phospho-STAT3 Levels Research Articles

Functional Evaluation of RNLS, a Gene Harboring Risk Variants for Type 1 Diabetes in European and African Ancestry Subjects

Genetic studies of type 1 diabetes (T1D) have identified more than 40 risk loci, yet the causative genes in each locus is widely unknown. Here, we investigated a candidate gene in the 10q23.31 risk locus—FAD-dependent amine oxidase (RNLS). Variants in RNLS gene are associated with T1D risk in both European and African ancestry populations, indicating common genetic contributions to T1D. Currently, our understanding of the function of RNLS is very limited and primarily within the context of a few human diseases, most notably hypertension, kidney disease, cardiovascular disease, and cancer. In previous studies a positive RNLS-STAT3 feedback loop has been suggested. With consideration to these reports, and the importance of JAK-STAT signalling in immune-mediated diseases we evaluated if RNLS functions in activation of STAT3. An IFN-β time course was conducted to understand if overexpression of RNLS affects STAT3 levels and phosphorylation levels of STAT3. While we observed no difference in steady-state levels of STAT3 in cells overexpressing RNLS, we did observe increased levels of phospho-STAT3 upon IFN-β stimulation (p: 0.0051). In addition, to understand the function of RNLS in the immune system, we used RNAseq to compare transcriptomes of Jurkat cells expressing endogenous RNLS vs. ones overexpressing RNLS. RNAseq analysis identified 390 genes that were differentially expressed, by more than two-fold, of which 74.1 (289/390) of genes were up-regulated and 25.9 (101/390) were down-regulated in cells overexpressing RNLS. Tissue specific gene network analysis revealed that genes that were up-regulated mainly functioned in pathways related to chromosome segregation and cell division while genes that were down-regulated by RNLS were involved in IFN and NF-kappa B signaling pathways. Overall our results established a role for RNLS in cell-cycle related pathways and immune signaling pathways, providing new mechanistic insights of the role of RNLS in T1D. Disclosure S. Onengut-Gumuscu: None. N. Vogler: None. M. Faidas: None. R.R. Pickin: None. E. Gersz: None. S.S. Rich: None.

Activation of STAT3 and Bcl-2 and reduction of reactive oxygen species (ROS) promote radioresistance in breast cancer and overcome of radioresistance with niclosamide.

Radiotherapy significantly improves the therapeutic outcomes and survival of breast cancer patients. However, the acquired resistance to this therapeutic modality is a major clinical challenge. Here we show that ionizing irradiation (IR)-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) at the Tyr705 residue and the induction of reactive oxygen species (ROS) in wild-type and radioresistant MDA-MB-231 and MDA-MB-468 triple-negative breast cancer (TNBC) cell lines. Comparing with radiosensitive parental TNBC cells, significantly low levels of ROS and higher protein levels of phospho-STAT3 and Bcl-2 were observed in TNBC cells with acquired radioresistance. Moreover, knockdown of STAT3 by shRNA sensitized the TNBC cells to IR. Niclosamide, a potent inhibitor of STAT3, overcame the radioresistance in TNBC cells via inhibition of STAT3 and Bcl-2 and induction of ROS. In combination with radiation, niclosamide treatment resulted in significant increase of ROS generation and induction of apoptosis in parental and radioresistant TNBC cells in vitro and TNBC xenograft tumors in vivo. These findings demonstrate that activation of STAT3 and Bcl-2 and reduction of ROS contribute to the development of radioresistance in TNBC, and niclosamide acts as a potent radiosensitizer via inhibiting STAT3 and Bcl-2 and increasing ROS generation in TNBC cells and xenograft tumors. Our findings suggest that niclosamide in combination with irradiation may offer an effective alternative approach for restoring the sensitivity of radioresistant TNBC cells to IR for improved therapeutic efficacy and outcomes.

