Promote STAT3 Phosphorylation Research Articles

IL6-STAT3-C/EBPβ-IL6 positive feedback loop in tumor-associated macrophages promotes the EMT and metastasis of lung adenocarcinoma

BackgroundLung cancer is one of the most common tumors in the world, and metastasis is one of the major causes of tumor-related death in lung cancer patients. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and are frequently associated with tumor metastasis in human cancers. However, the regulatory mechanisms of TAMs in lung cancer metastasis remain unclear.MethodsSingle-cell sequencing analysis of lung cancer and normal tissues from public databases and from 14 patients who underwent surgery at Zhongshan Hospital was performed. In vitro co-culture experiments were performed to evaluate the effects of TAMs on lung cancer migration and invasion. Changes in the expression of IL-6, STAT3, C/EBPΒ, and EMT pathway were verified using RT-qPCR, western blotting, and immunofluorescence. Dual luciferase reporter assays and ChIP were used to reveal potential regulatory sites on the transcription factor sets. In addition, the effects of TAMs on lung cancer progression and metastasis were confirmed by in vivo models.ResultsTAM infiltration is associated with tumor progression and poor prognosis. IL-6 secreted by TAMs can activate the JAK2/STAT3 pathway through autocrine secretion, and STAT3 acts as a transcription factor to activate the expression of C/EBPβ, which further promotes the transcription and expression of IL-6, forming positive feedback loops for IL6-STAT3-C/EBPβ-IL6 in TAMs. IL-6 secreted by TAMs promotes lung cancer progression and metastasis in vivo and in vitro by activating the EMT pathway, which can be attenuated by the use of JAK2/STAT3 pathway inhibitors or IL-6 monoclonal antibodies.ConclusionsOur data suggest that TAMs promote IL-6 expression by forming an IL6-STAT3-C/EBPβ-IL6 positive feedback loop. Released IL-6 can induce the EMT pathway in lung cancer to enhance migration, invasion, and metastasis. The use of IL-6-neutralizing antibody can partially counteract the promotion of LUAD by TAMs. A novel mechanism of macrophage-promoted tumor progression was revealed, and the IL6-STAT3-C/EBPβ-IL6 signaling cascade may be a potential therapeutic target against lung cancer.Graphical IL-6 secreted by TAM acts on itself to promote STAT3 phosphorylation, and pSTAT3 transfers into the nucleus, promotes the expression of C/EBPβ. C/EBPβ is able to further promote IL-6 expression, which forms positive feedback for IL-6 secretion. IL-6 secreted by TAMs acts on lung cancer to promote their metastasis through activation of EMT.

Blocking EGR1/TGF-β1 and CD44s/STAT3 Crosstalk Inhibits Peritoneal Metastasis of Gastric Cancer.

Peritoneal metastasis (PM) continues to limit the clinical efficacy of gastric cancer (GC). Early growth response 1 (EGR1) plays an important role in tumor cell proliferation, angiogenesis and invasion. However, the role of EGR1 derived from the tumor microenvironment in reshaping the phenotypes of GC cells and its specific molecular mechanisms in increasing the potential for PM are still unclear. In this study, we reported that EGR1 was significantly up-regulated in mesothelial cells from GC peritoneal metastases, leading to enhanced epithelial-mesenchymal transformation (EMT) and stemness phenotypes of GC cells under co-culture conditions. These phenotypes were achieved through the transcription and secretion of TGF-β1 by EGR1 in mesothelial cells, which could regulate the expression and internalization of CD44s. After being internalized into the cytoplasm, CD44s interacted with STAT3 to promote STAT3 phosphorylation and activation, and induced EMT and stemness gene transcription, thus positively regulating the metastasis of GC cells. Moreover, TGF-β1 secretion in the PM microenvironment was significantly increased compared with the matched primary tumor. The blocking effect of SHR-1701 on TGF-β1 was verified by inhibiting peritoneal metastases in xenografts. Collectively, the interplay of EGR1/TGF-β1/CD44s/STAT3 signaling between mesothelial cells and GC cells induces EMT and stemness phenotypes, offering potential as a therapeutic target for PM of GC.

