Laquinimod, a prototypic quinoline-3-carboxamide and aryl hydrocarbon receptor agonist, utilizes a CD155-mediated natural killer/dendritic cell interaction to suppress CNS autoimmunity.

BACKGROUND: Quinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1). METHODS: We explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed. RESULTS: We demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing. CONCLUSIONS: This study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance.

Nanoscale Tuning of VCAM-1 Determines VLA-4-Dependent Melanoma Cell Plasticity on RGD Motifs.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/µm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

Treatment with diphenyl-pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death.

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.

Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate.

UNLABELLED: The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. TRIAL REGISTRATION: ClinicalTrials.gov NCT01387711.

Integrin αE (CD103) is induced but plays no pathogenic role in psoriasiform skin lesions of TGFß1 transgenic mice.

T-cells expressing αE (CD103), an integrin induced by TGFß on T-cells in vitro, accumulate within epithelia in inflammatory disorders, including psoriasis. However, it is unclear, if and how αE (CD103) contributes to skin inflammation. Using two complementary approaches, we have investigated αE (CD103) in psoriasis-like skin inflammation of mice with transgenic epidermal expression of human TGFß1: αE (CD103) was inhibited by function-blocking antibodies in vivo, and double-mutants with additional αE (CD103)-depletion were generated in two different genetic backgrounds. Epidermal hTGFß1 expression was associated with prominent expression of αE (CD103) on infiltrating cells. However, neither treatment with αE (CD103)-blocking antibodies nor deficiency of αE (CD103) in double-mutant mice altered the psoriasis-like phenotype. In addition, histopathological and flow cytometric analyses revealed similar pathological skin alterations and lymphocyte subgroups in the different mouse strains. Thus, while αE (CD103) expression is indeed associated with hTGFß1 in vivo, it has little, if any, influence on the course of the psoriasis-like phenotype in K5.hTGFß1 transgenic mice.

The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

Inositoylated platelet-activating factor (Ino-C2-PAF) modulates dynamic lymphocyte-endothelial cell interactions and alleviates psoriasis-like skin inflammation in two complementary mouse models.

Psoriasis, a tumor necrosis factor alpha (TNFα)-governed inflammatory disorder with prominent dysregulation of cutaneous vascular functions, has evolved into a model disorder for studying anti-inflammatory therapies. We present experimental in vitro and in vivo data on 1-O-octadecyl-2-O-(2-(myo-inositolyl)-ethyl)-sn-glycero-3-(R/S)-phosphatidyl-choline (Ino-C2-PAF), the lead compound of a class of synthetic glycosylated phospholipids, in anti-inflammatory therapy. Ino-C2-PAF strongly induced apoptosis only in TNFα-stimulated, but not in untreated human vascular endothelial cells. Moreover, TNFα-induced endothelial adhesion molecules that mediated the rolling and firm adhesion of leukocytes (vascular cell adhesion protein-1 (VCAM-1), E-selectin, and ICAM-1) were selectively downregulated by Ino-C2-PAF. Similarly, expression of L-selectin, VCAM-1 receptor α4ß1 integrin , and lymphocyte function-associated antigen-1 on human peripheral blood mononuclear cells was reduced without induction of apoptosis. Functionally, these changes were accompanied by significant impairment of rolling and adhesion of human peripheral blood lymphocytes on TNFα-activated endothelial cells in a dynamic flow chamber system. When the therapeutic potential of Ino-C2-PAF was assessed in two complementary mouse models of psoriasis, K5.hTGFß1 transgenic and JunB/c-Jun-deficient mice, Ino-C2-PAF led to significant alleviation of the clinical symptoms and normalized the pathological cutaneous changes including vascularization. There were no overt adverse effects. These findings suggested that Ino-C2-PAF is a potential candidate in the therapy of inflammatory skin diseases that include abnormal vascular functions.

Inositol-C2-PAF down-regulates components of the antigen presentation machinery in a 2D-model of epidermal inflammation.

In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.

Doxorubicin-induced activation of NF-κB in melanoma cells is abrogated by inhibition of IKKß, but not by a novel IKKα inhibitor.

Drug resistance is arguably the most important challenge in cancer therapy. Here, doxorubicin induced profound of NF-κB activation in melanoma cells with a maximum (3.5-fold) at concentrations relevant in vivo. This was followed by transcriptional induction of several gene products involved in tumor progression. A novel IKKα inhibitor (BAY32-5915) was identified and characterized, and doxorubicin-induced NF-κB activation was assessed following inhibition of IKKα or IKKß by small-molecular compounds. While the IKKα inhibitor did not affect doxorubicin-induced NF-κB activation, this process was completely abrogated when the IKKß inhibitor, KINK-1, was used. Moreover, inhibition of IKKß, but not IKKα, led to significantly increased apoptosis in response to doxorubicin. Our results indicate that the net outcome of chemotherapy is difficult to predict and may even involve mechanisms conferring chemoresistance. In case of doxorubicin-induced NF-κB activation, blocking IKKß, but not IKKα, by small molecules can antagonize this activity and, thus, increase chemosensitivity.

