STAT3 Binding Research Articles

Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway

To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Mouse B2 SINE elements function as IFN-inducible enhancers.

Regulatory networks underlying innate immunity continually face selective pressures to adapt to new and evolving pathogens. Transposable elements (TEs) can affect immune gene expression as a source of inducible regulatory elements, but the significance of these elements in facilitating evolutionary diversification of innate immunity remains largely unexplored. Here, we investigated the mouse epigenomic response to type II interferon (IFN) signaling and discovered that elements from a subfamily of B2 SINE (B2_Mm2) contain STAT1 binding sites and function as IFN-inducible enhancers. CRISPR deletion experiments in mouse cells demonstrated that a B2_Mm2 element has been co-opted as an enhancer driving IFN-inducible expression of Dicer1. The rodent-specific B2 SINE family is highly abundant in the mouse genome and elements have been previously characterized to exhibit promoter, insulator, and non-coding RNA activity. Our work establishes a new role for B2 elements as inducible enhancer elements that influence mouse immunity, and exemplifies how lineage-specific TEs can facilitate evolutionary turnover and divergence of innate immune regulatory networks.

MiR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

αS1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of αS1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on αS1-casein synthesis in goat mammary epithelial cells (GMEC). αS1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on αS1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC. Compared with middle-lactation period, αS1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces αS1-casein abundance by targeting the 3'-untranslated region (3'UTR) of αS1-casein mRNA in GMEC. miR-380-3p enhances β-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes β-casein abundance through target gene αS1-casein, and activates β-casein transcription by enhancing the binding of STAT5 to β-casein gene promoter region. miR-380-3p decreases αS1-casein expression and increases β-casein expression by targeting αS1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.

The alcohol extracts of Sceptridium ternatum (Thunb.) Lyon exert anti-pulmonary fibrosis effect through targeting SETDB1/STAT3/p-STAT3 signaling

Ethnopharmacological relevancePulmonary fibrosis (PF) is a pathological process of irreversible scarring of lung tissues, with limited treatment means. Sceptridium ternatum (Thunb.) Lyon (STE) is a traditional Chinese herbal medicine that has a traditional use in relieving cough and asthma, resolving phlegm, clearing heat, and detoxicating in China. However, its role in PF has not been reported. Aim of the studyThis study aims to investigate the protective role of STE in PF and the underlying mechanisms. Materials and methodsSprague-Dawley (SD) rats were divided into control group, PF model group, positive drug (pirfenidone) group and STE group. After 28 days of STE administration in bleomycin (BLM)-induced PF rats, living Nuclear Magnetic Resonance Imaging (NMRI) was used to observe the structural changes of lung tissues. H&E and Masson's trichrome staining were used to observe PF-associated pathological alteration, and immunohistochemistry (IHC) staining, western blotting, and qRT-PCR were used to detect the expression of PF-related marker proteins in the lung tissues. ELISA was used to detect PF-associated biochemical criteria in the lung tissue homogenates. The proteomics technology was used to screen the different proteins. Co-immunoprecipitation, western blotting, and IHC staining were used to confirm the underlying targets of STE as well as its downstream signaling. UPLC-Triple-TOF/MS assay was used to explore the effective components in the alcohol extracts of STE. Autodock vina was used to detect the potential binding between the above effective components and SETDB1. ResultsSTE prevented PF by inhibiting the activation of lung fibroblasts and ECM deposition in BLM-induced PF rats. Mechanism analyses demonstrated that STE could inhibit the up-regulation of SETDB1 induced by BLM and TGF-β1, which further blocked the binding of SETDB1 and STAT3 as well as the phosphorylation of STAT3, ultimately preventing the activation and proliferation of lung fibroblasts. ConclusionSTE played a preventive role in PF by targeting the SETBD1/STAT3/p-STAT3 pathway, which may be a potential therapeutic agent for PF.

Retracted] FAM172A modulates apoptosis and proliferation of colon cancer cells via STAT1 binding to its promoter.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that lanes 1‑3 of the EMSA results shown in Fig.6 on p.1278 were strikingly similar to data that had already appeared in a different form in the following publication by different authors at different research institutes: QiuK, Li Z, Chen J, Wu S, Zhu X, GaoS, GaoJ, Ren G and Zhou X: EIN3 and ORE1 accelerate degreening during ethylene‑mediated leaf senescence by directly activating chlorophyll catabolic genes in Arabidopsis. PLoS Genet11: e1005399, 2015. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 35: 1273‑1280, 2016; DOI: 10.3892/or.2015.4485.

