Phosphorylation Status Of STAT3 Research Articles

IL-10RA governor the expression of IDO in the instruction of lymphocyte immunity.

Indoleamine 2,3-dioxygenase (IDO) impairs anti-pathogen and anti-tumour immunity. Mesenchymal stem cells (MSCs) modulate immunity via IDO but also suppress IFN-γ. While MSC IDO induction by IFN-γ is established, other drivers in this immunosuppressive setting remain unknown. Human bone marrow mesenchymal stem cells (MSCs) with IDO or IL-10RA knockdown were co-cultured with healthy donor T cells to assess immunosuppression. PDAC organoid anticancer activity was also tested in these co-cultures. Co-culturing MSCs with T cells in an IL-10RA-enriched environment enhances IDO expression, resulting in T cell suppression. Moreover, IL-10RA-positive MSCs collected from co-cultures with IL-10 supplementation show increased IDO expression. Conversely, MSCs with IL-10RA knockdown exhibit a significant reduction in IDO RNA and protein expression, as well as STAT3 phosphorylation status, which is a known upstream signalling pathway in IDO gene regulation, in T cell co-cultures. Down-regulation of IL-10RA also inhibits IDO activity in MSCs, resulting in reduced T cell suppression, and enabling the co-cultured T cells to kill PDAC organoids. Our research reveals IL-10RA as a pharmacological target in stromal cells for enhancing T cell-mediated PDAC eradication by downregulating IDO via blocked IL-10/IL-10RA signalling in MSCs. This advances IL-10RA interference in the tumour microenvironment (TME) to restore T cell cytotoxicity against cancers.

24P The STAT3 phosphorylation status discriminates tumor associated populations in myeloid cell tumor-infiltrating model

Tumor associated myeloid cells are major promoters of progression and metastasis. Monocytic and granulocytic myeloid-derived suppressor cells (M-MDSC and G-MDSC) together with tumor associated macrophages (TAM) are among the main contributors to tumor-induced immunosuppression. The influence of the tumor microenvironment on their differentiation is well-accepted but the specific molecular changes leading towards MDSC subsets and TAM are not well-characterized. Here, we describe the main differences between MDSC subsets and TAMs, focused on differential pathways by high-throughput proteomics.

EGCG Inhibits Adipose-Derived Mesenchymal Stem Cells Differentiation into Adipocytes and Prevents a STAT3-Mediated Paracrine Oncogenic Control of Triple-Negative Breast Cancer Cell Invasive Phenotype.

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.

Heat Shock Proteins and PD-1/PD-L1 as Potential Therapeutic Targets in Myeloproliferative Neoplasms.

Simple SummaryMyeloproliferative neoplasms (MPN), which are a heterogeneous group of rare disorders that affect blood cell production in bone marrow, present many significant challenges for clinicians. Though considerable progress has been made, in particular with the JAK1/2 inhibitor ruxolitinib, more effective alternative therapeutic approaches are needed. In the search for new and more efficient therapies, heat shock proteins, also known as stress proteins, and the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint axis have been found to be of great interest in hematologic malignancies. Here, we review the therapeutic potential of stress protein inhibitors in the management of patients diagnosed with MPN and summarize the accumulating evidence of the role of the PD-1/PD-L1 axis in MPN in order to provide perspectives on future therapeutic opportunities relative to the inhibition of these targets.Myeloproliferative neoplasms (MPN) are a group of clonal disorders that affect hematopoietic stem/progenitor cells. These disorders are often caused by oncogenic driver mutations associated with persistent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. While JAK inhibitors, such as ruxolitinib, reduce MPN-related symptoms in myelofibrosis, they do not influence the underlying cause of the disease and are not curative. Due to these limitations, there is a need for alternative therapeutic strategies and targets. Heat shock proteins (HSPs) are cytoprotective stress-response chaperones involved in protein homeostasis and in many critical pathways, including inflammation. Over the last decade, several research teams have unraveled the mechanistic connection between STAT signaling and several HSPs, showing that HSPs are potential therapeutic targets for MPN. These HSPs include HSP70, HSP90 (chaperoning JAK2) and both HSP110 and HSP27, which are key factors modulating STAT3 phosphorylation status. Like the HSPs, the PD-1/PD-L1 signaling pathway has been widely studied in cancer, but the importance of PD-L1-mediated immune escape in MPN was only recently reported. In this review, we summarize the role of HSPs and PD-1/PD-L1 signaling, the modalities of their experimental blockade, and the effect in MPN. Finally, we discuss the potential of these emerging targeted approaches in MPN therapy.

