STAT3 Activator Research Articles

The Long-Lasting Protective Effect of HGF in Cardiomyoblasts Exposed to Doxorubicin Requires a Positive Feed-Forward Loop Mediated by Erk1,2-Timp1-Stat3

Previous studies showed that the hepatocyte growth factor (HGF)–Met receptor axis plays long-lasting cardioprotection against doxorubicin anti-cancer therapy. Here, we explored the mechanism(s) underlying the HGF protective effect. DNA damage was monitored by histone H2AX phosphorylation and apoptosis by proteolytic cleavage of caspase 3. In doxorubicin-treated H9c2 cardiomyoblasts, the long-lasting cardioprotection is mediated by activation of the Ras/Raf/Mek/Erk (extracellular signal-regulated kinase 1,2) signaling pathway and requires Stat3 (signal transducer and activator of transcription 3) activation. The HGF protection was abrogated by the Erk1,2 inhibitor, PD98059. This translated into reduced Y705 phosphorylation and impaired nuclear translocation of Stat3, showing crosstalk between Erk1,2 and Stat3 signaling. An array of 29 cytokines, known to activate Stat3, was interrogated to identify the molecule(s) linking the two pathways. The analysis showed a selective increase in expression of the tissue inhibitor of metalloproteinases-1 (Timp1). Consistently, inhibition in cardiomyoblasts of Timp1 translation by siRNAs blunted both Stat3 activation and the cardioprotective effect of HGF. Thus, Timp1 is responsible for the generation of a feed-forward loop of Stat3 activation and helps cardiomyocytes to survive during the genotoxic stress induced by anthracyclines.

STAT3‑PTTG11 abrogation inhibits proliferation and induces apoptosis in malignant glioma cells

Pituitary tumor transforming gene 1 (PTTG11) is abundantly expressed in glioma. Our previous study demonstrated that the downregulation of PTTG11 gene expression significantly inhibited the proliferation, migration and invasion ability, and increased the apoptosis of SHG44 glioma cells. However, the molecular mechanisms that regulate PTTG11 and its actions remain elusive. In the present study, CCK-8 and flow cytometry assays were used to assess the proliferation/viability and apoptosis, respectively, of the human glioma U251 cell line. STAT3-PTTG1 signals were further evaluated by western blotting. The findings of the present study revealed that STAT3 induced PTTG11 expression, which subsequently induced downstream c-Myc and Bcl-2 expression while inhibiting Bax expression, thereby promoting cell viability and inhibiting apoptosis. PTTG11 suppression via siRNA inhibited the viability and increased the apoptosis of glioma cells induced by the STAT3 activator S3I-201. c-Myc and Bcl-2 expression was suppressed by PTTG11 inhibition. The findings of the present study suggest that the STAT3-PTTG11 signaling pathway may play an important role in glioma progression by regulating cell proliferation and apoptosis.

Alcohol alters IL-6 Signal Transduction in the CNS of Transgenic Mice with Increased Astrocyte Expression of IL-6.

Neuroimmune factors, including the cytokine interleukin-6 (IL-6), are important chemical regulators of central nervous system (CNS) function under both physiological and pathological conditions. Elevated expression of IL-6 occurs in the CNS in a variety of disorders associated with altered CNS function, including excessive alcohol use. Alcohol-induced production of IL-6 has been reported for several CNS regions including the cerebellum. Cerebellar actions of alcohol occur through a variety of mechanisms, but alcohol-induced changes in signal transduction, transcription, and translation are known to play important roles. IL-6 is an activator of signal transduction that regulates gene expression. Thus, alcohol-induced IL-6 production could contribute to cerebellar effects of alcohol by altering gene expression, especially under conditions of chronic alcohol abuse, where IL-6 levels could be habitually elevated. To gain an understanding of the effects of alcohol on IL-6 signal transduction, we studied activation/expression of IL-6 signal transduction partners STAT3 (Signal Transducer and Activator of Transcription), CCAAT-enhancer binding protein (C/EBP) beta, and p42/p44 mitogen-activated protein kinase (MAPK) at the protein level. Cerebella of transgenic mice that express elevated levels of astrocyte produced IL-6 in the CNS were studied. Results show that the both IL-6 and chronic intermittent alcohol exposure/withdrawal affect IL-6 signal transduction partners and that the actions of IL-6 and alcohol interact to alter activation/expression of IL-6 signal transduction partners. The alcohol/IL-6 interactions may contribute to cerebellar actions of alcohol, whereas the effects of IL-6 alone may have relevance to cerebellar changes occurring in CNS disorders associated with elevated levels of IL-6.

Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

Abstract Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans. IL-17 and IL-22 both mediate antifungal immunity yet activate distinct downstream signaling pathways. While much is known about IL-17-dependent immunity in OPC, the activities of IL-22 are less well delineated. We show that induction of Il22 is independent of Dectin-1, CARD9 and aryl hydrocarbon receptor (AhR) and is driven by IL-23 and the C. albicans pore forming peptide candidalysin. Despite similar induction requirements and cellular sources, IL-22 and IL-17 function non-redundantly during OPC and exert opposing roles in neutrophil recruitment. The IL-22 and IL-17 receptors are required in anatomically distinct locations; loss of IL-22RA1 in the oral basal epithelial layer (BEL) but not the suprabasal epithelial layer (SEL) causes susceptibility to OPC, whereas IL-17RA is needed in the SEL. Our data reveal that IL-22 is a major activator of STAT3 in the BEL during OPC. Moreover, loss of STAT3 in the BEL but not the SEL renders mice susceptible to OPC. Transcriptional profiling of RNASeq data linked IL-22/STAT3 to oral epithelial cell proliferation and survival, but also, unexpectedly, to driving an IL-17 gene signature. We show that IL-22 acts on the BEL to replenish the IL-17RA-expressing SEL, thereby restoring the ability of the oral epithelium to respond to IL-17. Consequently, IL-22 signaling in BEL ‘licenses’ IL-17R signaling in the oral epithelium, revealing spatially distinct yet cooperative activities of IL-22 and IL-17 in oral candidiasis. This work also suggests that oral thrush in Jobs’ syndrome patients may be caused by STAT3 impairments in the oral epithelium, not just Th17 cells.

CGAS–STING Cytosolic DNA Sensing Pathway Is Suppressed by JAK2-STAT3 in Tumor Cells

Deficiencies in DNA repair and DNA degrading nucleases can lead to accumulation of cytosolic DNA and the activation of STING pathways in tumor cells. The DNA sensors that recognize cytosolic DNA in tumor cells are not known. Here we show that double-stranded DNA and RNA:DNA hybrids bind cGAS and activate the cGAS-STING pathway in TRAMP-C2 prostate and other cancer cells. STING function in TRAMP-C2 cells was critical for rejection and immune cell infiltration of TRAMP-C2 tumors. In contrast, STING agonists failed to active STING and induce type I interferon (IFN) expression in many tested tumor cells. Inhibition of JAK2 and STAT3 restored type I IFN expression in these tumor cells. Intratumoral injections of IL-6, a known activator of JAK2 and STAT3, significantly reduced the anti-tumor effects of cGAMP. In summary, we show that IL-6 suppresses recognition of cytosolic DNA by the cGAS-STING pathway in tumor cells, which contributes to anti-tumor responses. Funding Statement: This work was supported by the NRF grant HUJ-CREATE-Cellular and Molecular Mechanisms of Inflammation and a ministry of education grant. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Mice were housed according to the IACUC guidelines of the National University of Singapore (R14-0204).

Sea Hare Hydrolysate-Induced Reduction of Human Non-Small Cell Lung Cancer Cell Growth through Regulation of Macrophage Polarization and Non-Apoptotic Regulated Cell Death Pathways.

Sea hare-derived compounds induce macrophage activation and reduce asthmatic parameters in mouse models of allergic asthma. These findings led us to study the role of sea hare hydrolysates (SHH) in cancer pathophysiology. SHH treatment-induced M1 macrophage activation in RAW264.7 cells, peritoneal macrophages, and THP-1 cells, as did lipopolysaccharide (LPS) (+ INF-γ), whereas SHH reduced interleukin (IL)-4 (+IL-13)-induced M2 macrophage polarization. In addition, SHH treatment inhibited the actions of M1 and M2 macrophages, which have anticancer and pro-cancer effects, respectively, in non-small cell lung cancer cells (A549 and HCC-366) and tumor-associated macrophages (TAMs). Furthermore, SHH induced G2/M phase arrest and cell death in A549 cells. SHH also downregulated STAT3 activation in macrophages and A549 cells, and the down-regulation was recovered by colivelin, a STAT3 activator. SHH-induced reduction of M2 polarization and tumor growth was blocked by colivelin treatment. SHH-induced cell death did not occur in the manner of apoptotic signaling pathways, while the death pattern was mediated through pyroptosis/necroptosis, which causes membrane rupture, formation of vacuoles and bleb, activation of caspase-1, and secretion of IL-1β in SHH-treated A549 cells. However, a combination of SHH and colivelin blocked caspase-1 activation. Z-YVAD-FMK and necrostatin-1, pyrotosis and necroptosis inhibitors, attenuated SHH’s effect on the cell viability of A549 cells. Taken together, SHH showed anticancer effects through a cytotoxic effect on A549 cells and a regulatory effect on macrophages in A549 cells. In addition, the SHH-induced anticancer effects were mediated by non-apoptotic regulated cell death pathways under STAT3 inhibition. These results suggest that SHH may be offered as a potential remedy for cancer immunotherapy.