Research of the inhibitory effect and the mechanisms of plumbagin on large cell lung cancer NL9980 cell line

Objective To investigate the effects of plumbagin on human NL9980 large-cell lung cancer cells and the role of interleukin-6/signal transducers and activators of transcription 3 (IL-6/STAT3) signaling pathway in the anticancer procedure of plumbagin. Methods Different concentrations of plumbagin were administrated in NL9980 cell line. Inhibitory effects of plumbagin on NL9980 cells were analyzed using methyl thiazol tetrazolium (MTT) assay. Groups of different drug doses (5.0, 7.5, 10.0 μmol/L) and blank control (without administration of plumbagin) were set with simple random sampling method. Cells were collected after 24 h of administration. Expression of IL-6 and STAT3 and the downstream genes B-cell lymphoma/leukemia-2 (bcl-2), vascular endothelial growth factor (VEGF), and Cyclin D1 were assessed by real-time quantitative polymerase chain reaction (Real-time PCR). Enzyme linked immunosorbent assay (ELISA) was used to measure secreted IL-6 levels and western blotting to measure intracellular phospho-STAT3 levels. Results Plumbagin significantly inhibited NL9980 cells via suppressing IL-6/STAT3 signaling. Expression of all genes was inhibited by plumbagin in a dose-dependent manner (F=32.170, P=0.000). As the concentration of plumbagin increased, the expression of IL-6 mRNA decreased significantly (F=173.067, P=0.000) to 0.50±0.02, 0.44±0.1, 0.34±0.04 respectively and IL-6 protein decreased to 4.71±0.02, 4.08±0.14, 2.01±0.07 significantlly (F=1 805.177, P=0.000). Besides, the expression of STAT3 mRNA (0.86±0.08, 0.72±0.03, 0.52±0.06, F=230.918, P=0.000) and protein (0.65±0.19, 0.45±0.22, 0.31±0.11, F=17.332, P=0.000) were reduced with the increase of concentration of plumbagin. Compared with the control group, expression of downstream genes bcl-2, VEGF and Cyclin D1 mRNA were decreased significantly after administration of plumbagin (F=33.698, P=0.000; F=337.743, P=0.000; F=180.136, P=0.000), and declined gradually as drug concentration increased. Moreover, bcl-2, VEGF, and Cyclin D1 mRNA levels positively correlated with levels of IL-6 and STAT3 gene expressions. Conclusion IL-6-mediated STAT3 suppression decreased bcl-2, VEGF, and Cyclin D1 expression. Thus, plumbagin may be an effective therapy for large-cell lung cancer that acts by targeting the IL-6/STAT3 signaling pathway. Key words: Plumbagin; Interleukin-6; Signal transducers and activators of transcription 3; Large cell lung cancer

P122 Vitamin D activates human intestinal fibroblasts

Vitamin D signals through the vitamin D receptor (VDR) which is a member of the nuclear receptor family of transcription factors that play an immunoregulatory role in the gut. Defective signalling due to vitamin D deficiency or decreased mucosal VDR levels has been related to Crohn’s disease (CD). We aim to analyse the acute effects of Vitamin D in the activation of human intestinal fibroblasts. Fibroblasts were isolated from non-damaged and damaged intestinal resection of CD patients and control patients (non-damaged intestine from colon cancer). Fibroblasts were treated with 1,25 Vitamin D3 (10 nM and 100 nM) for 24 h. Gene expression of pro-inflammatory cytokines and COL1A1 was quantified by qPCR and protein levels were determined by western blot. Statistical significance was measured by ANOVA. Vitamin D increased the mRNA expression of VDR in fibroblasts obtained from the inflamed and non-inflamed mucosa of CD patients (Figure 1A) and it increased the mRNA of CYP24A1, a VDR target (Figure 1B). Treatment with vitamin D rised in a dose-dependent manner COL1A1 mRNA expression in fibroblasts from CD patients (Figure 1C) and in parallel it induced the expression of pro-inflammatory cytokines (IL1β , IL6) (Figure 1D, 1E). Protein levels of phospho-NFκB and phospho-STAT3 were also higher in fibroblasts treated with Vitamin D from CD patients. Our study indicates that an acute treatment of Vitamin D activates an inflammatory pathway and a collagen I expression in human intestinal fibroblasts which may be involved in the initial response in the wound healing.