OTUD6A in tubular epithelial cells mediates angiotensin II-induced kidney injury by targeting STAT3.

Kidney fibrosis is a prominent pathological feature of hypertensive kidney diseases (HKD). Recent studies have highlighted the role of ubiquitinating/deubiquitinating protein modification in kidney pathophysiology. Ovarian tumor domain-containing protein 6 A (OTUD6A) is a deubiquitinating enzyme involved in tumor progression. However, its role in kidney pathophysiology remains elusive. We aimed to investigate the role and underlying mechanism of OTUD6A during kidney fibrosis in HKD. The results revealed higher OTUD6A expression in kidney tissues of nephropathy patients and mice with chronic angiotensin II (Ang II) administration than that from the control ones. OTUD6A was mainly located in tubular epithelial cells. Moreover, OTUD6A deficiency significantly protected mice against Ang II-induced kidney dysfunction and fibrosis. Also, knocking OTUD6A down suppressed Ang II-induced fibrosis in cultured tubular epithelial cells, whereas overexpression of OTUD6A enhanced fibrogenic responses. Mechanistically, OTUD6A bounded to signal transducer and activator of transcription 3 (STAT3) and removed K63-linked-ubiquitin chains to promote STAT3 phosphorylation at tyrosine 705 position and nuclear translocation, which then induced profibrotic gene transcription in epithelial cells. These studies identified STAT3 as a direct substrate of OTUD6A and highlighted the pivotal role of OTUD6A in Ang II-induced kidney injury, indicating OTUD6A as a potential therapeutic target for HKD.NEW & NOTEWORTHY Ovarian tumor domain-containing protein 6 A (OTUD6A) knockout mice are protected against angiotensin II-induced kidney dysfunction and fibrosis. OTUD6A promotes pathological kidney remodeling and dysfunction by deubiquitinating signal transducer and activator of transcription 3 (STAT3). OTUD6A binds to and removes K63-linked-ubiquitin chains of STAT3 to promote its phosphorylation and activation, and subsequently enhances kidney fibrosis.

IL20RB signaling enhances stemness and chemotherapy resistance in pancreatic cancer

ObjectivePancreatic cancer is an aggressive malignancy with high mortality, and cancer cell stemness and related drug resistance are considered important contributors to its poor prognosis. The objective of this study was to identify regulatory targets associated with the maintenance of pancreatic cancer stemness.Materials and MethodsPancreatic tumor samples were collected from patients at Sun Yat-sen University Cancer Center, followed by immunofluorescence analysis. Pancreatic cancer cell lines with Interleukin-20 receptor subunit beta (IL20RB) overexpression and knockdown were established, and clonal formation, spheroid formation and side population cell analysis were conducted. The effects of IL20RB knockdown on the tumor-forming ability of pancreatic cancer cells and chemotherapy resistance in vivo were explored.ResultsIL20RB expression was significantly upregulated in pancreatic cancer tissues, and was correlated with unfavorable prognosis. The IL20RB receptor promotes stemness and chemoresistance in both in vitro and in vivo models of pancreatic cancer. Mechanistically, IL20RB enhances the stemness and chemoresistance of pancreatic cancer by promoting STAT3 phosphorylation, an effect that can be counteracted by a STAT3 phosphorylation inhibitors. Additionally, Interleukin-19 derived from the microenvironment is identified as the primary ligand for IL20RB in mediating these effects.ConclusionOur findings demonstrate that IL20RB plays a crucial role in promoting stemness in pancreatic cancer. This discovery provides a potential therapeutic target for this lethal disease.Graphical abstract

SOX2-OT Binds with ILF3 to Promote Head and Neck Cancer Progression by Modulating Crosstalk between STAT3 and TGF-β Signaling.

