Signal Transducer And Activator Of Transcription 3 Inhibitor Research Articles

Design, synthesis and biological evaluation of novel potent STAT3 inhibitors based on related heterocycle-fused naphthoquinones for cancer therapy

Persistently activated signal transducer and activator of transcription 3 (STAT3) plays an important role in the development of multiple cancers, and therefore it is a potential therapeutic target for cancer treatment.

Histone deacetylase 5-induced deficiency of signal transducer and activator of transcription-3 acetylation contributes to spinal astrocytes degeneration in painful diabetic neuropathy.

Diabetes patients with painful diabetic neuropathy (PDN) show severe spinal atrophy, suggesting pathological changes of the spinal cord contributes to central sensitization. However, the cellular changes and underlying molecular mechanisms within the diabetic spinal cord are less clear. By using a rat model of type 1 diabetes (T1D), we noted an extensive and irreversible spinal astrocyte degeneration at an early stage of T1D, which is highly associated with the chronification of PDN. Molecularly, acetylation of astrocytic signal transducer and activator of transcription-3 (STAT3) that is essential for maintaining the homeostatic astrocytes population was significantly impaired in the T1D model, resulting in a dramatic loss of spinal astrocytes and consequently promoting pain hypersensitivity. Mechanistically, class IIa histone deacetylase, HDAC5 were aberrantly activated in spinal astrocytes of diabetic rats, which promoted STAT3 deacetylation by direct protein-protein interactions, leading to the PDN phenotypes. Restoration of STAT3 signaling or inhibition of HDAC5 rescued astrocyte deficiency and attenuated PDN in the T1D model. Our work identifies the inhibitory axis of HDAC5-STAT3 induced astrocyte deficiency as a key mechanism underlying the pathogenesis of the diabetic spinal cord that paves the way for potential therapy development for PDN.

STAT3 potentiates RNA polymerase I-directed transcription and tumor growth by activating RPA34 expression.

Deregulation of either RNA polymerase I (Pol I)-directed transcription or expression of signal transducer and activator of transcription 3 (STAT3) correlates closely with tumorigenesis. However, the connection between STAT3 and Pol I-directed transcription hasn't been investigated. The role of STAT3 in Pol I-directed transcription was determined using combined techniques. The regulation of tumor cell growth mediated by STAT3 and Pol I products was analyzed in vitro and in vivo. RNAseq, ChIP assays and rescue assays were used to uncover the mechanism of Pol I transcription mediated by STAT3. STAT3 expression positively correlates with Pol I product levels and cancer cell growth. The inhibition of STAT3 or Pol I products suppresses cell growth. Mechanistically, STAT3 activates Pol I-directed transcription by enhancing the recruitment of the Pol I transcription machinery to the rDNA promoter. STAT3 directly activates Rpa34 gene transcription by binding to the RPA34 promoter, which enhances the occupancies of the Pol II transcription machinery factors at this promoter. Cancer patients with RPA34 high expression lead to poor survival probability and short survival time. STAT3 potentiates Pol I-dependent transcription and tumor cell growth by activating RPA34 in vitro and in vivo.

Nanoparticle-Mediated Delivery of STAT3 Inhibitors in the Treatment of Lung Cancer.

Lung cancer is a common malignancy worldwide, with high morbidity and mortality. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that not only regulates different hallmarks of cancer, such as tumorigenesis, cell proliferation, and metastasis but also regulates the occurrence and maintenance of cancer stem cells (CSCs). Abnormal STAT3 activity has been found in a variety of cancers, including lung cancer, and its phosphorylation level is associated with a poor prognosis of lung cancer. Therefore, the STAT3 pathway may represent a promising therapeutic target for the treatment of lung cancer. To date, various types of STAT3 inhibitors, including natural compounds, small molecules, and gene-based therapies, have been developed through direct and indirect strategies, although most of them are still in the preclinical or early clinical stages. One of the main obstacles to the development of STAT3 inhibitors is the lack of an effective targeted delivery system to improve their bioavailability and tumor targetability, failing to fully demonstrate their anti-tumor effects. In this review, we will summarize the recent advances in STAT3 targeting strategies, as well as the applications of nanoparticle-mediated targeted delivery of STAT3 inhibitors in the treatment of lung cancer.