A Ferrocene Derivative Reduces Cisplatin Resistance in Breast Cancer Cells through Suppression of MDR-1 Expression and Modulation of JAK2/STAT3 Signaling Pathway.

Breast cancer is the most common kind of cancer among women in the world. Despite major cancer therapy successes in recent years, cancer cells usually develop mechanisms to survive chemotherapy- induced cell death. Therefore, new strategies are needed to reverse cancer chemoresistance. The aim of this study was to investigate the effect of a recently-synthesized ferrocene derivative named 1-ferrocenyl-3-(4-methylsulfonylphenyl)propen-1-one (FMSP) on cisplatin resistance in MCF-7 cells, focusing on its inhibitory effects on Multi-Drug Resistance-1 (MDR-1) and inflammatory-related STAT3 pathway. Cisplatin-resistant MCF-7 cells were developed and the effect of cisplatin and FMSP on cell viability was examined by MTT assay. RT-PCR and Western blotting analyses were performed to assess the gene and protein expression of MDR-1 as well as phosphorylation of JAK2 and STAT3. Overexpression of MDR1 as well as a marked increase in the level of phosphorylated STAT3 was observed in cisplatin-resistant MCF-7 (MCF-7R) cells. FMSP successfully reduced the MCF-7R cell viability and reversed both MDR1 expression and STAT3 phosphorylation status through which sensitivity of MCF-7R cells to cisplatin treatment was regained. Our results indicated that FMSP may be considered as a promising therapeutic agent for the prevention and management of chemoresistance in breast cancer cells.

Ectopic PD-L1 expression in JAK2 (V617F) myeloproliferative neoplasm patients is mediated via increased activation of STAT3 and STAT5.

Escalated PD-L1 expression has been identified during malignant transformation in a number of cancer types and helps cancer cells escape an effective anti-tumor immune response. The mechanisms underlying escalated production of PD-L1 in many cancers, however, are still far from clear. We studied PD-L1, STAT3 and STAT5 mRNA expression using qRT-PCR in 72 BCR/ABL1 negative myeloproliferative neoplasm (MPN) patients (39 polycythemia vera and 33 essential thrombocythemia). Furthermore, phosphorylation status of STAT3 and STAT5 was studied using immunoblotting in the same patients. All MPN patients were first screened for JAK2 (V617F) mutation by tetra-primer ARMS-PCR, followed by quantification of JAK2 (V617F) mutation burden in all V617F positive MPN patients by ASO-PCR. Patients were screened for BCR/ABL1 fusion gene transcripts to rule out Ph positive status. Our findings showed that mRNA levels of PD-L1 and STAT3 were significantly higher in JAK2 (V617F) MPN patients, while as STAT5 was insignificantly upregulated. STAT3 and STAT5 phosphorylation was seen to be higher in JAK2 (V617F) MPN patients compared to the JAK2 (WT) patients. Upregulation of PD-L1, STAT3 and STAT5 was significantly associated with JAK2 (V617F) percentage in MPN patients. PD-L1, STAT3 and STAT5 expression significantly and positively correlated with JAK2 (V617F) allele burden. In addition, significant coexpression of PD-L1 with STAT3 and STAT5 was observed in MPN patients. In summary, JAK2 (V617F) mutation is accompanied by increased PD-L1 expression and this PD-L1 over expression is mediated by JAK2 (V617F) mainly through STAT3, while as STAT5 may play a minor role.

Overexpression and Targeted Activation of the Protein Phosphatases SHP-1 Abrogates Survival Pathways in Large Granular Lymphocyte Leukemia (LGLL)

