The G allele of SNP rs3922 reduces the binding affinity between IGF2BP3 and CXCR5 correlating with a lower antibody production.

Effective vaccines that function through humoral immunity seek to produce high-affinity antibodies. Our previous research identified the single-nucleotide polymorphism rs3922G in the 3'UTR of CXCR5 as being associated with nonresponsiveness to the hepatitis B vaccine. The differential expression of CXCR5 between the dark zone (DZ) and light zone (LZ) is critical for organizing the functional structure of the germinal center (GC). In this study, we report that the RNA-binding protein IGF2BP3 can bind to CXCR5 mRNA containing the rs3922 variant to promote its degradation via the nonsense-mediated mRNA decay pathway. Deficiency of IGF2BP3 leads to increased CXCR5 expression, which results in the disappearance of CXCR5 differential expression between DZ and LZ, disorganized GCs, aberrant somatic hypermutations, and reduced production of high-affinity antibodies. Furthermore, the affinity of IGF2BP3 for the rs3922G-containing sequence is lower than that for the rs3922A counterpart, which may explain the nonresponsiveness to the hepatitis B vaccination. Together, our findings suggest that IGF2BP3 plays a crucial role in the production of high-affinity antibodies in the GC by binding to the rs3922-containing sequence to regulate CXCR5 expression.

Chimeric antigen receptor macrophages activated through TLR4 or IFN-γ receptors suppress breast cancer growth by targeting VEGFR2.

Chimeric antigen receptor macrophage (CAR-M) is a promising immunotherapy strategy of anti-tumor due to its high infiltration, direct phagocytosis of tumor cells, immunomodulation of tumor microenvironment (TME) and linkage of innate and adaptive immunity. Here a series of novelly designed CAR-Ms by targeting vascular endothelial growth factor receptor-2 (VEGFR2), which highly expressed in tumor cells and TME, were evaluated. Their activation signals were transduced by Tlr4 or Ifn-γ receptors either alone or in combination, which were designed to mediate M1 polarization of macrophages as the downstream of lipopolysaccharide or Ifn-γ that had been widely reported. Our results showed that VEGFR2-targeting CAR-Ms could be activated under the stimulation of VEGFR2-expressing cells. They exhibited higher expression of CD86, MHCII and TNF-α in vitro and enhanced tumor suppressive abilities in vivo. Implantation of these CAR-Ms into 4T1 breast cancer-bearing mice could obviously inhibit the progression of tumor without significant toxic side effects, especially the group of mmC in which constructed with Tlr4 as the intracellular domain of CAR. In conclusion, this research provides a promising design of CAR that induce macrophages activation by Tlr4 and/or Ifn-γ receptors, and these CAR-Ms could effectively inhibit tumor growth through targeting VEGFR2.

Sharpening up tumor microenvironment to enhance the efficacy of immune checkpoint blockade on head and neck cancer using a CpG-oligodeoxynucleotide.

Head and neck cancers are a type of life-threatening cancers characterized by an immunosuppressive tumor microenvironment. Only less than 20% of the patients respond to immune checkpoint blockade therapy, indicating the need for a strategy to increase the efficacy of immunotherapy for this type of cancers. Previously, we identified a type B CpG-oligodeoxynucleotide (CpG-ODN) called CpG-2722, which has the universal activity of eliciting an immune response in grouper, mouse, and human cells. In this study, we further characterized and compared its cytokine-inducing profiles with different types of CpG-ODNs. The antitumor effect of CpG-2722 was further investigated alone and in combination with an immune checkpoint inhibitor in a newly developed syngeneic orthotopic head and neck cancer animal model. Along with other inflammatory cytokines, CpG-2722 induces the gene expressions of interleukin-12 and different types of interferons, which are critical for the antitumor response. Both CpG-2722 and anti-programmed death (PD)-1 alone suppressed tumor growth. Their tumor suppression efficacies were further enhanced when CpG-2722 and anti-PD-1 were used in combination. Mechanistically, CpG-2722 shaped a tumor microenvironment that is favorable for the action of anti-PD-1, which included promoting the expression of different cytokines such as IL-12, IFN-ß, and IFN-γ, and increasing the presence of plasmacytoid dendritic cells, M1 macrophages, and CD8 positive T cells. Overall, CpG-2722 provided a priming effect for CD8 positive T cells by sharpening the tumor microenvironment, whereas anti-PD-1 released the brake for their tumor-killing effect, resulting in an enhanced efficacy of the combined CpG-2722 and anti-PD-1.

