Signal Transducer And Activator Of Transcription 3 Gene Research Articles

Abstract 2521: STAT3 is a central regulator of proliferation, invasion and metabolism in GBM tumor initiating cells

Abstract Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Currently, treatment for GBM involves surgical resection, chemotherapy, and radiation, resulting in a median survival of 15 months. This poor outcome highlights the need for improved therapeutic approaches to treat GBM patients. The signal transducer and activator of transcription 3 (STAT3) pathway is abnormally active in GBM, primarily in the mesenchymal subtype of GBM. STAT3 regulates various cellular processes including: proliferation, invasion and resistance to therapy. The Weiss lab has established a large collection of brain tumor initiating cell (BTIC) lines derived from GBM patients. These GBM BTIC lines are used to model the subpopulation of cells that are predicted to be the source of resistance and recurrence following treatment. Here, we are focused on the role of STAT3 as a central regulator of proliferation, invasion and metabolism in GBM BTIC lines. We show that activation of the STAT3 pathway using the ligand oncostatin M (OSM) or by stably expressing a constitutively active STAT3 mutant (STAT3C) in GBM BTIC lines leads to an increase in proliferation. STAT3 is known to regulate the expression of multiple matrix metalloproteinases (MMPs) that promote invasion. Using time-lapse imaging of 3D sphere invasion into matrigel, we observed that JAK2 (an activator of STAT3) and direct STAT3 inhibitors decrease invasion of GBM BTICs. Our analysis of gene expression data available from The Cancer Genome Atlas (TCGA) demonstrates that a glutamine metabolism gene expression signature correlates inversely with a STAT3 gene expression signature in GBM patient samples. Conversely, similar analysis reveals that a high glycolysis gene expression signature correlates with a high STAT3 gene expression signature. We hypothesize that targeting the STAT3 pathway will decrease proliferation and invasion while simultaneously sensitizing GBM BTIC lines to inhibition of glutamine metabolism. Glutaminase (GLS) is the first enzyme to act on glutamine, converting glutamine to glutamate, which is then further processed to α-ketoglutarate (αKG). αKG is important for the production of biosynthetic macromolecules that are essential for cell growth. Chemical inhibition of GLS decreases cell growth in culture with varying levels of sensitivity across multiple GBM BTIC lines. Interestingly, activation of the STAT3 pathway using OSM de-sensitizes GBM BTIC lines to GLS inhibition. Furthermore, chemical inhibition of JAK2 sensitizes a subset of GBM BTIC lines to GLS inhibition resulting in a further decrease in cell growth. Overall, we show that STAT3 is a central hub in GBM BTIC lines that modulates proliferation, invasion, and sensitivity to GLS inhibition. Currently, we are continuing these studies in vivo using GBM BTIC orthotopic xenografts in mice. Citation Format: Ian Restall, H. Artee Luchman, Samuel Weiss. STAT3 is a central regulator of proliferation, invasion and metabolism in GBM tumor initiating cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2521.

Inhibitory effect of STAT3 gene combined with CDDP on growth of human Wilms tumour SK-NEP-1 cells.

To investigate the effects of signal transducer and activator of transcription 3 (STAT3) combined with cisplatin (CDDP) on the growth of human Wilms tumour (WT) SK-NEP-1 cell subcutaneous xenografts in nude mice and the possible mechanisms. Human WT SK-NEP-1 cells were subcutaneously transplanted to establish the BALB/c nude mice xenograft model. Mice were randomly divided into five groups: blank control group, adenovirus control group (NC group), STAT3 group, CDDP group and STAT3 plus CDDP group (combination group). Tumour volume and tumour weight were observed during the therapeutic process. The expression levels of STAT3, glucose regulatory protein 78 (GRP78) and BCL2-associated X protein (BAX) were evaluated by immunohistochemical analysis. Compared with the STAT3 group or CDDP group, the tumour weight and volume was significantly reduced in the combination group (P<0.05). No statistical significance was found in NC group compared with the blank control group (P > 0.05). Immunohistochemical analysis showed that STAT3, GRP78 and BAX protein levels in the combination group were significantly higher than those in STAT3 group and CDDP group (P<0.05). Exogenous STAT3 and CDDP may synergistically inhibit the xenograft tumour growth through up-regulation of BAX protein via GRP78.

Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation

Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3+-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms.