Leptin Aggravates Reflux Esophagitis by Increasing Tissue Levels of Macrophage Migration Inhibitory Factor in Rats

Leptin, produced primarily by the adipose tissue, acts as a pro-inflammatory modulator, thereby contributing to the development of obesity-related disease. Although high levels of leptin in the obese are closely related to gastroesophageal reflux disease, the mechanism by which leptin influences esophageal inflammation remains unknown. Macrophage migration inhibitory factor (MIF) is produced by immune cells, such as T lymphocytes and macrophages, and MIF is known to induce the production of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6). We therefore investigated the mechanism whereby leptin aggravates reflux esophagitis, by focusing on esophageal tissue levels of MIF and CD3+ T lymphocytes, both of which are crucial for the reflux-induced epithelial damage. Esophageal inflammation was surgically induced in male Wistar rats by ligating the forestomach and narrowing the duodenum to facilitate gastroesophageal reflux, followed by administration of leptin or vehicle with an osmotic pump system for 1 week. We demonstrated that the administration of leptin exacerbated the reflux esophagitis with the apparent infiltration of CD3+ T lymphocytes and caused the significant increase in the esophageal tissue levels of MIF. Moreover, the leptin caused increases in the esophageal tissue levels of TNF-α, IL-1β and IL-6, downstream targets of MIF. Importantly, the increases in these pro-inflammatory cytokines were accompanied by increased protein levels of phospho-STAT3 and phospho-AKT, pivotal molecules of leptin signaling pathways. In conclusion, through enhancing the MIF-induced inflammatory signaling, leptin could contribute to the development of gastroesophageal reflux disease.

Novel imidazopyridine suppresses STAT3 activation by targeting SHP-1

The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthesized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16 on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.

Plumbagin suppresses the human large cell lung cancer cell lines by inhibiting IL-6/STAT3 signaling in vitro

BackgroundLarge cell lung cancer (LCLC) patients have a poor prognosis because their tumors are highly malignant and show resistance to chemotherapy and radiotherapy. Plumbagin has anticancer activity toward several tumor types, but its effects on LCLC are unknown. This study investigated the effects of plumbagin on human L9981 and NL9980 large cell lung cancer cells and the mechanisms underlying its action. MethodsThe effects of plumbagin on L9981 and NL9980 cells proliferative activity and invasion were analyzed using MTT and Boyden chamber assays, respectively. Exogenous IL-6 was used to detect the presence of the IL-6/STAT3 signaling pathway in LCLC cell lines. L9981 cells were harvested after plumbagin treatment at 9.0μmol/L (IC50), while NL9980 were harvested after treatment at 7.5μmol/L for 6, 24, and 48h, and the expression of IL-6, IL-6R, gp130, and STAT3 and the downstream genes Bcl-2, VEGF, and CycD1 was assessed by RT-PCR. ELISA was performed to determine secreted IL-6 levels, and intracellular phospho-STAT3 levels were measured by western blotting. ResultsAfter the introduction of exogenous IL-6, the mRNA expression of signaling genes and downstream genes was significantly increased in a concentration-dependent manner. Furthermore, plumbagin significantly inhibited the expression of the above mentioned genes in a dose-dependent and time-dependent manner. The mRNA expression levels of downstream genes were correlated with those of signaling genes. ConclusionPlumbagin was found to significantly inhibit the proliferation and invasion of L9981 and NL9980 cells, and may be an effective therapy for LCLC through targeting the IL-6/STAT3 signaling pathway.

Interleukin-37 alleviates airway inflammation and remodeling in asthma via inhibiting the activation of NF-κB and STAT3 signalings

Asthma is a common respiratory inflammatory disorder disease of childhood, and airway smooth muscle cells (ASMCs) play an important role in this disease. Recently, studies have found that interleukin (IL)-37 inhibits allergic airway inflammation of asthmatic mouse models. The aim of this study was to investigate the exact mechanism of IL-37 in asthma. In this study, we found recombinant human IL-37 protein significantly reduced ovalbumin (OVA)-induced airway hyperresponsiveness, inflammatory cell infiltration, the epithelial-mesenchymal-transition (EMT) process, and levels of IL-4, IL-6 and IL-13, but increased interferon (IFN)-γ expression. Moreover, IL-37 treatment remarkably inhibited transforming growth factor (TGF)-β1-induced cell proliferation, migration, EMT, and inflammatory response in ASMCs. IL-37 notably upregulated IκB expression and downregulated levels of NF-κB p65, phospho-NF-κB p65, STAT3 and phospho-STAT3 both in OVA-induced mice and in TGF-β1-stimulated ASMCs. The effects of IL-37 on TGF-β1-induced ASMCs were abrogated by STAT3 upregulation. Additionally, PDTC, a NF-κB inhibitor, showed the similar effects as IL-37 in ASMCs. In conclusion, IL-37 may alleviate airway inflammation and remodeling in asthma through suppressing the activation of NF-κB and STAT3.