Stimulation of pulmonary immune responses by the TLR2/6 agonist MALP-2 and effect on melanoma metastasis to the lung.

Given that metastasized melanoma is a fatal disease in most cases, it is tempting to develop strategies to a priori prevent metastasis. We have stimulated the pulmonary innate immune system by macrophage-activating lipopeptide-2 (MALP-2), a specific agonist at Toll-like receptor (TLR) 2/6, and investigated its impact on experimental melanoma metastasis. In C57BL/6 mice, intratracheal application of MALP-2 induced a profound influx of neutrophils and macrophages into the lung, which peaked after 24 h (sixfold increase) and returned to baseline within 72 h. Further analysis revealed that MALP-2 also markedly induced VCAM-1 expression on pulmonary blood vessels. In vitro experiments demonstrated that this adhesion molecule mediates binding of B16F10 melanoma cells. Furthermore, in vivo or in vitro treatment with MALP-2 did not significantly affect the ability of immune cells to lyse melanoma cells. As a consequence, notwithstanding the profound pulmonary immune response induction and in contrast to conclusions drawn from some previous publications, the net extent of experimental metastasis did not change significantly, regardless of the application regimen of MALP-2 prior to, concomitant with or after tumor cell inoculation. Melanoma cells stably transfected with green fluorescent protein allowed tracking of early events after tumor cell dissemination and showed that MALP-2-mediated TLR2/6 activation did not interfere with pulmonary melanoma cell arrest. Likewise, boosting the immune induction after establishment of metastases did not change the clinical outcome. These unexpected results vividly counsel caution regarding predictions of immunomodulating therapies, as multiple intertwined effects may influence the net outcome.

Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions.

Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-ß1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein-coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.

Inhibition of platelet GPIb alpha and promotion of melanoma metastasis.

Platelet glycoprotein Ibalpha (GPIb alpha) is part of the receptor complex GPIb-V-IX, which has a critical role in hemostasis, especially through interactions with the subendothelial von Willebrand factor. As there is accumulating evidence for a contribution of platelet receptors to hematogenous tumor metastasis, GPIb alpha is an interesting molecule to study in this context. We have investigated the effect of GPIb alpha inhibition by monovalent Fab fragments on experimental pulmonary metastasis in a syngeneic mouse model using C57BL/6 mice and B16F10 melanoma cells. The early fate of green fluorescent protein (GFP)-transfected melanoma cells under GPIb alpha blockade was also assessed, as was the effect of GPIb alpha inhibition on pulmonary metastasis in mice lacking P-selectin. Surprisingly and, to our knowledge previously unreported, GPIb alpha inhibition led to a significant increase in pulmonary metastasis, and assessment of the early fate of circulating GFP-labeled B16F10 showed improved survival and pulmonary arrest of tumor cells shortly after GPIb alpha inhibition, indicating that inhibition of a platelet protein can, in some cases, promote metastasis of a malignant tumor. In contrast, GPIb alpha blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIb alpha in the initial, P-selectin-dependent steps of metastasis. These findings suggest that GPIb alpha contributes to the control of tumor metastasis, in addition to its role in hemostasis.

NF-kappaB inhibition through proteasome inhibition or IKKbeta blockade increases the susceptibility of melanoma cells to cytostatic treatment through distinct pathways.

Metastasized melanoma is almost universally resistant to chemotherapy. Given that constitutive or drug-induced upregulation of NF-kappaB activity is associated with this chemoresistance, NF-kappaB inhibition may increase the susceptibility to antitumoral therapy. On the cellular level, two principles of NF-kappaB inhibition, proteasome inhibition by bortezomib and IkappaB kinase-beta (IKKbeta) inhibition by the kinase inhibitor of NF-kappaB-1 (KINK-1), significantly increased the antitumoral efficacy of camptothecin. When combined with camptothecin, either of the two NF-kappaB-inhibiting principles synergistically influenced progression-related in vitro functions, including cell growth, apoptosis, and invasion through an artificial basement membrane. In addition, when C57BL/6 mice were intravenously injected with B16F10 melanoma cells, the combination of cytostatic treatment with either of the NF-kappaB-inhibiting compounds revealed significantly reduced pulmonary metastasis compared to either treatment alone. However, on the molecular level, nuclear translocation of p65, cell cycle analysis, and expression of NF-kappaB-dependent gene products disclosed distinctly different molecular mechanisms, resulting in the same functional effect. That proteasome inhibition and IKKbeta inhibition affect distinct molecular pathways downstream of NF-kappaB, both leading to increased chemosensitivity, is previously unreported. Thus, it is conceivable that switching the two principles of NF-kappaB inhibition, once resistance to one of the agents occurs, will improve future treatment regimens.

INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling.

BACKGROUND: The gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells. RESULTS: Markedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1. CONCLUSION: These results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.