Cardioprotection from emulsified isoflurane postconditioning is lost in rats with streptozotocin-induced diabetes due to the impairment of Brg1/Nrf2/STAT3 signalling.

Isoflurane postconditioning (IsoPostC) attenuates myocardial ischaemia/reperfusion injury (IRI). Signal transducer and activator of transcription-3 (STAT3) is critical in ischaemic postconditioning cardioprotection, which can be regulated by the Brahma-related gene (Brg1) and nuclear factor-erythroid 2-related factor 2 (Nrf2), although they are both reduced in diabetic hearts. We hypothesized that reduced Brg1/Nrf2 and STAT3 activation may jeopardize IsoPostC-mediated cardioprotection in diabetic hearts. In the present study, Langendorff-perfused, non-diabetic (control) and 8-week-old streptozotocin-induced Type1 diabetic rat hearts were subjected to 30min of global ischaemia and 120min of reperfusion without or with IsoPostC, which was achieved by administering emulsified isoflurane (2.0%, v/v) in Krebs-Henseleit (KH) solution immediately at the onset of reperfusion for 10min and switching to KH solution perfusion alone thereafter. Cultured H9C2 cells were exposed to normal glucose (NG, 5.5mM) or high glucose (HG, 30mM) and subjected to hypoxia/reoxygenation (HR) in the presence or absence of IsoPostC. Diabetic rats displayed larger post-ischaemic myocardial infarction and more severe haemodynamic dysfunction, associated with increased myocardial oxidative stress and reduced cardiac Brg1, Nrf2 and STAT3 phosphorylation/activation (p-STAT3), compared with controls. These changes were reversed/prevented by IsoPostC in control but not in diabetic rats. In H9C2 cells exposed to NG but not HG, IsoPostC significantly attenuated HR-induced cellular injury and superoxide anion production with increased Brg1, Nrf2 and p-STAT3. These beneficial effects of IsoPostC were abolished by Brg1, Nrf2 or STAT3 gene knockdown. Brg1 or Nrf2 gene knockdown abolished IsoPostC-induced STAT3 activation. N-acetylcysteine restored Brg1, Nrf2 and p-STAT3, and IsoPostC-induced protection in H9C2 cells exposed to HG and HR. In conclusion, IsoPostC confers cardioprotection through Brg1/Nrf2/STAT3 signalling, and impairment of this pathway may be responsible for the loss of IsoPostC cardioprotection in diabetes.

Two host microRNAs influence WSSV replication via STAT gene regulation.

MicroRNAs (miRNAs) have important roles in post-transcriptional regulation of gene expression. During viral infection, viruses utilize hosts to enhance their replication by altering cellular miRNAs. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays crucial roles in the antiviral responses. In this study, two miRNAs (miR-9041 and miR-9850) from Macrobrachium rosenbergii were found to promote white spot syndrome virus (WSSV) replication. The up-regulation of miR-9041 or miR-9850 suppresses STAT expression in the gills of M. rosenbergii, which subsequently down-regulates the expression of its downstream dynamin (Dnm) genes: Dnm1, Dnm2, and Dnm3. Knockdown of miR-9041 and miR-9850 restricts WSSV replication by up-regulating STAT and Dnm gene expression. The silencing of STAT, Dnm1, Dnm2, or Dnm3 led to an increase of the number of WSSV copies in shrimp. The injection of recombinant Dnm1, Dnm2, or Dnm3 proteins could inhibit WSSV replication in vivo. Overall, our research indicates the roles of host miRNAs in the enhancement of WSSV replication by regulating the host JAK/STAT pathway.

Interaction between STAT3 gene polymorphisms and smoking on Crohn's disease susceptibility: a case-control study in a Chinese Han population.