Abstract 1651: Discovery of oridonin-based proteolysis targeting chimera (PROTAC) compounds as putative protein degraders and potent anticancer agents for triple-negative breast cancer

Abstract Natural products provide a molecular starting point for the development of novel therapeutic agents including a plethora of anti-cancer drugs currently in use. Oridonin is a natural diterpene compound enriched in medicinal herb Rabdosia rubescens, and its unique anti-cancer properties have been extensively studied. However, clinical applications of oridonin are limited due to its relatively low potency. Previously, we developed a class of druglike nitrogen-enriched oridonin derivatives, including CYD0618 and others, with thiazole modifications as effective anticancer agents to inhibit breast cancer growth in vitro and in vivo. Although breast cancer screening and treatments have improved over the last half-century, aggressive subtypes like triple-negative breast cancer (TNBC) are refractory to frontline therapies, in which the STAT3 transcription factor plays a key role in developing therapeutic resistance and recurrence. Currently, there are no FDA-approved direct STAT3 inhibitors in clinical use. We reported that CYD0618 inhibits STAT3 signaling, likely through a direct interaction with STAT3. With proteolysis targeting chimera (PROTAC) technology, our team designed novel oridonin- and CYD0618-based PROTAC compounds including PW0965 and PW1087 in an attempt to achieve STAT3 binding and degradation. We determined anticancer effects of PW0965 and PW1087 on proliferation, colony formation, apoptosis and cell motility of triple-negative breast cancer (TNBC) cells MDA-MB-231 and BT549. We found that both compounds significantly inhibited the proliferation of TNBC cells. Compared with oridonin, both compounds more effectively induced apoptosis of TNBC cells. Additionally, MDA-MB-231 and BT549 cells treated with these PROTACs showed significantly reduced migration and invasion compared to control cells. PW0965 and PW1087 treatment reduced protein levels of STAT3 and its activation at Y705 residue in TNBC cells as early as 4 hours of treatment. This observed reduction in STAT3 protein on Western blot exhibited dose and time dependence. Collectively, our results demonstrate that oridonin and its analog-based PROTACs exhibit excellent potency against TNBC cells, likely through protein degradation of STAT3. The potency of PW0965 and PW1087 warrants further in vitro and in vivo investigation in cancer cells and tumor xenograft models. The molecular profiles of these PROTACs, including their interactions with STAT proteins and protein degradation, are currently under investigation. Citation Format: Ruixia Ma, Jun Li, Gabrielle R. Vontz, Pingyuan Wang, Haiying Chen, Jia Zhou, Qiang Shen. Discovery of oridonin-based proteolysis targeting chimera (PROTAC) compounds as putative protein degraders and potent anticancer agents for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1651.

PYCR1 promotes the malignant progression of lung cancer through the JAK-STAT3 signaling pathway via PRODH-dependent glutamine synthesize

BackgroundLung cancer is a serious threat to human life. It is of great significance to elucidate the pathogenesis of lung cancer and search for new markers. This study evaluate the clinical value of pyrroline-5-carboxylate reductase 1 (PYCR1) and explore its role and mechanisms in the malignant progression of lung cancer. MethodsPYCR1 expression and its relationship with prognosis were analyzed using a bioinformatics database. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were utilized to examine the expression of PYCR1 in lung cancer tissues and peripheral blood. PYCR1-overexpressing lung cancer cells were constructed, then the cell proliferative, migration, and invasion ability was examined by the MTT and Transwell assays. siRNA against PRODH and STAT3 inhibitor sttatic was used to further elucidate the underlying mechanisms. Luciferase and CHIP assays were carried out for validate the how PYCR1 regulated PD-L1 expression via STAT3. Xenograft experiment was performed to determine the role of PYCR1 in vivo. ResultsDatabase analysis showed that PYCR1 expression was significantly increased in lung cancer tissues, and its high expression predicted poor prognosis. Lung cancer tissue and peripheral blood of patients showed obviously increased PYCR1 expression, and the sensitivity and specificity of serum PYCR1 in the diagnosis of lung cancer were 75.7% and 60%, respectively. PYCR1 overexpression enhanced the proliferative, migration, and invasion abilities of lung cancer cells. Both PRODH silence and stattic effectively attenuated the function of PYCR1. Animal experiment and IHC data indicated that PYCR1 could activated STAT3 phosphorylation and PD-L1, as well as suppressed T cell infiltration in lung cancer. Finally, we also validated that PYCR1 promoted PD-L1 transcription by elevating STAT3 binding to the gene promoter. ConclusionPYCR1 has certain value in the diagnosis and prognosis of lung cancer. Moreover, through regulating JAK-STAT3 signaling pathway, PYCR1 significantly participated in process of lung cancer progression via the metabolism link between proline and glutamine, indicating that PYCR1 might be also a novel therapeutic target.