Atorvastatin ameliorates lipid overload-induced mitochondrial dysfunction and myocardial hypertrophy by decreasing fatty acid oxidation through inactivation of the p-STAT3/CPT1 pathway

Although statins are shown to have cardiac pleiotropic effects independent of lowering cholesterol, the underlying mechanism remains unclear. Mitochondrial dysfunction induced by increased fatty acid oxidation (FAO) is the culprit in the development of cardiac hypertrophy and dysfunction. This study was to explore whether the cardiac pleiotropic effects of atorvastatin were associated with FAO regulation, with a specific focus on carnitine palmitoyltransferase 1 (CPT1). High-fat diet (HFD)-fed mice and palmitic acid (PA)-stimulated neonatal rat primary cardiomyocytes (NRCMs) were treated with atorvastatin, with or without FAO modulators, signal transducer and activator of transcription 3 (STAT3) agonist, and inhibitor. Atorvastatin (3 mg/kg) did not reduce serum cholesterol levels in HFD-fed mice but ameliorated mitochondrial dysfunction and cardiac hypertrophy. In vitro, atorvastatin and the FAO inhibitor alleviated PA-induced mitochondrial dysfunction and cardiomyocyte hypertrophy. However, the FAO enhancer eliminated atorvastatin’s protective effects. Furthermore, atorvastatin decreased CPT1 and FAO levels and prevented STAT3 phosphorylation and nuclear translocation. STAT3 inhibitor had the same inhibitory effects as atorvastatin on CPT1, FAO levels, and cardiomyocyte hypertrophy, whereas STAT3 agonist disrupted these effects of atorvastatin. Our results demonstrate that atorvastatin decreases myocardial FAO by inactivating the p-STAT3/CPT1 signaling pathway, which improves lipid overload-induced mitochondrial dysfunction and cardiac hypertrophy in a cholesterol-independent manner. This is the first study to explore the cardiac pleiotropic effects of atorvastatin with respect to FAO. However, whether atorvastatin regulates FAO in the cardiac hypertrophy model induced by other variables has not been investigated in this work, and this is expected to be performed in the future.

Pyrimethamine, a STAT3 Inhibitor, Synergizes with Venetoclax and Shows Efficacy in Hypomethylating Agent Resistant MDS and AML

