Suppressor Of Cytokine Signaling Proteins Research Articles

Quercetin Impedes Th17 Cell Differentiation to Mitigate Arthritis Involving PPARγ-Driven Transactivation of SOCS3 and Redistribution Corepressor SMRT from PPARγ to STAT3.

Quercetin (QU) is one of the most abundant flavonoids in plants and has attracted the attention of researchers because of its remarkable antirheumatoid arthritis (RA) effects and extremely low adverse reactions. However, the underlying mechanism needs further study. Flow cytometry, immunofluorescence, enzyme linked immunosorbent assay （ELISA）, and quantitative real-time polymerase chain reaction （qRT-PCR） reveal the obvious inhibitory effects of QU on Th17 cell differentiation in arthritic mice. More importantly, QU markedly limits the development of Th17 cell polarization, which is virtually compromised by the treatment with peroxisome proliferator activated receptor γ (PPARγ) inhibitor GW9662 and knockdown of PPARγ. Additionally, molecular dynamics simulation and immunofluorescence exhibit QU directly binds to PPARγ and increases PPARγ nuclear translocation. Besides, QU confers its moderation effect on suppressor of cytokine signaling protein (SOCS3)/signal transducer and activator of transcription 3 (STAT3) axis partially depending on PPARγ. Furthermore, coimmunoprecipitation shows QU redistributes the corepressor silencing mediator for retinoid and thyroid-hormone receptors (SMRT) from PPARγ to STAT3. Finally, the inhibition of Th17 response and the antiarthritic effect of QU are nullified by GW9662 treatment in arthritic mice. QU targets PPARγ and consequently inhibits Th17 cell differentiation by dual inhibitory activity of STAT3 to exert antiarthritic effect. The findings facilitate its development and put forth a stage for uncovering the mechanism of other naturally occurring compounds with chemical structures similar to QU.

Structure-Activity Relationship Investigations of Novel Constrained Chimeric Peptidomimetics of SOCS3 Protein Targeting JAK2.

SOCS3 (suppressor of cytokine signaling 3) protein suppresses cytokine-induced inflammation and its deletion in neurons or immune cells increases the pathological growth of blood vessels. Recently, we designed several SOCS3 peptidomimetics by assuming as template structures the interfacing regions of the ternary complex constituted by SOCS3, JAK2 (Janus Kinase 2) and gp130 (glycoprotein 130) proteins. A chimeric peptide named KIRCONG chim, including non-contiguous regions demonstrated able to bind to JAK2 and anti-inflammatory and antioxidant properties in VSMCs (vascular smooth muscle cells). With the aim to improve drug-like features of KIRCONG, herein we reported novel cyclic analogues bearing different linkages. In detail, in two of them hydrocarbon cycles of different lengths were inserted at positions i/i+5 and i/i+7 to improve helical conformations of mimetics. Structural features of cyclic compounds were investigated by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopies while their ability to bind to catalytic domain of JAK2 was assessed through MST (MicroScale Thermophoresis) assay as well as their stability in biological serum. Overall data indicate a crucial role exerted by the length and the position of the cycle within the chimeric structure and could pave the way to the miniaturization of SOCS3 protein for therapeutic aims.

Long Non-coding RNA GAS5/miR-520-3p/SOCS3 Axis Regulates Inflammatory Response in Lipopolysaccharide-Induced Macrophages.