Long non-coding RNA (lncRNA) is involved in the progression of head and neck squamous cell carcinoma (HNSCC). The molecular mechanism of lncRNA SOX2-OT in HNSCC remains unclear. Therefore, we aimed to elucidate the oncogenic role of SOX2-OT in HNSCC. QRT-PCR analysis was performed in 61 pairs of HNSCC cancer tissues, adjacent normal tissues, and 68 plasma samples confirmed that lncRNA SOX2-OT was overexpressed in cancer tissues and plasma samples, which served as a poor prognostic factor for HNSCC. The FISH assay demonstrated that SOX2-OT was localized in the nucleus and cytoplasm of HNSCC cell lines. Further, the cell function assay confirmed that SOX2-OT promoted cell proliferation and metastasis in vitro and in vivo. RNA pulldown and RIP assay results revealed that SOX2-OT bonds with ILF3 in HNSCC, and the rescue assay confirmed that SOX2-OT played an oncogenic role depending on ILF3 protein expression. Ingenuity pathway analysis and Western blotting indicated that SOX2-OT regulated HNSCC progression by promoting STAT3 phosphorylation and modulating the crosstalk between STAT3 and TGF-β signaling. These results reveal evidence for the role of SOX2-OT in HNSCC progression and metastasis by binding to ILF3, which may serve as a therapeutic target and prognostic biomarker in HNSCC.

ROS-mediated MAPK activation aggravates hyperoxia-induced acute lung injury by promoting apoptosis of type II alveolar epithelial cells via the STAT3/miR-21–5p axis

Inhibition of type II alveolar epithelial (AE-II) cell apoptosis is a critical way to cure hyperoxia-induced acute lung injury (HALI). It has been reported that miR-21–5p could reduce H2O2-induced apoptosis in AE-II cells. However, the upstream molecular mechanism remains unclear. Herein, we established a cellular model of HALI by exposing AE-II cells to H2O2 treatment. It was shown that miR-21–5p alleviated H2O2-induced apoptosis in AE-II cells. ROS inhibition decreased apoptosis of H2O2-evoked AE-II cells via increasing miR-21–5p expression. In addition, ROS induced MAPK and STAT3 phosphorylation in H2O2-treated AE-II cells. MAPK inactivation reduces H2O2-triggered AE-II cell apoptosis. MAPK activation inhibits miR-21–5p expression by promoting STAT3 phosphorylation in H2O2-challenged AE-II cells. Furthermore, STAT3 activation eliminated MAPK deactivation-mediated inhibition on the apoptosis of AE-II cells under H2O2 condition. In conclusion, ROS-mediated MAPK activation promoted H2O2-triggered AE-II cell apoptosis by inhibiting miR-21–5p expression via STAT3 phosphorylation, providing novel targets for HALI treatment.

BDNF alleviates Parkinson's disease by promoting STAT3 phosphorylation and regulating neuronal autophagy.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the gradual death of dopaminergic neurons. Brain-derived neurotrophic factor (BDNF) and its receptors are widely distributed throughout the central nervous system, which can promote the survival and growth of neurons and protect neurons. This study revealed that BDNF promotes STAT3 phosphorylation and regulates autophagy in neurons. The PD mouse model was established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, SH-SY5Y cells were treated with 1-methyl-4-phenyl-pyridinium (MPP+) to establish a PD cell model. The level of BDNF was low in PD model mice and SH-SY5Y cells treated with MPP+. BDNF enhanced the levels of p-TrkB, P-STAT3, PINK1, and DJ-1. BDNF promoted autophagy, inhibited the level of p-α-syn (Ser129) and enhanced cell proliferation. The autophagy inhibitor 3-Methyladenine (3-methyladenine, 3-MA) reversed the protective effects of BDNF on neurons. BiFC assay results showed that there was a direct physical interaction between BDNF and STAT3, and coimmunoprecipitation experiments indicated an interaction between STAT3 and PI3K. The PI3K agonist Recilisib activated the PI3K/AKT/mTOR pathway, promoted autophagy, and alleviated neuronal cell damage. BDNF alleviates PD pathology by promoting STAT3 phosphorylation and regulating neuronal autophagy in SH-SY5Y cells and cultured primary neurons. Finally, BDNF has neuroprotective effects on PD model mice.