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arise after accumulation of multiple genetic and epigenetic alterations in hematopoietic stem cells (HSCs). Alterations in HSCs promote malignant transformation and give rise to leukemic stem cells (LSCs). Current therapies for MDS and AML, including recently FDA approved agents such as venetoclax (Ven), a Bcl-2 inhibitor, have failed to eliminate LSCs, therefore, molecular targeting of aberrant LSCs may be a potential strategy for developing targeted drugs capable of eliminating LSCs. Studies have shown that aberrant HSCs exhibit multiple disrupted cellular pathways, including overexpression of the signal transducer and activator of transcription 3 (STAT3) (Shastri, 2017). Elevated levels of STAT3 are observed in MDS and AML patient samples. Interestingly, an increased level of STAT3 in MDS and AML patients is predictive of a significantly adverse prognosis, thus supporting a critical role of STAT3 signaling in MDS and AML. We have previously shown that an antisense oligonucleotide inhibitor of STAT3, gets rapidly incorporated into HSCs and induces apoptosis and downregulation of STAT3 when compared to health controls (Shastri, JCI 2018). An initial screen to identify compounds that could target STAT3 revealed that pyrimethamine (PYR), an FDA approved anti-parasitic drug, inhibits STAT3 transcriptional activity at concentrations readily achieved in patients (Nelson, 2011). Therefore, we are further evaluating the efficacy of PYR for treatment of MDS and AML. HMA and Ven resistance is a critical issue in MDS/AML and can occur due to the persistence of relapse associated aberrant stem and progenitor cells that overexpress STAT3. Hence, we are evaluating the efficacy of PYR in a hypomethylating agent (HMA) and venetoclax (Ven) resistant model. Our in vitro studies show that PYR can inhibit viability by inducing apoptosis in multiple leukemic cells within its therapeutic index. Western blot analysis reveals that STAT3 protein is overexpressed in a Ven resistant MOLM13 AML line, and in a HMA resistant cell THP1 AML cell line. In HMA and Ven resistant leukemia cell lines, PYR can significantly decrease proliferation when combined with azacytidine (P= 0.0109) (Fig 1A) or venetoclax (P= 0.0271) treatments. Remarkably, PYR and Ven drug combination show a strong synergistic interaction in HMA resistant cell line (Z score = 14.523) and Ven resistant cell line (Z score = 11.065). An in vivo study to evaluate the efficacy of PYR shows that PYR treatment leads to a significant decrease of blasts counts in a cell-line derived xenograft (CDX) leukemic mouse model (Fig 1B) compared to a PBS control. In addition, primary MDS patient samples treated with PYR show increased myeloid differentiation compared to healthy controls. Our results show that MDS/AML cell lines, including HMA and Ven resistant leukemia cell lines, are sensitive to PYR treatment, in a dose dependent manner within the therapeutic index of STAT3 inhibition (5-10 uM). Interestingly, PYR with Ven drug combination shows significant reduction in cell proliferation and likely synergy. Our current findings support PYR synergy with Ven and the combination as a possible therapeutic option for patients with MDS/AML after prior treatment with hypomethylating agents for MDS and AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Selectively Targeting STAT3 Using a Small Molecule Inhibitor is a Potential Therapeutic Strategy for Pancreatic Cancer.

Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging. To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, in vitro kinase assay, RNA sequencing, and selective cell growth inhibition assessment. In vivo studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan-Meier analyses were used to assess survival. WB436B selectively bound to STAT3 over other STAT families protein, and in vitro antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis in vivo and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain. Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.

Synthesis and evaluation of naphthalene derivatives as potent STAT3 inhibitors and agents against triple-negative breast cancer growth and metastasis.

Triple-negative breast cancer (TNBC) represents the worst prognostic subtype of breast cancer and lacks targeted therapeutic drugs. Signal transducer and activator of transcription 3 (STAT3) is overexpressed and constitutively activated in TNBCs and associated with poor patient outcomes. However, no agents targeting STAT3 have been successfully developed and marketed. Selective Estrogen Receptor Modulators (SERMs) have been reported as potential inhibitors of the IL-6/STAT3 signaling pathway. Naphthalene compounds have good pharmacological activity and significant anti-cancer activity. In this study, we synthesized a new series of naphthalene derivatives with the general structure of SERM and evaluated their effects on TNBC and STAT3 signals. A new series of compounds based on the scaffold of SERMs and an amino group were designed and screened based on the structure-activity relationship by MTT assay. The binding activity of SMY002 to STAT3 was predicted and validated by docking and SPR. The STAT3 signaling target and anti-cancer effects of SMY002 were evaluated with three TNBC cell lines and the mice transplanted tumor model. Among the compounds, SMY002 displayed the most potent activity, which could directly interact with STAT3 SH2-domain, and strongly inhibit the phosphorylation, dimerization, nuclear distribution, transcriptional activity, and target genes expression of STAT3. Furthermore, SMY002 markedly suppressed migration, invasion, survival, growth, and metastasis of TNBC cells in vitro and in vivo via down-regulating the expression of Cyclin D1 and MMP9. SMY002 can significantly inhibit the growth and metastasis of TNBC cells by targeting the STAT3 signal.