Sepsis is mainly caused by infection, and inflammation plays a vital role in the progression of sepsis. Increasing evidence shows the regulatory mechanism of long non-coding RNA growth arrest-specific 5 (GAS5) in inflammatory response. However, the potential role of GAS5 in sepsis was not really clear. Here, we set to investigate the role and mechanism of GAS5 in the inflammatory response of lipopolysaccharide (LPS)-induced macrophages in vitro. Levels of GAS5, microRNA-520-3p, suppressor of cytokine signaling 3 (SOCS3) and inflammatory cytokines tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 and IL-1β were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Nitric oxide (NO) release was measured through flow cytometry. The levels of TNF-α, IL-6, and IL-1β were also detected using ELISA. Dual-luciferase reporter and RNA pull-down assays were performed to clarify the relationship between miR-520-3p and GAS5 or SOCS3. Western blot was carried out to assess SOCS3 protein expression in macrophages. GAS5 level was remarkably decreased in sepsis serum and it was inversely related to the severity of sepsis. Upregulation of GAS5 repressed inflammatory response in LPS-induced macrophages, and the inhibitory effect was returned by miR-520-3p mimics. Moreover, miR-520-3p inhibitor downregulated the levels of inflammatory factors of TNF-α, IL-6, and IL-1β, as well as suppressed NO release. Mechanically, GAS5 functioned as a sponge of miR-520-3p and miR-520-3p directly targeted SOCS3. GAS5 regulated inflammatory response by the miR-520-3p/SOCS3 axis in LPS-induced macrophages, which furnished a novel therapeutic idea in clinical treatment of inflammation-induced sepsis.

Lithium Chloride Promotes Milk Protein and Fat Synthesis in Bovine Mammary Epithelial Cells via HIF-1α and β-Catenin Signaling Pathways.

Lithium is one of the trace elements with many physiological properties, such as being anti-cancer, anti-viral, and anti-inflammatory. However, little is known about its effect on milk synthesis during lactation. Therefore, we selected different concentrations (5mM, 10mM, and 20mM) of lithium chloride (LiCl) and assessed the effect of LiCl on bovine mammary epithelial (MAC-T) cells that underwent 4days of differentiation induction. Moreover, we analyzed the effect of LiCl on the expression of genes related to milk fat and milk protein synthesis. Herein, LiCl (5-20mM) significantly increased the expression of β-casein, promoted mRNA expression and phosphorylated protein expression of the signal transduction molecule and activator of transcription 5β (STAT5-β), and inhibited mRNA and protein expression of suppressor of cytokine signaling 2 (SOCS2). In contrast, 5 and 10mM LiCl significantly inhibited expression of SOCS3. LiCl at concentration of 5-20mM enhanced phosphorylation level of mTOR protein; at 10mM and 20mM, LiCl significantly promoted expression and phosphorylation of downstream ribosomal protein S6 kinase beta-1 (S6K1) protein. Considering milk fat synthesis, mRNA expression of acetyl CoA carboxylase (ACC) and lipoprotein lipase (LPL) genes was considerably increased in the presence of LiCl (5-20mM). Additionally, increased protein expression levels of stearoyl-CoA desaturase (SCD), peroxisome proliferator-activated receptor-γ (PPARγ), and sterol regulatory element-binding protein 1 (SREBP1) were observed at all LiCl concentrations tested. Subsequently, LiCl (5-20mM) significantly promoted protein expression and phosphorylation of β-catenin, while 10mM and 20mM of LiCl significantly promoted protein expression of hypoxia-inducible factor-1α (HIF-1α). Collectively, it has been shown that 10mM LiCl can effectively activate HIF-1α, β-catenin, and β-catenin downstream signaling pathways. Conversely, at 10mM, LiCl inhibited SOCS2 and SOCS3 protein expression through JAK2/STAT5, mTOR, and SREBP1 signaling pathways, improving synthesis of milk protein and fat. Therefore, LiCl can be used as a potential nutrient to regulate milk synthesis in dairy cows.

Expression of SOCS1 Protein in Endotoxin-Tolerant Mouse Model and Its Regulation Mechanism by mir-150.

In order to investigate the expression of Suppressor of Cytokine Signaling 1 (SOCS1) and its regulatory mechanism by mir-150 in a lipopolysaccharide (LPS) tolerant mouse model of endotoxin, a total of 60 male BALB/C mice were randomly divided into 2 groups. The LPS is used to construct the endotoxin resistant mouse model and the mice are included in the model group (n = 30), 0.9% sodium chloride injection is used to construct the normal control group (n = 30). And tumor necrosis factor-α (TNF-α) is determined by Elisa to determine whether the model was successfully constructed. The correlation between SOCS1 protein and mir-150 is analyzed by the Pearson correlation coefficient. In the experiments, the results show that the expression of TNF-α in the macrophage fluid of the model group is significantly decreased (P < 0.05), indicating that the endotoxin tolerance mouse model is successfully constructed, so the secretion of TNF-α is reduced.