Dexmedetomidine Pretreatment Protects Against Myocardial Ischemia/Reperfusion Injury by Activating STAT3 Signaling.

Myocardial infarction is a common perioperative complication, and blood flow restoration causes ischemia/reperfusion injury (IRI). Dexmedetomidine (DEX) pretreatment can protect against cardiac IRI, but the mechanism is still insufficiently understood. In vivo, myocardial ischemia/reperfusion (30 minutes/120 minutes) was induced via ligation and then reperfusion of the left anterior descending coronary artery (LAD) in mice. Intravenous infusion of 10 μg/kg DEX was performed 20 minutes before ligation. Moreover, the α2-adrenoreceptor antagonist Yohimbine and STAT3 inhibitor Stattic were applied 30 minutes ahead of DEX infusion. In vitro, hypoxia/reoxygenation (H/R) with DEX pretreatment for 1 hour was performed in isolated neonatal rat cardiomyocytes. In addition, Stattic was applied before DEX pretreatment. In the mouse cardiac ischemia/reperfusion model, DEX pretreatment lowered the serum creatine kinase-MB isoenzyme (CK-MB) levels (2.47 ± 0.165 vs 1.55 ± 0.183; P < .0001), downregulated the inflammatory response ( P ≤ .0303), decreased 4-hydroxynonenal (4-HNE) production and cell apoptosis ( P = .0074), and promoted the phosphorylation of STAT3 (4.94 ± 0.690 vs 6.68 ± 0.710, P = .0001), which could be blunted by Yohimbine and Stattic. The bioinformatic analysis of differentially expressed mRNAs further confirmed that STAT3 signaling might be involved in the cardioprotection of DEX. Upon H/R treatment in isolated neonatal rat cardiomyocytes, 5 μM DEX pretreatment improved cell viability ( P = .0005), inhibited reactive oxygen species (ROS) production and calcium overload (both P ≤ .0040), decreased cell apoptosis ( P = .0470), and promoted STAT3 phosphorylation at Tyr705 (0.102 ± 0.0224 vs 0.297 ± 0.0937; P < .0001) and Ser727 (0.586 ± 0.177 vs 0.886 ± 0.0546; P = .0157), which could be abolished by Stattic. DEX pretreatment protects against myocardial IRI, presumably by promoting STAT3 phosphorylation via the α2-adrenoreceptor in vivo and in vitro.

Proline/serine-rich coiled-coil protein 1 inhibits macrophage inflammation and delays atherosclerotic progression by binding to Annexin A2.

Atherosclerosis (AS), the main pathological basis of life-threatening cardiovascular disease, is essentially caused by chronic macrophage inflammation. Overexpression of proline/serine-rich coiled-coil protein 1 (PSRC1) reduces macrophage inflammatory responses and delays AS development. However, the exact mechanism of PSRC1 is unclear. Proteins interacting with PSRC1 were screened by proteomics in RAW264.7 cells, followed by RT-qPCR, immunoprecipitation and immunofluorescence to explore the specific mechanistic pathways affecting inflammation. CRISPR-Cas9 constructs for PSRC1-/- ApoE-/- (DKO) mice and high-fat diet-fed ApoE-/- and DKO mice were used for AS models for in vivo experiments. Upstream transcription factors of PSRC1 were predicted by ATAC-seq, ChIP-seq and UCSC, and the regulatory mechanism was verified by ChIP-qPCR and dual luciferase assays. Peripheral blood serum and monocytes were collected from coronary artery disease (CAD) patients and non-CAD patients. Increased binding of ANXA2 to PSRC1 in macrophages under oxidized low-density lipoprotein stimulation and decreased release of ANXA2 to the extracellular compartment were observed. Knockdown of ANXA2 in AS model mice delayed AS progression. Knockdown of ANXA2 in DKO mice reversed the AS-promoting effect of PSRC1 knockdown. Mechanistically, ANXA2 promotes STAT3 phosphorylation, which in turn promotes inflammatory responses. In addition, SP1 is a PSRC1 upstream repressive transcription factor, and the SP1 inhibitor mithramycin (Mith) elevated PSRC1 expression and exerted anti-AS effects in AS model mice. Patients with CAD had considerably greater serum levels of ANXA2 than those without CAD, and Mith reduced the secretion of ANXA2 in peripheral blood monocytes of CAD patients. In macrophages, PSRC1 can interact with ANXA2 to inhibit its extracellular release and delay AS development. SP1 is an upstream transcription factor of PSRC1 and inhibits the transcription of PSRC1. The SP1 inhibitor Mith can elevate PSRC1 levels and slow AS progression while reducing ANXA2 release from monocytes in CAD patients. Mith is expected to be a new agent for AS treatment.