Exposure to short-chain chlorinated paraffins induces astrocyte activation via JAK2/STAT3 signaling pathway

In the last few decades, short-chain chlorinated paraffins (SCCPs) have become the most heavily produced monomeric organohalogen compounds, and have been reported to induce multiple organ toxicity. However, the effects of SCCPs on the central nervous system are unknown. In the present study, we show that SCCP exposure induced astrocyte proliferation and increased the expression of two critical markers of astrocyte activation, glial fibrillary acidic protein and inducible nitric oxide synthase, in vivo and in vitro. SCCP exposure also increased inflammatory factory gene expression. Moreover, SCCP treatment triggered Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signalling, as shown by increased phosphorylation and STAT3 translocation to the nucleus. Both JAK2 and STAT3 inhibition effectively attenuated SCCP-induced astrocyte activation. Finally, JAK2 inhibition significantly rescued STAT3 phosphorylation and nuclear translocation. Taken together, JAK2/STAT3 pathway activation contributed to SCCP-induced astrocyte activation. These data will help elucidate the molecular mechanism underlying SCCP-induced neurotoxicity.

Genomic distribution of signal transducer and activator of transcription (STAT) family in colorectal cancer.

JAK/STAT pathway has been widely acknowledged in the development of human cancers. However, the role of different phosphorylated STAT proteins translocating into nucleus in transcription activation of target genes is not fully understood. In present research, ChIP-seq was carried on to investigate the genome-wide distribution of the activated STAT1, STAT2, STAT3, STAT5 and STAT6 in colorectal cancer HCT-116 cells. Our observations indicated that the homodimers rather than heterodimers of STAT protein predominantly occupied on genomic DNA. STAT3 accounted for the largest proportion among all STAT proteins HCT-116 cells. Furthermore, the biased binding motif targeted by different STAT homodimers suggested the distinct biological functions. Here, we noticed that NR5A2 was a specific co-activator of STAT3 by DNA motif analysis. Co-IP assay determined that NR5A2 indeed interacted with STAT3 homodimer rather than other homodimers or heterodimers. NR5A2 knockdown resulted in a reduced binding affinity of STAT3 homodimer in the original regions. Taken together, we characterize the genome-wide landscape of activated STAT proteins, and reveal the differences of binding patterns as well as the target genes and associated functions between homodimer and heterodimer of STAT proteins in HCT-116 cells. We also present some new findings and possible mechanisms regarding the role of NR5A2 on STAT3 in CRC. Our findings may provide new insights into the design of STAT inhibitors to treat CRC and other diseases.

STAT3 and Its Targeting Inhibitors in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) usually originates from the precancerous lesions of oral mucosa and accounts for approximately 90% of oral cancers. Current therapeutic approaches do not yet meet the needs of patients, and the 5-year survival rate of patients with OSCC is only 50%. Recent studies have revealed that the signal transducer and activator of transcription 3 (STAT3) plays a key role in the development and progression of OSCC. STAT3 is overexpressed and constitutively activated in OSCC cells, and promotes cancer cell proliferation and anti-apoptosis, migration and invasion, angiogenesis, radiotherapy resistance, and immune escape, as well as stem cell self-renewal and differentiation by regulating the transcription of its downstream target genes. Inhibitors of the STAT3 signaling pathway have shown the promising anticancer effects in vitro and in vivo, and STAT3 is expected to be a molecular target for the treatment of OSCC. In this review, we highlight the oncogenic significance of STAT3 in OSCC with an emphasis on the therapeutic approaches and effective small molecule inhibitors targeting STAT3. Finally, we also propose the potential research directions in the expectation of developing more specific STAT3 inhibitors for OSCC treatment.

Empagliflozin rescues primary murine cardiac fibroblasts, but not cardiomyocytes, from hypoxia-reoxygenation injury through STAT3-mediated cascades