MFG-E8 alleviates oxygen-glucose deprivation-induced neuronal cell apoptosis by STAT3 regulating the selective polarization of microglia

Background: Ischemic stroke is a complex pathological process, involving inflammatory reaction, energy metabolism disorder, free radical injury, cell apoptosis and other aspects. Accumulating evidences have revealed that MFG-E8 had a protective effect on multiple organ injuries. However, the comprehensive function and mechanism of MFG-E8 in ischemic brain remain largely unclear.Methods: BV-2 cells were treated with recombinant murine MFG-E8 (rmMFG-E8) or/and Colivelin TFA after exposing for 4 h with oxygen glucose deprivation (OGD). Cell viability and apoptosis were assessed by MTT assay and Flow cytometry. RT-qPCR and Western blot assays were applied to examine the expression levels of MFG-E8, apoptosis-related proteins and M1/M2 polarization markers.Results: Our results demonstrated that OGD significantly inhibited microglial viability and facilitated apoptosis. In addition, we found that OGD downregulated MFG-E8 expression, and MFG-E8 inhibited OGD-induced microglial apoptosis and promoted microglial M2 polarization. In terms of mechanism, we proved that MFG-E8 regulated OGD-induced microglial M1/M2 polarization by inhibiting p-STAT3 and SOCS3 expressions, which was reversed by STAT3 activator (Colivelin TFA). Finally, we verified MFG-E8 alleviated OGD-induced neuronal cell apoptosis by M2 polarization of BV-2 cells.Conclusions: We demonstrated that MFG-E8 reduced neuronal cell apoptosis by enhancing activation of microglia via STAT3 signaling. Therefore, we suggested that MFG-E8 might provide a novel mechanism for ischemic stroke.

Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40.

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.

Abstract P5-07-11: Mammary gland specific Stat3 deletion during involution results in distinct histological changes and higher collagen deposition