Disruption of the MYC Superenhancer Complex by Dual Targeting of FLT3 and LSD1 in Acute Myeloid Leukemia.

This work establishes the synergistic efficacy of combined FLT3 and LSD1 inhibition in FLT3-ITD AML by disrupting STAT5 and GFI1 binding at the MYC blood-specific superenhancer complex.

Rabeprazole destroyed gastric epithelial barrier function through FOXF1/STAT3-mediated ZO-1 expression.

Rabeprazole is a representative of proton pump inhibitors and widely used in anti-ulcer treatment. However, the effect of Rabeprazole on gut barrier function remains to be identified. In this study, we show that ZO-1 expression is decreased in patients receiving Rabeprazole by immunofluorescence (IF) analysis. Western blotting (WB) and real-time PCR (qPCR) results demonstrate that Rabeprazole treatment leads to a significant downregulation of ZO-1 expression through inhibition of the FOXF1/STAT3 pathway, leading to destroy barrier function, which illustrates a novel pathway that Rabeprazole regulates barrier function in gastric epithelial cells. Mechanistically, Rabeprazole treatment led to a downregulation of STAT3 and FOXF1 phosphorylation, leading to inhibit nuclear translocation and decrease the binding of STAT3 and FOXF1 to ZO-1 promoter, respectively. Most important, endogenous FOXF1 interacted with STAT3, and this interaction was dramatically abolished by Rabeprazole stimulation. Overexpression of STAT3 and FOXF1 in GES-1 cells reversed the inhibitory effect of Rabeprazole on ZO-1 expression, respectively. These finding extended the function of Rabeprazole and established a previously unappreciated mechanism by which the Rabeprazole/FOXF1/STAT3 axis facilitated ZO-1 expression to regulate barrier function, and a comprehensive consideration and evaluation was required in treatment of patients.

Isophysalin A Inhibits the Stemness of Breast Cancer Cells Possibly via Stat3 and IL-6 Signaling.

Breast cancer stem cells (BCSCs) are involved in carcinogenesis of the breast and contribute to therapeutic resistance. In the present study, we found that isophysalin A acts as a potent cancer stem cell inhibitor and investigated the anti-CSC mechanism of action of isophysalin A on breast cancer. The effect of isophysalin A on BCSCs was examined using a mammosphere formation, a colony formation and a cell migration assay, as well as CD44 (Cluster of differentiation 44)high/CD24 (Cluster of differentiation 24)low analysis, an apoptosis assay, quantitative real-time PCR, western blotting, an electrophoretic mobility shift assay, and a cytokine profiling assay. Isophysalin A inhibited cell proliferation, colony formation, cell migration, CSC formation, and mammosphere proliferation and increased BCSC apoptosis. The subpopulation of CD44high/CD24low was decreased by isophysalin A, which also reduced the DNA binding of Stat3 and the total and nuclear protein expression levels of Stat3 and phosphorylated Stat3. Furthermore, the mRNA and media IL-6/IL-8 levels of the mammosphere were also reduced by isophysalin A. Isophysalin A inhibited the Stat3 and IL-6 signaling pathways and induced CSC death; thus, isophysalin A may be a potential natural inhibitor of BCSCs.

Interferon Gamma-Inducible NAMPT in Melanoma Cells Serves as a Mechanism of Resistance to Enhance Tumor Growth.