Mouse 4T1 Breast Cancer Cell-Derived Exosomes Induce Proinflammatory Cytokine Production in Macrophages via miR-183.

Tumor-associated macrophages (TAMs) play a critical role in the tumor inflammatory microenvironment and facilitate tumor growth and metastasis. Most types of tumors aberrantly express microRNAs (miRNAs), which can be transferred between cells by exosomes and can regulate gene expression in recipient cells, but it remains unclear whether tumor-derived miRNAs are transferred by exosomes and regulate the TAM phenotype. We report that mouse 4T1 breast cancer cell-derived exosomes enhanced TAM expression of IL-1ß, IL-6, and TNF-α and that inhibition of 4T1-cell exosome secretion through short hairpin RNA-mediated Rab27a/b depletion repressed tumor growth and metastasis and markedly downregulated IL-1ß, IL-6, and TNF-α in a 4T1 breast tumor model. Furthermore, miRNA expression profiling revealed that three miRNAs (miR-100-5p, miR-183-5p, and miR-125b-1-3p) were considerably more abundant in 4T1 cell exosomes than in mouse bone marrow-derived macrophages, indicating potential exosome-mediated transfer of the miRNAs, and, notably, miR-183-5p was found to be transferred from 4T1 cells to macrophages through exosomes. Moreover, PPP2CA was verified as an miR-183-5p target gene, and PPP2CA downregulation enhanced NF-κB signaling and promoted macrophage expression of IL-1ß, IL-6, and TNF-α. Lastly, when miR-183-5p was downregulated in exosomes through miR-183-5p sponge expression in 4T1 cells, these 4T1-derived exosomes triggered diminished p65 phosphorylation and IL-1ß, IL-6, and TNF-α secretion, and the miRNA downregulation also led to repression of tumor growth and metastasis in the 4T1 breast tumor model in vivo. Thus, miR-183-5p expressed in tumor cells was transferred to macrophages by exosomes and promoted the secretion of proinflammatory cytokines by inhibiting PPP2CA expression, which contributed to tumor progression in a breast cancer model.

MicroRNA-3613-3p functions as a tumor suppressor and represents a novel therapeutic target in breast cancer.

BACKGROUND: MicroRNAs have been reported to participate in tumorigenesis, treatment resistance, and tumor metastasis. Novel microRNAs need to be identified and investigated to guide the clinical prognosis or therapy for breast cancer. METHOD: The copy number variations (CNVs) of MIR3613 from Cancer Genome Atlas (TCGA) or Cancer Cell Line Encyclopedia (CCLE) were analyzed, and its correlation with breast cancer subtypes or prognosis was investigated. The expression level of miR-3613-3p in tumor tissues or serum of breast cancer patients was detected using in situ hybridization and qPCR. Gain-of-function studies were performed to determine the regulatory role of miR-3613-3p on proliferation, apoptosis, and tumor sphere formation of human breast cancer cells MDA-MB-231 or MCF-7. The effects of miR-3613-3p on tumor growth or metastasis in an immunocompromised mouse model of MDA-MB-231-luciferase were explored by intratumor injection of miR-3613-3p analogue. The target genes, interactive lncRNAs, and related signaling pathways of miR-3613-3p were identified by bioinformatic prediction and 3'-UTR assays. RESULTS: We found that MIR3613 was frequently deleted in breast cancer genome and its deletion was correlated with the molecular typing, and an unfavorable prognosis in estrogen receptor-positive patients. MiR-3613-3p level was also dramatically lower in tumor tissues or serum of breast cancer patients. Gain-of-function studies revealed that miR-3613-3p could suppress proliferation and sphere formation and promote apoptosis in vitro and impeded tumor growth and metastasis in vivo. Additionally, miR-3613-3p might regulate cell cycle by targeting SMS, PAFAH1B2, or PDK3 to restrain tumor progression. CONCLUSION: Our findings indicate a suppressive role of miR-3613-3p in breast cancer progression, which may provide an innovative marker or treatment for breast cancer patients.