GDF15 Promotes Cell Growth, Migration, and Invasion in Gastric Cancer by Inducing STAT3 Activation.

Growth differentiation factor 15 (GDF15) has been reported to play an important role in cancer and is secreted and involved in the progression of various cancers, including ovarian cancer, prostate cancer, and thyroid cancer. Nevertheless, the functional mechanism of GDF15 in gastric cancer is still unclear. Immunohistochemical staining was performed to estimate the expression of GDF15 in 178 gastric cancer tissues. The biological role and action mechanism of GDF15 were investigated by examining the effect of GDF15 knockdown in AGS and SNU216 gastric cancer cells. Here, we report that the high expression of GDF15 was associated with invasion depth (p = 0.002), nodal involvement (p = 0.003), stage III/IV (p = 0.01), lymphatic invasion (p = 0.05), and tumor size (p = 0.049), which are related to poor survival in gastric cancer patients. GDF15 knockdown induced G0/G1 cell cycle arrest and remarkably inhibited cell proliferation and reduced cell motility, migration, and invasion compared to the control. GDF15 knockdown inhibited the epithelial-mesenchymal transition by regulating the STAT3 phosphorylation signaling pathways. Taken together, our results indicate that GDF15 expression is associated with aggressive gastric cancer by promoting STAT3 phosphorylation, suggesting that the GDF15-STAT3 signaling axis is a potential therapeutic target against gastric cancer progression.

OTUD1 promotes pathological cardiac remodeling and heart failure by targeting STAT3 in cardiomyocytes.

Rationale: Understanding the molecular mechanisms of deleterious cardiac remodeling is important for the development of treatments for heart failure. Recent studies have highlighted a role of deubiquitinating enzymes in cardiac pathophysiology. In the present study, we screened for alteration of deubiquitinating enzymes in experimental models of cardiac remodeling, which indicated a potential role of OTU Domain-Containing Protein 1 (OTUD1). Methods: Wide-type or OTUD1 knockout mice with chronic angiotensin II infusion and transverse aortic constriction (TAC) were utilized to develop cardiac remodeling and heart failure. We also overexpressed OTUD1 in mouse heart with AAV9 vector to validate the function of OTUD1. LC-MS/MS analysis combined with Co-IP was used to identify the interacting proteins and substrates of OTUD1. Results: We found that OTUD1 is elevated in mouse heart tissues following chronic angiotensin II administration. OTUD1 knockout mice were significantly protected against angiotensin II-induced cardiac dysfunction, hypertrophy, fibrosis and inflammatory response. Similar results were obtained in the TAC model. Mechanistically, OTUD1 bounds to the SH2 domain of STAT3 and causes deubiquitination of STAT3. Cysteine at position 320 of OTUD1 exerts K63 deubiquitination to promote STAT3 phosphorylation and nuclear translocation, thereby increasing STAT3 activity to induce inflammatory responses, fibrosis, and hypertrophy in cardiomyocytes. Finally, OTUD1 overexpression by AAV9 vector increases Ang II-induced cardiac remodeling in mice and OTUD1-regulated responses can be inhibited by blocking STAT3. Conclusion: Cardiomyocyte OTUD1 promotes pathological cardiac remodeling and dysfunction by deubiquitinating STAT3. These studies have highlighted a novel role of OTUD1 in hypertensive heart failure and identified STAT3 as a target of OTUD1 in mediating these actions.

