STAT3 Knockdown Research Articles

A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways.

Natural products and their derivatives have long been used as pharmacological agents in the fight against cancer. Human natural killer (NK) cells are critical in our immune system in that they are capable of destroying tumor cells directly. However, there are few reports that elucidate the role of natural products in activating NK cells. In this study, we discovered that a synthetic disaccharide derivative of diphyllin, 4-O-{[2′′,3′′,4′′-tri-O-acetyl-α-D-arabinopyranosyl-(1′′→4′)]-2′,3′-di-O-acetyl-α-L-rhamnopyranosyl}diphyllin (TAARD), can alone stimulate interferon (IFN)-γ secretion in primary human NK cells and the NKL cell line. Additionally, it had an additive effect with IL-12 or IL-15 on IFN-γ production, but little adverse effects on NK cells. Mechanistically, TAARD induced the phosphorylation of NF-κB and STAT3, resulting in their binding on the IFNG promoter, which was dependent on TLR1 and TLR3 signaling, respectively. STAT3 and NF-κB knockdown with lentivirus shRNA as well as the NF-κB-specific inhibitor, N-tosyl-l-phenylalaninechloromethyl ketone, significantly suppressed TAARD-induced IFN-γ generation in primary NK cells. Blockade of TLR1 and TLR3 with neutralizing antibodies considerably decreased TAARD-induced activation of NF-κB and STAT3, respectively, as well as IFN-γ generation in NK cells. Collectively, our data suggest that TAARD can induce NK cell IFN-γ production through TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering its potential use as an agent for cancer prevention or treatment.

Abnormally expressed JunB transactivated by IL-6/STAT3 signaling promotes uveal melanoma aggressiveness via epithelial-mesenchymal transition.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, and it carries a high risk of metastasis and mortality. Various proinflammatory cytokines have been found to be significantly increased in the aqueous humor or vitreous fluid of UM patients; however, the role of these cytokines in UM metastasis remains elusive. In the present study, we found that long-term interleukin (IL)-6 exposure promoted the migration and invasion of UM cells, diminished cell–cell adhesion, and enhanced focal adhesion. Moreover, IL-6 treatment decreased the membranous epithelial marker TJP1 and increased the cytoplasmic mesenchymal marker Vimentin. Further investigation demonstrated that JunB played a critical role in IL-6-induced UM epithelial–mesenchymal transition (EMT). In UM cells, the expression of JunB was significantly up-regulated during the IL-6-driven EMT process. Additionally, JunB induction occurred at the transcriptional level in a manner dependent on phosphorylated STAT3, during which activated STAT3 directly bound to the JunB promoter. Importantly, the knockdown of STAT3 prevented the IL-6-induced EMT phenotype as well as cell migration and invasion, whereas JunB overexpression recovered the attenuated aggressiveness of UM cells. Similarly, with IL-6 stimulation, the stable overexpression of JunB strengthened the migratory and invasive capabilities of UM cells and induced the EMT-promoting factors (Snail, Twist1, matrix metalloproteinase (MMP)-2, MMP-14, and MMP-19). Analysis of The Cancer Genome Atlas (TCGA) database indicated that JunB was positively correlated with IL-6 and STAT3 in UM tissues. The present study proposes an IL-6/STAT3/JunB axis leading to UM aggressiveness by EMT, which illustrates the negative side of inflammatory response in UM metastasis.