The aim of this study was to investigate the impact of the signal transducer and activator of transcription 3 (STAT3) gene and additional STAT3 gene-smoking interaction on Crohn's disease (CD) risk based on a Chinese population. A total of 1012 participants (491 men, 521 women), were selected, including 502 CD patients and 510 normal controls. The mean age of all participants was 42.3±11.2years. Logistic regression model was used to examine the association between single nucleotide polymorphism (SNP) of STAT3 and CD risk; the odds ratio (OR) and 95% confident interval (95% CI) were calculated. Generalized multifactor dimensionality reduction was employed to analyze the interaction among several SNPs. Logistic analysis showed the significant association between genotypes of variants in two SNP and decreased CD risk, after covariates adjustment. The carriers of homozygous mutant of two SNP polymorphism revealed decreased CD risk than those with wild-type homozygotes; OR (95% CI) was 0.75 (0.59-0.93) and 0.68 (0.57-0.91), respectively. There was a significant two-locus model (p=0.0107) involving rs744166 and smoking, indicating a potential gene-environment interaction between rs744166 and smoking. Overall, the cross-validation consistency was 10/10, and the testing accuracy was 62.17%, and never smokers with TC or CC genotype have the lowest CD risk, compared to smokers with TT genotype; OR (95% CI) was 0.52 (0.31-0.82), after covariate adjustment. Our results support an important association of rs744166 and rs4796793 with decreased CD risk, and additional interaction between rs744166 and smoking.

STAT4 rs7574865 polymorphism in Iranian patients with rheumatoid arthritis

ObjectivesThe gene encoding signal transducer and activator of transcription 4 (STAT4) has been documented to be associated with rheumatoid arthritis (RA) in several populations. The aim of this study was to determine the association of the STAT4 gene polymorphism (rs7574865) with RA in the Iranian population. MethodsThis study included 612 Iranian RA patients and 389 healthy control subjects. For all samples, DNA was genotyped for STAT4 G>T (rs7574865) polymorphism using the MGB TaqMan Allelic Discrimination method. ResultsIn our survey, the frequencies of GG, GT, and TT genotypes were 50%, 41.3%, and 8.7% in RA patients, and 51.4%, 37.5%, and 11.1% in controls (P>0.05), respectively. STAT4 rs7574865 alleles were not associated with the disease risk in the Iranian population (P>0.05). ConclusionsAlthough most studies have proposed STAT4 polymorphisms as a susceptible risk factor for RA, we did not detect any significant association between the STAT4 polymorphism and RA in our study. Population-based studies help to identify genetic determinants of disease susceptibility. Possible explanations for differences in susceptible genetic factors among populations could be different genetic predispositions and contribution of gene–gene or gene–environment interactions.

The effects of STAT1 dysfunction on the gut

Introduction: Mutations in the signal transducer and activator of transcription1 (STAT1) have been associated with a variety of clinical patterns. Interestingly patients with heterozygous mutations in the DNA binding domain (DBD) of STAT1 suffer acute and chronic colitis. Methods: To further analyze the role of STAT1 deficiency in intestinal inflammation, we employed protein expression analysis of total and activated STAT1 in intestinal biopsy samples from 2 patients with heterozygous mutations in the DBD of the STAT1 gene. Results: Both patients showed clinical and histological features of colitis. Total and activated STAT1 were decreased in duodenal and colonic enterocytes, and total STAT1 was found to be mislocalized in aggregates subapically. In addition, intestinal biopsy samples showed decreased numbers of lymphocytes. Patient-derived lymphoblasts demonstrated lack of viability and high susceptibility for cell death. Conclusion: STAT1 expression and distribution in the gut of patients with mutations in the DBD are abnormal, suggesting a primary role of STAT1 dysfunction in enterocytes in addition to the secondary effect of aberrant inflammation. Statement of novelty: Colitis associated with STAT1 mutations appears to have unique features distinct from typical inflammatory bowel disease.

Abstract A103: Cell-autonomous activation of the interferon/STAT1 pathway in response to genotoxic treatment

