Overexpression of ribophorin II is required for viability of nasopharyngeal cancer cells by regulating JAK1/STAT3 activation.

OBJECTIVE: This study aimed to elucidate the role of ribophorin II (RPN2) in nasopharyngeal cancer (NPC) cell survival and death. RPN2 expression was upregulated in 22 human NPC specimens and 5-8F and CNE1 cells compared with that in adjacent normal tissues and normal nasopharyngeal NP69 cells. MATERIALS AND METHODS: CCK-8 and colony formation assays indicated that the silencing of RPN2 hindered the proliferation and growth of 5-8F and CNE1 cells. RESULTS: RPN2 expression was upregulated in 22 human NPC specimens as well as in 5-8F and CNE1 cells compared with that in adjacent normal tissues and NP69 cells. CCK-8 and colony formation assays indicated that the silencing of RPN2 reduced the proliferation and growth of 5-8F and CNE1 cells. Annexin V/PI flow cytometry and Bcl-2/Bax analysis showed that RPN2 silencing led to increased apoptosis. Moreover, JAK1 was found to interact with RPN2, and total JAK1, STAT3, and phosphorylated STAT3 levels were dramatically decreased in cells with RPN2 silencing. Furthermore, the nuclear localization of STAT3 was blocked by the silencing of RPN2. The administration of the STAT3 activator colivelin could offset the inhibitory effect of RPN2 silencing on the survival and apoptosis of NPC cells. CONCLUSION: RPN2 is upregulated in NPC tissues or cells, and RPN2 silencing repressed NPC cell proliferation and elicited apoptosis. RPN2 overexpression is possibly associated with JAK1/STAT3 silencing and activation. Finally, RPN2 represents a promising target for NPC treatment.

Cutaneous JAK Expression in Vitiligo.

BACKGROUND: The Janus kinase-signal transducer and activator of transcription signaling pathway has been suggested as a promising therapeutic target in vitiligo. However, limited data is available on the cutaneous expression of JAK in vitiligo. AIM: This study is designed to analyze the cutaneous expression patterns of JAK1, 2, and 3 in vitiligo and investigate their relation to the disease clinical parameters. METHODS: This case-control study recruited 24 patients having active vitiligo and 20 age, sex, and skin type-matched healthy volunteers. Skin biopsies were obtained from patients (lesional, perilesional and nonlesional) and controls for assessment of JAK1, 2, and 3 expression using RT-PCR. RESULTS: JAK1 and JAK3 were overexpressed in patients' skin compared to control skin and showed a stepwise pattern of upregulation from control to nonlesional, perilesional and lesional skin. However, JAK3 showed much stronger expression. In contrast JAK2 expression showed no significant difference in any of lesional, perilesional or nonlesional skin compared to control skin. JAK1 and JAK3 expression levels showed no correlation with neither the disease activity nor severity. CONCLUSION: JAK1 and more prominently JAK3 are upregulated in vitiliginous skin and possibly contribute to the pathogenesis of the disease. Accordingly, selective JAK3/1 inhibition may provide a favorable therapeutic opportunity for vitiligo patients.This study is registered on the ClinicalTrials.gov Identifier: NCT03185312.

Inhibition of JAK-STAT Signaling by Baricitinib Reduces Interferon-γ-Induced CXCL10 Production in Human Salivary Gland Ductal Cells.

Sjögren's syndrome (SS) is a chronic autoimmune disease targeting salivary and lacrimal glands. C-X-C motif chemokine ligand 10 (CXCL10) expression is upregulated in lip salivary glands (LSGs) of primary SS (pSS) patients, and CXCL10 involved in SS pathogenesis via immune-cell accumulation. Moreover, interferon (IFN)-γ enhances CXCL10 production via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We investigated the effects of baricitinib, a selective JAK1/2 inhibitor, on both IFN-γ-induced CXCL10 production and immune-cell chemotaxis. We used immunohistochemical staining to determine the expression levels and localization of JAK1 and JAK2 in LSGs of SS patients (n = 12) and healthy controls (n = 3). We then evaluated the effect of baricitinib in an immortalized normal human salivary gland ductal (NS-SV-DC) cell line. Immunohistochemical analysis of LSGs from pSS patients revealed strong JAK1 and JAK2 expression in ductal and acinar cells, respectively. Baricitinib significantly inhibited IFN-γ-induced CXCL10 expression as well as the protein levels in an immortalized human salivary gland ductal-cell clone in a dose-dependent manner. Additionally, western blot analysis showed that baricitinib suppressed the IFN-γ-induced phosphorylation of STAT1 and STAT3, with a stronger effect observed in the case of STAT1. It also inhibited IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggested that baricitinib suppressed IFN-γ-induced CXCL10 expression and attenuated immune-cell chemotaxis by inhibiting JAK/STAT signaling, suggesting its potential as a therapeutic strategy for pSS.

Long noncoding RNA nuclear-enriched abundant transcript 1 regulates proliferation and apoptosis of neuroblastoma cells treated by cisplatin by targeting miR-326 through Janus kinase/signal transducer and activator of transcription 3 pathway.