Introduction: Aberrant phosphorylation-mediated signaling is a well-established condition involved in the onset and progression of virtually all types of cancer. That not only depends on the over-activation of protein kinases, but also on the lack of the proper counterbalance of protein phosphatases, because of their decreased expression or activity. In turn, this is likely to be due to genetic and epigenetic alterations, or interactions with cellular inhibitors and inhibitory post-translational modifications, respectively. In Large Granular Lymphocyte Leukemia (LGLL), a rare lymphoproliferative disorder characterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, CLPD-NK), constitutive activation of phosphorylation-regulated survival pathways, especially the JAK2/STAT3 axis, but also PI3K/Akt and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since such pathways are modulated by the tyrosine phosphatase SHP-1 under normal conditions, it is conceivable that putative alterations of this phosphatase, and consequent inability to disrupt hyperactive pathways, play a role in LGLL pathogenesis. In this study, we assessed the ability of small molecules inducing the expression and stimulating the activity of SHP-1 to abrogate the survival pathways in LGL by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were collected from untreated patients with LGLL (both T-LGLL and CLPD-NK). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were further separated from PBMCs by using immunomagnetic beads (Miltenyi Biotec). LGLs were incubated either with acetyl-11-keto-β-boswellic acid (AKBA), a pentacyclic triterpene capable of inducing SHP-1 expression, or SC-78, a regorafenib-like derivative specifically stimulating SHP-1 activity, or both at increasing concentrations and discrete time points. After such treatments, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the abundance of SHP-1 and the phosphatase activity thereof. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both AKBA and SC-78 proved effective at eliciting a significant level of apoptosis in the low micromolar range, exhibiting an additive effect when used in combination. Moreover, both compounds activated SHP-1 activity, as demonstrated by in-vitro phosphatase assays. As to the phosphorylation-dependent signals affected by SHP-1 activation, we observed a marked decrease in the phosphorylation status of STAT3, resulting in the downregulation of the STAT3-induced expression of downstream target genes (Mcl-1, survivin, Cyclin D and S1P5), as well as the dephosphorylation of Akt and p65, which confirmed the role of SHP-1 as a functional antagonist of survival pathways in LGLs. Conclusion: Taken together, our results support the newly emerging evidence that the targeted activation of SHP-1, which is already recognized as a tumor suppressor in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening new options for the treatment of LGLL. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens

Therapeutic options to treat primary glioblastoma (GBM) tumors are scarce. GBM tumors with epidermal growth factor receptor (EGFR) mutations, in particular a constitutively active EGFRvIII mutant, have extremely poor clinical outcomes. GBM tumors with concurrent EGFR amplification and active phosphatase and tensin homolog (PTEN) are sensitive to the tyrosine kinase inhibitor erlotinib, but the effect is not durable. A persistent challenge to improved treatment is the poorly understood role of cellular, metabolic, and biophysical signals from the GBM tumor microenvironment on therapeutic efficacy and acquired resistance. The intractable nature of studying GBM cell in vivo motivates tissue engineering approaches to replicate aspects of the complex GBM tumor microenvironment. Here, we profile the effect of erlotinib on two patient-derived GBM specimens: EGFR + GBM12 and EGFRvIII GBM6. We use a three-dimensional gelatin hydrogel to present brain-mimetic hyaluronic acid (HA) and evaluate the coordinated influence of extracellular matrix signals and EGFR mutation status on GBM cell migration, survival and proliferation, as well as signaling pathway activation in response to cyclic erlotinib exposure. Comparable to results observed in vivo for xenograft tumors, erlotinib exposure is not cytotoxic for GBM6 EGFRvIII specimens. We also identify a role of extracellular HA (via CD44) in altering the effect of erlotinib in GBM EGFR + cells by modifying STAT3 phosphorylation status. Taken together, we report an in vitro tissue engineered platform to monitor signaling associated with poor response to targeted inhibitors in GBM.

MiR-125b-1-3p Exerts Antitumor Functions in Lung Carcinoma Cells by Targeting S1PR1.

Background:MicroRNAs (miRNAs) have been extensively studied over the decades and have been identified as potential molecular targets for cancer therapy. To date, many miRNAs have been found participating in the tumorigenesis of non-small cell lung cancer (NSCLC). The present study was designed to evaluate the functions of miR-125b-1-3p in NSCLC cells.Methods:MiR-125b-1-3p expression was detected in tissue samples from 21 NSCLC patients and in NSCLC cell lines using the real-time polymerase chain reaction. A549 cell lines were transfected with a miR-125b-1-3p mimic or miR-125b-1-3p antisense. Cell counting kit-8, wound healing, Matrigel invasion assays, and flow cytometry were used to assess the effects of these transfections on cell growth, migration, invasion, and apoptosis, respectively. Western blotting was used to detect apoptosis-related proteins, expression of S1PR1, and the phosphorylation status of STAT3. Significant differences between groups were estimated using Student's t-test or a one-way analysis of variance.Results:MiR-125b-1-3p was downregulated in NSCLC samples and cell lines. Overexpression of miR-125b-1-3p inhibited NSCLC cell proliferation (37.8 ± 9.1%, t = 3.191, P = 0.013), migration (42.3 ± 6.7%, t = 6.321, P = 0.003), and invasion (57.6 ± 11.3%, t = 4.112, P = 0.001) and simultaneously induced more NSCLC cell apoptosis (2.76 ± 0.78 folds, t = 3.772, P = 0.001). MiR-125b-1-3p antisense resulted in completely opposite results. S1PR1 was found as the target gene of miR-125b-1-3p. Overexpression of miR-125b-1-3p inhibited S1PR1 protein expression (27.4 ± 6.1% of control, t = 4.083, P = 0.007). In addition, S1PR1 siRNA decreased STAT3 phosphorylation (16.4 ± 0.14% of control, t = 3.023, P = 0.015), as in cells overexpressing miR-125b-1-3p (16.7 ± 0.17% of control, t = 4.162, P = 0.026).Conclusion:Our results suggest that miR-125b-1-3p exerts antitumor functions in NSCLC cells by targeting S1PR1.

Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3<sup/>.

Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity.

Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient’s breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals and osteopenia. Leptin was shown to play an important role in bone growth. It can also regulate the function of chondrocytes. Changes in leptin and Ob-R levels in AIS patients have been reported in several studies. The underlying mechanisms between the dysfunction of peripheral leptin signaling and abnormal chondrocytes remain unclear; The following parameters were evaluated in AIS patients and the control groups: total serum leptin levels; Ob-R expression in the plasma membrane of primary chondrocytes; JAK2 and STAT3 phosphorylation status. Then, we inhibited the lysosome and proteasome and knocked down clathrin heavy chain (CHC) expression in primary chondrocytes isolated from girls with AIS and evaluated Ob-R expression. We investigated the effects of leptin combined with a lysosome inhibitor or CHC knockdown in primary chondrocytes obtained from AIS patients; Compared with the controls, AIS patients showed similar total serum leptin levels, reduced JAK2 and STAT3 phosphorylation, and decreased cartilage matrix synthesis in the facet joint. Lower metabolic activity and lower membrane expression of Ob-R were observed in primary chondrocytes from the AIS group than in the controls. Lysosome inhibition increased the total Ob-R content but had no effect on the membrane expression of Ob-R or leptin’s effects on AIS primary chondrocytes. CHC knockdown upregulated the membrane Ob-R levels and enhanced leptin’s effects on AIS primary chondrocytes; The underlying mechanism of chondrocytes that are hyposensitive to leptin in some girls with AIS is low plasma membrane Ob-R expression that results from an imbalance between the rate of receptor endocytosis and the insertion of newly synthesized receptors into the membrane.

Antitumoral Activity Of Nitric Oxide-Releasing Compounds

Despite significant improvements in the conventional anti-ovarian cancer therapies, tumor cell resistance to various cytostatic drugs remains a relevant problem. Therefore, the new cancer treatment strategies are being developed. Among many agents that have been studied for their potential anti-cancer activity, the most promising are the nitric oxide (NO) donors-syntethic compounds that release NO in vivo and/or in vitro. We have evaluated the effect of NO donors on the SK-OV-3 and OVCAR-3 ovarian cancer cell lines. We assessed some of the cancer cells' specific features: the uncontrolled proliferation, over-activation of particular signaling proteins, high resistance to therapeutics and elevated expression and secretion of invasiveness/metastatic factors. Two members of NONOates family were used: SPER/NO and DETA/NO. Cancer cell lines were cultured with different concentrations of NO donors. The cytotoxic, pro-apoptotic activity of NO donors and their impact on the phosphorylation status of STAT-3 and AKT in cells were determined. The expression of VEGF-A, MMPs and TGF-β was also evaluated. NO donors inhibited ovarian cancer cells growth making them also more susceptible to the cisplatin cytotoxic activity. Moreover, both NO donors induced apoptosis of cells and decreased activity of signaling proteins (STAT3 and AKT). Similarly, SPER/NO and DETA/NO lowered the secretion of pro-metastatic factors, responsible for cancer cells invasiveness. The obtained results show that both NO donors demonstrated a wide range of action on both ovarian cancer cell lines. Therefore, they have a high potential of being a supporting compounds in the cancer therapies.

Synergy of leptin/STAT3 with HER2 receptor induces tamoxifen resistance in breast cancer cells through regulation of apoptosis-related genes.

