:9. static spread. Br J Dermatol 147: 621–624. Sidbury R, Neuschler N, Neuschler E, Sun P, Wang XQ, Urosevic M, Dummer R, Conrad C, Beyeler M, Laine E, Miller RL et al. (2003). Topically applied imiquimod Burg G et al. (2005). Disease-independent skin recruit- inhibits vascular tumor growth in vivo. J Invest Dermatol ment and activation of plasmacytoid predendritic cells 121: 1205–1209. following imiquimod treatment. J Natl Cancer Inst 97: Sidky YA, Borden EC, Weeks CE, Reiter MJ, Hatcher JF, 1143–1153. Bryan GT. (1992). Inhibition of murinetumor growth by Urosevic M, Maier T, Benninghoff B, Slade HB, Burg G, an interferon-inducing imidazoquinoline. Cancer Res 52: Dummer R. (2003). Mechanisms underlying imiquimod- 3528–3533. induced regression of basal cell carcinomas in vivo. Arch Smith KJ, Germain M, Skelton H. (2001). Squamous cell Dermatol 139: 1325–1332. carcinoma in situ (Bowen’s disease) in renal transplant Urosevic M, Oberholzer PA, Maier T, Hafner J, Laine E, patients treated with 5% imiquimod and 5% 5-fluorouracil SladeHB et al. (2004). Imiquimod treatment induces therapy. Dermatol Surg 27: 561–564. expression of opioid growth factor receptor: a novel Stanley MA. (2002). Imiquimod and the imidazoquinolones: tumor antigen induced by interferon-a? Clin Cancer Res mechanism of action and therapeutic potential. Clin Exp 10: 4959–4970. Dermatol 27: 571–577. Vidal D, Matias-Guiu X, Alomar A. (2004). Efficacy of Stary G, Bangert C, Tauber M, Strohal R, Kopp T, Stingl G. imiquimod for the expression of Bcl-2, Ki67, p53 and basal (2007). Tumoricidal activity of TLR7/8-activated inflamma- cell carcinoma apoptosis. Br J Dermatol 151: 656–662. tory dendritic cells. J Exp Med 204: 1441–1451. Wagner TL, Ahonen CL, Couture AM, Gibson SJ, Miller RL, Steinmann A, Funk JO, Schuler G, von den Driesch P. (2000). Smith RM et al. (1999). Modulation of TH1 and TH2 Topical imiquimod treatment of a cutaneous melanoma cytokine production with the immune response modifiers, metastasis. J Am Acad Dermatol 43: 555–556. R-848 and imiquimod. Cell Immunol 191: 10–19. Stephanou A, Latchman DS. (2005). Opposing actions of Wagner TL, Horton VL, Carlson GL. (1997). Induction of STAT-1 and STAT-3. Growth Factors 23: 177–182. cytokines in Cynomolgus monkeys by the immune response Sterry W, Ruzicka T, Herrera E, Takwale A, Bichel J, Andres K modifiers, imiquimod, S-27609 and S-28463. Cytokine 9: et al. (2002). Imiquimod 5% cream for the treatment of 837–845. superficial and nodular basal cell carcinoma: randomized Weeks CE, Gibson SJ. (1994). Induction of interferon and studies comparing low-frequency dosing with and without other cytokines by imiquimod and its hydroxylated meta- occlusion. Br J Dermatol 147: 1227–1236. boliteR-842 in human blood cells in vitro. J Interferon Stockfleth E, Meyer T, Benninghoff B, Christophers E. (2001). Cytokine Res 14: 81–85. Successful treatment of actinic keratosis with imiquimod cream Wille-Reece U, Flynn BJ, Lore K, Koup RA, Miles AP, Saul 5%:areportofsixcases.Br J Dermatol 144: 1050–1053. A et al. (2006). Toll-like receptor agonists influence the Stockfleth E, Meyer T, Benninghoff B, Salasche S, Papado- magnitude and quality of memory T cell responses after poulos L, Ulrich C et al. (2002). A randomized, double- prime-boost immunization in nonhuman primates. J Exp blind, vehicle-controlled study to assess 5% imiquimod Med 203: 1249–1258. cream for the treatment of multiple actinic keratoses. Arch Wolf IH, Cerroni L, Kodama K, Kerl H. (2005). Treatment Dermatol 138: 1498–1502. of lentigo maligna (melanoma in situ) with theim- Suchin KR, Junkins-Hopkins JM, Rook AH. (2002). Treat- mune response modifier imiquimod. Arch Dermatol 141: ment of stage IA cutaneous T-cell lymphoma with topical 510–514.

Oncogene TLR7 and TLR8 as targets in cancer therapy MP Scho¨n and M Scho¨n 199 Wolf IH, Smolle J, Binder B, Cerroni L, Richtig E, cream and the pulsed-dye laser. Br J Dermatol 152: Kerl H. (2003). Topical imiquimod in the treatment 376–377. of metastatic melanoma to skin. Arch Dermatol 139: Zuber AK, Brave A, Engstrom G, Zuber B, Ljungberg K, 273–276. Fredriksson M et al. (2004). Topical delivery of imiquimod to Zeitouni NC, Dawson K, Cheney RT. (2005). Treatment a mousemodelas a noveladjuvant for human immuno- of cutaneous metastatic melanoma with imiquimod 5% deﬁciency virus (HIV) DNA. Vaccine 22: 1791–1798.

Oncogene