ABSTRACT
Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.
Subject(s)
Immune Tolerance , Neoplasms/immunology , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cancer Vaccines/immunology , Gene Deletion , Immunotherapy , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Toll-Like Receptors/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.
Subject(s)
Adjuvants, Immunologic/pharmacology , Influenza Vaccines/pharmacology , Influenza, Human/immunology , Mitochondria/drug effects , Orthomyxoviridae Infections/immunology , Animals , Antibodies, Viral/immunology , Dendritic Cells/immunology , Female , Gene Expression , Humans , Immunity, Innate/drug effects , Influenza Vaccines/immunology , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred BALB C , Mitochondria/genetics , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Stress, Physiological , Toll-Like ReceptorsABSTRACT
Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage in Tlr7-/- mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, and Tlr7 deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount of Bacteroides and Lactobacillus, respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.
Subject(s)
Ethanol/toxicity , Interleukins/metabolism , Intestinal Mucosa/metabolism , Liver Diseases, Alcoholic/drug therapy , Membrane Glycoproteins/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 7/metabolism , Administration, Oral , Animals , Bacteroides/drug effects , Disease Models, Animal , Fatty Liver/complications , Fatty Liver/genetics , Fatty Liver/metabolism , Female , Gastrointestinal Microbiome/drug effects , Inflammation/complications , Inflammation/genetics , Inflammation/metabolism , Intestinal Mucosa/drug effects , Lactobacillus/drug effects , Ligands , Liver Diseases, Alcoholic/genetics , Liver Diseases, Alcoholic/metabolism , Liver Diseases, Alcoholic/physiopathology , Membrane Glycoproteins/agonists , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs , Pancreatitis-Associated Proteins/genetics , Pancreatitis-Associated Proteins/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , Signal Transduction/genetics , Tight Junctions/drug effects , Tight Junctions/pathology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/genetics , Interleukin-22ABSTRACT
Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm/drug effects , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Adenosine Triphosphatases/metabolism , Animals , Antibodies, Monoclonal , Breast/drug effects , Breast/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Mice , NIH 3T3 Cells , Nuclear Proteins/metabolism , Paclitaxel/pharmacology , Polycomb Repressive Complex 1/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolismABSTRACT
In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.
Subject(s)
Benzamides/pharmacology , NF-kappa B/metabolism , Thiazoles/pharmacology , Animals , Benzamides/chemistry , Cell Line , Dose-Response Relationship, Drug , Humans , Mice , Molecular Structure , Structure-Activity Relationship , Thiazoles/chemistryABSTRACT
Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.
Subject(s)
Adenine/analogs & derivatives , CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular/drug effects , Lung Neoplasms/drug therapy , Membrane Glycoproteins/agonists , Phosphatidic Acids/pharmacology , Toll-Like Receptor 7/agonists , Adenine/pharmacology , Administration, Intranasal , Animals , CD8-Positive T-Lymphocytes/pathology , Female , Immunity, Cellular/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Receptors, Antigen, T-Cell/immunology , Toll-Like Receptor 7/immunologyABSTRACT
The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/ß-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/ß-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/ß-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic ß-catenin. Moreover, TPA increased the association of ß-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/ß-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/ß-catenin signaling.
Subject(s)
Carcinogens/toxicity , Casein Kinase 1 epsilon/metabolism , Casein Kinase Idelta/metabolism , Skin Neoplasms/pathology , Tetradecanoylphorbol Acetate/toxicity , Wnt Signaling Pathway/drug effects , Animals , Axin Protein/metabolism , Carcinogenesis/chemically induced , Carcinogenesis/pathology , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase Idelta/antagonists & inhibitors , Cell Line, Tumor , Disease Models, Animal , Fibroblasts , HEK293 Cells , Humans , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Mice , Phosphorylation , Protein Stability/drug effects , Purines/pharmacology , Skin Neoplasms/chemically induced , Transcription Factor 4 , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.
Subject(s)
Adjuvants, Immunologic/chemical synthesis , Ligands , Monosaccharides/chemistry , Toll-Like Receptor 7/agonists , Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/pharmacology , Animals , Binding Sites , Cell Line , Dimerization , Humans , Interleukin-6/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Molecular Docking Simulation , Protein Structure, Tertiary , RAW 264.7 Cells , Structure-Activity Relationship , Toll-Like Receptor 7/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.