Abstract 4319: Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling

Abstract Inactivating mutations of the breast cancer type 1 susceptibility protein (BRCA1) have been implicated in breast and ovarian cancer (OC) initiation. Having observed differential response of OC cells to histone deacetylase inhibitors (HDACi) dependent on the presence of a functional BRCA1, we mapped promoters marked by acetylation of lysine 9 or lysine 27 of histone H3 (H3K9ac, H3K27ac) by ChIP-sequencing, and measured gene expression by RNA-sequencing in the isogenic OC cell lines UWB1.289 carrying an inactivating mutation of BRCA1 (BRCA1-null) and UWB1.289 cells with restored BRCA1 gene (BRCA1+). Significant differences in promoter levels of H3K9 and H3K27 acetylation were associated with marked alteration of gene expression levels between BRCA-null and BRCA1+ cells and in response to the HDACi, entinostat. Pathway analyses revealed cellular movement, cellular development, cellular growth and proliferation, TGFβ1, TNF, and INF-γ among the top cellular/molecular functions and upstream regulators enriched by the absence of BRCA1 among H3K9ac differentially marked and differentially expressed genes. Importantly, the same pathways and regulators were enriched in response to entinostat in BRCA1+ cells, indicating that changes in gene expression affecting these functions and regulators in BRCA1-null cells depend on promoter-associated histone acetylation. Integrated analyses revealed that TGFβ1 and IFN-γ were also activated in BRCA1-null/or low vs. BRCA1-normal ovarian tumors in the TCGA database. IFN-γ target genes were upregulated at baseline and responded less to IFN-γ stimulation in BRCA1-null vs. BRCA1+ cells. Binding of HDAC1 to promoters of IFN-γ target genes was increased in BRCA1-null cells. Similarly, the BRCA1-null breast cancer cell line HCC1937 responded less to IFN-γ stimulation compared with HCC1937 cells in which BRCA1 was restored. The knockdown of STAT1, the main IFN-γ signaling transduction molecule, inhibited BRCA1+ cell proliferation, but did not affect BRCA1-null cells. We conclude that the transcriptome of BRCA1-null cells and tumors is altered through changes in histone acetylation affecting cancer-related pathways, including IFN-γ cellular responses. These changes could alter tumor progression in BRCA-null OC and impact response to immunotherapy. Citation Format: Horacio Cardenas, Jessica Thomes Pepin, Guanglong Jiang, Salvatore Condello, Kenneth P. Nephew, Yunlong Liu, Daniela Matei. Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4319.

Interleukin-6–mediated trans-signaling inhibits transforming growth factor-β signaling in trabecular meshwork cells

Glaucoma is one of the major causes of blindness, and transforming growth factor-β2 (TGF-β2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-β2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-β in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-β signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-β2 stimulation. To elucidate the relationship between TGF-β2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-β-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-β-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-β-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-β were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-β-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-β signaling in HTM cells.

Interleukin-5 suppresses Vascular Endothelial Growth Factor-induced angiogenesis through STAT5 signaling

Interleukin-5 (IL-5) is best known as key regulator in eosinophil-associated diseases such as asthma. While a connection to vascular changes in eosinophil-associated lung diseases is still elusive, recent evidence suggests that IL-5 may have an atheroprotective role. Here, we report an unexpected anti-angiogenic potential of IL-5 on vascular endothelial cells in vitro. IL-5 significantly inhibited fundamental functions of human lung microvascular endothelial cells (HMVEC-L) in vessel formation including VEGF-induced endothelial cell proliferation, migration and tube formation. Knockdown (KD) of STAT5 abolished the direct anti-angiogenic effect of IL-5 on VEGF-induced endothelial cell proliferation, migration and tube formation.

STAT3-siRNA induced B16.F10 melanoma cell death: more association with VEGF downregulation than p-STAT3 knockdown

STAT3 knockdown by small interfering RNA (siRNA) has been described to inhibit carcinogenic growth in various types of tumors. Earlier we have reported delivery of siRNA by oleic acid- and stearic acid-modified-polyethylenimine and enhancement of silencing of STAT3 by small interfering RNA (siRNA) in B16.F10 melanoma cell lines and consequent tumor suppression. Present investigation mainly focused on the downstream events involved in B16.F10 melanoma cell death and consequent tumor suppression following knockdown of p-STAT3 by siRNA. Lipid-substituted polyethylenimine (PEI)-p-STAT3-siRNA were prepared and characterized by measuring its N/P ratio, zeta potential, size, association and dissociation with siRNA. B16.F10 melanoma cells were treated with six different concentrations of PEI-p-STAT3-siRNA (200, 100, 50, 25, 12.5 and 6.25 nM). Downregulation of p-STAT3 and VEGF were studied using western blot and ELISA in association with the melanoma cell death. PEI-p-STAT3-siRNA hydrodynamic diameter ranged from 110 to 270 nm. PEI assisted p-STAT3-siRNA delivery exhibited increased uptake by B16.F10, when analyzed by fluorescent and confocal microscopy along with flowcytometry. It induced concentration-dependent knockdown of the p-STAT3 that also downregulated VEGF expression in similar fashion and induced B16.F10 cell death. Higher concentrations of p-STAT3-siRNA appear to significantly downregulate the VEGF expression via p-STAT3 knockdown. Decreasing survival of B16.F10 cells with the increasing concentration of p-STAT3-siRNA significantly correlated with VEGF downregulation, not with p-STAT3 expression. Data suggest that VEGF downregulation following knockdown of p-STAT3 may be a key event in survival reduction in B16.F10 melanoma cells and.