Neuroblastoma is a common malignancy and frequently affects children, leading to a low survival rate. Long noncoding RNAs (lncRNAs) are reported to be closely related to cancer progression. The purpose of this study was to explore a novel mechanism of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in neuroblastoma. NEAT1 was upregulated in neuroblastoma cell lines (IMR32 and SK-N-SH). Overexpression of NEAT1 increased proliferation inhibited by cisplatin and decreased apoptosis promoted by cisplatin. MicroRNA-326 (miR-326) was a target of NEAT1 and miR-326 reintroduction abolished the effects of NEAT1 overexpression on cell proliferation and apoptosis. Moreover, NEAT1 overexpression activated Janus kinase/signal transducer and activator of transcription 3 (JAK1/STAT3) signaling pathway through absorbing miR-326. Besides, NEAT1 overexpression promoted tumor growth in vivo through stimulating the expression of p-JAK1 and p-STAT3 but inhibiting miR-326 expression. NEAT1 accelerated proliferation and weakened apoptosis of neuroblastoma cells treated by cisplatin by targeting miR-326 through activating JAK1/STAT3 signaling pathway, suggesting that NEAT1 was a potential biomarker against neuroblastoma.

Selective Janus Kinase 1 Inhibition Is a Promising Therapeutic Approach for Lupus Erythematosus Skin Lesions.

Background: Cutaneous lupus erythematosus (CLE) is an interferon (IFN) -driven autoimmune skin disease characterized by an extensive cytotoxic lesional inflammation with activation of different innate immune pathways. Aim of our study was to investigate the specific role of Janus kinase 1 (JAK1) activation in this disease and the potential benefit of selective JAK1 inhibitors as targeted therapy in a preclinical CLE model. Methods: Lesional skin of patients with different CLE subtypes and healthy controls (N = 31) were investigated on JAK1 activation and expression of IFN-associated mediators via immunohistochemistry and gene expression analyses. The functional role of JAK1 and efficacy of inhibition was evaluated in vitro using cultured keratinocytes stimulated with endogenous nucleic acids. Results were confirmed in vivo using an established lupus-prone mouse model. Results: Proinflammatory immune pathways, including JAK/STAT signaling, are significantly upregulated within inflamed CLE skin. Here, lesional keratinocytes and dermal immune cells strongly express activated phospho-JAK1. Selective pharmacological JAK1 inhibition significantly reduces the expression of typical proinflammatory mediators such as CXCL chemokines, BLyS, TRAIL, and AIM2 in CLE in vitro models and also improves skin lesions in lupus-prone TREX1-/- -mice markedly. Conclusion: IFN-associated JAK/STAT activation plays a crucial role in the pathophysiology of CLE. Selective inhibition of JAK1 leads to a decrease of cytokine expression, reduced immune activation, and decline of keratinocyte cell death. Topical treatment with a JAK1-specific inhibitor significantly improves CLE-like skin lesions in a lupus-prone TREX1-/- -mouse model and appears to be a promising therapeutic approach for CLE patients.

Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation.

MicroRNAs mediates gene expression in various diseases. Studies have shown that aberrant expression of miRNAs affected cerebral protection. In this study, we have investigated the effects of microRNA-708 (miR-708) on cell survival of oxygen and glucose-deprived reoxygenation (OGD/R) human neuroblastoma cells (SH-SY5Y) and explored whether miR-708 inhibited neuronal death by targeting JAK1. In vitro model of ischemia was used to investigate the neuroprotective functions of miR-708. MiR-708 mimics/siJAK1 transfected SH-SY5Y cells were treated with OGD. After 48h of reoxygenation, cell viability and cell survival were determined by EdU and FITC/PI double staining flow cytometry, respectively. Luciferase activity assay was performed to validate the role of JAK1 as a direct target of miR-708. qRT-PCR and Immunofluorescence assays were used to determine the expression of JAK1, MAP2 and NEUN in miR-708 mimics transfected SH-SY5Y cells. To explore the mechanisms involved in cell growth promotion by JAK1, morphological changes in cells were detected upon knockdown of JAK1, and the expression levels of JAK1, Bax, Bcl-2, cleaved-caspase-3, STAT3 and Mcl-1 were determined by Western blotting. The expression of miR-708 significantly decreased in cells treated with OGD/R. MiR-708 directly targeted JAK1 3'UTR to down-regulate JAK1 mRNA expression, whereas the expression of MAP2 and NEUN was upregulated. Previous studies have demonstrated that the suppression of JAK1 inhibited apoptosis phenocopied function of the miR-708 overexpression in OGD/R SH-SY5Y cells. miR-708 decreased the rate of apoptosis of OGD/R SH-SY5Y cells by suppressing the expression of JAK1.