(1) Background: Immune cells infiltrate the tumor microenvironment and secrete inflammatory cytokines, including interferons (IFNs), to drive antitumor responses and promote tumor clearance. However, recent evidence suggests that sometimes, tumor cells can also harness IFNs to enhance growth and survival. The essential NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) gene is constitutively expressed in cells during normal homeostasis. However, melanoma cells have higher energetic demands and elevated NAMPT expression. We hypothesized that interferon gamma (IFNγ) regulates NAMPT in tumor cells as a mechanism of resistance that impedes the normal anti-tumorigenic effects of IFNγ. (2) Methods: Utilizing a variety of melanoma cells, mouse models, Crispr-Cas9, and molecular biology techniques, we explored the importance of IFNγ-inducible NAMPT during melanoma growth. (3) Results: We demonstrated that IFNγ mediates the metabolic reprogramming of melanoma cells by inducing Nampt through a Stat1 binding site in the Nampt gene, increasing cell proliferation and survival. Further, IFN/STAT1-inducible Nampt promotes melanoma in vivo. (4) Conclusions: We provided evidence that melanoma cells directly respond to IFNγ by increasing NAMPT levels, improving their fitness and growth in vivo (control n = 36, SBS KO n = 46). This discovery unveils a possible therapeutic target that may improve the efficacy of immunotherapies involving IFN responses in the clinic.

AKT Blocks SIK1-Mediated Repression of STAT3 to Promote Breast Tumorigenesis.

AKT binds and phosphorylates SIK1 to overcome SIK1-mediated repression of STAT3, indicating that STAT3 is a potential therapeutic target in breast cancer with hyperactive AKT signaling.

Short-term exposure to dimethyl fumarate (DMF) inhibits LPS-induced IκBζ expression in macrophages.

Background: The pharmacological activity of dimethyl fumarate (DMF) in treating psoriasis and multiple sclerosis (MS) is not fully understood. DMF is hydrolysed to monomethyl fumarate (MMF) in vivo, which is believed to account for the therapeutic effects of DMF. However, previous studies have provided evidence that DMF also enters the circulation. Given that DMF is short-lived in the blood, whether DMF has a therapeutic impact is still unclear. Methods: Lipopolysaccharide (LPS)-mediated RAW264.7 cell activation was used as a model of inflammation to explore the anti-inflammatory effects of short-term DMF exposure in vitro. Whole blood LPS stimulation assay was applied to compare the anti-inflammatory effects of DMF and MMF in vivo. Griess assay was performed to examined nitrite release. The expression of pro-inflammatory cytokines and transcription factors were measured by quantitative PCR (qPCR), ELISA and Western blot. Depletion of intracellular glutathione (GSH) was evaluated by Ellman's assay. Luciferase reporter assays were performed to evaluate DMF effects on Nrf2-ARE pathway activation, promoter activity of Nfkbiz and mRNA stability of Nfkbiz. Binding of STAT3 to the IκBζ promoter were examined using Chromatin immunoprecipitation (ChIP) assay. Results: Short-term exposure to DMF significantly inhibited the inflammatory response of RAW264.7 cells and suppressed LPS-induced IκBζ expression. Importantly, oral DMF but not oral MMF administration significantly inhibited IκBζ transcription in murine peripheral blood cells. We demonstrated that the expression of IκBζ is affected by the availability of intracellular GSH and regulated by the transcription factor Nrf2 and STAT3. DMF with strong electrophilicity can rapidly deplete intracellular GSH, activate the Nrf2-ARE pathway, and inhibit the binding of STAT3 to the IκBζ promoter, thereby suppressing IκBζ expression in macrophages. Conclusion: These results demonstrate the rapid anti-inflammatory effects of DMF in macrophages, providing evidence to support the direct anti-inflammatory activity of DMF.

Activation of OSM-STAT3 Epigenetically Regulates Tumor-Promoting Transcriptional Programs in Cervical Cancer.

Despite improvements in preventative strategies, such as regular screenings with Pap tests and human papillomavirus (HPV) tests as well as HPV vaccinations, effective treatment for advanced cervical cancer remains poor. Deregulation of STAT3 is an oncogenic factor that promotes tumorigenesis and epithelial-to-mesenchymal transition (EMT) in various cancers. Oncostatin M (OSM), a pleiotropic cytokine, induces STAT3 activation, exacerbating cervical cancer. However, the mechanism by which the OSM-STAT3 axis epigenetically regulates tumor-progression-related genes in cervical cancer is not well understood. Here, we show that OSM-mediated STAT3 activation promotes pro-tumorigenic gene expression programs, with chromatin remodeling in cervical cancer. Reanalysis of scRNA-seq data performed in cervical cancer uncovered an interaction between the oncostatin M receptor (OSMR) on tumor cells and OSM induced by tumor-associated macrophages (TAMs). Our gene expression profiling (bulk RNA-seq) shows that OSM-induced genes were involved in hypoxia, wound healing, and angiogenesis, which were significantly inhibited by SD-36, a STAT3-selective degrader. Additionally, ATAC-seq experiments revealed that STAT3 binding motifs were preferentially enriched in open chromatin regions of the OSM-STAT3-regulated genes. Among the 50 candidate genes that were regulated epigenetically through the OSM-STAT3 axis, we found that the expression levels of NDRG1, HK2, PLOD2, and NPC1 were significantly correlated with those of OSMR and STAT3 in three independent cervical cancer cohorts. Also, higher expression levels of these genes are significantly associated with poor prognosis in cervical cancer patients. Collectively, our findings demonstrate that the OSM-STAT3 signaling pathway regulates crucial transcriptomic programs through epigenetic changes and that selective inhibition of STAT3 may be a novel therapeutic strategy for patients with advanced cervical cancer.