The three branches of the unfolded protein response exhibit differential significance in breast cancer growth and stemness.

The unfolded protein response (UPR) is widely activated in cancers. The mammalian UPR encompasses three signaling branches, namely inositol-requiring enzyme-1α (IRE1α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6α (ATF6α). The functional significance of each branch in tumorigenesis is incompletely understood, especially in cancer stem cells (CSCs). Here, we report that inhibition and silencing of the three UPR sensors has differential effects on breast cancer growth and the CSC population. The levels of PERK and ATF6α strongly correlate with the expression of sex determining region Y (SRY)-box 2 (SOX2), a pluripotency regulator, in human breast cancer tissues. UPR activation is also elevated in the CSC-enriched mammospheres. Inhibition of the UPR sensors or excess ER stress markedly reduces the formation and maintenance of mammospheres, suggesting that an appropriate level of UPR activation is critical for the CSC survival. Mechanistically, transcription factors from UPR and pluripotency pathways interact and reciprocally influence each other. A transcription modulator, CCAAT-enhancer-binding protein delta (C/EBPδ), interacts with pluripotency regulator, SOX2, and UPR transcription factors, thus likely serving as a link to coordinate UPR and pluripotency maintenance in CSCs. Our findings demonstrate that UPR is critical for both cancer growth and pluripotency, and highlight the differential role and complexity of the three UPR branches in tumorigenesis.

ICAM3 mediates tumor metastasis via a LFA-1-ICAM3-ERM dependent manner.

ICAM3 was reported to promote metastasis in tumors. However, the underlying mechanism remains elusive. Here, we disclosed that the expression of ICAM3 was closely correlated with the TNM stage of human breast and lung cancer, as well as the dominant overexpression in high aggressive tumor cell lines (231 and A549 cells). Moreover, the knockdown of ICAM3 inhibited tumor metastasis whereas the ectopic expression of ICAM3 promoted tumor metastasis both in vitro and in vivo. In addition, exploration of the underlying mechanism demonstrated that ICAM3 not only binds to LFA-1 with its extracellular domain and structure protein ERM but also to lamellipodia with its intracellular domain which causes a tension that pulls cells apart (metastasis). Furthermore, ICAM3 extracellular or intracellular mutants alternatively abolished ICAM3 mediated tumor metastasis in vitro and in vivo. As a therapy strategy, LFA-1 antibody or Lifitegrast restrained tumor metastasis via targeting ICAM3-LFA-1 interaction. In summary, the aforementioned findings suggest a model of ICAM3 in mediating tumor metastasis. This may provide a promising target or strategy for the prevention of tumor metastasis.

Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7-9.

Activation of TLR7-9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7-9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7-9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7-9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7-9.

Toll-like receptor 9 and 21 have different ligand recognition profiles and cooperatively mediate activity of CpG-oligodeoxynucleotides in zebrafish.

CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9, whereas mammalian genomes lack TLR21. Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpG-ODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.

Ifit1 Protects Against Lipopolysaccharide and D-galactosamine-Induced Fatal Hepatitis by Inhibiting Activation of the JNK Pathway.

Treatment of mice with lipopolysaccharide (LPS) and the liver-specific transcriptional inhibitor D-(+)-galactosamine (GalN) induces fatal hepatitis, which is mediated by tumor necrosis factor α (TNF-α) and characterized by massive hepatic apoptosis. Previous studies suggest that GalN increases the sensitivity to LPS/TNF-α, probably by blocking the transcription of protective factors, but the identity of most of these factors is still unclear. Here, we report that Ifit1 protects against LPS/GalN-induced fatal hepatitis. Forced expression of Ifit1 in hepatocytes significantly diminished TNF-α-mediated apoptosis. Moreover, targeted expression of Ifit1 in the liver by recombinant adeno-associated virus serotype 8 protected mice from LPS/GalN-induced lethal hepatitis, which was associated with the inhibition of TNF-α-mediated activation of the c-Jun N-terminal kinase (JNK)-Bim cascade. Furthermore, Ifit1 bound to a scaffolding protein Axin and inhibited its function to mediate JNK activation. Together, our data demonstrate that Ifit1 is a novel protective factor that inhibits LPS/GalN-induced (TNF-α-mediated) fatal hepatitis, suggesting that Ifit1 is a potential therapeutic target for treatment of inflammatory liver diseases.

Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation.

Although Oct4 is known as a critical transcription factor involved in maintaining "stemness", its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression.

Tumor-associated macrophages regulate murine breast cancer stem cells through a novel paracrine EGFR/Stat3/Sox-2 signaling pathway.

The cancer stem cell (CSC) hypothesis has gained significant recognition as a descriptor of tumorigenesis. Additionally, tumor-associated macrophages (TAMs) are known to promote growth and metastasis of breast cancer. However, it is not known whether TAMs mediate tumorigenesis through regulation of breast CSCs. Here, we report that TAMs promote CSC-like phenotypes in murine breast cancer cells by upregulating their expression of Sox-2. These CSC-like phenotypes were characterized by increased Sox-2, Oct-4, Nanog, AbcG2, and Sca-1 gene expression, in addition to increased drug-efflux capacity, resistance to chemotherapy, and increased tumorigenicity in vivo. Downregulation of Sox-2 in tumor cells by siRNA blocked the ability of TAMs to induce these CSC-like phenotypes and inhibited tumor growth in vivo. Furthermore, we identified a novel epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (Stat3)/Sox-2 paracrine signaling pathway between macrophages and mouse breast cancer cells that is required for macrophage-induced upregulation of Sox-2 and CSC phenotypes in tumor cells. We showed that this crosstalk was effectively blocked by the small molecule inhibitors AG1478 or CDDO-Im against EGFR and Stat3, respectively. Therefore, our report identifies a novel role for TAMs in breast CSC regulation and establishes a rationale for targeting the EGFR/Stat3/Sox-2 signaling pathway for CSC therapy.

Targeting carnitine palmitoyl transferase 1A (CPT1A) induces ferroptosis and synergizes with immunotherapy in lung cancer.

Despite the successful application of immune checkpoint therapy, no response or recurrence is typical in lung cancer. Cancer stem cells (CSCs) have been identified as a crucial player in immunotherapy-related resistance. Ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, is highly regulated by cellular metabolism remolding and has been shown to have synergistic effects when combined with immunotherapy. Metabolic adaption of CSCs drives tumor resistance, yet the mechanisms of their ferroptosis defense in tumor immune evasion remain elusive. Here, through metabolomics, transcriptomics, a lung epithelial-specific Cpt1a-knockout mouse model, and clinical analysis, we demonstrate that CPT1A, a key rate-limiting enzyme of fatty acid oxidation, acts with L-carnitine, derived from tumor-associated macrophages to drive ferroptosis-resistance and CD8+ T cells inactivation in lung cancer. Mechanistically, CPT1A restrains ubiquitination and degradation of c-Myc, while c-Myc transcriptionally activates CPT1A expression. The CPT1A/c-Myc positive feedback loop further enhances the cellular antioxidant capacity by activating the NRF2/GPX4 system and reduces the amount of phospholipid polyunsaturated fatty acids through ACSL4 downregulating, thereby suppressing ferroptosis in CSCs. Significantly, targeting CPT1A enhances immune checkpoint blockade-induced anti-tumor immunity and tumoral ferroptosis in tumor-bearing mice. The results illustrate the potential of a mechanism-guided therapeutic strategy by targeting a metabolic vulnerability in the ferroptosis of CSCs to improve the efficacy of lung cancer immunotherapy.

Targeting tumor cell-to-macrophage communication by blocking Vtn-C1qbp interaction inhibits tumor progression via enhancing macrophage phagocytosis.

