Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation.

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

Identification of Schlafen-11 as a Target of CD47 Signaling That Regulates Sensitivity to Ionizing Radiation and Topoisomerase Inhibitors.

Knockdown or gene disruption of the ubiquitously expressed cell surface receptor CD47 protects non-malignant cells from genotoxic stress caused by ionizing radiation or cytotoxic chemotherapy but sensitizes tumors in an immune competent host to genotoxic stress. The selective radioprotection of non-malignant cells is mediated in part by enhanced autophagy and protection of anabolic metabolism pathways, but differential H2AX activation kinetics suggested that the DNA damage response is also CD47-dependent. A high throughput screen of drug sensitivities indicated that CD47 expression selectively sensitizes Jurkat T cells to inhibitors of topoisomerases, which are known targets of Schlafen-11 (SLFN11). CD47 mRNA expression positively correlated with schlafen-11 mRNA expression in a subset of human cancers but not the corresponding non-malignant tissues. CD47 mRNA expression was also negatively correlated with SLFN11 promoter methylation in some cancers. CD47 knockdown, gene disruption, or treatment with a CD47 function-blocking antibody decreased SLFN11 expression in Jurkat cells. The CD47 signaling ligand thrombospondin-1 also suppressed schlafen-11 expression in wild type but not CD47-deficient T cells. Re-expressing SLFN11 restored radiosensitivity to a CD47-deficient Jurkat cells. Disruption of CD47 in PC3 prostate cancer cells similarly decreased schlafen-11 expression and was associated with a CD47-dependent decrease in acetylation and increased methylation of histone H3 in the SLFN11 promoter region. The ability of histone deacetylase or topoisomerase inhibitors to induce SLFN11 expression in PC3 cells was lost when CD47 was targeted in these cells. Disrupting CD47 in PC3 cells increased resistance to etoposide but, in contrast to Jurkat cells, not to ionizing radiation. These data identify CD47 as a context-dependent regulator of SLFN11 expression and suggest an approach to improve radiotherapy and chemotherapy responses by combining with CD47-targeted therapeutics.

Natural Killer Cell Recruitment and Activation Are Regulated by CD47 Expression in the Tumor Microenvironment.

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro Cd47 -/- NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47 -/- than in wild-type mice. Melanoma-bearing Cd47 -/- mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47-/- NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.

Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy.

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells, but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

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.

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation.

Modulating tissue responses to stress is an important therapeutic objective. Oxidative and genotoxic stresses caused by ionizing radiation are detrimental to healthy tissues but beneficial for treatment of cancer. CD47 is a signaling receptor for thrombospondin-1 and an attractive therapeutic target because blocking CD47 signaling protects normal tissues while sensitizing tumors to ionizing radiation. Here we utilized a metabolomic approach to define molecular mechanisms underlying this radioprotective activity. CD47-deficient cells and cd47-null mice exhibited global advantages in preserving metabolite levels after irradiation. Metabolic pathways required for controlling oxidative stress and mediating DNA repair were enhanced. Some cellular energetics pathways differed basally in CD47-deficient cells, and the global declines in the glycolytic and tricarboxylic acid cycle metabolites characteristic of normal cell and tissue responses to irradiation were prevented in the absence of CD47. Thus, CD47 mediates signaling from the extracellular matrix that coordinately regulates basal metabolism and cytoprotective responses to radiation injury.

CD47-dependent regulation of H2S biosynthesis and signaling in T cells.