Subject(s)
Adenine/analogs & derivatives , Adjuvants, Immunologic/pharmacology , Gold/chemistry , Mannose/chemistry , Metal Nanoparticles/administration & dosage , Small Molecule Libraries/pharmacology , Splenomegaly/prevention & control , Toll-Like Receptor 7/agonists , Adenine/chemistry , Adenine/pharmacology , Adjuvants, Immunologic/chemistry , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Immunization , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Ligands , Metal Nanoparticles/chemistry , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Small Molecule Libraries/chemistry , Splenomegaly/immunology , Splenomegaly/pathology , Toll-Like Receptor 7/immunologyABSTRACT
Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/ß-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/ß-catenin pathway. Prodigiosin blocked Wnt/ß-catenin signaling by targeting multiple sites of this pathway, including the low-density lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3ß (GSK3ß). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3ß and suppressed ß-catenin-stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3ß, active ß-catenin, and cyclin D1. Through its ability to inhibit Wnt/ß-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Prodigiosin/pharmacology , Prodigiosin/therapeutic use , Wnt Signaling Pathway/drug effects , Animals , Apoptosis/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin D1/genetics , Dishevelled Proteins/genetics , Female , HEK293 Cells , Humans , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Tumor Burden/drug effects , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/geneticsABSTRACT
We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Membrane Glycoproteins/immunology , Myeloid Differentiation Factor 88/immunology , Orthomyxoviridae Infections/immunology , Toll-Like Receptor 4/immunology , Toll-Like Receptor 7/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adjuvants, Immunologic/pharmacology , Animals , Antibodies, Viral/immunology , Antigens, Viral/immunology , Female , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza Vaccines/immunology , Lung/immunology , Lung/virology , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology , Th1 Cells/immunology , Th2 Cells/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 7/genetics , VaccinationABSTRACT
Tumor-specific molecules are needed across diverse areas of oncology for use in early detection, diagnosis, prognosis and therapy. Large and growing public databases of transcriptome sequencing data (RNA-seq) derived from tumors and normal tissues hold the potential of yielding tumor-specific molecules, but because the data are new they have not been fully explored for this purpose. We have developed custom bioinformatic algorithms and used them with 296 high-grade serous ovarian (HGS-OvCa) tumor and 1,839 normal RNA-seq datasets to identify mRNA isoforms with tumor-specific expression. We rank prioritized isoforms by likelihood of being expressed in HGS-OvCa tumors and not in normal tissues and analyzed 671 top-ranked isoforms by high-throughput RT-qPCR. Six of these isoforms were expressed in a majority of the 12 tumors examined but not in 18 normal tissues. An additional 11 were expressed in most tumors and only one normal tissue, which in most cases was fallopian or colon. Of the 671 isoforms, the topmost 5% (n = 33) ranked based on having tumor-specific or highly restricted normal tissue expression by RT-qPCR analysis are enriched for oncogenic, stem cell/cancer stem cell, and early development loci--including ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. Many of the 33 isoforms are predicted to encode proteins with unique amino acid sequences, which would allow them to be specifically targeted for one or more therapeutic strategies--including monoclonal antibodies and T-cell-based vaccines. The systematic process described herein is readily and rapidly applicable to the more than 30 additional tumor types for which sufficient amounts of RNA-seq already exist.
Subject(s)
Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/therapy , RNA, Messenger/genetics , Transcriptome , Female , High-Throughput Nucleotide Sequencing , Humans , Ovarian Neoplasms/genetics , Real-Time Polymerase Chain ReactionABSTRACT
The bladder presents an attractive target for topical drug delivery. The barrier function of the bladder mucosa (urothelium) presents a penetration challenge for small molecules and nanoparticles. We found that focal mechanical injury of the urothelium greatly enhances the binding and penetration of intravesically-administered cell-penetrating peptide CGKRK (Cys-Gly-Lys-Arg-Lys). Notably, the CGKRK bound to the entire urothelium, and the peptide was able to penetrate into the muscular layer. This phenomenon was not dependent on intravesical bleeding and was not caused by an inflammatory response. CGKRK also efficiently penetrated the urothelium after disruption of the mucosa with ethanol, suggesting that loss of barrier function is a prerequisite for widespread binding and penetration. We further demonstrate that the ability of CGKRK to efficiently bind and penetrate the urothelium can be applied toward mucosal targeting of CGKRK-conjugated nanogels to enable efficient and widespread delivery of a model payload (rhodamine) to the bladder mucosa.