KINK-1, a novel small-molecule inhibitor of IKKbeta, and the susceptibility of melanoma cells to antitumoral treatment.

BACKGROUND: Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-kappaB) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-kappaB is regulated by inhibitory kappaB (IkappaB) proteins, which are in turn phosphorylated by the IkappaB kinase (IKK) complex. METHODS: The effect on NF-kappaB activity of a novel small-molecule inhibitor of the beta subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-kappaB-1) was assessed by measuring phosphorylation of the alpha subunit of IkappaB by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-kappaB using immunofluorescence. Regulation of NF-kappaB-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-kappaB activity in melanoma cells. It reduced the expression of NF-kappaB-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-kappaB activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 171 to 192 mg, respectively, P < .001 from t tests comparing mean lung weight of double-treated mice to that in mice treated with either compound alone). CONCLUSION: Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.

Proteinase-activated receptor-2 (PAR2): a tumor suppressor in skin carcinogenesis.

The proteinase-activated receptor PAR(2) has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. However, the role of PAR(2) in cutaneous cancerogenesis is still unknown. Here we could show a protective role of PAR(2) in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. Tumors started to appear after eight weeks. After 13 weeks, PAR(2)-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild type (eight tumors per mouse). Analysis of possible signal transduction pathways activated upon PAR(2) stimulation in HaCaT keratinocytes showed an involvement of extracellular signal-regulated kinase 1/2 and profound epidermal growth factor receptor transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta1. Thus, our results provide early experimental evidence for a tumor-protective role of PAR(2).

The antitumoral mode of action of imiquimod and other imidazoquinolines.

Imiquimod, the lead compound of the imidazoquinoline family of nucleoside analogues, has shown good efficacy against a variety of tumors of different origin. The mode of action of imiquimod and related compounds, which we have begun to understand in some detail in recent years, is complex and interesting inasmuch as it appears to comprise several presumably mutually enhancing components. Predominant amongst its actions is the induction of pro-inflammatory cytokines through agonistic activity towards Toll-like receptor (TLR)-7 and TLR-8, and consecutively, activation of the central transcription factor NF-kappaB. This activity stimulates the production of pro-inflammatory cytokines, chemokines and other mediators resulting in activation of antigen-presenting cells and the mounting of a profound Th1-weighted antitumoral cellular immune response. In addition, there are a number of secondary effects on the molecular and cellular level that can be explained through the activation of NF-kappaB. The pro-inflammatory activity of imiquimod appears to be augmented by suppression of a negative regulatory feedback mechanism which normally limits inflammatory responses. This is achieved independent of TLR-7 and TLR-8 through interference with adenosine receptor signaling pathways, particularly the A(2A) subtype, and receptor-independent reduction of adenylyl cyclase activity. Finally, at higher, albeit therapeutically relevant concentrations, imiquimod exerts a pro-apoptotic activity against tumor cells. Induction of apoptosis by imiquimod appears to be dependent on Bcl-2 proteins and involves caspase activation. The combination of multiple, presumably synergistic anti-tumoral functions by a single compound represents an interesting principle of pathogenesis-oriented, anti-neoplastic therapy.

The small antitumoral immune response modifier imiquimod interacts with adenosine receptor signaling in a TLR7- and TLR8-independent fashion.

Imiquimod, a small-molecule immune response modifier of the imidazoquinoline family, has shown profound antitumoral and antiviral efficacy both in vitro and in clinical applications in vivo. It has been demonstrated that this activity is mediated through the Toll-like receptor (TLR)7- and TLR8-signaling cascade resulting in the secretion of proinflammatory cytokines and, consecutively, induction of a tumor-directed cellular immune response. In addition, imiquimod exerts a direct proapoptotic activity in tumor cells. We demonstrate here that imiquimod induces activation of the transcription factor NF-kappaB and the downstream production of proinflammatory cytokines in the absence of TLR7 and TLR8. In Chinese hamster ovary cells stably transfected with the human adenosine receptor subtypes, we then show in radioligand-binding competition experiments that imiquimod binds to adenosine receptors at concentrations relevant in clinical settings, with highest affinities to the A(1) and A(2A) subtypes. The effect on the receptor-mediated activation of adenylyl cyclase was also studied, and these experiments revealed that imiquimod acts as an adenosine receptor antagonist. In addition, imiquimod had an inhibitory effect on adenylyl cyclase activity downstream from the receptor. Finally, using transformed human keratinocytes, we provide experimental evidence that imiquimod and A(2A) adenosine receptor-specific compounds similarly induce proinflammatory cytokines in the absence of immune cells. Thus, imiquimod appears to suppress an important feedback mechanism of inflammation by antagonism of adenosine receptor-dependent increase of cAMP and a concomitant receptor-independent inhibition of cAMP production. These novel mechanisms presumably act synergistic with the positive induction of proinflammatory cytokines and can, at least in part, explain the profound inflammation observed in some patients in vivo.