Pharmacological concentrations of H2S donors inhibit some T cell functions by inhibiting mitochondrial function, but evidence is also emerging that H2S at physiological concentrations produced via chemical sources and endogenously is a positive physiological mediator of T cell function. Expression of the H2S biosynthetic enzymes cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) is induced in response to T cell receptor signaling. Inhibiting the induction of these enzymes limits T cell activation and proliferation, which can be overcome by exposure to exogenous H2S at submicromolar concentrations. Exogenous H2S at physiological concentrations increases the ability of T cells to form an immunological synapse by altering cytoskeletal actin dynamics and increasing the reorientation of the microtubule-organizing center. Downstream, H2S enhances T cell receptor-dependent induction of CD69, CD25, and Interleukin-2 (IL-2) gene expression. The T cell stimulatory activity of H2S is enhanced under hypoxic conditions that limit its oxidative metabolism by mitochondrial and nonenzymatic processes. Studies of the receptor CD47 have revealed the first endogenous inhibitory signaling pathway that regulates H2S signaling in T cells. Binding of the secreted protein thrombospondin-1 to CD47 elicits signals that block the stimulatory activity of exogenous H2S on T cell activation and limit the induction of CSE and CBS gene expression. CD47 signaling thereby inhibits T cell receptor-mediated T cell activation.

Orthotopic model of canine osteosarcoma in athymic rats for evaluation of stereotactic radiotherapy.

OBJECTIVE: To develop an orthotopic model of canine osteosarcoma in athymic rats as a model for evaluating the effects of stereotactic radiotherapy (SRT) on osteosarcoma cells. ANIMALS: 26 athymic nude rats. PROCEDURES: 3 experiments were performed. In the first 2 experiments, rats were injected with 1 × 10(6) Abrams canine osteosarcoma cells into the proximal aspect of the tibia (n = 12) or distal aspect of the femur (6). Tumor engraftment and progression were monitored weekly via radiography, luciferase imaging, and measurement of urine pyridinoline concentration for 5 weeks and histologic evaluation after euthanasia. In the third experiment, 8 rats underwent canine osteosarcoma cell injection into the distal aspect of the femur and SRT was administered to the affected area in three 12-Gy fractions delivered on consecutive days (total radiation dose, 36 Gy). Percentage tumor necrosis and urinary pyridinoline concentrations were used to assess local tumor control. The short-term effect of SRT on skin was also evaluated. RESULTS: Tumors developed in 10 of 12 tibial sites and all 14 femoral sites. Administration of SRT to rats with femoral osteosarcoma was feasible and successful. Mean tumor necrosis of 95% was achieved histologically, and minimal adverse skin effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The orthotopic model of canine osteosarcoma in rats developed in this study was suitable for evaluating the effects of local tumor control and can be used in future studies to evaluate optimization of SRT duration, dose, and fractionation schemes. The model could also allow evaluation of other treatments in combination with SRT, such as chemotherapy or bisphosphonate, radioprotectant, or parathyroid hormone treatment.

Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock*.

BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.

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.

A sandwich ELISA for the detection of Wnt5a.

Wnt5a is a noncanonical member of the Wnt family of signaling molecules that has been linked to various physiological and pathological processes including cell differentiation, cell migration, cell growth, vascular remodeling, cancer and chronic inflammation. To understand the role of Wnt5a in these processes, it is necessary to determine the function and expression level of Wnt5a. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We found that a rabbit anti-human Wnt5a is a suitable capture antibody for establishing a sandwich ELISA. We used two systems to detect Wnt5a: (1) goat anti-mouse Wnt5a and horseradish peroxidase (HRP) conjugated F(ab')(2) donkey anti-goat IgG as detection and enzyme-linked antibodies respectively, or (2) biotinylated goat anti-mouse Wnt5a and HRP-streptavidin as detection antibody and enzyme-linked avidin respectively. A sandwich ELISA using either of these systems failed to detect recombinant mouse (rm)-Wnt5a diluted in Hank's balanced salt solution supplemented with Ca(2+) and Mg(2+) and 1% bovine serum albumin (HBBS+, 1% BSA). Addition of polyethylene glycol (PEG) to the HBBS+, buffer during the binding stage of rm-Wnt5a, afforded the detection of rm-Wnt5a. The use of PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a. The relationship between the ELISA signal and concentration of Wnt5a was linear with an R(2) of 0.9934. In summary, we have developed a specific and sensitive sandwich ELISA that detects rm-Wnt5a.

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.