Japanese encephalitis virus induces apoptosis by inhibiting Foxo signaling pathway

Japanese encephalitis virus (JEV) infection induces brain tissue disease characterized by neuron death. however, little is known about the underlying mechanism. Using RNA sequencing, we profiled global mRNA expression changes in response to in vitro and in vivo JEV infection. Integration analysis of in vitro and in vivo mRNA transcriptome revealed that JEV infection regulated apoptosis-related Foxo signaling pathway. Foxo expression was reduced by JEV infection in vitro and in vivo. Knockdown of Foxo promoted apoptosis, while its overexpression reduced apoptosis in JEV-infected Neuro-2a cells. JEV infection in Neuro-2a cells decreased the expression of Foxo downstream genes including pro-apoptotic protein Bim, anti-apoptotic protein Bcl-6 and p21. Overexpression of anti-apoptotic proteins Bcl-6 and p21 repressed JEV-induced apoptosis. These findings suggest that Foxo primarily exerts an anti-apoptotic function via Bcl-6 and p21 in JEV-infected Neuro-2a cells. A STAT3 binding site was identified in the promoter region of Foxo by TFBIND software and confirmed by ChIP and reporter assays. JEV infection reduced STAT3 expression as well as its binding at the Foxo promoter compared to mock infection in Neuro-2a cells. Moreover, STAT3 knockdown reduced Foxo promoter activity and Foxo expression. Therefore, JEV reduced Foxo expression, at least in part, by downregulating STAT3. Taken together, we found that JEV induced cell apoptosis by inhibiting STAT3-Foxo-Bcl-6/p21 pathway, which provides a novel insight into JEV-caused encephalitis.

Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy

Inhibition of vascular endothelial growth factor (VEGF) in retinopathy of prematurity (ROP) raises concerns for premature infants because VEGF is essential for retinovascular development as well as neuronal and glial health. This study tested the hypothesis that endothelial cell-specific knockdown of VEGF receptor 2 (VEGFR2), or downstream STAT3, would inhibit VEGF-induced retinopathy without delaying physiologic retinal vascular development. We developed an endothelial cell-specific lentiviral vector that delivered shRNAs to VEGFR2 or STAT3 and a green fluorescent protein reporter under control of the VE-cadherin promoter. The specificity and efficacy of the lentiviral vector-driven shRNAs were validated in vitro and in vivo. In the rat oxygen-induced retinopathy model highly representative of human ROP, the effects of endothelial cell knockdown of VEGFR2 or STAT3 were determined on intravitreal neovascularization (IVNV), physiologic retinal vascular development [assessed as area of peripheral avascular/total retina (AVA)], retinal structure, and retinal function. Targeted knockdown of VEGFR2 or STAT3 specifically in retinal endothelial cells by subretinal injection of lentiviral vectors into postnatal day 8 rat pup eyes efficiently inhibited IVNV, and knockdown of VEGFR2 also reduced AVA and increased retinal thickness without altering retinal function. Taken together, our results support specific knockdown of VEGFR2 in retinal endothelial cells as a novel therapeutic method to treat retinopathy.

Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins.

Hypoxia-mediated tumor progression, metastasis, and drug resistance are major clinical challenges in ovarian cancer. Exosomes released in the hypoxic tumor microenvironment may contribute to these challenges by transferring signaling proteins between cancer cells and normal cells. We observed that ovarian cancer cells exposed to hypoxia significantly increased their exosome release by upregulating Rab27a, downregulating Rab7, LAMP1/2, NEU-1, and also by promoting a more secretory lysosomal phenotype. STAT3 knockdown in ovarian cancer cells reduced exosome release by altering the Rab family proteins Rab7 and Rab27a under hypoxic conditions. We also found that exosomes from patient-derived ascites ovarian cancer cell lines cultured under hypoxic conditions carried more potent oncogenic proteins-STAT3 and FAS that are capable of significantly increasing cell migration/invasion and chemo-resistance in vitro and tumor progression/metastasis in vivo. Hypoxic ovarian cancer cells derived exosomes (HEx) are proficient in re-programming the immortalized fallopian tube secretory epithelial cells (FT) to become pro-tumorigenic in mouse fallopian tubes. In addition, cisplatin efflux via exosomes was significantly increased in ovarian cancer cells under hypoxic conditions. Co-culture of HEx with tumor cells led to significantly decreased dsDNA damage and increased cell survival in response to cisplatin treatment. Blocking exosome release by known inhibitor Amiloride or STAT3 inhibitor and treating with cisplatin resulted in a significant increase in apoptosis, decreased colony formation, and proliferation. Our results demonstrate that HEx are more potent in augmenting metastasis/chemotherapy resistance in ovarian cancer and may serve as a novel mechanism for tumor metastasis, chemo-resistance, and a point of intervention for improving clinical outcomes.