Abstract Introduction Empagliflozin (EMPA) is a selective sodium glucose co-transporter 2 inhibitor (SGLT2i) and an established drug against type 2 diabetes mellitus. Cardiovascular clinical outcome trials on EMPA have shown a reduced mortality rate related to major cardiovascular events, but the precise mechanism of action remains elusive. We have previously proved that chronic EMPA administration reduces infarct size in an in vivo murine model of ischemia / reperfusion injury, irrespective of the diabetic status. The cardioprotective effect of EMPA was attributed to the increase of Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation. Purpose In this study we aimed to decipher the cell specific effects of EMPA on primary adult ventricular murine cardiomyocytes (pAVMCs) and primary murine cardiac fibroblasts (pMCFs), subjected to hypoxia/reoxygenation (HYP/REO) and to 1) investigate the effect of EMPA on cell death, and 2) reveal the mechanism of cardioprotection. Methods PAVMCs and pMCFs were isolated from adult C57BL/6 murine hearts (n=4–6) and were incubated with EMPA (500nM–100nM) for 24 hours. Cells were then subjected to 3 hours of HYP (99% N2), followed by 1 hour of REO. STATTIC, a selective STAT3 inhibitor was used (500nM–1μM) to determine the contribution of STAT3 on the observed effect. MTT assay was performed at the end of REO to determine cell viability. To unravel the mechanism of cardioprotection, the experimental protocol was repeated, and the cells were either stained with dihydroethidium (DHE) dye to determine relative oxidative stress changes or were harvested for isolation of total protein content. We focused on STAT3 and Akt kinase as key mediators of the cardioprotective survivor activating factor enhancement (SAFE) and the reperfusion injury salvage kinase (RISK) pathways. We also investigated the expression of Cardiotrophin-1 (CT-1), as a potential upstream mediator of STAT3 phosphorylation. Results The evaluation of cell death revealed that pMCFs viability was significantly increased upon EMPA treatment. Added to this, STATTIC co-treatment with EMPA blunted the protective effect on pMCFs, which indicates that EMPA's protection is STAT3-mediated. Oxidative stress remained unaltered by the treatments. Investigation of the molecular pathways that are responsible for the increased viability of pMCFs revealed that EMPA treatment induces an increased phosphorylation of STAT3 and an increase in CT-1 expression, which are both reversed by STATTIC. Akt phosphorylation and expression remained unchanged by EMPA treatment. Notably, the effect of EMPA on pAVMCs' viability was negligible. Conclusion(s) EMPA does not protect pAVMCs, from HYP/REO injury. EMPA rescues the pMCFs in a STAT-3 dependent manner which indicates that EMPA's cardioprotective effect is cell specific and implicates the SAFE pathway. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim International GmbHGrant title: “Investigation of the distinct effects of Empagliflozin on different cell populations of the healthy myocardium”

NINJ1 triggers extravillous trophoblast cell dysfunction through blocking the STAT3 signaling pathway.

Trophoblasts are the most important parts of the placenta in early pregnancy. Trophoblast cell dysfunction can induce embryo implantation insufficiency, thereby resulting in multiple diseases, including recurrent spontaneous abortion (RSA). A previous study indicates higher nerve injury-induced protein 1 (NINJ1) RNA levels in the villi tissues of RSA patients. This study aimed to investigate the effect of NINJ1 on trophoblast behaviors and pregnancy loss. Fresh villi tissues were obtained from with RSA patients and patients with artificial selective abortion for personal reasons, and NINJ1 expression in these tissues was detected. Extravillous trophoblast cell line HTR-8/SVneo was transfected with small-interfering RNA targeting NINJ1 or NINJ1 overexpression vector to perform loss-/gain-of-function experiments. Spontaneous abortion (SA) was induced by mating CBA/J females with DBA/2 males and the pregnant females were intraperitoneally injected with adenovirus vector carrying NINJ1 short hairpin RNA. NINJ1 mRNA and protein levels were higher in the villi tissues of RSA patients than those of artificial selective abortion patients. NINJ1 knockdown promoted trophoblast cell proliferation, migration and invasion but inhibited cell apoptosis. Moreover, conditioned medium from NINJ1-depleted trophoblasts promoted the angiogenesis of human umbilical vein endothelial cells. NINJ1 knockdown also promoted activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway in trophoblasts, and STAT3 inhibitor reversed NINJ1 knockdown-induced effects on trophoblast behaviors. Furthermore, pregnancy loss was attenuated by NINJ1 inhibition. NINJ1 contributes to the development of SA and triggers trophoblast cell dysfunction through inhibiting the STAT3 pathway.