Inhibition of miR-483-5p improves the proliferation, invasion and inflammatory response of triple-negative breast cancer cells by targeting SOCS3.

microRNAs (miRs) have been indicated to serve oncogenic or tumor suppressor roles. However, the role of miR-483-5p in breast cancer and its associated molecular mechanisms remain unclear. In the present study, compared with adjacent normal tissues and MCF-10a cells, the expression level of miR-483-5p was upregulated in triple-negative breast cancer (TNBC) tissues and TNBC cell lines. Bioinformatic analysis and luciferase reporter assay confirmed the presence of miR-483-5p binding sites in the 3'-untranslated region of suppressor of cytokine signaling 3 (SOCS3). In addition, the expression level of SOCS3 protein in TNBC tissues was markedly lower compared with in adjacent tissues, and miR-483-5p expression was negatively correlated with SOCS3 expression in TNBC tissues. Cell proliferation and flow cytometry assays indicated that knockdown of miR-483-5p inhibited the proliferation and promoted apoptosis in the TNBC cell line BT-549. This effect was markedly attenuated by SOCS3 small interfering (si)RNA transfection. Additionally, wound healing and Transwell assays demonstrated that SOCS3 siRNA reversed the inhibitory effects of miR-483-5p inhibitor on the migration and invasion of BT-549 cells. Moreover, the decrease in miR-483-5p expression significantly reduced the secretion of TNF-α, IL-6, IL-1β and monocyte chemoattractant protein-1 in BT-549 cells, while SOCS3 siRNA could partially reverse this effect. Additionally, SOCS3 overexpression reversed the effects of miR-483-5p mimic on the proliferation, migration, invasion and inflammation of BT-549 cells. Taken together, these data demonstrated that the inhibition of miR-483-5p could inhibit the proliferation, migration, invasion and inflammatory response, while promoting the apoptosis of TNBC cells by negatively regulating SOCS3. miR-483-5p may be a potential target for TNBC therapy.

Opposite effects of miR-155 in the initial and later stages of lipopolysaccharide (LPS)-induced inflammatory response

Although microRNA-155 (miR-155) is considered a pro-inflammatory mediator, cumulative evidence indicates that it also has anti-inflammatory effects in macrophages and dendritic cells. In this study, we identified the dramatic expression changes of more than half of potential miR-155-targeted genes upon lipopolysaccharide (LPS) stimulation; 223 genes were down-regulated and 85 genes were up-regulated, including suppressor of cytokine signaling 1 (SOCS1) and transforming growth factor-β-activated kinase 1-binding protein 2 (TAB2), two well-known genes involved in miR-155-mediated regulation of the Toll-like receptor 4 (TLR4) signaling pathway. We also found that miR-155 acted as an anti-inflammatory mediator in the initial stage of LPS-induced inflammatory response mainly through repressing TAB2 protein translation, and as a pro-inflammatory mediator by down-regulating SOCS1 in the later stage. Meanwhile, overexpression of TAB2 3' untranslated region (UTR) in macrophages promoted the development of endotoxin tolerance by competing for binding with miR-155, which resulted in an elevated expression level of SOCS1 protein. These findings provide new insights for understanding the regulatory mechanisms in fine-tuning of LPS-induced innate immune response.

Comprehensive analysis of suppressor of cytokine signaling proteins in human breast Cancer