Background: Cancer cells are capable of evading clearance by macrophages through overexpression of anti-phagocytic surface proteins known as "don't eat me" signals. Monoclonal antibodies that antagonize the "don't-eat-me" signaling in macrophages and tumor cells by targeting phagocytic checkpoints have shown therapeutic promises in several cancer types. However, studies on the responses to these drugs have revealed the existence of other unknown "don't eat me" signals. Moreover, identification of key molecules and interactions regulating macrophage phagocytosis is required for tumor therapy. Methods: CRISPR screen was used to identify genes that impede macrophage phagocytosis. To explore the function of Vtn and C1qbp in phagocytosis, knockdown and subsequent functional experiments were conducted. Flow cytometry were performed to explore the phagocytosis rate, polarization of macrophage, and immune microenvironment of mouse tumor. To explore the underlying molecular mechanisms, RNA sequencing, immunoprecipitation, mass spectrometry, and immunofluorescence were conducted. Then, in vivo experiments in mouse models were conducted to explore the probability of Vtn knockdown combined with anti-CD47 therapy in breast cancer. Single-cell sequencing data from the Gene Expression Omnibus from The Cancer Genome Atlas database were analyzed. Results: We performed a genome-wide CRISPR screen to identify genes that impede macrophage phagocytosis, followed by analysis of cell-to-cell interaction databases. We identified a ligand-receptor pair of Vitronectin (Vtn) and complement C1Q binding protein (C1qbp) in tumor cells or macrophages, respectively. We demonstrated tumor cell-secreted Vtn interacts with C1qbp localized on the cell surface of tumor-associated macrophages, inhibiting phagocytosis of tumor cells and shifting macrophages towards the M2-like subtype in the tumor microenvironment. Mechanistically, the Vtn-C1qbp axis facilitated FcγRIIIA/CD16-induced Shp1 recruitment, which reduced the phosphorylation of Syk. Furthermore, the combination of Vtn knockdown and anti-CD47 antibody effectively enhanced phagocytosis and infiltration of macrophages, resulting in a reduction of tumor growth in vivo. Conclusions: This work has revealed that the Vtn-C1qbp axis is a new anti-phagocytic signal in tumors, and targeting Vtn and its interaction with C1qbp may sensitize cancer to immunotherapy, providing a new molecular target for the treatment of triple-negative breast cancer.

Exosomal miRNA-223-3p derived from tumor associated macrophages promotes pulmonary metastasis of breast cancer 4T1 cells.

Research about the effect of exosomes derived from tumor associated macrophages (TAM-exos) in the distant organ metastasis of breast cancer is limited. In this study, we found that TAM-exos could promote the migration of 4T1 cells. Through comparing the expression of microRNAs in 4T1 cells, TAM-exos, and exosomes from bone marrow derived macrophages (BMDM-exos) by sequencing, miR-223-3p and miR-379-5p were screened out as two noteworthy differentially expressed microRNAs. Furthermore, miR-223-3p was confirmed to be the reason for the improved migration and metastasis of 4T1 cells. The expression of miR-223-3p was also increased in 4T1 cells isolated from the lung of tumor-bearing mice. Cbx5, which has been reported to be closely related with metastasis of breast cancer, was identified to be the target of miR-223-3p. Based on the information of breast cancer patients from online databases, miR-223-3p had a negative correlation with the overall survival rate of breast cancer patients within a three-year follow-up, while Cbx5 showed an opposite relationship. Taken together, miR-223-3p in TAM-exos can be delivered into 4T1 cells and exosomal miR-223-3p promotes pulmonary metastasis of 4T1 cells by targeting Cbx5.

Functional interaction of heat shock protein 90 and Beclin 1 modulates Toll-like receptor-mediated autophagy.

Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll-like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent (EC(50) 100 nM) and time-dependent (t(50) 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3- and TLR4-mediated autophagy. In addition, S. typhimurium infection-induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR-mediated autophagy.

Oral DNA vaccines target the tumor vasculature and microenvironment and suppress tumor growth and metastasis.

Four novel oral DNA vaccines provide protection against melanoma, colon, breast, and lung carcinoma in mouse models. Vaccines are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs and respectively target vascular endothelial growth factor receptor-2, transcription factor Fos-related antigen-1, anti-apoptosis protein survivin and Legumain, an asparaginyl endopeptidase specifically overexpressed on tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). These vaccines are all capable of inducing potent cell-mediated protective immunity against self-antigens, resulting in marked suppression of tumor growth and dissemination. Key mechanisms induced by these DNA vaccines include efficient suppression of angiogenesis in the tumor vasculature and marked activation of cytotoxic T cells, natural killer cells, and antigen-presenting dendritic cells. The vaccine targeting Legumain establishes the new paradigm whereby a reduction in the density of TAMs in the TME decreases the release of factors potentiating tumor growth and angiogenesis. This, in turn, remodels the TME and decreases its immunosuppressive milieu and thereby potentiates the DNA vaccine's ability to effectively suppress tumor cell proliferation, vascularization, and metastasis. It is anticipated that such research efforts will lead to novel DNA-based vaccines that will be effective for the treatment of cancer.