Down-regulation of STAT3 induces the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells

BackgroundSignal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers. Aberrant activity of STAT3 correlates with tumor growth, invasion and metastasis, which makes it a potential therapeutic target of cancer. To explore the biological role of STAT3 in esophageal cancer, we used small hairpin RNA to knockdown the expression of the STAT3 gene in the esophageal carcinoma ECA109 cell line and the cell apoptosis, cell cycle and cell migration were investigated.MethodsThe cell apoptosis was tested using DNA ladder, mitochondrial membrane potential assay, TUNEL assay, annexin V-PI staining. Cell cycle phases were estimated using flow cytometry analysis. The mRNA and proteins related to apoptosis and cell cycle were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. And cell migration was investigated by in vitro Transwell assay. The data were analyzed with two-sample Student’s t test and ANOVA followed by the LSD post hoc test.ResultsOur results showed that knockdown of STAT3 in ECA109 cells induced noticeable apoptotic morphological changes like cell shrinkage, apoptotic vacuoles, membrane blebbing time-dependently. In addition, DNA ladder, TUNEL assay, Annexin V-PI staining and declined level of cleaved Caspase-3 indicated that down-regulation of STAT3 could induce apoptosis in ECA109 cells. Flow cytometry analysis displayed the induction of G1-phase cell cycle arrest of ECA109 cells by STAT3 decreasing, consistent with the descend of c-Myc and cyclin D1 in protein levels. Furthermore, STAT3 knockdown suppressed the expression of matrix metalloproteinases-9, sushi domain containing 2 and urokinase plasminogen activator in ECA109 cells and inhibited cell migration ability.ConclusionsKnockdown of STAT3 could induce the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells, and inhibit the migration ability of cells as well.

Effects of Interleukin-6 on posterior capsular opacification

The purpose of this work was to determine the effects of interleukin-6 (IL-6) on the development of posterior capsular opacification (PCO) in vitro and in vivo. Western blot and real-time PCR were used to test the IL-6-induced epithelial-mesenchymal transition (EMT) marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM) markers fibronectin (Fn) and type I collagen (COL-1), transforming growth factor β2 (TGF-β2), and the activation and role of the JAK/STAT3 signaling pathway in human lens epithelial cells (HLECs). Immunocytofluorescence staining was performed to detect gp130 and IL-6Rα expression in HLECs. Rat PCO models were then established to examine the impact of STAT3 knockdown by shRNA adeno-associated virus on PCO development, and immunohistochemical staining was performed to detect the expression of Fn in the anterior and posterior capsule in vivo. We found that IL-6 promotes the expression of Fn, COL-1, TGF-β2, p-JAK2 and p-STAT3 in HLECs but exerts little effect on α-SMA. The JAK/STAT3 inhibitor WP1066 effectively suppressed the IL-6-induced expression of Fn and COL-1 in lens epithelial cells. STAT3 knockdown effectively inhibited the development of PCO in rats and significantly reduced the expression of Fn in the anterior and posterior capsule. These data suggest that IL-6 contributes to the development of PCO by promoting TGF-β2 activation and ECM synthesis through a JAK/STAT3 signaling-dependent mechanism. Furthermore, inhibiting JAK/STAT3 signaling effectively impairs both PCO development in rats and ECM synthesis in the lens capsule.