BackgroundAbnormal expression of suppressor of cytokine signaling (SOCS) proteins regulates tumor angiogenesis and development in cancers. In this study, we aimed to perform a comprehensive bioinformatic analysis of SOCS proteins in breast invasive carcinoma (BRCA).MethodsThe gene expression, methylation level, copy number, protein expression and patient survival data related to SOCS family members in BRCA patients were obtained from the following databases: Oncomine, The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA), Gene Expression Profiling Interactive Analysis (GEPIA), PCViz, cBioPortal and Kaplan-Meier plotter. Correlation analyses, identification of interacting genes and construction of regulatory networks were performed by functional and pathway enrichment analyses, weighted gene coexpression network analysis (WGCNA) and gene set enrichment analysis (GSEA).ResultsData related to 1109 BRCA tissues and 113 normal breast tissue samples were extracted from the TCGA database. SOCS2 and SOCS3 exhibited significantly lower mRNA expression levels in BRCA tissues than in normal tissues. BRCA patients with high mRNA levels of SOCS3 (p < 0.01) and SOCS4 (p < 0.05) were predicted to have significantly longer overall survival (OS) times. Multivariate analysis showed that SOCS3 was an independent prognostic factor for OS. High mRNA expression levels of SOCS2 (p < 0.001), SOCS3 (p < 0.001), and SOCS4 (p < 0.01), and a low expression level of SOCS5 (p < 0.001) were predicted to be significantly associated with better recurrence-free survival (RFS). Multivariate analysis showed that SOCS2 was an independent prognostic factor for RFS. Lower expression levels of SOCS2 and SOCS3 were observed in patients with tumors of more advanced clinical stage (p < 0.05). Functional and pathway enrichment analyses, together with WGCNA and GSEA, showed that SOCS3 and its interacting genes were significantly involved in the JAK-STAT signaling pathway, suggesting that JAK-STAT signaling might play a critical role in BRCA angiogenesis and development. Western blot results showed that overexpression of SOCS3 inhibited the activity of the JAK-STAT signaling pathway in vitro.ConclusionsSOCS family proteins play a very important role in BRCA. SOCS3 may be a prognostic factor and SOCS2 may be a potential therapeutic target in breast cancer.

COP9 Signalosome Subunit 3 Restricts Neuroinflammatory Responses During Cerebral Ischemia/Reperfusion Injury Through Stabilizing Suppressor of Cytokine Signaling 3 Protein.

BackgroundThe suppressor of cytokine signaling 3 (SOCS3) is a specific negative regulator of signal transducer and activator of transcription 3 (STAT3) signaling, which is predominantly activated to induce neuroinflammatory response in microglia and functions essential roles during cerebral ischemia-reperfusion (I/R) injury. Constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a signaling platform controlling protein stability by remodeling of cullin-RING ubiquitin ligases, which is recently reported to specifically recognize proteins with SOCS-box domains. However, whether SOCS3 is related to COP9 signalosome in neuroinflammation during cerebral I/R injury is completely unclear.MethodsMice subjected to transient middle cerebral artery occlusion (MCAO) and reperfusion, and BV2 microglia cells treated with oxygen-glucose deprivation and reoxygenation (OGD/R) were used to mimic cerebral I/R injury. Western blot, qRTPCR, immunofluorescence, and co-Immunoprecipitation assays were performed to explore the regulatory mechanism of SOCS3 on neuroinflammation and the relationship of SOCS3 and COP9 signalosome during cerebral I/R injury.ResultsSOCS3 expression is significantly upregulated in microglia during OGD/R treatment, and overexpression of SOCS3 suppresses OGD/R-induced STAT3 activation and inflammatory factor expression. Furthermore, we find that COP9 signalosome subunit 3 (CSN3) interacts with SOCS3 protein to enhance its stability, thereby resulting in restricting OGD/R-induced STAT3 activation and inflammatory response. Moreover, we find that knockdown of CSN3 evidently accelerates STAT3 activation, and aggravates cerebral I/R injury in vivo.ConclusionCSN3 restricts neuroinflammatory responses during cerebral I/R injury through stabilizing SOCS3 protein and indicates that CSN3 a potential therapeutic target for cerebral I/R injury.

Effect of Shenkang on renal fibrosis and activation of renal interstitial fibroblasts through the JAK2/STAT3 pathway