Abstract Rationale: African American women (AAW) have a higher incidence of triple negative breast cancer (TNBC), and higher mortality from breast cancer when compared to Caucasian women (CW). Epidemiological studies have shown that lack, or short duration, of breast-feeding, a more common practice among AAW than CW, may be one of the factors that increase the risk of TNBC. The pregnancy-lactation-involution cycle is a dynamic process where upon pregnancy, breast undergoes extensive proliferation and differentiation for milk production followed by apoptosis as it involutes to near pre-pregnant state. Prolonged breastfeeding results in gradual involution (GI) whereas no or short duration of breastfeeding leads to forced and abrupt involution (AI) of breast. We modeled GI and AI in wild-type FVB/N mice and reported that mammary glands of abruptly involuted mice had expansion of luminal progenitor (LP) cells over time and hyperplastic precancerous changes within 120 days postpartum1. In addition, there was increased Stat3 (Signal Transducer and Activator of Transcription 3) activation (pStat3Y705) in AI glands. Persistent Stat3 activation is linked to tumor cell proliferation, survival, invasion, and tumor-promoting inflammation2. In mice, Stat3 is required for the initiation and acute phase response during mammary gland involution and is crucial for the proliferation of LP cells3-5. We hypothesized that Stat3 activation plays a key role in the development of hyperplasia and fibrosis observed following AI.Methods: We bred Stat3Fl/Flmice with MMTV-Cre mice to induce mammary epithelial specific deletion of Stat3. Experimental (MMTV-Cre+;Stat3Fl/Fl) and control females (Stat3Fl/Fl) were bred once at 8 weeks of age and litter sizes were normalized to 6pups/dam within 24 hours of partum. With day of partum as day0, all 6 pups were removed on day7 and AI was initiated. Mammary glands were harvested on postpartum day28, day56, and day120. Florescence activated cell sorting (FACS) was performed to determine distribution of mammary epithelial subpopulations. Histological effects of Stat3 deletion on mammary gland involution was assessed via staining using haematoxylin and eosin (H&E), Masson’s Trichrome and immunostaining using anti-Ki67 and anti-Stat3 antibodies. Results: Stat3 deletion failed to abrogate the hyperplasia and collagen deposition we initially observed in our AI mice with intact Stat3 as determined by H&E and Trichrome stains. In addition, collagen deposition was further increased following Stat3 deletion, when compared to control cohort. FACS analysis demonstrated that Stat3 deletion resulted in reduction of LP cell population only at early timepoint (day28) but this population rebounded in glands by later timepoints. Stat3 deletion temporarily increased cell proliferation by Ki-67 staining at day28 and 56, with no difference observed between controls at day120. Conclusion: This is the first study showing results of mammary epithelial specific Stat3 deletion on long-term effect of AI. Contrary to our hypothesis, Stat3 deletion did not reduce the hyperplastic changes we observed following abrupt involution of mammary glands. Our findings indicate that Stat3 signaling may limit collagen formation within the mammary gland during abrupt involution. Stat3 is important for the maintenance of LP cells at early timepoints. Further studies are underway to determine the critical downstream events following Stat3 activation that limits long-term effects of abrupt involution. 1. Basree et. al. 2019, in press, Breast Cancer Research 2. Yu et. al. 2014 (PMID: 25342631) 3. Chapman et. al. 1999 (PMID: 10521404) 4. Hughes et. al. 2012 (PMID: 22081431)5. Staniszewska et. al. 2012 (PMID: 23285109) Citation Format: Allen Zhang, Neelam Shinde, Christopher S Koivisto, Mustafa M Basree, Resham S Mawalkar, Hee K Kim, Gustavo Leone, Sarmila Majumder, Bhuvaneswari Ramaswamy. Mammary gland specific Stat3 deletion during involution results in distinct histological changes and higher collagen deposition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-07-11.

MiR-142-5p/DAX1-dependent regulation of P450c17 contributes to triclosan-mediated testosterone suppression

Triclosan (TCS) is a potent antibacterial and antifungal compound that is extensively used in various daily products. TCS is also considered as an underlying endocrine disruptor and has anti-androgenic effects. In our previous work, we found that TCS suppressed testicular steroidogenesis via the miR-6321/JNK/Nur77 cascade, but roles of the abnormal expression of miR-142-5p and P450c17 in this molecular event were still unknown. Therefore, to verify the hypothesis that miR-142-5p and P450c17 might significantly function in other manner in testosterone decline after TCS exposure, Sprague-Dawley rats and the rat Leydig cell line were used in this study. Results showed that after TCS exposure, testicular histomorphology was abnormally changed and testosterone level was declined. Overexpressed miR-142-5p by TCS directly targeted the JAK1/STAT1 pathway. Bidirectional Co-IP assays and the use of STAT1 activator demonstrated that STAT1 could interact with and regulate Sp1. The activity, mRNA level, and protein expression of DNMT1 and DNMT3β were all decreased after TCS treatment. Sp1 silencing, ChIP, and qPCR assays showed that Sp1 regulated DNMT1 expressions by directly binding to the promoter region of DNMT1. Though the DNA methylation status of the DAX1 promoter was not affected, TCS induced the transcription and translation of DAX1 by DNMT1, in turn leading to the inhibition of steroidogenic P450c17. Taken together, TCS-induced miR-142-5p inhibits P450c17 by the JAK1/STAT1 pathway and downstream Sp1/DNMT1/DAX1 cascade, finally facilitating the decrease in testosterone levels.

A Potential Nutraceutical Candidate Lactucin Inhibits Adipogenesis through Downregulation of JAK2/STAT3 Signaling Pathway-Mediated Mitotic Clonal Expansion.