Dual roles of IL-22 at ischemia-reperfusion injury and acute rejection stages of rat allograft liver transplantation.

Interleukin-22 (IL-22) is a recently identified regulator of inflammation, but little is known about its role in liver transplantation. Therefore, in this study, we explored the roles and the underlying mechanisms of IL-22 in acute allograft rejection by using a rat allogeneic liver transplantation model. Results showed that allograft liver transplantation led to damage of the parent liver and to significantly increased IL-22 expression in the allograft liver and plasma of the recipient rats compared with the rats who received isografts. Moreover, the significantly increased IL-22 expression was accompanied by markedly increased level of phospho-STAT3 in the allogeneic liver tissues after transplantation. Of note, neutralization of the IL-22 protein in recipient rats significantly worsened the function of the allograft liver at 1 day post-transplantation (ischemia-reperfusion injury, IRI) but improved the function at 7 days post-transplantation (acute rejection, AR). At IRI stage, IL-22 protected liver function through the increase of anti-apoptosis and pro-regeneration cytokines. However, IL-22 led to the increase of pro-inflammation factors at AR stage, accompanied by the marked increase of the Th17 and the marked decrease of Treg cells in allograft recipient rats through modulating the expression of chemokines for different cell types, which however were reversed by in vivo IL-22 neutralization. Results indicate the dual roles of IL-22 and suggest the differential potential clinical application of IL-22 at different stage of allograft liver transplantation.

Zinc improves learning and memory abilities of fetal growth restriction rats and promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 axis activity.

Fetal growth restriction (FGR) is a well-known risk factor for cognitive dysfunction, especially for learning and memory abilities. However, knowledge about prevention and treatment methods of learning and memory abilities of fetal are limit. Here, Morris water maze and passive avoidance tests showed zinc supplementation could protect the impairment of the learning and memory abilities caused by FGR. As accumulating evidence suggested that insufficiency of placental trophoblast cell invasion was closely related to FGR fetal neurodevelopmental dysplasia, we further explored the relationship between zinc supplementation during pregnancy and placental trophoblast. Microarray identified 346 differently expressed genes in placental tissues with and without zinc supplementation, and GO and KEGG analyses showed these differently expressed genes were highly enriched in cell invasion and migration and STAT3 pathway. Protein-protein interaction(PPI) analysis found that STAT3 interacted with matrix metalloproteinase-2/9 (MMP-2/9). In vivo, western blot results authenticated that the expression levels of phospho-STAT3, STAT3, MMP-2 and MMP-9 were up-regulated in placental tissues after zinc treatment. To validate whether zinc could promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 activity. In vitro, Transwell assay was performed, and we observed that abilities of invasion and migration were obviously increased in zinc treated trophoblast cells. And phospho-STAT3, STAT3, MMP-2 and MMP-9 expression levels were correspondingly increased in zinc treated trophoblast cells, which were dose-dependent. Moreover, gain–of-function and loss-of-function of STAT3 confirmed that zinc promotes cell invasion and migration via regulating STAT3 mediated up-regulation of MMP-2/9 activity. We propose that activation of MMP-2/9 mediated by STAT3 may contribute to invasion and migration of trophoblast cells, which improved neurodevelopmental impairment of FGR rats probably via contributing to placental development. Our findings are the first to show a possible mechanism of reversing neurodevelopmental impairment of FGR rats by zinc supplementation, holding promise for the development of novel therapeutic modalities for learning and memory abilities impairment caused by FGR.