Abstract Identification of the mechanisms of tumor resistance remains an unmet need in oncology. Among the reasons for this challenging issue is the lack of reliable preclinical models that represent inter and intra-patient tumor heterogeneity observed in the clinic. Patient-derived xenografts (PDXs) consist in grafting human cancer samples into immunocompromised mice directly after patient surgery. Since each PDX faithfully recapitulates patient's tumor genetics, these preclinical models well represent the intrinsic heterogeneity of cancer. We took advantage of the large collection of breast cancer PDXs held at XenTech to address the mechanisms of tumor response to treatment vs tumor recurrence. Upon receiving chemotherapy, some breast cancer PDXs (the 'responders') undergo tumor shrinkage, whereas others continue to grow (the 'non-responders'). Comparative transcriptomic profiling of laser-microdissected cancer cells showed that the reduction of tumor volume in responders was closely associated to the over-expression of genes related to the interferon (IFN)/signal transducer and activator of transcription 1 (STAT1) pathway. Using mouse versus human cytokine arrays we observed that activation of this pathway was associated with the secretion of ligands of human origin (cancer) rather than with ligands secreted from the nude mice residual immune system. Next, we screened a large number of cell lines to identify in vitro cell models able to mimic cell-autonomous induction of the IFN/STAT1 signature after genotoxic treatment. Both an immortalized cell line and a primary culture dissociated from a responder PDX were shown to activate the IFN/STAT1 pathway and to express the cognate gene signature after treatment with mafosfamide (the active metabolite of cyclophosphamide used in the clinic). Expression analyses (qPCR) confirmed induction of IFN type I elements by cell models. Conditioned medium collected from mafosfamide-treated cancer cells was able to activate luciferase reporter genes harboring ISRE (interferon stimulated response elements) and GAS (gamma interferon activated sequence) response elements, meaning that active ligands of the IFN/STAT1 pathways were secreted. Accordingly, STAT1 gene silencing (siRNA) resulted in markedly attenuated gene signature expression after mafosfamide treatment. The addition of conditioned medium significantly reduced mafosfamide-induced cancer cell death suggesting that the over-expression of the IFN/STAT1 pathway may ultimately have protective effect on cancer cell viability. In conclusion, this study supports that cell-autonomous activation of the IFN/STAT1 pathway is a surrogate biomarker of initial tumor shrinkage in response to genotoxics, and suggests that it may play a role in tumor resistance to treatment. Citation Format: Julie Gaston, Laura Cheradame, Marie-Emmanuelle Legrier, Olivier Déas, Myriam Lassalle, Enora Le Ven, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. Cell-autonomous activation of the interferon/STAT1 pathway in response to genotoxic treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A103.

An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses

Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 clade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5′-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in the disk abalone. Further, this study provides a basis for structural and functional exploration of STAT members in the invertebrate JAK/STAT signaling pathway.

Distal regulatory element of the STAT1 gene potentially mediates positive feedback control of STAT1 expression.

We previously identified a distal regulatory element located approximately 5.5-kb upstream of the signal transducer and activator of transcription 1 (STAT1) gene, thereafter designating it as 5.5-kb upstream regulatory region (5.5URR). In this study, we investigated the functional roles of 5.5URR in the transcriptional regulation of STAT1 gene. A chromosome conformation capture assay indicated physical interaction of 5.5URR with the STAT1 core promoter. In luciferase reporter assays, 5.5URR-combined STAT1 core promoter exhibited significant increase in reporter activity enhanced by forced STAT1 expression or interferon (IFN) treatment, but STAT1 core promoter alone did not. The 5.5URR contained IFN-stimulated response element and GAS sites, which bound STAT1 complexes in electrophoretic mobility shift assays. Consistently, chromatin immunoprecipitation (ChIP) assays of HEK293 cells with Halo-tagged STAT1 expression indicated the association of Halo-tagged STAT1 with 5.5URR. ChIP assays with IFN treatment demonstrated that IFNs promoted the recruitment of Halo-tagged STAT1 to 5.5URR. Forced STAT1 expression or IFN treatment increased the expression of endogenous STAT1 and other IFN signaling pathway components, such as STAT2, IRF9 and IRF1, besides IFN-responsive genes. Collectively, the results suggest that 5.5URR may provide a regulatory platform for positive feedback control of STAT1 expression possibly to amplify or sustain the intracellular IFN signals.

Identification, gene expression and immune function of the novel Bm-STAT gene in virus-infected Bombyx mori