Tamoxifen is a major treatment modality for estrogen receptor positive breast cancer, but the occurrence of resistance remains a problem. Recently, obesity-related leptin has been found to interfere with tamoxifen in breast cancer MCF-7 cells. In the present study we investigated the effect of leptin on three tamoxifen-treated breast cancer cell types (i.e., MDA-MB-231, MCF-7 and MCF-7/HER2). The effect of tamoxifen/leptin treatment was evaluated using a MTT cell viability assay. mRNA expression was assessed by real time PCR and protein expression by Western blotting. WWOX, Survivin and BCL2 gene promoter activities were evaluated by chromatin immunoprecipitation. Cell viability assays revealed that estrogen receptor negative MDA-MB-231 cells were resistant, that estrogen receptor positive MCF-7 cells were sensitive and that MCF-7/HER2 cells were relatively resistant to tamoxifen, while leptin co-administration 'rescued' MCF-7 and, especially, MCF-7/HER2 cells from the anti-proliferative effect of tamoxifen. The cell lines also exhibited a different phosphorylation status of STAT3, a transcription factor that is activated by the obesity related leptin receptor b (Ob-Rb). Most importantly, chromatin immunoprecipitation assays revealed differential STAT3 binding to the anti-apoptotic BCL2 and pro-apoptotic WWOX gene promoters in MCF-7 and MCF-7/HER2 cells, leading to concomitant modifications of its mRNA/protein expression levels, thus providing a selective advantage to HER2 over-expressing MCF-7/HER2 cells after treatment with tamoxifen and tamoxifen plus leptin. Our study provides novel evidence indicating that synergy between the leptin/Ob-Rb/STAT3 signalling pathway and the HER2 receptor protects tamoxifen-treated HER2 over-expressing cells from the inhibitory effect of tamoxifen through differential regulation of apoptosis-related genes.

Regulation of Natural Killer Cell Responses By Dendritic Cells Via Lymphotoxin-Alpha, Interleukin-12, and Tumor Growth Factor-Beta

Dendritic cells (DC) induce T-cell responses in cancer patients. However, less is known about the role of DC-vaccines in shaping natural killer (NK) cell responses. In this study, we sought to investigate the activity of NK cells following vaccination with DC. Patients with chronic lymphocytic leukemia (CLL) and melanoma were vaccinated with tumor-antigen loaded monocytes-derived DC and NK cell activity was analyzed before and two weeks after vaccination. Compared with pre-vaccination samples, NK cells expressed reduced levels of several activation markers and produced less IFNγ (p=0.03). To further explore the mechanism of the reduced NK cell responses, DC were differentiated from monocytes in GM-CSF, IL-4 and LPS and thereafter co-cultured with autologous NK cells in vitro. In these experiments, DC-mediated inhibition of NK cells was independent of cell-contact but dependent on the phosphorylation status of STAT3 in DC. Compared with untreated DC, STAT3-inhibited-DC produced higher amounts of lymphotoxin-alpha (LTα, 5.5±5.0 vs. 38.1±25.9 pg/ml; p=0.005) and IL-12 (29.6±39.0 vs. 176.8±96.8 pg/ml; p=0.005) and reduced amounts of tumor growth factor-beta (TGFβ, 495.0±138.7 vs. 300.4±141.8 pg/ml; p=0.03) and did not suppress NK cell proliferation (p=0.03), cytotoxicity (p=0.0003), and IFNγ production (p=0.02). Neutralization of TGFβ resulted in elevated production of LTα (2.25±4.5 vs. 251.5±12.0 pg/ml; p=0.03) and IL-12 (0.25±0.5 vs. 15.0±1.82 pg/ml; p=0.02) by DC and addition of recombinant LTα or IL-12 in co-cultures of DC and NK cells was indispensable to maintain NK cell activity. Compared with LPS, DC matured with a cocktail of R848, poly I:C, and IFNγ showed reduced levels of pSTAT3 and produced higher levels of LTα and IL-12 and did not inhibit NK cell activity. These results show that LTα, IL-12 and TGFβ are involved in the cross-talk between NK cells and DC. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer. DisclosuresÖsterborg:Celgene: DMC memeber of Celgene-initiated phase 3 trials Other.