Non-canonical STAT3 function reduces REDD1 transcription.

The transcription factor STAT3 is a potent activator of transcription, but evidence exists that STAT3 can also repress gene expression. However, little is known about the molecular mechanisms involved in STAT3-dependent gene repression. Notably, STAT3 reduces the expression of the stress-induced mTOR inhibitor REDD1 by reducing REDD1 mRNA transcription. Here, we determined the functional domains of STAT3 responsible for the reduction of REDD1 mRNA and protein expression. Within STAT3, the N-terminal domain and tyrosine 705 are crucial for STAT3-dependent reduction of REDD1 expression. Interestingly, binding of STAT3 to canonical STAT-binding sides within the REDD1 promoter is not necessary for STAT3-mediated reduction of REDD1 expression. Still, STAT3 is recruited to the REDD1 promoter upon stimulation with IL-6, and reduces REDD1 promoter activity. The reduction of REDD1 expression is specific for STAT3, as neither expression nor activation of STAT1 reduces REDD1 mRNA and protein expression. In summary, we present a novel, non-canonical STAT3-dependent mechanism for reducing gene expression. This transcriptional repression increases the functions of STAT3 proteins beyond classical transcriptional activation of cytokine-regulated target genes to a more complex function in modulating gene expression in immunity and cellular stress.

IL-6/STAT3 Signaling Axis Enhances and Prolongs Pdcd1 Expression in Murine CD8 T Cells.

CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.

Identification of type II interferons and receptors in an osteoglossiform fish, the arapaima Arapaima gigas

In mammals, type II interferon (IFN; i.e. IFN-γ) signalling transduces through its specific receptors IFN-γR1 and IFN-γR2. In an osteoglossiform fish, the arapaima Arapaima gigas, three type II IFNs, IFN-γ-like, IFN-γ and IFN-γrel, and their four possible receptor subunits IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 were identified in this study. The three type II IFN genes are composed of four exons and three introns, and they all contain IFN-γ signature motif and signal peptide, with the presence of potential nuclear localization signal (NLS) in IFN-γ-like and IFN-γ. The IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 are composed of seven exons and six introns, with predicted IFN-γR1-1 and IFN-γR1-2 proteins containing JAK1 and STAT1 binding sites, and IFN-γR2-1 and IFN-γR2-2 containing JAK2 binding sites. Gene synteny analysis showed that the type II IFN and their receptor loci are duplicated in arapaima. All these genes were expressed constitutively in all organs/tissues examined, and responded to the stimulation of polyI:C. The prokaryotic recombinant IFN-γ-like, IFN-γ and IFN-γrel proteins can significantly induce the upregulation of immune-related genes in trunk kidney leucocytes. The ligand-receptor relationship analyses revealed that recombinant IFN-γ-like, IFN-γ, and IFN-γrel transduce downstream signalling through IFN-γR1-1/IFN-γR2-1, IFN-γR1-2/IFN-γR2-2, and IFN-γR1-1, respectively, in xenogeneic cells with the overexpression of original or chimeric receptors. In addition, tyrosine (Y) 366 and Y377 in the intracellular region may be essential for the function of IFN-γR1-2 and IFN-γR1-1, respectively. The finding of type II IFN system in A. gigas thus provides different knowledge in understanding the diversity and evolution of type II IFN ligand-receptor relationships in vertebrates.

