TLR signaling inhibitor, phenylmethimazole, in combination with tamoxifen inhibits human breast cancer cell viability and migration.

Heightened co-expression and dysregulated signaling associated with Toll-like receptor 3 (TLR3) and Wnt5a is an integral component of solid tumors and hematological malignancies. Our previous findings in pancreatic cancer and melanoma suggest that inhibition of these pathways by a TLR3 signaling inhibitor, phenylmethimazole (C10), results in significantly decreased IL-6 levels, STAT3 phosphorylation, minimal cancer cell migration and reduced cancer cell growth in vitro and in vivo. In this study, we extended our earlier observations by performing studies in human breast cancer cells. We found that human MCF-7 breast cancer cells express high basal levels of TLR3 and Wnt5a RNA. C10 treatment resulted in significantly decreased TLR3 and Wnt5a expression levels. This functionally translated into significantly reduced IL-6 levels and STAT3 phosphorylation in vitro. In addition, the inhibition of this signaling cascade by C10 further resulted in decreased cell viability and migration of MCF-7 cells. Strikingly, the combination of C10 and tamoxifen, the standard of care therapy for breast cancer, further decrease cancer cell growth better than either agent alone. These data support the novel finding that inhibition of TLR3 signaling in combination with tamoxifen, may increase the effectiveness of current treatments of breast cancer.

AIM2, an IFN-inducible cytosolic DNA sensor, in the development of benign prostate hyperplasia and prostate cancer.

UNLABELLED: Close links have been noted between chronic inflammation of the prostate and the development of human prostatic diseases such as benign prostate hyperplasia (BPH) and prostate cancer. However, the molecular mechanisms that contribute to prostatic inflammation remain largely unexplored. Recent studies have indicated that the IFN-inducible AIM2 protein is a cytosolic DNA sensor in macrophages and keratinocytes. Upon sensing DNA, AIM2 recruits the adaptor ASC and pro-CASP1 to assemble the AIM2 inflammasome. Activation of the AIM2 inflammasome cleaves pro-interleukin (IL)-1ß and pro-IL-18 and promotes the secretion of IL-1ß and IL-18 proinflammatory cytokines. Given that human prostatic infections are associated with chronic inflammation, the development of BPH is associated with an accumulation of senescent cells with a proinflammatory phenotype, and the development of prostate cancer is associated with the loss of IFN signaling, the role of AIM2 in mediating the formation of prostatic diseases was investigated. It was determined that IFNs (α, ß, or γ) induced AIM2 expression in human prostate epithelial cells and cytosolic DNA activated the AIM2 inflammasome. Steady-state levels of the AIM2 mRNA were higher in BPH than in normal prostate tissue. However, the levels of AIM2 mRNA were significantly lower in clinical tumor specimens. Accordingly, constitutive levels of AIM2 mRNA and protein were lower in a subset of prostate cancer cells as compared with BPH cells. Further, the cytosolic DNA activated the AIM2 inflammasome in the androgen receptor-negative PC3 prostate cancer cell line, suggesting that AIM2-mediated events are independent of androgen receptor status. IMPLICATIONS: The AIM2 inflammasome has a fundamental role in the generation of human prostatic diseases.

Differential roles for the interferon-inducible IFI16 and AIM2 innate immune sensors for cytosolic DNA in cellular senescence of human fibroblasts.

The IFN-inducible IFI16 and AIM2 proteins act as innate immune sensors for cytosolic double-stranded DNA (dsDNA). On sensing dsDNA, the IFI16 protein induces the expression of IFN-ß whereas the AIM2 protein forms an inflammasome, which promotes the secretion of IL-1ß. Given that the knockdown of IFI16 expression in human diploid fibroblasts (HDF) delays the onset of cellular senescence, we investigated the potential roles for the IFI16 and AIM2 proteins in cellular senescence. We found that increased IFI16 protein levels in old (vs. young) HDFs were associated with the induction of IFN-ß. In contrast, increased levels of the AIM2 protein in the senescent (vs. old) HDFs were associated with increased production of IL-1ß. The knockdown of type I IFN-α receptor subunit, which reduced the basal levels of the IFI16 but not of the AIM2, protein delayed the onset of cellular senescence. Accordingly, increased constitutive levels of IFI16 and AIM2 proteins in ataxia telangiectasia mutated (ATM) HDFs were associated with the activation of the IFN signaling and increased levels of IL-1ß. The IFN-ß treatment of the young HDFs, which induced the expression of IFI16 and AIM2 proteins, activated a DNA damage response and also increased basal levels of IL-1ß. Interestingly, the knockdown of AIM2 expression in HDFs increased the basal levels of IFI16 protein and activated the IFN signaling. In contrast, the knockdown of the IFI16 expression in HDFs decreased the basal and dsDNA-induced activation of the IFN signaling. Collectively, our observations show differential roles for the IFI16 and AIM2 proteins in cellular senescence and associated secretory phenotype.