Diterpenoid DGA induces apoptosis via endoplasmic reticulum stress caused by changes in glycosphingolipid composition and inhibition of STAT3 in glioma cells

Glioma is one of the most common malignant primary brain tumors, with poor prognosis and high recurrence. There are currently few drugs approved for brain tumors; thus, it is necessary to develop new effective drugs. Natural diterpenoids have important biological activities, including antiinflammatory, antioxidative, and antitumor effects. In this study, 7α,14β-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA), a diterpenoid compound modified from glaucocalyxin A, inhibited the proliferation of many tumor cells, especially glioma. Flow cytometry analysis showed that DGA induced apoptosis in glioma cells. DGA also inhibited xenograft tumors in nude mice. It affected the expression of ceramide synthases (CerS) in glioma cells; CerS1 decreased, and CerS2 and CerS5 increased, resulting in a change in the composition of glycosphingolipids containing varying acyl chain lengths. In glioma cells treated with DGA, the gene transcription of activating transcription factor 4 (ATF4), X-box binding protein-1 (XBP1), and C/EBP-homologous protein (CHOP) in unfolded protein response pathways was upregulated. Meanwhile, the ratio of proapoptotic protein Bcl-2–associated X protein (BAX) to antiapoptotic protein B-cell lymphoma 2 (Bcl-2) also increased. This suggested that an imbalance of glycosphingolipids caused by DGA induced severe endoplasmic reticulum stress and triggered cell apoptosis. Moreover, Western blotting showed DGA inhibited the signal transducers and activators of transcription 3 (STAT3) signaling pathway by reducing the phosphorylation of STAT3 and its upstream kinases, which also promoted the apoptosis of glioma cells. Together, these results explored the anticancer activities of DGA and highlighted it as a potential candidate for treating glioma.

Analysis of CF patient survival confirms STAT3 as a CF-modifying gene with changing impact over time.

The signal transducer and activator of transcription 3 (STAT3) has been identified as one of the cystic fibrosis (CF) modifying genes. In this study, we aimed to assess the association between STAT3 genotype and CF patient survival over several decades and to investigate the effect of STAT3 inhibition on epithelial CFTR expression. We analyzed the informative genetic marker STAT3Sat for its association with survival in 174 p.Phe508del-CFTR homozygous CF patients treated at the CF center in Hannover spanning birth cohorts from >3 decades (1959-1994). Furthermore, we treated two epithelial cell lines with STAT3 inhibitors and monitored changes of CFTR protein expression by western blot. Only for p.Phe508del-CFTR homozygous patients born prior to 1975, survival was significantly influenced by STAT3sat genotype (P = 0.023). The expression levels of STAT3 and CFTR positively correlated in epithelial cell lines (P = 0.01). Our results in different birth cohorts identified a time-dependent impact of STAT3 genotype on CF patients' survival and found that improved symptomatic treatment of later-born CF patients obviates STAT3's modifying influence. Consistent with our previous results, STAT3-specific inhibition resulted in increased CFTR expression in the epithelial cell line 16HBE14o-. Thus, care should be taken when CF-modifying genes are studied in cross-sectional cohorts as the impact of modifying genes might not be invariant in the light of changing therapeutic regimens.

Mechanism of the promotion of GEFS+ by the STAT3-mediated expression of interleukin-6.