BackgroundActivation of renal fibroblasts is a critical mechanism in the process of renal fibrosis. As a commonly used herbal formula, Shenkang (SK) has been found to attenuate renal fibrosis and renal parenchyma destruction. However, the effect of SK on renal fibroblast activation in unilateral ureteral obstruction (UUO) mice and its molecular mechanism remain undetermined. The present study was performed to elucidate the effect of SK on renal fibroblast activation and renal fibrosis, as well as the potential underlying mechanism, in both NRK-49F cells and UUO mice.MethodsNRK-49F cells were stimulated with 10 ng/ml TGF-β1 for 48 h. After SK treatment, the CCK-8 method was used to evaluate cell viability. Thirty-six C57BL/6 mice were randomly divided into the sham group, UUO group, angiotensin receptor blocker (ARB) group, and SK high-, moderate- and low-dose groups. UUO was induced in mice except those in the sham group. Drugs were administered 1 day later. On the 13th day, the fractional anisotropy (FA) value was determined by MRI to evaluate the degree of renal fibrosis. After 14 days, serum indexes were assessed. Hematoxylin and eosin (HE) and Sirius red staining were used to observe pathological morphology and the degree of fibrosis of the affected kidney. Western blotting and PCR were used to assess the expression of related molecules in both cells and animals at the protein and gene levels.ResultsOur results showed that SK reduced extracellular matrix (ECM) and α-smooth muscle actin (α-SMA) expression both in vitro and in vivo and attenuated renal fibrosis and the pathological lesion degree after UUO, suppressing JAK2/STAT3 activation. Furthermore, we found that SK regulated the JAK2/STAT3 pathway regulators peroxiredoxin 5 (Prdx5) in vitro and suppressor of cytokine signaling protein 1 (SOCS1) and SOCS3 in vivo.ConclusionsThese results indicated that SK inhibited fibroblast activation by regulating the JAK2/STAT3 pathway, which may be a mechanism underlying its protective action in renal fibrosis.

SPTBN1 inhibits inflammatory responses and hepatocarcinogenesis via the stabilization of SOCS1 and downregulation of p65 in hepatocellular carcinoma.

Background: Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein for transforming growth factor beta (TGF-β) signaling, is recognized as a tumor suppressor in the development of hepatocellular carcinoma (HCC); however, the underlying molecular mechanisms of this tumor suppression remain obscure.Methods: The effects on expression of pro-inflammatory cytokines upon the inhibition or impairment of SPTBN1 in HCC cell lines and liver tissues of Sptbn1+/- and wild-type (WT) mice were assessed by analyses of quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), enzyme linked immunosorbent assay (ELISA), Western blotting and gene array databases from HCC patients. We investigated the detailed molecular mechanisms underlying the inflammatory responses by immunoprecipitation-Western blotting, luciferase reporter assay, chromatin immunoprecipitation quantitative real time PCR (ChIP-qPCR), immunohistochemistry (IHC) and electrophoretic mobility shift assay (EMSA). The proportion of myeloid-derived suppressor cells in liver, spleen, bone marrow and peripheral blood samples from WT and Sptbn1+/- mice were measured by fluorescence-activated cell sorting (FACS) analysis. Further, the hepatocacinogenesis and its correlation with inflammatory microenvironment by loss of SPTBN1/SOCS1 and induction of p65 were analyzed by treating WT and Sptbn1+/- mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).Results: Loss of SPTBN1 in HCC cells upregulated the expression of pro-inflammatory cytokines including interleukin-1α (IL-1α), IL-1β, and IL-6, and enhanced NF-κB transcriptional activation. Mechanistic analyses revealed that knockdown of SPTBN1 by siRNA downregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an E3 ligase of p65, and subsequently upregulated p65 accumulation in the nucleus of HCC cells. Restoration of SOCS1 abrogated this SPTBN1 loss-associated elevation of p65 in HCC cells. In human HCC tissues, SPTBN1 gene expression was inversely correlated with gene expression of IL-1α, IL-1β and IL-6. Furthermore, a decrease in the levels of SPTBN1 gene, as well as an increase in the gene expression of IL-1β or IL-6 predicted shorter relapse free survival in HCC patients, and that HCC patients with low expression of SPTBN1 or SOCS1 protein is associated with poor survival. Heterozygous loss of SPTBN1 (Sptbn1+/-) in mice markedly upregulated hepatic expression of IL-1α, IL-1β and IL-6, and elevated the proportion of myeloid-derived suppressor cells (MDSCs) and CD4+CD25+Foxp3+ regulatory T cells (Foxp3+Treg) cells in the liver, promoting hepatocarcinogenesis of mouse fed by DDC.Conclusions: Our findings provided evidence that loss of SPTBN1 in HCC cells increases p65 protein stability via the inhibition of SOCS1 and enhances NF-κB activation, stimulating the release of inflammatory cytokines, which are critical molecular mechanisms for the loss of SPTBN1-induced liver cancer formation. Reduced SPTBN1 and SOCS1 predict poor outcome in HCC patients.