Phenylmethimazole blocks palmitate-mediated induction of inflammatory cytokine pathways in 3T3L1 adipocytes and RAW 264.7 macrophages.

Visceral adipocytes and associated macrophages produce and release excessive amounts of biologically active inflammatory cytokines via the portal and systemic vascular system, which induce insulin resistance in insulin target tissues such as fat, liver, and muscle. Free fatty acids (FFAs) absorbed via the portal system or released from adipocytes also induce insulin resistance. In this report, we show that phenylmethimazole (C10) blocks basal IL6 and leptin production as well as basal Socs-3 expression in fully differentiated 3T3L1 cells (3T3L1 adipocytes) without affecting insulin-stimulated AKT signaling. In addition, C10 inhibits palmitate-induced IL6 and iNos up-regulation in both 3T3L1 adipocytes and RAW 264.7 macrophages, LPS-induced NF-κB and IFN-ß activation in 3T3L1 cells, and LPS-induced iNos, Ifn-ß, Il1ß, Cxcl10, and Il6 expression in RAW 264.7 macrophages. C10 also blocks palmitate-induced Socs-3 up-regulation and insulin receptor substrate-1 (IRS-1) serine 307 phosphorylation in 3T3L1 adipocytes. Additionally, we show for the first time that although palmitate increases IRS-1 serine 307 phosphorylation in 3T3L1 adipocytes, AKT serine 473 phosphorylation is enhanced, not reduced, by palmitate. These results suggest that through inhibition of FFA-mediated signaling in adipocytes and associated macrophages, as well as possibly other insulin target cells/tissues (i.e. non-immune cells), C10 might be efficacious to prevent or reverse cytokine-induced insulin resistance seen in obesity-related insulin resistance and type 2 diabetes mellitus.

Interferon-inducible p200-family proteins as novel sensors of cytoplasmic DNA: role in inflammation and autoimmunity.

Deregulated innate immune responses that result in increased levels of type I interferons (IFNs) and stimulation of IFN-inducible genes are thought to contribute to chronic inflammation and autoimmunity. One family of IFN-inducible genes is the Ifi200 family, which includes the murine (eg, Ifi202a, Ifi202b, Ifi203, Ifi204, Mndal, and Aim2) and human (eg, IFI16, MNDA, IFIX, and AIM2) genes. Genes in the family encode structurally related proteins (the p200-family proteins), which share at least one partially conserved repeat of 200-amino acid (200-AA) residues. Consistent with the presence of 2 consecutive oligonucleotide/oligosaccharide-binding folds in the repeat, the p200-family proteins can bind to DNA. Additionally, these proteins (except the p202 proteins) also contain a pyrin (PYD) domain in the N-terminus. Increased expression of p202 proteins in certain strains of female mice is associated with lupus-like disease. Interestingly, only the Aim2 protein is conserved between the mouse and humans. Several recent studies have provided evidence that the Aim2 and p202 proteins can recognize DNA in cytoplasm and the Aim2 protein upon sensing DNA can form a caspase-1-activating inflammasome. In this review, we discuss how the ability of p200-family proteins to sense cytoplasmic DNA may contribute to the development of chronic inflammation and associated diseases.

Phenylmethimazole decreases Toll-like receptor 3 and noncanonical Wnt5a expression in pancreatic cancer and melanoma together with tumor cell growth and migration.

PURPOSE: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo. EXPERIMENTAL DESIGN: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated. RESULTS: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation. CONCLUSIONS: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.

Wnt5a is expressed in murine and human atherosclerotic lesions.

Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.