Genes in the signal transducer and activator of transcription (STAT) family are vital for activities including gene expression and immune response. To investigate the functions of the silkworm Bombyx mori STAT (Bm-STAT) gene in antiviral immunity, two Bm-STAT gene isoforms, Bm-STAT-L for long form and Bm-STAT-S for short form, were cloned. Sequencing showed that the open reading frames were 2313bp encoding 770 amino acid residues for Bm-STAT-L and 2202bp encoding 734 amino acid residues for Bm-STAT-S. The C-terminal 42 amino acid residues of Bm-STAT-L were different from the last 7 amino acid residues of Bm-STAT-S. Immunofluorescence showed that Bm-STAT was primarily distributed in the nucleus. Transcription levels of Bm-STAT in different tissues were determined by quantitative PCR, and the results revealed Bm-STAT was mainly expressed in testes. Western blots showed two bands with molecular weights of 70kDa and 130kDa in testes, but no bands were detected in ovaries by using anti-Bm-STAT antibody as the primary antibody. Expression of Bm-STAT in hemolymph at 48h post infection with B. mori macula-like virus (BmMLV) was slightly enhanced compared with controls, suggesting a weak response induced by infection with BmMLV. Hemocyte immunofluorescence showed that Bm-STAT expression was elevated in B. mori nucleopolyhedrovirus (BmNPV)-infected cells. Moreover, resistance of BmN cells to BmNPV was reduced by downregulation of Bm-STAT expression and increased by upregulation. Resistance of BmN cells to BmCPV was not significantly improved by upregulating Bm-STAT expression. Therefore, we concluded that Bm-STAT is a newly identified insect gene of the STAT family. The JAK-STAT pathway has a more specialized role in antiviral defense in silkworms, but JAK-STAT pathway is not triggered in response to all viruses.

Investigation of genetic susceptibility to nonspecific digestive disorder between TYK2, JAK1, and STAT3 genes in rabbits

Tyrosine kinase 2 (TYK2), Janus kinase 1 (JAK1), and signal transducer and activator of transcription 3 (STAT3) all play critical roles in innate and acquired immunity. In this study, we investigated the association between the TYK2, JAK1, and STAT3 genes and genetic susceptibility to nonspecific digestive disorder (NSDD) in rabbits. Five coding single-nucleotide polymorphisms (cSNPs) in TYK2 were detected in New Zealand White (NZW) rabbits with direct sequencing, and two of them, c.1477 (C>T) and c.2013 (C>T), from two different linkage disequilibrium blocks, were further genotyped. A case–control association analysis revealed that allele C (c.1477) in TYK2 increases the risk of NSDD (odds ratio [OR]: 1.21; 95% confidence interval [CI] 1.036–2.145; P=0.021), whereas allele C (c.2013) plays a potentially protective role against NSDD (OR: 0.41; 95% CI 0.2996–0.5705; P<0.001). A gene×gene interaction analysis with a multifactor dimensionality reduction (MDR) method indicated that the combination of c.1477 from TYK2 and c.831 from STAT3 is associated with the susceptibility to NSDD (OR: 7.2567; 95% CI: 4.7306–11.1317; P<0.0001). Together, our data demonstrate that the TYK2 gene is associated with NSDD in rabbits. The TYK2, JAK1, and STAT3 combination model may be used to assist breeders in selecting rabbits.

Exploring Polymorphism in 3’ UTR Region of STAT1 Gene in different Buffalo Breeds

Study was aimed to screen the adult female of different buffalo breeds maintained at organized herd of ICAR-NDRI, Karnal (Haryana) and from field (Punjab, Himachal Pradesh and Chhattisgarh state) for the presence or absence of important genetic variations in 3’UTR region of Signal Transducer and Activator of Transcription 1 (STAT1) gene, known to be associated with mammary gland development and with milk fat% and milk yield in cows. Animals from three breeds of riverine buffaloes Murrah, Gojri and Chhattisgarhi were studied using PCR-RFLP technique with HaeIII restriction enzyme. It revealed monomorphic patterns in the 3’UTR region STAT1 gene studied in all the three breeds of riverine buffaloes, indicating absence of genetic variation at this loci and suggesting that the loci is fixed for buffaloes in contract to exotic cattle breeds.

Novel genetic variants associated with mRNA expression of signal transducer and activator of transcription 3(STAT3) gene significantly affected goat growth traits

The signal transducer and activator of transcription 3 (STAT3) has been shown to play key roles in cell development and apoptosis. In this study, we reported three novel single nucleotide polymorphisms (SNPs) within the STAT3 gene: AC_000176:g.45204C>T (SNP1), g.62058A>G (SNP2), and g.62230C>T (SNP3) in the second, tenth, and eleventh introns, among two Chinese native goat breeds. By using the forced polymerase chain reaction–restriction fragment length polymorphism technique, the SNPs were genotyped and we determined the minor allelic frequencies of Xinong Saanen dairy (XNSN) goats and Hainan black (HN) goats. Haplotype frequencies and linkage disequilibrium (LD) coefficients demonstrated that the CAC and CGT haplotypes had the highest frequencies, and the SNP2 and SNP3 loci had strong LD values in these breeds. Statistical analyses indicated a positive association between either a single SNP locus or and combined genotypes of two- or three-loci associations with some growth traits (P<0.05 or P<0.01). Next, we found the different genotypes were significantly associated with mRNA expression levels of the STAT3 gene. Together, we show that the expression of STAT3 is associated with goat growth traits, which will be beneficial for animal breeding in the future.