SOCS3–microtubule interaction via CLIP-170 and CLASP2 is critical for modulation of endothelial inflammation and lung injury

Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.

Long Non-Coding RNA NORAD Inhibits Breast Cancer Cell Proliferation and Metastasis by Regulating miR-155-5p/SOCS1 Axis

PurposeNon-coding RNA activated by DNA damage (NORAD) has been reported to be a cancer-related long non-coding RNA (lncRNA) implicated in the progression of several cancers; however, its role in breast cancer (BC) has not yet been clarified.MethodsQuantitative real-time polymerase chain reaction was used to examine NORAD, microRNA (miR)-155-5p, and suppressor of cytokine signaling 1 (SOCS1) mRNA expression levels. Western blotting was used to analyze SOCS1 protein expression. The malignancy of BC cells was assessed using the cell counting kit-8 (CCK-8), BrdU, and Transwell assays. Bioinformatics analysis, RNA immunoprecipitation assay, and dual-luciferase reporter gene assays were used to verify the targeted relationship between NORAD and miR-155-5p. Additionally, the regulatory effects of NORAD and miR-155-5p on SOCS1 expression were determined by western blotting.ResultsNORAD expression was significantly reduced in BC cell lines and tissues, and its low expression was associated with poor tumor tissue differentiation. NORAD overexpression repressed BC cell proliferation, migration, and invasion, whereas its knockdown produced the opposite effects. Additionally, miR-155-5p was found to be a target of NORAD, and the biological functions of miR-155-5p and NORAD were counteractive. MiR-155-5p was confirmed to target SOCS1, and SOCS1 was found to be positively regulated by NORAD.ConclusionNORAD suppresses miR-155-5p to upregulate SOCS1, thereby repressing the proliferation, migration, and invasion of BC cells.

Suppressor of cytokine signaling proteins 1 and 3 and hepatitis B virus infection

Suppressor of cytokine signaling proteins 1 and 3 and hepatitis B virus infection

Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer

BackgroundDysfunction of microRNAs (miRNAs) is a major cause of aberrant expression of inflammatory cytokines and contributes to macrophage polarization. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity, whereas M2 macrophages display regulatory functions in tissue repair and remodeling and promote Th2 immune responses. Previous studies have shown that miRNA let-7 is associated with cellular differentiation and that the expression of let-7b-5p is significantly augmented in M2 macrophages. However, the mechanism by which let-7b-5p regulates macrophage differentiation in prostate cancer (PCa) remains largely unknown.MethodsHuman macrophages were induced by blood monocytes from healthy male donors, and M1 macrophages were polarized by stimulating them overnight with 100 ng/ml of lipopolysaccharides and 100 ng/ml of IFN-γ. Conditioned medium from PC-3 cells was used to induce prostatic macrophages (M-CMs) in vitro, and we then transfected let-7b-5p mimics or inhibitors into M1 and M-CMs for 72 h. The expression of cluster of differentiation 206 (CD206) in each group was detected with the High-Throughput Connotation of Imaging System. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of the inflammatory cytokines IL-10, IL-12, IL-13, TNF-alpha, and let-7b in macrophages. SOCS1 protein levels were evaluated by ELISA, and the phosphorylation difference in STAT family member proteins was analyzed using CST signal-pathway chip. Phagocytosis by macrophages and the effect of macrophages on the proliferation of prostate cancer PC-3 cells were evaluated with phagocytosis assay or the Cell Counting Kit-8 (CCK-8) and colony formation assay. The relationship between SOCS1 and let-7b-5p was confirmed with a dual-luciferase reporter.ResultsThe expression of cluster of differentiation 206 (CD206, a M2-like macrophage surface molecule) was significantly increased in M1 macrophages treated with let-7b-5p mimics, while CD206 expression was decreased in M-CMs treated with let-7b-5p inhibitors. Overexpression or knockdown of let-7b-5p significantly affected the expression of inflammatory factors in macrophages—including interleukin 10 (IL-10), IL-12, IL-13, and tumor necrosis factor alpha. Let-7b-5p downregulated the expression of suppressor of cytokine signaling 1 (SOCS1) and increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a proteins in M-CMs and M1 macrophages with let-7b-5p mimics relative to the other groups. In addition, with the elevated expression of let-7b-5p, the phagocytosis by macrophages showed a commensurate and significant decrease. As a result, M-CMs treated with let-7b-5p inhibitors reduced the proliferation of PC-3 PCa cells.ConclusionsCollectively, these data indicated that let-7b-5p may regulate M2 polarization through the SOCS1/STAT pathway and that reversal of M2 differentiation by let-7b-5p inhibitors enhanced macrophage phagocytosis, ultimately inhibiting the proliferation of PCa cells.