Prolonged Activation of STAT3 Mediates the IL6‐Induced Loss of Stress Fibers and Increase in Endothelial Permeability

Interleukin 6 (IL6) promotes endothelial barrier breakdown in vitro, as well as, in vivo. However, the signaling mechanisms that mediate this response are not fully understood. IL6 trans‐signaling promotes a prolonged activation of JAK/STAT3 in human umbilical vein endothelial cells (HUVECs) that lasts for at least 24 hours, leading to a sustained increase in endothelial permeability. IL6‐induced STAT3 phosphorylation and loss of barrier function is prevented when cells are pre‐treated with the JAK inhibitor, ruxolitinib, suggesting that JAK activity is required for these responses. Furthermore, siRNA‐mediated STAT3 knockdown attenuates the loss of IL6‐induced loss of barrier function, demonstrating a critical role for STAT3 in this response. Immunofluorescence analysis showed that this change in barrier function correlates with a marked loss of junctional ZO‐1 (but not changes in ZO‐1 protein expression). Phalloidin staining demonstrates that IL‐6 also promotes a JAK, and STAT3, dependent loss of actin stress fibers and a marked reduction of the cortical actin signal. A prolonged activation of STAT3 is required for this response, because treatment of HUVECs with ruxolitinib up to 4 hours after activation of IL6 trans‐signaling, completely reverses the IL6‐induced loss of barrier function. STAT3 activation kinetics are regulated by a negative feedback loop that is mediated by the suppressor of cytokine signaling 3 (SOCS3). Consistently, siRNA‐mediated SOCS3 knockdown increases IL6‐induced STAT3 phosphorylation in HUVECs and exacerbates the IL6‐induced increase in permeability. Collectively, our data shows that prolonged activation of STAT3 is required for the IL6‐induced barrier breakdown, and loss of junctional ZO‐1, cortical actin and actin stress fibers in endothelial cells.Support or Funding InformationThis project was supported by an American Heart Association Science Development Grant 13SDG17100110 and funds from the Department of Molecular and Cellular Physiology, Albany Medical Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

STAT5 inhibition induces TRAIL/DR4 dependent apoptosis in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, aggressive, heterogeneous, Non-Hodgkin's lymphoma with poor prognosis and inadequate response to current therapies. Recent sequencing studies indicate a prevalence of activating mutations in the JAK/STAT signaling pathway. Oncogenic mutations in STAT5B, observed in approximately one third of cases of multiple different PTCL subtypes, correlate with inferior patient outcomes. Therefore, interest in the development of therapeutic strategies for targeting STAT5 in PTCL is warranted. In this study, we show that the drug pimozide inhibits STAT5 in PTCL, leading to apoptotic cell death by means of the TRAIL/DR4 dependent extrinsic apoptotic pathway. Pimozide induced PTCL cell death is caspase 8 dependent, increases the expression of the TRAIL receptor, DR4, on the surface of pre-apoptotic PTCL cells, and enhances TRAIL induced apoptosis in a TRAIL dependent manner. In parallel, we show that mRNA and protein levels of intrinsic pathway BCL-2 family members and mitochondrial membrane potential remain unaffected by STAT5 knockdown and/or inhibition. In primary PTCL patient samples, pimozide inhibits STAT5 activation and induces apoptosis. Our data support a role for STAT5 inhibition in PTCL and implicate potential utility for inhibition of STAT5 and activation of the extrinsic apoptotic pathway as combination therapy in PTCL.

Mesenchymal Stem Cells Inhibited Dendritic Cells Via the Regulation of STAT1 and STAT6 Phosphorylation in Experimental Autoimmune Uveitis.

We have previously reported that MSCs inhibited experimental autoimmune uveitis (EAU) in rodent models induced by either uveitogenic antigens or antigen-specific T cells. In this study, we explored the inhibitory mechanisms of MSCs on dendritic cells (DCs) in EAU. We collected the DCs from the lymph nodes of MSC treated or untreated EAU rats, as well as bone marrow derived DCs cultured in vitro with or without MSC treatment. The levels of costimulatory molecules of CD80, CD86, CD40, OX40L and suppressors of cytokine signaling (SOCS1, SOCS2, and SOCS3) on these DCs were analyzed by flow Cytometry. The expression of CCR-7 and MMP-9 was examined by real time PCR and western blots. Total proteins of STAT1 and STAT6 signaling molecules and their phosphorylation were examined by western blots. ShRNA of STAT1 and STAT6 were respectively employed to explore the influence of STAT1 and STAT6 knockdown on DCs. MSC treatment down-regulated the expression of CD80, CD86, CD40, and OX40L, as well as CCR-7 and MMP-9, but increased the levels of SOCS1, SOCS 2, and SOCS3 on DCs. STAT1 phosphorylation was reduced while STAT6 phosphorylation was enhanced in MSC treated DCs. Moreover, MSC treatment and STAT1 shRNA equally reduced CCR-7 and MMP-9 levels in DCs, and inhibited the proliferation of R16-specific T cells. In contrast, knockdown of STAT6 in DCs by STAT6 shRNA increased the expression of CD80 and CD86 and accelerated the proliferation of R16-specific T cells. MSCs inhibit DC maturation by regulating Stat1 and Stat6 phosphorylation in EAU.