Dihydrotanshinone I: A Target for STAT3 in the Therapy of Tamoxifen‐Resistant Breast Cancer

AbstractBreast cancer is the most prevalent tumour in women. Over two‐thirds of breast cancers are estrogen receptor (ER)‐positive breast cancers. These breast cancer patients are prone to tamoxifen resistance during breast cancer treatment. There is still a lack of effective clinical treatment for tamoxifen‐resistant breast cancer. Dihydrotanshinone I (DHTS) promotes various types of apoptosis in tumour cells. Our network pharmacology study revealed that DHTS has a strong binding capacity to STAT3. Tamoxifen‐resistant breast cancers are often accompanied by STAT3 activation. However, there are no reports of DHTS for tamoxifen‐resistant breast cancer. Subsequent in vitro experiments also confirmed that DHTS promoted apoptosis in MCF‐7‐TamR cells, down‐regulated the performance of STAT3, pSTAT3, and BCL‐2, and activated the production of apoptosis‐associated PARP1 and cleaved caspase3. This process could be rescued by overexpression of BCL‐2 downstream of STAT3 and was not rescued by either the cytokine EGF or the cytokine PDGF, which promote STAT3 phosphorylation. We hypothesized that DHTS targeting STAT3 promotes apoptosis in MCF‐7‐TamR cells, mainly by affecting STAT3 phosphorylation. Our research approach described above may provide new ideas for the treatment of tamoxifen‐resistant breast cancer.

Cryptococcus neoformans Infection Induces IL-17 Production by Promoting STAT3 Phosphorylation in CD4+ T Cells.

Cryptococcus neoformans infection in the central nervous system is a severe infectious disease with poor outcomes and high mortality. It has been estimated that there are 220,000 new cases each year. Over 90% of C. neoformans meningitis cases were diagnosed in AIDS patients with CD4+ T cell count <100 cells/μl; however, the mechanism of cryptococcal meningitis in patients with normal immune functions remains unclear. IL-17 is a pro-inflammatory cytokine and plays an important role in anti-fungal immunity. Here we report that significantly high levels of IL-17 were predominantly detected in the cerebrospinal fluid of patients with either AIDS- or non-AIDS-associated C. neoformans meningitis but not in patients with tuberculous meningitis or non-neurosyphilis. Antifungal therapy minimized the IL-17 level in the cerebrospinal fluid. An in vitro mechanistic study showed that C. neoformans stimulation of healthy peripheral blood mononuclear cells prompted IL-17 production, and CD4+ T cells were the predominant IL-17-producing cells. IL-17 production by C. neoformans stimulation was STAT3 signaling dependent. Inhibition of STAT3 phosphorylation attenuated the C. neoformans-mediated IL-17 expression. Our data highlighted the significance of CD4+ T cells in antifungal immunity and suggested IL-17 as a diagnostic biomarker of C. neoformans infection and STAT3 as a checkpoint for antifungal targeted therapies.

P2RY13 Exacerbates Intestinal Inflammation by Damaging the Intestinal Mucosal Barrier via Activating IL-6/STAT3 Pathway.

The pathogenesis of ulcerative colitis (UC) is unclear, while genetic factors have been confirmed to play an important role in its development. P2RY13 is a G protein-coupled receptor (GPCRs), which are involved in the pathogenesis of inflammation and immune disorders. According to GEO database analysis, we first observed that the expression of P2Y13 was increased in UC patients. Therefore, we sought to determine the role of P2Y13 in the development of colitis. Our data showed that P2RY13 was highly expressed in the inflamed intestinal tissues of UC patients. In mice, pharmacological antagonism of P2Y13 can significantly attenuate the intestinal mucosal barrier disruption. In LPS-induced NCM460 cell, knockdown or pharmacological inhibition of P2RY13 increased the expression of intestinal tight junction protein and reduced apoptosis. In addition, we found that the effect of P2Y13 on colitis is related to the activation of the IL-6/STAT3 pathway. Activation of P2Y13 increases IL-6 expression and promotes STAT3 phosphorylation and nuclear transport. Deletion of the STAT3 gene in the intestinal epithelial cells of mice significantly mitigated the exacerbation of colitis due to P2Y13 activation. Thus, P2Y13 can aggravate intestinal mucosal barrier destruction by activating the IL-6/STAT3 pathway. P2Y13 might be a potential drug target for UC.

PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell–specific PKM2−/− mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe−/−). Controls were littermate PKM2fl/flLysMCre– or PKM2fl/flLysMCre–Apoe−/− mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell–specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

Abstract 107: PKM2 Promotes Neutrophil Activation And Cerebral Thrombo-inflammation: Therapeutic Implications For Ischemic Stroke

In recent years compelling evidence has emerged that implicates role of metabolic reprogramming in the modulation of thrombosis and inflammation. The dimeric form of the metabolic enzyme pyruvate kinase muscle 2 (PKM2) enters nucleus and exerts protein kinase activity. We found that nuclear PKM2 levels were upregulated in neutrophils after the onset of ischemic stroke both in humans and in mice. Therefore, we evaluated the role of PKM2 in promoting thrombo-inflammation and ischemic brain injury.We generated novel myeloid cell-specific PKM2 -/- mice on wild-type ( PKM2 fl/fl LysMCre + ) and hyperlipidemic background ( PKM2 fl/fl LysMCre + Apoe -/- ). We observed that genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps following cerebral ischemia/reperfusion. In the filament and autologous clot/rtPA stroke models, irrespective of sex, deletion of PKM2 in myeloid cells either in wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrin(ogen), platelet (CD41- positive) deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. Utilizing small molecule inhibitor (ML265) that inhibits PKM2 dimerization, we evaluated ex vivo thrombosis using human whole blood in a microfluidic flow chamber system. We observed a fivefold reduction in the thrombus growth rate in ML265-treated group. Similarly, ML265 treatment in mice resulted into significantly reduced poststroke neutrophil hyperactivation and improved short-term and long-term functional outcomes following stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery following reperfusion.

ETV4 transcriptionally activates HES1 and promotes Stat3 phosphorylation to promote malignant behaviors of colon adenocarcinoma.

Colon adenocarcinoma (COAD) is the commonest type of colorectal cancer with high morbidity and mortality worldwide. ETS variant 4 (ETV4) is a member of the ETS transcription factors and is frequently involved in the progression of many cancers. This study focused on the relevance of ETV4 to the progression of COAD. ETV4 was highly expressed in the collected COAD tissues and acquired cells and indicated advanced Dukes staging in patients. Knockdown of ETV4 in COAD cells weakened proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) activity of cells. The downstream genes of ETV4 were predicted, and a Gene Ontology (GO) analysis was conducted to identify the key molecule involved. ETV4 bound to the promoter sequence of HES1 and activated its transcription. Further overexpression of HES1 restored the malignant behaviors of COAD cells. HES1 was also found to promote phosphorylation of Stat3. Similar results were reproduced in vivo where downregulation of ETV4 blocked the growth of xenograft tumors in nude mice. This study demonstrated that ETV4 encourages malignant development of COAD through activating HES1 transcription and Stat3 phosphorylation. This study may offer novel insights into COAD therapy.

TROY signals through JAK1-STAT3 to promote glioblastoma cell migration and resistance

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a discouraging prognosis. Its aggressive and highly infiltrative nature renders the current standard treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the signaling pathways that regulate GBM migration/invasion and resistance is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNF receptor superfamily, increases with glial tumor grade, inversely correlates with patient overall survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Here, we have identified for the first time that TROY interacts with JAK1. Increased TROY expression increases JAK1 phosphorylation. In addition, increased TROY expression promotes STAT3 phosphorylation and STAT3 transcriptional activity that is dependent upon JAK1. TROY-mediated activation of STAT3 is independent of its ability to stimulate activity of NF-κB. Inhibition of JAK1 activity by ruxolitinib or knockdown of JAK1 expression by siRNA significantly inhibits TROY-induced STAT3 activation, GBM cell migration, and decreases resistance to temozolomide. Taken together, our data indicate that the TROY signaling complex may represent a potential therapeutic target with the distinctive capacity to exert effects on multiple pathways mediating GBM cell invasion and resistance.