Abstract 1709: New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) includes heterogenous molecular subtypes, e.g., basal-like, immunomodulatory, mesenchymal, mesenchymal stem cell and luminal androgen receptor subtypes. Chemotherapy is the standard care for patients with early TNBC, but advanced or metastatic disease has unfavorable prognosis. PARP inhibitors and immune checkpoint inhibitors are effective for patients with BRCA gene mutation and PD-L1-positive TNBC, respectively. However, their clinical benefits seem to be limited. Thus, we are investigating new therapy targeted on immune modulation in TNBC. Materials and methods: First, we performed microarray expression analysis (Agilent Technologies, Inc.) of small RNA extracted from 11 breast cancer specimens. Second, miRNA subset was compared in TNBC and non-TNBC, and several genes were identified as predicted targets for interaction of miRNA in TNBC. Third, we examined a targeted gene and protein expressions in cancer tissues by real-time qPCR and immunohistochemical staining. Tissues samples were obtained from breast cancer patients with TNBC and non-TNBC, who were scheduled to receive neoadjuvant chemotherapy (NAC). Statistical significance is evaluated by chi-squared test. This study is approved at IRB in Kyorin University School of Medicine and also supported by Grant-in-Aid for Scientific Research C of Japan Society for the Promotion of Science. Results: From comprehensive analysis, SOCS1 (suppressor of cytokine signaling 1), IL-13 and several genes were predicted as interactive targets of miRNA in TNBC. We focused on SOCS1 gene amplification and its protein overexpression in breast cancer. Most patients were treated with NAC of anthracycline- and/or taxane-based regimens. Their pathological response was graded in surgical specimens according to classification of the Japanese Breast Cancer Society. From 55 tissue specimens stained by anti-SOCS1 antibody, there was no difference among staining intensity graded at score 0-3 and pathological response of chemotherapy. However, gene amplification of SOCS1 was not statistically different but relatively lower in 6 cases with pathologically partial or complete response (grade 2 or 3) than in 11 cases with no pathological response (grade 1a or 1b). Discussions: SOCS1 is well known as a negative regulator of JAK-STAT pathway. SOCS1 is also related to suppression of T cell activation and modulation of M1 macrophage. Our data suggest that down regulation of SOCS1 gene influences clinical benefit of chemotherapy as well as host immune response like tumor-infiltrating lymphocytes (TIL). We will examine relationship between SOCS1 protein expression and TIL in breast cancer and also examine cell proliferation and T cell activation in TNBC and non-TNBC cell lines under presence and absence of SOCS1 gene expression. Conclusion: SOCS1 is a promising molecular target to modulate immune editing in TNBC. Citation Format: Shigeru Imoto, Tomohiro Chiba, Sota Asaga, Hirotsugu Isaka, Shigehiro Yokoi, Yoshiharu Ishizaka, Ai Tsuchiya, Hiroshi Kamma. New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1709.