Abstract P5-07-01: Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity

Abstract Since the neoplastic phenotype of a cell is largely driven by its gene expression patterns, increasing attention is focused on transcription factors that regulate critical mediators of tumor formation and metastatic progression like the oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). Whereas normal cells have transient activation of STAT3 due to tight control by negative regulators, cancer cells frequently have inappropriate constitutive activation of STAT3 which drives increased expression of genes involved in tumorigenesis. However, little is known about proteins that interact with STAT3 to modulate its function. To identify novel STAT3-interacting proteins, we performed liquid chromatography tandem mass spectrometry-based profiling of STAT3-containing complexes immunoprecipitated from the triple-negative breast cancer cell lines MDA-MB-468 and SUM159PT, which have constitutively active STAT3. We identified granulin (GRN) as a novel STAT3-interacting protein and validated the STAT3-GRN interaction in breast cancer cells by co-immunoprecipitation. To investigate the functional effect of GRN on STAT3 activity, we silenced GRN using small interfering RNA. We found that GRN was necessary for constitutive and maximal cytokine-induced STAT3 transcriptional activity in breast cancer cells. GRN modulated cytokine-induced STAT3 function by enhancing STAT3 DNA binding and increasing the time-integrated amount of STAT3 activation and nuclear translocation. Silencing GRN mirrored the effect of silencing STAT3 on reducing the viability, clonogenesis, and migratory capacity of triple-negative breast cancer cells. Furthermore, GRN mRNA levels were significantly and positively correlated with STAT3 gene expression signatures indicative of STAT3 activation as well as with reduced overall survival in breast cancer patients. These studies used a proteomics approach to identify GRN as a novel STAT3 interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. Citation Format: Jennifer E Yeh, Simion Kreimer, Sarah R Walker, Andrea Richardson, Alexander R Ivanov, David A Frank. Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-01.

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer.

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer.

STAT1-mediated down-regulation of Bcl-2 expression is involved in IFN-γ/TNF-α-induced apoptosis in NIT-1 cells.

Tumor necrosis factor (TNF)-α and interferon (IFN)-γ are the major pro-inflammatory cytokines involved in beta-cell destruction. The fate of islet beta-cells in the cytokine-induced intrinsic mitochondrial apoptotic pathway is determined by the interaction between members of the Bcl-2 family. However, the mechanism through which beta-cell apoptosis is regulated remains unclear. In this study, we treated the murine beta-cell line NIT-1 with TNF-α and IFN-γ and then investigated the regulation of signal transducer and activator of transcription-1 (STAT-1) and expression of the members of the Bcl-2 family in this apoptotic pathway. Results showed that TNF-α and IFN-γ synergistically reduced NIT-1 cell viability. In addition, the decrease in cell growth was due to apoptosis as shown by apoptotic body formation, detected by confocal laser microscope, and a significant increase in Annexin-Vup+ cell percentage, detected by flow cytometry. Combination treatment with TNF-α and IFN-γ caused a remarkable increase in the release of cytochrome c, and in the activation of caspase-9 and caspase-3, as well as, an obvious enhancement in STAT-1 phosphorylation; the treatment, however, resulted in the down-regulation in Bcl-2 expression. The enhancement in STAT-1 activity and a down-regulation in Bcl-2 expression was also observed in MIN6 cells, another murine beta-cell derived line, after cells exposure to the combination of TNF-α and IFN-γ treatment. Knockdown of STAT-1 gene expression by siRNA or inhibition of STAT-1 activation with fludarabine reversed Bcl-2 down-expression and led to a significant decrease in apoptosis in TNF-α- and IFN-γ-treated NIT-1 cells. Taken together, our results suggest that STAT1-mediated down-regulation of Bcl-2 is involved in NIT-1 cell apoptosis induced by combination treatment with TNF-α and IFN-γ.