The prevalence of obesity has increased dramatically worldwide in the past ~50 years. Searching for safe and effective anti-obesity strategies are urgently needed. Lactucin, a plant-derived natural small molecule, is known for anti-malaria and anti-hyperalgesia. The study is to investigate whether lactucin plays a key role in adipogenesis. To this end, in vivo male C57BL/6 mice fed a high-fat diet (HFD) were treated with 20 mg/kg/day of lactucin or vehicle by gavage for seven weeks. Compared with vehicle-treated controls, Lactucin-treated mice showed lower body mass and mass of adipose tissue. Consistently, in vitro 3T3-L1 cells were treated with 20 μM of lactucin. Compared to controls, lactucin-treated cells showed significantly less lipid accumulation during adipocyte differentiation and lower levels of lipid synthesis markers. Mechanistically, we showed the anti-adipogenic property of lactucin was largely limited to the early stage of adipogenesis. Lactucin-treated cells fail to undergo mitotic clonal expansion (MCE). Further studies demonstrate that lactucin-induced MCE arrests might result from reduced phosphorylation of JAK2 and STAT3. We then asked whether activation of JAK2/STAT3 would restore the inhibitory effect of lactucin on adipogenesis with pharmacological STAT3 activator colivelin. Our results revealed similar levels of lipid accumulation between lactucin-treated cells and controls in the presence of colivelin, indicating that inactivation of STAT3 is the limiting factor for the anti-adipogenesis of lactucin in these cells. Together, our results provide the indication that lactucin exerts an anti-adipogenesis effect, which may open new therapeutic options for obesity.

Long noncoding RNA FOXD2-AS1 enhances chemotherapeutic resistance of laryngeal squamous cell carcinoma via STAT3 activation

Laryngeal squamous cell carcinoma (LSCC) is a common head and neck cancer. Despite recently improved management of LSCC, chemotherapy resistance of patients remains a challenge. In this study, we identified that long noncoding RNA FOXD2-AS1 regulates LSCC therapeutic resistance by augmenting LSCC stemness. LSCC chemotherapy-resistant patients showed increased FOXD2-AS1 expression compared with that in chemotherapy-sensitive patients, which predicted poor prognosis. Gain- or loss-of-function experiments showed that upregulated FOXD2-AS1 maintained cancer stemness, reducing the response to chemotherapy, while FOXD2-AS1 downregulation had the opposite effects. FOXD2-AS1 acted as a scaffold for STAT3 and PRMT5, promoting STAT3 transcriptional activity, which is essential to maintain cancer stemness and promote chemotherapeutic resistance. Interfering with FOXD2-AS1 using short hairpin RNA rescued LSCC’s chemotherapeutic sensitivity. Thus, FOXD2-AS1 promotes LSCC chemotherapeutic resistance and is an upstream activator of STAT3, making FOXD2-AS1 a potential therapeutic target to improve the chemotherapy effect in LSCC patients.

Cardiovascular Effects of Pharmacological Targeting of Sphingosine Kinase 1.

High blood pressure is a risk factor for cardiovascular diseases. Ang II (angiotensin II), a key pro-hypertensive hormone, mediates target organ consequences such as endothelial dysfunction and cardiac hypertrophy. S1P (sphingosine-1-phosphate), produced by Sphk1 (sphingosine kinase 1), plays a pivotal role in the pathogenesis of hypertension and downstream organ damage, as it controls vascular tone and regulates cardiac remodeling. Accordingly, we aimed to examine if pharmacological inhibition of Sphk1 using selective inhibitor PF543 can represent a useful vasoprotective and cardioprotective anti-hypertensive strategy in vivo. PF543 was administered intraperitoneally throughout a 14-day Ang II-infusion in C57BL6/J male mice. Pharmacological inhibition of Sphk1 improved endothelial function of arteries of hypertensive mice that could be mediated via decrease in eNOS (endothelial nitric oxide synthase) phosphorylation at T495. This effect was independent of blood pressure. Importantly, PF543 also reduced cardiac hypertrophy (heart to body weight ratio, 5.6±0.2 versus 6.4±0.1 versus 5.9±0.2 mg/g; P<0.05 for Sham, Ang II+placebo, and Ang II+PF543-treated mice, respectively). Mass spectrometry revealed that PF543 elevated cardiac sphingosine, that is, Sphk1 substrate, content in vivo. Mechanistically, RNA-Seq indicated a decreased expression of cardiac genes involved in actin/integrin organization, S1pr1 signaling, and tissue remodeling. Indeed, downregulation of Rock1 (Rho-associated coiled-coil containing protein kinase 1), Stat3 (signal transducer and activator of transcription 3), PKC (protein kinase C), and ERK1/2 (extracellular signal-regulated kinases 1/2) level/phosphorylation by PF543 was observed. In summary, pharmacological inhibition of Sphk1 partially protects against Ang II-induced cardiac hypertrophy and endothelial dysfunction. Therefore, it may represent a promising target for harnessing residual cardiovascular risk in hypertension.