EAF2 and p53 Co-Regulate STAT3 Activation in Prostate Cancer

The tumor suppressor genes EAF2 and p53 are frequently dysregulated in prostate cancers. Recently, we reported that concurrent p53 nuclear staining and EAF2 downregulation were associated with high Gleason score. Combined loss of EAF2 and p53 in a murine model induced prostate tumors, and concurrent knockdown of EAF2 and p53 in prostate cancer cells enhanced proliferation and migration, further suggesting that EAF2 and p53 could functionally interact in the suppression of prostate tumorigenesis. Here, RNA-seq analyses identified differentially regulated genes in response to concurrent knockdown of p53 and EAF2. Several of these genes were associated with the STAT3 signaling pathway, and this was verified by significantly increased p-STAT3 immunostaining in the Eaf2−/−p53−/− mouse prostate. STAT3 knockdown abrogated the stimulation of C4-2 cell proliferation by concurrent knockdown of EAF2 and p53. Furthermore, immunostaining of p-STAT3 was increased in human prostate cancer specimens with EAF2 downregulation and/or p53 nuclear staining. Our findings suggest that simultaneous inactivation of EAF2 and p53 can act to activate STAT3 and drive prostate tumorigenesis.

Overcoming enzalutamide-resistance by specific knockdown of transcription-factor STAT5 in prostate cancer

Overcoming enzalutamide-resistance by specific knockdown of transcription-factor STAT5 in prostate cancer

The CXCR4–STAT3–IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide

Chronic lymphocytic leukemia (CLL) cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12)–CXCR4–STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12–CXCR4–STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12–CXCR4–S727–STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

Effects of knockdown of Stat5 on invasion and epithelial mesenchymal transition of thyroid carcinoma TT cells

Objective To investigate the effects of knockdown of signal transducer and activator of transcription 5 (Stat5) on invasion and epithelial mesenchymal transition (EMT) of thyroid carcinoma TT cells. Methods Thyroid carcinoma TT cells were cultured and divided into Stat5-small interfering RNA (siRNA) group and NC-siRNA group, transfected with Stat5 siRNA and negative control NC-siRNA respectively. After transfection, the numbers of invasive cells were determined by Transwell model and the expressions of matrix metalloproteinase (MMP) family genes, EMT marker genes, angiogenesis genes in the cells were determined by fluorescent quantitative PCR. Results The numbers of invasive cells in Stat5-siRNA group were significantly lower than those in NC-siRNA group (42.39±5.82 vs. 64.41±8.49, t=-4.784, P=0.001; 51.23±6.38 vs. 78.54±9.52, t=-5.329, P<0.001; 60.35±8.35 vs. 98.42±11.25, t=-6.076, P<0.001) after the transfection of siRNA for 12 hours, 18 hours and 24 hours. Twenty-four hours after transfection of siRNA, the mRNA expressions of MMP1, MMP2, MMP9, MMP13, N-cadherin, Vimentin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin (Ang)-1 and Ang-2 in Stat5-siRNA group were significantly lower than those in NC-siRNA group (0.42±0.07 vs. 1.03±0.15, t=-8.240, P<0.001; 0.35±0.06 vs. 1.01±0.13, t=-10.307, P<0.001; 0.29±0.05 vs. 1.05±0.18, t=-9.097, P<0.001; 0.54±0.08 vs. 0.98±0.12, t=-6.822, P<0.001; 0.38±0.06 vs. 1.04±0.18, t=-7.778, P<0.001; 0.29±0.04 vs. 1.06±0.12, t=-12.612, P<0.001; 0.36±0.07 vs. 1.06±0.14, t=-10.000, P<0.001; 0.43±0.08 vs. 0.97±0.12, t=-8.372, P<0.001; 0.25±0.03 vs. 1.03±0.12, t=-14.100, P<0.001; 0.19±0.03 vs. 0.99±0.13, t=-13.408, P<0.001). The mRNA expression of E-cadherin was significantly higher than that in NC-siRNA group (2.88±0.42 vs. 0.98±0.12, t=9.726, P<0.001). Conclusion Knockdown of Stat5 inhibits the invasion and EMT of thyroid carcinoma TT cells. Key words: Thyroid neoplasms; Neoplasm invasiveness; Signal transducer and activator of transcription 5; Epithelial mesenchymal transition