Red nucleus interleukin-6 participates in the maintenance of neuropathic pain through JAK/STAT3 and ERK signaling pathways

We previously reported that interleukin-6 (IL-6) in the red nucleus (RN) is up-regulated at 3weeks after spared nerve injury (SNI), and plays facilitated role in the later maintenance of neuropathic pain. The current study aimed to reveal the roles of different signaling pathways, including Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase/protein kinase B (PI3K/AKT), in RN IL-6-mediated pain modulation. In accord with the increase of IL-6 in the RN following SNI, the protein levels of phospho-STAT3 (p-STAT3), p-ERK and p-JNK were also up-regulated in the RN contralateral to the nerve injury side at 3weeks after SNI. The increases of p-STAT3 and p-ERK (but not p-JNK) were associated with IL-6 and could be blocked by anti-IL-6 antibody. Microinjection of JAK2 inhibitor AG490, ERK inhibitor PD98059 and also JNK inhibitor SP600125 into the RN significantly increased the paw withdrawal threshold (PWT) and alleviated SNI-induced mechanical allodynia. Further studies showed that microinjection of recombinant rat IL-6 (rrIL-6, 20ng) into the RN of normal rats significantly decreased the PWT of rats and increased the local protein levels of p-STAT3 and p-ERK, but not p-JNK. Pre-treatment with AG490 and PD98059 could prevent IL-6-induced mechanical allodynia. Whereas, p-p38 MAPK and p-AKT did not show any expression changes in the RN of rats with SNI or rats treated with rrIL-6. These results suggest that RN IL-6 participates in the later maintenance of SNI-induced neuropathic pain and plays facilitated role through activating JAK/STAT3 and ERK signaling pathways.

Short-term treatment with eicosapentaenoic acid improves inflammation and affects colonic differentiation markers and microbiota in patients with ulcerative colitis

Patients with long-standing ulcerative colitis (UC) have an increased colorectal cancer (CRC) risk. In this pilot study we evaluated the effect of Eicosapentaenoic acid as free fatty acid (EPA-FFA) supplementation on mucosal disease activity, colonic differentiation markers and microbiota composition in UC patients. Twenty long-standing UC patients in stable clinical remission and with fecal calprotectin (FC) > 150 µg/g were enrolled (T0) and supplemented with EPA-FFA 2 g/daily for 90 days (T3). Endoscopic and histologic disease activities were measured by Mayo and Geboes scores, respectively. HES1, KLF4, STAT3, IL-10 and SOCS3 levels were determined using western blotting and qRT-PCR, while phospho-STAT3 levels were assessed by western blotting. Goblet cells were stained by Alcian blue. Microbiota analyses were performed on both fecal and colonic samples. Nineteen patients completed the study; seventeen (89.5%) were compliant. EPA-FFA treatment reduced FC levels at T3. Patients with FC > 150 µg/g at T3 (n = 2) were assumed as non-responders. EPA-FFA improved endoscopic and histological inflammation and induced IL-10, SOCS3, HES1 and KLF4 in compliant and responder patients. Importantly, long-term UC-driven microbiota composition was partially redressed by EPA-FFA. In conclusion, EPA-FFA supplementation reduced mucosal inflammation, promoted goblet cells differentiation and modulated intestinal microbiota composition in long-standing UC patients.

Abstract 2636: Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal form of primary breast cancer. Inflammatory signaling pathways are active in IBC, but the function of the immune response remains elusive. ErbB receptors play a role in IBC. ERBB2 is amplified in 50% of IBCs, and ERBB3 is also mutated in IBC. Currently, Lapatinib, a dual ErbB inhibitor, is used in IBC patients with ERBB2 amplification. Although immune cell inflammatory signaling may promote IBC tumor growth and metastasis, the presence of cytotoxic tumor-associated lymphocytes has also been associated with a more favorable breast cancer prognosis. The contradictory nature of the immune data highlights the need to elucidate the relationship between the immune response and subsequent treatments. Given the active IBC immune component and the recent clinical benefit of immune checkpoint inhibitors in cancer treatment, IBC’s may represent a clinically unique breast cancer population that may benefit from immunomodulating agents. Interestingly, Myc is a downstream effector of ErbB signaling, and Myc is thought to regulate the expression of immune checkpoint proteins CD47 and PD-L1. The presence of PD-L1-positive IBC immune infiltrate suggests that IBC’s may benefit from therapies that disrupt PD-L1 signaling together with ErbB inhibitors. METHODS: IHC was used to examine immune checkpoint signaling and characterize the tumor-immune infiltrate. Tumor tissues were also characterized using an RNA-seq panel that examined the expression of 377 immune-related genes. Cell lines (BT474, SKR3, AU565, and SUM225) were treated with Lapatinib and Neratinib to examine the relationship between growth factor receptors and downstream immune signaling pathways. RESULTS: RNA-seq revealed the expression of specific immunosuppressive signaling pathways in tumors. Treatment of breast cancer cells with ErbB inhibitors resulted in a decrease in the levels of PD-L1. Treatment with Lapatinib and Neratinib diminished PD-L1 in all cell lines. Levels of Phospho-Stat3 decreased in BT474 and Sum225 but not in SKBR3 and AU565, implying that PD-L1 is regulated by another mechanism (ERK-MYC). CONCLUSION: Our results provide mechanistic insight into ErbB receptor activation and the expression of downstream signaling molecules (Stat3, Myc, PD-L1, and others). In addition, our unique RNA-seq immune signature reveals the expression of several genes that may serve as biomarkers of inhibitory immune signaling pathways. Our laboratory is currently examining the correlation between PD-L1 expression and the activation of ErbB2 in model systems and clinical trials using ErbB inhibitors in combination with PD-L1 inhibitors. Our immune panel gene signature may serve as a useful diagnostic test that, in conjunction with traditional ErbB testing, can identify patients that will benefit from combination therapy of an ErbB inhibitor and an immune checkpoint inhibitor. Note: This abstract was not presented at the meeting. Citation Format: Christopher A. Hamm, Sumin Zhao, Cesar A. Santa-Maria, Massimo Cristofanilli, Neil L. Spector, Sarah Bacus. Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2017-2636