STAT3 activates the anti-apoptotic form of caspase 9 in oncovirus-infected B lymphocytes

The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 – and – expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.

A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation.

Mumps virus (MuV) is a neurotropic non-segmented, negative-stranded, enveloped RNA virus in the Paramyxovirus family. The 15.4 kb genome encodes seven genes, including the V/P, which encodes, among other proteins, the V protein. The MuV V protein has been shown to target the cellular signal transducer and activator of transcription proteins STAT1 and STAT3 for proteasome-mediated degradation. While MuV V protein targeting of STAT1 is generally accepted as a means of limiting innate antiviral responses, the consequence of V protein targeting of STAT3 is less clear. Further, since the MuV V protein targets both STAT1 and STAT3, specifically investigating viral antagonism of STAT3 targeting is challenging. However, a previous study reported that a single amino acid substitution in the MuV V protein (E95D) inhibits targeting of STAT3, but not STAT1. This provided us with a unique opportunity to examine the specific role of STAT 3 in MuV virulence in an in vivo model. Here, using a clone of a wild type MuV strain expressing the E95D mutant V protein, we present data linking inhibition of STAT3 targeting with the accelerated clearance of the virus and reduced neurovirulence in vivo, suggesting its role in promoting antiviral responses. These data suggest a rational approach to virus attenuation that could be exploited for future vaccine development.

Targeted blockade of interleukin 9 inhibits tumor growth in murine model of pancreatic cancer.

Interleukin 9 (IL-9) has been implicated in the pathogenesis of several tumor types, but the role of anti-IL-9 in pancreatic cancer remains unclear. We aimed to explore the mechanism and effects of blockading IL-9 in a pancreatic cancer mouse model. Panc02 cells were injected subcutaneously into mice to establish a mouse model. The mice were randomly categorized into 3 groups - the control group, the immunoglobulin G (IgG) group and the anti-IL-9 group - corresponding to intravenous tail injection of phosphate-buffered saline (PBS), IgG isotype antibody and anti-IL-9 antibody, respectively. Then, the expression of IL-9, interleukin-9 receptor (IL-9r), Janus kinase 1 (Jak1), Jak3, and signal transducer and activator of transcription 3 (Stat3) mRNA was tested with quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Interleukin 9 in the tumor tissue was detected using enzyme-linked immunosorbent assay (ELISA). Western blotting and immunocytochemistry were performed to detect STAT3 and phosphorylation signal transducers and activators of transcription-3 (pSTAT3). Matrix metalloproteinase 2 (MMP2), MMP9 and vascular endothelial growth factor (VEGF) levels were assessed using immunocytochemistry. Tumor weight in the anti-IL-9 group was significantly lower than in the other groups (p < 0.05). There was a remarkable survival benefit in the anti-IL-9 group compared to the other groups (p < 0.05). The concentration of IL-9 in tumor tissue was significantly downregulated in the anti-IL-9-treated mice (p < 0.05). The expression of Jak1 and Jak3 mRNA and pSTAT3, MMP2 and MMP9 proteins in the anti-IL-9 group was lower than that of the PBS or IgG groups (p < 0.05), but the STAT3 and VEGF protein levels showed no significant difference (p < 0.05). Anti-IL-9 antibody could effectively restrain the growth of pancreatic cancer in mice, and this effect may partly occur by blocking the STAT3 pathway.

Prolactin Promotes Fibrosis and Pancreatic Cancer Progression.

Pancreatic ductal adenocarcinoma (PDAC) is associated with significant fibrosis. Recent findings have highlighted the profibrotic activity of tissue-resident macrophages in the pancreatic cancer microenvironment. Here, we show that neoplastic pancreatic epithelium, as well as a subset of tissue-resident macrophages, expresses the prolactin-receptor (PRLR). High mobility group box 1-induced prolactin expression in the pancreas maintained FAK1 and STAT3 phosphorylation within the epithelium and stroma. Gain-of-function and loss-of-function experiments demonstrated the essential role of prolactin in promoting collagen deposition and fibrosis. Finally, the signaling cascade downstream of prolactin/PRLR activated STAT3 rather than STAT5 in PDAC. These findings suggest that targeting prolactin together with IL6, a known major activator of STAT3, could represent a novel therapeutic strategy for treating pancreatic cancer. SIGNIFICANCE: Prolactin is a key factor in the cross-talk between the stroma and neoplastic epithelium, functioning to promote fibrosis and PDAC progression.