Knockdown of STAT3 expression in SKOV3 cells by biodegradable siRNA–PLGA/CSO conjugate micelles

Biodegradable and biocompatible poly(d,l-lactic-co-glycolic acid) (PLGA)was conjugated to the 5′-thiol end of signal transducer and activator of transcription 3 (STAT3) small interfering RNA (STAT3–siRNA) via a disulfide bond. In aqueous environments, these siRNA–PLGA conjugates can spontaneously form core/shell type spherical micelles with a particle size of about 200nm. A biodegradable, low molecular weight cationic polymer, chitosan oligosaccharide (CSO), was added to the siRNA–PLGA micelles at different nitrogen to phosphate (N/P) ratios to form stable, spherical siRNA–PLGA/CSO micelles with sizes of 150–180nm. The siRNA–PLGA/CSO micelles were produced via ionic complexation between negatively charged siRNA and positively charged CSO on the outer shell of the micelles. The siRNA–PLGA/CSO micelles exhibited superior cellular uptake and STAT3 gene silencing efficiency in SKOV3 ovarian cancer cells when compared with siRNA/CSO complexes at the same N/P ratios with no significant differences with lipofectamine 2000. Furthermore, the siRNA–PLGA/CSO micelles showed that the efficiencies of cellular uptake and STAT3 gene silencing gradually increased with increasing N/P ratios. The siRNA–PLGA/CSO micelles also inhibited the growth of SKOV3 cells, as well as, promoted apoptosis of the cells. These results indicate that siRNA–PLGA/CSO micelles can be utilized as a novel and efficient siRNA carrier to treat a variety of diseases.

Abstract 13879: Ischemic Postconditioning Confers Cardioprotection through Adiponectin-dependent Mitochondrial STAT3 Activation in Mice

Introduction: Signal transducer and activator of transcription 3 (STAT3) plays a key role in postconditioning (IPo) mediated protection against myocardial ischemia reperfusion injury, but the mechanism by which IPo activates STAT3 is unknown. Adiponectin (APN), a protein with anti-ischemic properties, activates STAT3. We hypothesized that IPo activates mitochondrial STAT3 (MitoSTAT3) via APN signaling. Methods and Results: Wild type (WT) and APN knockout (KO) mice were either sham operated or subjected to 30 min of coronary artery occlusion followed by 2 hours of reperfusion with or without IPo (3 cycles of 10 seconds reperfusion and 10 seconds reocclusion; n=8/group). At the end of reperfusion, KO mice exhibited more severe myocardial injury evidenced as increased infarct size (% of area at risk) 49.2±2.0 vs WT 39.4±3.5, P &lt;0.01; plasma troponin I (ng/ml): KO 72.8±7.6 vs WT 45.7±4.0, P &lt;0.01; worse cardiac function (lower dP/dt max and end-systolic pressure-volume relation, P &lt;0.05); more severely impaired mitochondrial function (reductions in complex IV and complex V protein expression) and more severe reduction of MitoSTAT3 phosphorylation (activation) at site Ser727, P &lt;0.01. IPo significantly attenuated post-ischemic cardiac injury and dysfunction with a concomitant increase in phosphorylated MitoSTAT3 and attenuation of mitochondrial dysfunction in WT (all P &lt;0.05) but not in KO mice. In cultured cardiac H9C2 cells, hypoxic postconditioning (HPo, 3 cycles of 5 min hypoxia and 5 min reoxygenation) significantly attenuated hypoxia/reoxygenation (HR, 3 hours hypoxia/3 hours reoxygenation) induced cell injury (increased apoptotic cell death as % of HR): HR 100.2±0.4 vs HPo 78.2±4.8, P &lt;0.05) and reduced mitochondrial transmembrane potential (% total cells, HR 37.2±4.9 vs HPo 23.5±3.7, P &lt;0.01). APN, adiponectin receptor 1 (AdipoR1), or STAT3 gene knockdown but not AdipoR2 gene knockdown, respectively, abolished HPo cellular protection (all P &lt;0.05 vs. HPo). APN supplementation (10μg/ml) restored HPo protection in cells with APN knockdown but not in cells with AdipoR1or STAT3 gene knockdown. Conclusion: Adiponectin and AdipoR1 signaling are required for IPo to activate myocardial mitochondrial STAT3 to confer cardioprotection.