Chemical and genetic inhibition of STAT3 sensitizes hepatocellular carcinoma cells to sorafenib induced cell death.

Hepatocellular carcinoma (HCC) has become the second leading cause of cancer related death, with an increasing death rate in recent years. For advanced HCC, sorafenib is the first-line FDA approved drug, with no more than 3 months' overall survival advantage. Recently, a novel strategy has been proposed to improve sorafenib efficacy through enhancing the ability of sorafenib to induce cell death. STAT3 plays a key role in cancer development and recurrence by promoting cell proliferation, survival and immune evasion through its well-established function as a transcription factor in cancer. Notably, STAT3 transcription activity, indicated by its phosphorylation on Y705 is heterogeneous in different liver cancer cell lines. And sorafenib attenuates STAT3 phosphorylation on Y705. However, the role of STAT3 in sorafenib induced cell death is still largely unknown. Here, we show that liver cancer cells also exhibit heterogeneous sensitivities to sorafenib induced cell death, which co-relates with the STAT3-Y705 phosphorylation levels and JAK1/2 expression levels in Hep3B, Huh7 and HepG2 cells. Furthermore, overexpression or knockdown of STAT3 could switch HCC cells between resistant and sensitive to sorafenib induced cell death, which could be partially due to its regulation on Mcl-1, an anti-apoptotic protein. Finally, both inhibitors of STAT3 SH2 domain (S3i-201) or STAT3 upstream kinases JAKs (JAK inhibitor I) could synergistically enhance sorafenib induced cell death. Taken together, these data strongly suggest that STAT3 is not only a downstream effector of sorafenib, but also a key regulator of cellular sensitivity to sorafenib induced cell death, which provide support for the notion to develop STAT3-targeting drugs to improve clinical efficacy of sorafenib in liver cancer.

Proscillaridin A Promotes Oxidative Stress and ER Stress, Inhibits STAT3 Activation, and Induces Apoptosis in A549 Lung Adenocarcinoma Cells.

Cardiac glycosides are natural compounds used for the treatment of cardiovascular disorders. Although originally prescribed for cardiovascular diseases, more recently, they have been rediscovered for their potential use in the treatment of cancer. Proscillaridin A (PSD-A), a cardiac glycoside component of Urginea maritima, has been reported to exhibit anticancer activity. However, the cellular targets and anticancer mechanism of PSD-A in various cancers including lung cancer remain largely unexplored. In the present study, we found that PSD-A inhibits growth and induces apoptosis in A549 lung adenocarcinoma cells. The anticancer activity of PSD-A was found to be associated with the activation of JNK, induction of ER stress, mitochondrial dysfunction, and inhibition of STAT3 activation. PSD-A induces oxidative stress as evidenced from ROS generation, GSH depletion, and decreased activity of TrxR1. PSD-A-mediated ER stress was verified by increased phosphorylation of eIF2α and expression of its downstream effector proteins ATF4, CHOP, and caspases-4. PSD-A triggered apoptosis by inducing JNK (1/2) activation, increasing bax/bcl-2 ratio, dissipating mitochondrial membrane potential, and inducing cleavage of caspases and PARP. Further study revealed that PSD-A inhibits both constitutive and inducible STAT3 activations and decreases STAT3 DNA-binding activity. Moreover, PSD-A-mediated inhibition of STAT3 activation was found to be associated with increased SHP-1 expression, decreased phosphorylation of Src, and binding of PSD-A with STAT3 SH2 domain. Finally, STAT3 knockdown by shRNA inhibited growth and enhanced apoptotic efficacy of PSD-A. Taken together, the data suggest that PSD-A could be developed into a potential therapeutic agent against lung adenocarcinoma.