C-X-C motif chemokine receptor 4 promotes tumor angiogenesis in gastric cancer via activation of JAK2/STAT3.

C-X-C motif chemokine receptor 4 (CXCR4) overexpression promotes gastric cancer growth and metastasis. In this study, we determined its role in regulating tumor angiogenesis. We overexpressed CXCR4 in gastric cancer cells and examined the effects of conditioned medium on endothelial cell proliferation, migration, and tube formation. The effects of CXCR4 overexpression on vascular endothelial growth factor (VEGF) expression and signal transducer and activator of transcription 3 (STAT3) activation were analyzed. In vivo xenograft studies were done to confirm the role of CXCR4 in tumor angiogenesis. Conditioned medium from CXCR4-overexpressing gastric cancer cells stimulated endothelial cell proliferation, migration, and tube formation. Such effects were blocked by addition of a neutralizing anti-VEGF antibody. CXCR4 induced VEGF production and JAK2/STAT3 activation and enhanced STAT3 binding to VEGF promoter in gastric cancer cells. Delivery of a dominant negative variant of STAT3 significantly impaired CXCR4-induced upregulation of VEGF. Overexpression of CXCR4 facilitated tumor growth and angiogenesis in SGC7901 xenograft tumors, which was associated with increased levels of phospho-STAT3. CXCR4 contributes to tumor angiogenesis in gastric cancer by inducing STAT3-dependent VEGF expression and represents a promising therapeutic target for this malignancy.

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

Disulfiram suppresses cancer stem-like properties and STAT3 signaling in triple-negative breast cancer cells

In the presence of copper (Cu), disulfiram (DSF) suppresses properties associated with cancer stem cells (CSCs) in breast cancer, but the mechanism of action is poorly understood. In the present study, we observed that DSF/Cu treatment induced apoptosis, mediated by caspase-3 activation in triple-negative breast cancer (TNBC) cells. DSF/Cu treatment also specifically targeted CSC-like cell populations, marked by the inhibition of ALDH1 activity, the suppression of CD44+/CD24-and CD49f+/CD24 + subpopulations, and the subsequent impairment of mammosphere formation. These effects were functionally associated with a significant impact on the STAT3 signaling pathway, characterized by the downregulation of phospho-STAT3, cyclin D1 and survivin. In an MDA-MB-231-derived xenograft model, DSF administration significantly downregulated ALDH1A1, CD44 and phospho-STAT3 levels. These findings show for the first time that DSF suppresses stem-like properties in TNBC by targeting the STAT3 signaling pathway.

STAT3 Activation Impairs the Stability of Th9 Cells.