Subject(s)
Cell-Penetrating Peptides/administration & dosage , Drug Delivery Systems , Mucous Membrane/drug effects , Nanoparticles/administration & dosage , Urinary Bladder/drug effects , Administration, Intravesical , Animals , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mucous Membrane/injuries , Urinary Bladder/injuriesABSTRACT
WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/ß-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/ß-catenin signaling through FZD7 to maintain an undifferentiated phenotype.
Subject(s)
Embryonic Stem Cells/cytology , Frizzled Receptors/physiology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Cell Line , Frizzled Receptors/metabolism , Humans , Mice , Signal Transduction , Wnt3A Protein/metabolismABSTRACT
Although initially responsive to chemotherapy, many patients with ovarian cancer subsequently develop relapsed and potentially fatal metastatic disease, which is thought to develop from cancer stem cells (CSCs) that are relatively resistant to conventional therapy. Here, we show that CSCs express a type I receptor tyrosine kinase-like orphan receptor (ROR1), which is expressed during embryogenesis and by many different cancers, but not normal postpartum tissues. Ovarian cancers with high levels of ROR1 had stem cell-like gene-expression signatures. Furthermore, patients with ovarian cancers with high levels of ROR1 had higher rates of relapse and a shorter median survival than patients with ovarian cancers that expressed low-to-negligible amounts of ROR1. We found that ROR1-positive (ROR1(+)) cells isolated from primary tumor-derived xenografts (PDXs) also expressed aldehyde dehydrogenase 1 (ALDH1) and had a greater capacity to form spheroids and to engraft immune-deficient mice than did ROR1-negative (ROR1(Neg)) ovarian cancer cells isolated from the same tumor population. Treatment with UC-961, an anti-ROR1 mAb, or shRNA silencing of ROR1 inhibited expression of the polycomb ring-finger oncogene, Bmi-1, and other genes associated with the epithelial-mesenchymal transition. Moreover, shRNA silencing of ROR1, depletion of ROR1(+) cells, or treatment with UC-961 impaired the capacity of ovarian cancer cells to form spheroids or tumor xenografts. More importantly, treatment with anti-ROR1 affected the capacity of the xenograft to reseed a virgin mouse, indicating that targeting ROR1 may affect CSC self-renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which contributes to their capacity to form tumors, making ROR1 a potential target for the therapy of patients with ovarian cancer.
Subject(s)
Molecular Targeted Therapy/methods , Neoplastic Stem Cells/metabolism , Ovarian Neoplasms/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Kaplan-Meier Estimate , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Microscopy, Confocal , Neoplastic Stem Cells/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/prevention & control , Prognosis , RNA Interference , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Transplantation, HeterologousABSTRACT
UNLABELLED: Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross-protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor 4 (TLR4) and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4-MD2 complex, and 1V270 is a phospholipid-conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2-associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous and heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that intramuscular (i.m.) administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long-lasting, and balanced Th1- and Th2-type immunity; demonstrates efficacy in a variety of murine influenza virus vaccine models assaying homologous, heterologous, and heterosubtypic challenge viruses; and has an excellent safety profile. IMPORTANCE: Novel adjuvants are needed to enhance immunogenicity and increase the protective breadth of influenza virus vaccines to reduce the seasonal disease burden and ensure pandemic preparedness. We show here that the combination of synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic challenge models. Combining TLR4 and TLR7 ligands balances Th1- and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. The combined adjuvant has an attractive safety profile and the potential to augment seasonal-vaccine breadth, contribute to a broadly neutralizing universal vaccine formulation, and improve response time in an emerging pandemic.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Cross Protection , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 7/agonists , Adjuvants, Immunologic/chemical synthesis , Animals , Antibodies, Viral/immunology , Humans , Influenza Vaccines/administration & dosage , Influenza, Human/virology , Ligands , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Th1 Cells/immunology , Th2 Cells/immunology , Toll-Like Receptor 4/immunology , Toll-Like Receptor 7/immunologyABSTRACT
Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.