Recent Advances in the Development of Toll-like Receptor Agonist-Based Vaccine Adjuvants for Infectious Diseases.

Vaccines are powerful tools for controlling microbial infections and preventing epidemic diseases. Efficient inactive, subunit, or viral-like particle vaccines usually rely on a safe and potent adjuvant to boost the immune response to the antigen. After a slow start, over the last decade there has been increased developments on adjuvants for human vaccines. The development of adjuvants has paralleled our increased understanding of the molecular mechanisms for the pattern recognition receptor (PRR)-mediated activation of immune responses. Toll-like receptors (TLRs) are a group of PRRs that recognize microbial pathogens to initiate a host's response to infection. Activation of TLRs triggers potent and immediate innate immune responses, which leads to subsequent adaptive immune responses. Therefore, these TLRs are ideal targets for the development of effective adjuvants. To date, TLR agonists such as monophosphoryl lipid A (MPL) and CpG-1018 have been formulated in licensed vaccines for their adjuvant activity, and other TLR agonists are being developed for this purpose. The COVID-19 pandemic has also accelerated clinical research of vaccines containing TLR agonist-based adjuvants. In this paper, we reviewed the agonists for TLR activation and the molecular mechanisms associated with the adjuvants' effects on TLR activation, emphasizing recent advances in the development of TLR agonist-based vaccine adjuvants for infectious diseases.

MTDH/AEG-1-based DNA vaccine suppresses lung metastasis and enhances chemosensitivity to doxorubicin in breast cancer.

The gene MTDH/AEG-1 is overexpressed in more than 40% of breast cancer patients, and it is associated with poor clinical outcomes. Previous studies have indicated that MTDH/AEG-1 could promote metastatic lung-seeding and enhance chemoresistance. Therefore, MTDH/AEG-1 could be a candidate target against breast cancer lung metastasis. We demonstrated that MTDH/AEG-1-based DNA vaccine, delivered by attenuated Salmonella typhimurium, could evoke strong CD8(+) cytotoxic-T-cell mediated immune responses against breast cancer. This vaccine showed anti-tumor growth and metastasis efficacy in a prophylactic setting. Importantly, in a therapeutic model, MTDH/AEG-1 vaccine was proved to increase chemosensitivity to doxorubicin and inhibit breast cancer lung metastasis. This vaccine could also prolong the life span of tumor-bearing mice without significant side effects in vivo. These results suggested that this novel DNA vaccine was effective in the inhibition of breast cancer growth and metastasis, and this vaccine in combination with chemotherapies offered new strategies for the clinical therapeutics of breast cancer metastasis.

Synthetic enzyme inhibitor: a novel targeting ligand for nanotherapeutic drug delivery inhibiting tumor growth without systemic toxicity.

Unresolved problems associated with ligand-targeting of liposomal nanoparticles (NPs) to solid tumors include variable target receptor expression due to genetic heterogeneity and insufficient target specificity, leading to systemic toxicities. This study addresses these issues by developing a novel ligand-targeting strategy for liposomal NPs using RR-11a, a synthetic enzyme inhibitor of Legumain, an asparaginyl endopeptidase. Cell-surface expression of Legumain is driven by hypoxic stress, a hallmark of solid tumors. Legumain-targeted RR-11a-coupled NPs revealed high ligand-receptor affinity, enhanced solid-tumor penetration and uptake by tumor cells. Treatment of tumor-bearing mice with RR-11a-coupled NPs encapsulating doxorubicin resulted in improved tumor selectivity and drug sensitivity, leading to complete inhibition of tumor growth. These antitumor effects were achieved while eliminating systemic drug toxicity. Therefore, synthetic enzyme inhibitors, such as RR-11a, represent a new class of compounds that can be used for highly specific ligand-targeting of NPs to solid tumors. FROM THE CLINICAL EDITOR: This study addresses the problems associated with ligand-targeting of liposomal nanoparticles to solid tumors with variable target receptor expression. A novel and efficacious targeting strategy has been developed towards a synthetic enzyme inhibitor of Legumain. The authors demonstrate successful tumor growth inhibiting effect while eliminating systemic drug toxicity in an animal model using this strategy.