Th9 cells regulate multiple immune responses, including immunity to pathogens and tumors, allergic inflammation, and autoimmunity. Despite ongoing research into Th9 development and function, little is known about the stability of the Th9 phenotype. In this study, we demonstrate that IL-9 production is progressively lost in Th9 cultures during several rounds of differentiation. The loss of IL-9 is not due to an outgrowth of cells that do not secrete IL-9, as purified IL-9 secretors demonstrate the same loss of IL-9 in subsequent rounds of differentiation. The loss of IL-9 production correlates with increases in phospho-STAT3 levels within the cell, as well as the production of IL-10. STAT3-deficient Th9 cells have increased IL-9 production that is maintained for longer in culture than IL-9 in control cultures. IL-10 is responsible for STAT3 activation during the first round of differentiation, and it contributes to instability in subsequent rounds of culture. Taken together, our results indicate that environmental cues dictate the instability of the Th9 phenotype, and they suggest approaches to enhance Th9 activity in beneficial immune responses.

Expansion of polymorphonuclear myeloid-derived suppressor cells in patients with end-stage renal disease may lead to infectious complications

Myeloid-derived suppressor cells (MDSCs) are recently identified immune suppressive cells in multiple chronic inflammations. Here, we investigated MDSCs in patients with end-stage renal disease (ESRD) and their clinical significance in these patients and healthy individuals (49each). Polymorphonuclear and mononuclear MDSCs were investigated by flow cytometry. Patients with ESRDbefore hemodialysis presented a significantly higherlevel of polymorphonuclear MDSCs. Depletion ofpolymorphonuclear-MDSCs resolved T cell IFN-γ responses. By co-culture, T cell proliferation and the production of IFN-γ were abrogated by the addition of polymorphonuclear MDSCs in a dose-dependent manner. Both of these effects were reversed by a reactive oxygen species inhibitor. The levels of reactive oxygen species were higher in polymorphonuclear MDSCs derived frompatients with ESRD than from normal individuals. ThemRNA level of NOX2, the key protein complex responsiblefor reactive oxygen species production, washigher in ESRD-related polymorphonuclear MDSCs. The phospho-STAT3 level, a key activator of MDSCs, washigherin ESRD-related polymorphonuclear MDSCs. Finally, the polymorphonuclear MDSC level before and after hemodialysis was positively related to infectious diseases. Patients with ESRD were dichotomized into 2 groups bythe amount of polymorphonuclear MDSCs. Patients with high levels of polymorphonuclear MDSCs presented with a higher incidence of infectious events. Thus, polymorphonuclear MDSCs were elevated in ESRD patients with strong immune-suppressive capability through a phospho-STAT3/reactive oxygen species pathway. Hence, polymorphonuclear MDSCs might increase the risk of infectious complications.

Combined effects of atorvastatin and aspirin on growth and apoptosis in human prostate cancer cells.

Epidemiologic studies indicate the use of either statins or aspirin have beneficial effects in prostate cancer patients. The present study was undertaken to evaluate the effects and mechanisms of atorvastatin and aspirin alone or in combination in human prostate cancer cells cultured invitro and grown as xenograft tumors in severe combined immune-deficient (SCID) mice. The growth and apoptosis in prostate cancer cells were determined by the trypan blue exclusion and propidium iodide staining assays. Activation of the nuclear factor κB (NF-κB) was measured by luciferase reporter assay, and the levels of phospho-signal transducer and activator of transcription (Stat)3 and phospho-extracellular signal-regulated kinase (Erk)1/2 were determined by western blot analysis. Mice were injected subcutaneously with PC-3 cells in Matrigel. After 4-6 weeks, mice with PC-3 tumors received i.p.injections of vehicle, atorvastatin (5mg/kg), aspirin (80mg/kg), or atorvastatin (5mg/kg) + aspirin (80mg/kg) three times a week for 30days. Our results demonstrated the combination of atorvastatin and aspirin had more potent effects on growth inhibition and apoptosis stimulation in prostate cancer cells than either drug alone. Mechanistic studies indicated the induction of apoptosis in PC-3 cells was associated with strong inhibition of NF-κB and decreases in the levels of phospho-Stat3 and phospho-Erk1/2. Results of the present study demonstrated a strong combined effect of atorvastatin and aspirin on inhibiting the growth of prostate cancer cells invitro and invivo. The findings provide a strong rationale for clinical evaluation of the combination of atorvastatin and aspirin in patients with prostate cancer.

IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells.

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.