Antitumor Efficacy of the Anti-Interleukin-6 (IL-6) Antibody Siltuximab in Mouse Xenograft Models of Lung Cancer

Interleukin-6 (IL-6) can activate downstream signaling pathways in lung cancer cells, such as the STAT3 pathway, and is reported to be produced by tumor cells with activating EGFR mutations. We examined IL-6/STAT3 in lung cancer tumor tissues and the effects of siltuximab, a neutralizing antibody to human IL-6, in mouse models of lung cancer. IL-6 and STAT3 activation levels were compared with tumor histology and presence of KRAS mutations in snap-frozen, non-small-cell lung cancer tumors. The effects of siltuximab alone or in combination with erlotinib were examined in mouse xenograft models constructed using three cell line xenograft models and one primary explant mouse model. We examined the influence of cancer-associated fibroblasts (CAFs) on tumor growth and siltuximab effects. IL-6 levels were higher in tumors of squamous cell versus adenocarcinoma histology and were not associated with presence of KRAS mutations. Tyrosine phosphorylation status of STAT3 did not correlate with tumor IL-6 levels. Serine phosphorylation of STAT3 was correlated with KRAS mutation status. Both tumor and stromal cells contributed to total IL-6 within tumors. Siltuximab had minimal effect as a single agent in xenografts with tumor cells alone; however, in models coadministered with CAFs, siltuximab had more potent effects on tumor inhibition. We observed no effects of combined erlotinib and siltuximab. IL-6 is elevated in subsets of human NSCLCs, especially with squamous cell histology. Tumors supported by stromal production of IL-6 seem to be the most vulnerable to tumor growth inhibition by siltuximab.

Galiellalactone Is a Direct Inhibitor of the Transcription Factor STAT3 in Prostate Cancer Cells

The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing phosphorylated STAT3 (pSTAT3). However, the molecular mechanism of this STAT3-inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-Sepharose beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and the nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (Cys-367, Cys-468, and Cys-542). Here we demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.

Gene Silencing of SOCS3 by siRNA Intranasal Delivery Inhibits Asthma Phenotype in Mice

Suppresors of cytokine signaling (SOCS) proteins regulate cytokine responses and control immune balance. Several studies have confirmed that SOCS3 is increased in asthmatic patients, and SOCS3 expression is correlated with disease severity. The objective of this study was to evaluate if delivering of SOCS3 short interfering RNA (siRNA) intranasally in lungs could be a good therapeutic approach in an asthma chronic mouse model. Our results showed that intranasal treatment with SOCS3-siRNA led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion, a reduction in lung collagen, which are prominent features of airway remodeling. The mechanism implies JAK/STAT and RhoA/Rho-kinase signaling pathway, because we found a decreasing in STAT3 phosphorylation status and down regulation of RhoA/Rho-kinase protein expression. These results might lead to a new therapy for the treatment of chronic asthma.

Hyperosmotic stress sustains cytokine-stimulated phosphorylation of STAT3, but slows its nuclear trafficking and impairs STAT3-dependent transcription

Persistent STAT3 phosphorylation and nuclear retention are hallmarks of a range of pathologies suggesting the importance of STAT3 transcriptional responses in disease progression. Since hyperosmotic stress (HOS) is a hallmark of diseases such as diabetes and asthma, we analysed the impact of HOS on cytokine-stimulated STAT3 signalling. In contrast to transient STAT3 Y705 and S727 phosphorylation in murine embryonic fibroblasts (MEFs) stimulated by the interleukin-6 family cytokine, leukemia inhibitory factor (LIF), under non-stress conditions, HOS induced by sorbitol treatment increased STAT3 S727 but not Y705 phosphorylation. Strikingly, combined LIF+HOS treatment stimulated persistent STAT3 Y705 and S727 phosphorylation and nuclear localisation, but STAT3 nuclear accumulation was slowed during HOS, likely reflecting the mislocalisation of Ran and importin-α3 during HOS that also reduced the nuclear localisation of classical importin-α/β-recognised nuclear import cargoes. Strikingly, combined LIF+HOS exposure, even though stimulating STAT3 phosphorylation and nuclear accumulation did not elicit a transcriptional output, as demonstrated by qPCR analyses of its target genes SOCS3 and c-Fos. Our analysis thus shows for the first time that HOS can disconnect nuclear, phosphorylated STAT3 from transcriptional outcomes, and emphasizes the importance of assessing STAT3 target gene changes in addition to STAT3 phosphorylation status and localisation.

The JAK2-Selective Inhibitor NS-018 Preferentially Suppresses CFU-GM Colony Formation By Bone-Marrow Cells From High-Risk Myelodysplastic Syndrome Patients

BackgroundCell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. MethodsBone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. ResultsWe first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. ConclusionOur results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. [Display omitted] Disclosures:Kodama:Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.