DTNA promotes HBV-induced hepatocellular carcinoma progression by activating STAT3 and regulating TGFβ1 and P53 signaling

ObjectiveHepatitis B virus (HBV) infection causes liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) development, but the underlying mechanism remains poorly understood. This study aimed to investigate the roles and molecular mechanisms of Dystrobrevin-α (DTNA) in HBV-induced liver cirrhosis and HCC pathogenesis. MethodsDTNA expression was bioinformatically analyzed using the GEO database. DTNA expression was silenced by transfection with shRNAs. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry respectively. The expression of genes in mRNA or protein levels was assessed by quantitative RT-PCR and western blotting. The interaction between proteins was predicted with the String and GCBI online softwares, and then confirmed by co-immunoprecipitation. Animal models were established by injecting nude mice with AVV8-HBV1.3 vector. ResultsBioinformatics analysis showed a significantly increase in DTNA expression in HBV-positive liver cirrhosis and HCC patients. HBV infection caused a significantly increase in DTNA expression in HCC cell lines HepAD38 and HepG2.2.15. DTNA knockdown suppressed proliferation and promoted apoptosis of HBV-infected HepAD38 and HepG2.2.15 cells. HBV induced elevated expression of fibrosis-related genes Collagen II and TGFβ1 in LO-2 cells, which were suppressed by DTNA knockdown. DTNA directly binded with STAT3 protein to promote STAT3 phosphorylation and TGFβ1 expression and repress P53 expression in HBV-infected HepAD38 and LO-2 cells. The DTNA/STAT3 axis was activated during HBV-induced fibrosis, cirrhosis and HCC development in mouse model. ConclusionDTNA binds with and further activates STAT3 to induce TGFβ1 expression and repress P53 expression, thus promoting HBV-induced liver fibrosis, cirrhosis and hepatocellular carcinoma progression.

Glial Endozepines Reverse High-Fat Diet-Induced Obesity by Enhancing Hypothalamic Response to Peripheral Leptin.

Research on energy homeostasis has focused on neuronal signaling; however, the role of glial cells has remained little explored. Glial endozepines exert anorexigenic actions by mechanisms which remain poorly understood. In this context, the present study was designed to decipher the mechanisms underlying the anorexigenic action of endozepines and to investigate their potential curative effect on high-fat diet-induced obesity. We carried out a combination of physiological, pharmacological, and molecular analyses together to dissect the underlying mechanisms of endozepine-induced hypophagia. To evaluate the potential anti-obesity effect of endozepines, different model of obesity were used, i.e., ob/ob and diet-induced obese mice. We show that the intracerebral administration of endozepines enhances satiety by targeting anorexigenic brain circuitry and induces STAT3 phosphorylation, a hallmark of leptin signaling. Strikingly, endozepines are entirely ineffective at reducing food intake in the presence of a circulating leptin antagonist and in leptin-deficient mice (ob/ob) but potentiate the reduced food intake and weight loss induced by exogenous leptin administration in these animals. Endozepines reversed high fat diet-induced obesity by reducing food intake and restored leptin-induced STAT3 phosphorylation in the hypothalamus. Interestingly, we observed that glucose and insulin synergistically enhance tanycytic endozepine expression and release. Finally, endozepines, which induce ERK activation necessary for leptin transport into the brain in cultured tanycytes, require tanycytic leptin receptor expression to promote STAT3 phosphorylation in the hypothalamus. Our data identify endozepines as potential anti-obesity compounds in part through the modulation of the LepR-ERK-dependent tanycytic leptin shuttle.