Limonin enhances the radiosensitivity of nasopharyngeal carcinoma cells via attenuating Stat3-induced cell stemness

The inhibitory effects of limonin have been disclosed in various tumors, however, its roles in nasopharyngeal carcinoma (NPC) progression are never been revealed. In the current work, we collected NPC cells with a higher stemness compared with bulk cells through isolating the side population (SP) cells. It was found that limonin exhibited a stronger inhibitory effect on SP cells than that in bulk cells, which was evident by a lower IC50 value. Additionally, limonin attenuated the stemness and migration ability of SP cells with the higher stemness, characterized as decreasing the spheroid formation ability, expression of stemness markers and migration ability. Moreover, the proportion of SP cells in G0 phase was remarkably higher than that in bulk cells. Notably, upon limonin treatment, the proportion of SP cells in G0 was decreased and S/G2/M increased. Furthermore, limonin enhanced the radiosensitivity of NPC cells. The mechanistic studies based on RNA-sequencing analysis revealed that limonin inhibited the gene transcription driven by Stat3 (signal transducer and activator of transcription 3) and an activator of Stat3 (Colivelin or IL-6) rescued the inhibitory effects of limonin. Therefore, these results demonstrate that limonin could reduce the stemness of NPC cells and thus the radiosensitivity through suppressing Stat3 transcriptional activity.

Claudin-9 enhances the metastatic potential of hepatocytes via Tyk2/Stat3 signaling.

We have previously identified a tight junction protein claudin-9 (CLDN9) as an upregulated gene in hepatocellular carcinoma (HCC) through an immunohistochemistry analysis. Here, we explore its function and clinical relevance in human HCC. Stable transfection of the hepatocyte line HL7702 with CLDN9 was confirmed by the real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence. The impact of CLDN9 on the cell invasion and migration was assessed in vitro by a transwell assay and wound-healing experiment. Western blotting was used to determine the activation state of the Tyk2 (tyrosine kinase 2)/Stat3 (signal transducer and activator of transcription 3) pathway. Moreover, we used a Tyk2-RNAi assay to silence the expression of Tyk2 in CLDN9 expressing hepatocytes; subsequently, the impact of the Tyk2/Stat3 signaling pathway on the cell invasion and migration in vitro was assessed by a transwell assay and a wound-healing experiment. Furthermore, an immunohistochemistry method was utilized to explore the expression levels of CLDN9 and p-Stat3 in the HCC tissues and histologically non-neoplastic hepatic tissues. We confirmed that the expression of CLDN9 significantly enhanced the metastatic ability of hepatocytes in vitro, and the activation of the Stat3 pathway by Tyk2 was an important mechanism by which CLDN9 promoted hepatocyte aggressiveness in HCC. As an HCC proto-oncogene, CLDN9 affected the Stat3 signaling pathway via Tyk2 and ultimately enhanced the metastatic ability of hepatocytes.

ROR\u03b1 suppresses interleukin-6-mediated hepatic acute phase response

Acute liver failure (ALF) is characterized by loss of liver function in response to sustained augmentation of the acute-phase response (APR) in the liver, which can progress even to death. Although the inflammatory interleukin-6 (IL-6)–axis is a crucial factor that drives the hepatic APR by releasing diverse acute-phase proteins (APPs), therapeutic strategies to block the IL-6–STAT3-mediated APR are not well developed. Here, we show that the nuclear receptor retinoic acid-related orphan receptor α (RORα) limits APR-mediated liver injury by inhibiting the hepatic IL-6–STAT3 signaling pathway. Administration of JC1-40, an RORα activator, diminished diethylnitrosamine-induced acute liver injury and repressed transcriptional expression of APPs such as CXCL1 and LCN2 in mice. IL-6-mediated activation of STAT3 was repressed after RORα activation by either adenoviral infusion of RORα or JC1-40 treatment in primary hepatocytes. Activation of RORα decreased transcriptional expression of IL-6 receptor α, an upstream activator of STAT3, both in vitro and in vivo. This may be one mechanism underlying the RORα-mediated inhibition of STAT3. Taken together, our results suggest that RORα is a regulator of the hepatic IL-6–STAT3 signaling pathway and may be a new therapeutic target for treating APR-associated inflammatory ALF.