CSIG-02. FOSL1 PROMOTES GLIOMA STEM CELLS TRAITS THROUGH INTERACTING WITH ITS BINDING PROTEIN PHOSPHORYLATED STAT3

Abstract BACKGROUND Glioma stem cells (GSCs) have key roles in tumorigenesis, progression, and resistance to conventional treatments for glioblastoma. We reported that TRPM7 channels, which fused with C-terminal kinase, endow glioma cells with stem cell-like capacities of self-renewal. Here, we investigated the role of TRPM7 downstream target gene FOSL1’s post transcription in glioma stemness. METHODS 1) The immunohistochemical staining for FOSL1 was performed using the rabbit polyclonal anti-FOSL1 antibody in glioma tissue arrays from 76 Chinese patients and 10 normal individuals. The staining intensity of cells in TMA was evaluated in three different fields (≥100 cells/field). The semi-quantitative HSCORE was calculated for FOSL1. 2) Upon suppression of FOSL1 by siRNA FOSL1, GSC markers, CD133 expression was evaluated by flow cytometry using PE anti-human CD133 antibody, while ALDH1 enzymatic activity was assessed using an Aldefluor kit, in A172 and U87MG glioma cell lines and patient-derived xenoline PDX-L14 of proneural subtype. 3) To assay FOSL1 gene promoter activity, the 5’-flanking region of the human FOSL1 gene (1201bp) was inserted into the firefly luciferase reporter vector pGL3-Basic between MluI and BglII sites. For mutant construct, STAT3 binding sites were mutated using Q5 site-directed mutagenesis kit. RESULTS 1) Quantification of the IHC results showed that FOSL1’s positive nuclear staining was significantly higher in GBM (n=13, p=0.0005), grade III (n=12, p=0.0116), and grade II astrocytoma (n=51, p=0.0485) compared to that of normal brain tissue (n=10). 2) GSC markers CD133 and ALDH1 were positively associated with FOSL1 in glioma. 3) Phosphorylated STAT3 directly binds to the FOSL1 promoter and the activation levels of STAT3 have a positive effect on the transcriptional activity of FOSL1. CONCLUSION Our results demonstrated the role of TRPM7-regulated STAT3 activation directly regulated FOSL1 gene transcription, which shed new light on TRPM7/FOSL1-directed therapeutics in glioma. (This study was supported by NIH NIGMS GM121230)

When Smart Beta Meets Machine Learning and Portfolio Optimization

<h3>Abstract</h3> Exposure of human cells to interferon-γ (IFNγ) results in a mitotically heritable yet reversible state called long-term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here we discovered that in primed cells, both interferon-responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re-exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ-induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK-STAT signalling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that memory of interferon exposure constitutes a STAT1-mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

Selenoprotein I deficiency in T cells promotes differentiation into tolerant phenotypes while decreasing Th17 pathology.

Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase contributing to cellular metabolism and the synthesis of glycosylphosphatidylinositol (GPI) anchors. SELENOI knockout (KO) in T cells has been shown to impair metabolic reprogramming during T cell activation and reduce GPI-anchored Thy-1 levels, which are both crucial for Th17 differentiation. This suggests SELENOI may be important for Th17 differentiation, and we found that SELENOI was indeed up-regulated early during the activation of naïve CD4+ T cells in Th17 conditions. SELENOI KO reduced RORγt mRNA levels by decreasing SOX5 and STAT3 binding to promoter and enhancer regions in the RORC gene encoding this master regulator of Th17 cell differentiation. Differentiation of naïve CD4+ T cells into inflammatory versus tolerogenic Th cell subsets was analyzed and results showed that SELENOI deficiency skewed differentiation away from pathogenic Th17 cells (RORγt+ and IL-17A+ ) while promoting tolerogenic phenotypes (Foxp3+ and IL-10+ ). Wild-type and T cell-specific SELENOI KO mice were subjected to experimental autoimmune encephalitis (EAE), with KO mice exhibiting diminished clinical symptoms, reduced CNS pathology and decreased T cell infiltration. Flow cytometry showed that SELENOI T cell KO mice exhibited lower CD4+ RORγt+ and CD4+ IL-17A+ T cells and higher CD4+ CD25+ FoxP3+ T cells in CNS tissues of mice subjected to EAE. Thus, the metabolic enzyme SELENOI is up-regulated to promote RORγt transcription that drives Th17 differentiation, and SELENOI deficiency shifts differentiation toward tolerogenic phenotypes while protecting against pathogenic Th17 responses.