Subject(s)
Mutation/genetics , Mutation/physiology , Neoplasms/genetics , Phosphoproteins/genetics , RNA Splice Sites/genetics , Ribonucleoprotein, U2 Small Nuclear/genetics , Transcriptome/genetics , Humans , Neoplasms/metabolism , RNA Splicing Factors , Sequence Analysis, RNAABSTRACT
Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands. Therefore, such conjugates could represent a versatile platform for the development of vaccines against cancer and infectious diseases.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Dendritic Cells/immunology , Inflammation/immunology , Macrophages/immunology , Polysaccharides/chemistry , Toll-Like Receptor 7/physiology , Animals , Bone Marrow/drug effects , Bone Marrow/immunology , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/cytology , Dendritic Cells/drug effects , Female , Humans , Immunization , Inflammation/drug therapy , Inflammation/pathology , Ligands , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Toll-like receptor (TLR) stimulation has been implicated as a major contributor to chronic inflammation. Among these receptors, TLR4 has been described as a key regulator of endogenous inflammation and has been proposed as a therapeutic target. Previously, we discovered by high-throughput screening a group of substituted pyrimido[5,4-b]indoles that activated a nuclear factor-κB reporter in THP-1 human monocytic cells. A biologically active hit compound was resynthesized, and derivatives were prepared to assess structure-activity relationships. The derived compounds activated cells in a TLR4/myeloid differentiation protein 2 (MD2)-dependent and CD14-independent manner, using the myeloid differentiation primary response 88 and Toll/IL-1 receptor domain-containing adapter-inducing interferon-ß pathways. Two lead compounds, 1Z105 and 1Z88, were selected for further analysis based on favorable biologic properties and lack of toxicity. In vivo pharmacokinetics indicated that 1Z105 was orally bioavailable, whereas 1Z88 was not. Oral or parenteral doses of 1Z105 and 1Z88 induced undetectable or negligible levels of circulating cytokines and did not induce hepatotoxicity when administered to galactosamine-conditioned mice, indicating good safety profiles. Both compounds were very effective in preventing lethal liver damage in lipopolysaccharide treated galatosamine-conditioned mice. Orally administered 1Z105 and parenteral 1Z88 prevented arthritis in an autoantibody-driven murine model. Hence, these low molecular weight molecules that target TLR4/MD2 were well tolerated and effective in reducing target organ damage in two different mouse models of sterile inflammation.
Subject(s)
Inflammation/drug therapy , Lymphocyte Antigen 96/physiology , Toll-Like Receptor 4/physiology , Animals , Arthritis, Experimental/prevention & control , Galactosamine/toxicity , Hep G2 Cells , High-Throughput Screening Assays , Humans , Ligands , Lipopolysaccharide Receptors/physiology , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/physiology , Signal Transduction , Structure-Activity RelationshipABSTRACT
BACKGROUND: Kaposi's sarcoma (KS) is the most frequently occurring cancer in Mozambique among men and the second most frequently occurring cancer among women. Effective therapeutic treatments for KS are poorly understood in this area. There is an unmet need to develop a simple but accurate tool for improved monitoring and diagnosis in a resource-limited setting. Standardized clinical photographs have been considered to be an essential part of the evaluation. METHODS: When a therapeutic response is achieved, nodular KS often exhibits a reduction of the thickness without a change in the base area of the lesion. To evaluate the vertical space along with other characters of a KS lesion, we have created an innovative imaging system with a consumer light-field camera attached to a miniature "photography studio" adaptor. The image file can be further processed by computational methods for quantification. RESULTS: With this novel imaging system, each high-quality 3D image was consistently obtained with a single camera shot at bedside by minimally trained personnel. After computational processing, all-focused photos and measurable 3D parameters were obtained. More than 80 KS image sets were processed in a semi-automated fashion. CONCLUSIONS: In this proof-of-concept study, the feasibility to use a simple, low-cost and user-friendly system has been established for future clinical study to monitor KS therapeutic response. This 3D imaging system can be also applied to obtain standardized clinical photographs for other diseases.