Snake Cathelicidin Derived Peptide Inhibits Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus and has reemerged in recent years with epidemic potential. ZIKV infection may result in severe syndromes such as neurological complications and microcephaly in newborns. Therefore, ZIKV has become a global public health threat and currently there is no approved specific drug for its treatment. Animal venoms are important resources of novel drugs. Cathelicidin-BF (BF-30) is a defensive peptide identified from Bungarus fasciatus snake venom and has been shown to be an excellent template for applicable peptide design. In this study, we found that ZY13, one of the peptidic analogs of BF-30, inhibits ZIKV infection in vitro and in vivo. Mechanistic studies revealed that ZY13 can directly inactivate ZIKV and reduce the production of infectious virions. Further studies also indicated that administration of ZY13 strengthen the host antiviral immunity via AXL-SOCS (suppressor of cytokine signaling protein) pathway. Additionally, the results of mouse experiment suggest that ZY13 efficiently restrict ZIKV infection and improve the growth defects of ZIKV-infected mouse pups. Together, our findings not only demonstrate that ZY13 might be a candidate for anti-ZIKV drug, but also indicated the importance of animal venom peptides as templates for antivirals development.

Effects of miR-152-Mediated Targeting of SOCS3 on Hepatic Insulin Resistance in Gestational Diabetes Mellitus Mice

BackgroundThe present study explored the effects of miR-152-mediated targeting of suppressor of cytokine signaling 3 (SOCS3) on hepatic insulin resistance (HIR) in mice with gestational diabetes mellitus (GDM). Healthy SPF C57BL/6J mice were selected to establish a GDM model. MethodsMice were divided into seven groups as follows: Normal group, Model group, NC-mimic group, miR-152 mimic group, NC-pcDNA3.0 group, pcDNA3.0-SOCS group, and miR-152 mimic + pcDNA3.0-SOCS3 group. The relationship between miR-152 and SOCS3 expression was analyzed by a dual-luciferase reporter system. Islet cell morphology and expression of miR-152 and SOCS3 mRNA and protein in the islet tissue were detected by hematoxylin and eosin (HE) staining, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. Fasting blood glucose (FBG) level was measured by a glucose meter while triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and insulin levels were determined by an automatic biochemical analyzer. Insulin resistance index (HOMA-IR) was calculated by the formula FBG × FINS/22.5. ResultsThe results showed that the levels of miR-152, SOCS3, FBG, fasting insulin (FINS), TG, and TC increased, and HDL-C content decreased in other groups as compared with those in the Normal group (all p < 0.05). Oral glucose tolerance test (OGTT), FBG, FINS, TG, TC, and HDL-C values showed an opposite trend in miR-152 mimic and pcDNA3.0-SOCS3 groups as compared with the Model group (all p < 0.05). ConclusionsmiR-152 can inhibit HIR in GDM mice by downregulating the expression of SOCS3.

Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

M6A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

N6-methyladenosine (m6A) RNA modification maintained by N6-methyltransferases and demethylases is involved in multiple biological functions. Methyltransferase like 3 (METTL3) is a major N6-methyltransferase. However, the role of METTL3 and its installed m6A modification in colorectal tumorigenesis remains to be fully elucidated. METTL3 is highly expressed as indicated in colorectal cancer samples in the TCGA and Oncomine databases, implying its potential role in colon tumorigenesis. SW480 cell line with stable METTL3 knockout (METTL3-KO) was generated using CRISPR/Cas9 and were confirmed by the loss of METTL3 expression and suppression of m6A modification. The proliferation of METTL3-KO cells was significantly inhibited compared with that of control cells. METTL3-KO decreased the decay rate of suppressor of cytokine signaling 2 (SOCS2) RNA, resulting in elevated SOCS2 protein expression. m6A-RNA immunoprecipitation-qPCR (MeRIP-qPCR) revealed that SOCS2 mRNA was targeted by METTL3 for m6A modification. Similar to METTL3-KO SW480 cells, SW480 cells treated with 3-deazaadenosine, an RNA methylation inhibitor, exhibited elevated SOCS2 protein expression. Increased levels of SOCS2 in METTL3-KO SW480 cells were associated with decreased expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), contributing to the inhibition of cell proliferation. The underlying associations among METTL3, SOCS2, and LGR5 were further confirmed in SW480 cells transfected with si-METTL3 and in tumor samples from patients with colorectal cancer. Taken together, our data demonstrate that an increased level of METTL3 may maintain the tumorigenicity of colon cancer cells by suppressing SOCS2.