BackgroundGenetic epilepsy with febrile seizures plus (GEFS+) is generally considered an ion channelopathy. To date, there have been few studies on inflammation associated with various types of epilepsy, and it remains unclear whether the inflammatory mechanism plays a key role in epilepsy.MethodsIn order to explore the role of the regulatory mechanism of immune factor expression in the pathogenesis of GEFS+, the present study detected the expression level of relevant immune factors such as interleukin-6 (IL-6) in peripheral blood of GEFS+ mice.ResultsThe cluster of differentiation 4+/cluster of differentiation 8+ (CD4+/CD8+) ratio in the GEFS+ mice was decreased, while the signal transducer and activator of transcription 3 (STAT3) was also activated and the IL-6 was upregulated. Inhibit of STAT3 can lead to the GEFS+ asymptomatically due to the downregulated IL-6, IL-1β, and complement factor H (CFH) levels. Suppression of STAT3 can also inhibited the epileptic seizures, the CD8+ T cells were declined after the IL-6 was neutralized.ConclusionsThe purpose of this study was to analyze and compare the effect of STAT3 expression and activation differences on GEFS+ attack, and to clarify the relationship between various cytokines and GEFS+ outbreak. Inhibiting the expression of pro-inflammatory factors can further prevent GEFS+ attack, which supports that IL-6 is one of the important factors that aggravate the clinical symptoms of GEFS+. We expected to provide a theoretical basis for immunosuppressive therapy of GEFS+ and a new way for its clinical treatment.

Design, synthesis, and biological characterization of a potent STAT3 degrader for the treatment of gastric cancer.

Gastric cancer is a common malignant tumor that threatens human health, and its occurrence and development mechanism is a complex process involving multiple genes and multiple signals. Signal transducer and activator of transcription 3 (STAT3) has been elucidated as a promising target for developing anticancer drugs in gastric cancer. However, there is no FDA-approved STAT3 inhibitor yet. Herein, we report the design and synthesis of a class of STAT3 degraders based on proteolysis-targeting chimeras (PROTACs). We first synthesized an analog of the STAT3 inhibitor S3I-201 as a ligand, using the cereblon (CRBN)/cullin 4A E3 ligase ligand pomalidomide to synthesize a series of PROTACs. Among them, the SDL-1 achieves the degradation of STAT3 protein in vitro, and exhibits good anti-gastric cancer cell proliferation activity, inhibits invasion and metastasis of MKN1 cell, and induces MKN1 cell apoptosis and arrests cell cycle at the same time. Our study shows that SDL-1 is a potent STAT3 degrader and may serve as a potential anti-gastric cancer drug, providing ideas for further development of drugs for clinical use.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10−5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10−34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US. Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-β1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays. Herein, we found that verapamil could inhibit TGF-β1/Ca2 + ⁄calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-β1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-β1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-β1-induced TIMP1 expression in fibroblasts. Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

The STAT3 inhibitor GPB730 enhances the sensitivity to enzalutamide in prostate cancer cells

Enzalutamide is a second-generation anti-androgen which has shown increased survival in patients with metastatic prostate cancer. However, some patients do not respond to this therapy or will develop resistance to treatment over time. Signal Transducer and Activator of Transcription 3 (STAT3) is known to be involved in castration-resistant prostate cancer and to interact with androgen receptor (AR)-signaling. This study aims to investigate the combination enzalutamide and the small molecule STAT3 inhibitor GPB730 for enhanced therapeutic effect in advanced prostate cancer in vitro. The prostate cancer cell lines LNCaP (androgen dependent) and C4–2 (androgen insensitive) were used. The effect of enzalutamide and GPB730, alone and in combination, was investigated on viability and IC50 values calculated. Enzalutamide and GPB730 treated LNCaP and C4–2 cells were subjected to western blot and QPCR analyses in order to investigate the expression of AR, STAT3 and down-stream targets. C4–2 were less sensitive to growth inhibition by enzalutamide than LNCaP cells. GPB730 enhanced the growth inhibitory effect of enzalutamide in LNCaP and C4–2 cells. The addition of GPB730 to enzalutamide decreased the IC50 values for enzalutamide by 3.3-fold for LNCaP and by 12-fold for C4–2. In C4–2 cells, GPB730 alone decreased PSA expression and enhanced the enzalutamide induced decrease in NKX3.1 expression. GPB730 and enzalutamide in combination enhanced inhibition of c-myc and survivin expression. This study suggests that enzalutamide may be combined with the STAT3 inhibitor GPB730 in order to enhance the efficacy of enzalutamide, offering a new therapeutic approach in advanced prostate cancer.