Recent progress and perspective in JAK inhibitors for rheumatoid arthritis: from bench to bedside.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. However, the combined use of synthetic disease-modifying anti-rheumatic drug (DMARD) such as methotrexate and a biological DMARD targeting tumour necrosis factor (TNF) has revolutionized treatment of RA. Clinical remission is a realistic target to treat and the maintenance of remission has produced significant improvements in structural and function outcomes. However, biological DMARDs are limited to intravenous or subcutaneous uses and orally available small but strong products have been developed. The multiple cytokines and cell surface molecules bind to receptors, resulting in the activation of various signalling, including phosphorylation of kinase proteins. Among multiple kinases, Janus kinase (JAK) plays pivotal roles in the pathological processes of RA. Tofacitinib, a small product targeting JAK, inhibits phosphorylation of JAK1 and JAK3, subsequent Stat1 and expression of Stat1-inducible genes, which contribute to efficient propagation of its anti-inflammatory effects for the treatment of RA. The primary targets of tofacitinib are dendritic cells, CD4(+) T cells such as Th1 and Th17 and activated B cells which leads to multi-cytokine targeting. Six global phase 3 studies revealed that oral administration of 5 or 10 mg tofacitinib was significantly effective than placebo with or without methotrexate in active RA patients with methotrexate-naïve, inadequately responsive to methotrexate or TNF-inhibitors. Therapeutic efficacy of tofacitinib was observed in a short term after administration and was as strong as adalimumab, a TNF-inhibitor. The most commonly observed adverse events were related to infection, hematologic, hepatic and renal disorders and association of tofacitinib with carcinogenicity and infections remains debated. Further investigation on post-marketing survey would help us understand the positioning of this drug.

The pseudokinase SgK223 promotes invasion of pancreatic ductal epithelial cells through JAK1/Stat3 signaling.

BACKGROUND: Characterization of molecular mechanisms underpinning development of pancreatic ductal adenocarcinoma (PDAC) may lead to the identification of novel therapeutic targets and biomarkers. SgK223, also known as Pragmin, is a pseudokinase and scaffolding protein closely related to SgK269/PEAK1. Both proteins are implicated in oncogenic tyrosine kinase signaling, but their mechanisms and function remain poorly characterized. METHODS: Expression of SgK223 in PDAC and PDAC cell lines was characterized using gene expression microarrays, mass spectrometry (MS)-based phosphoproteomics and Western blotting. SgK223 was overexpressed in human pancreatic ductal epithelial (HPDE) cells via retroviral transduction, and knocked down in PDAC cells using siRNA. Cell proliferation was determined using a colorimetric cell viability assay, and cell migration and invasion using transwells. Expression of markers of epithelial-mesenchyme transition (EMT) was assayed by quantitative PCR. SgK223 and Stat3 signaling was interrogated by immunoprecipitation, Western blot and gene reporter assays. The functional role of specific kinases and Stat3 was determined using selective small molecule inhibitors. RESULTS: Elevated site-selective tyrosine phosphorylation of SgK223 was identified in subsets of PDAC cell lines, and increased expression of SgK223 detected in several PDAC cell lines compared to human pancreatic ductal epithelial (HPDE) cells and in PDACs compared to normal pancreas. Expression of SgK223 in HPDE cells at levels comparable to those in PDAC did not alter cell proliferation but led to a more elongated morphology, enhanced migration and invasion and induced gene expression changes characteristic of a partial EMT. While SgK223 overexpression did not affect activation of Erk or Akt, it led to increased Stat3 Tyr705 phosphorylation and Stat3 transcriptional activity, and SgK223 and Stat3 associated in vivo. SgK223-overexpressing cells exhibited increased JAK1 activation, and use of selective inhibitors determined that the increased Stat3 signaling driven by SgK223 was JAK-dependent. Pharmacological inhibition of Stat3 revealed that Stat3 activation was required for the enhanced motility and invasion of SgK223-overexpressing cells. CONCLUSIONS: Increased expression of SgK223 occurs in PDAC, and overexpression of SgK223 in pancreatic ductal epithelial cells promotes acquisition of a migratory and invasive phenotype through enhanced JAK1/Stat3 signaling. This represents the first association of SgK223 with a particular human cancer, and links SgK223 with a major signaling pathway strongly implicated in PDAC progression.

IL6/JAK1/STAT3 Signaling Blockade in Endometrial Cancer Affects the ALDHhi/CD126+ Stem-like Component and Reduces Tumor Burden.

Cancer stem-like cells (CSC) may be critical to maintain the malignant behavior of solid and hematopoietic cancers. Recently, patients with endometrial cancer whose tumors expressed high levels of aldehyde dehydrogenase (ALDH), a detoxifying enzyme characteristic of many progenitor and stem cells, exhibited a relative reduction in survival compared with patients with low levels of ALDH. Given evidence of its role as a CSC marker, we hypothesized that high level of ALDH activity (ALDH(hi)) in a tumor might positively correlate with the presence of stem- and progenitor-like tumor cells in this disease setting. In support of this hypothesis, ALDH could be used to enrich for CSC in endometrial cancer cell lines and primary tumors, as illustrated by the increased tumor-initiating capacity of ALDH(hi) cells in immunodeficient mice. ALDH(hi) cells also exhibited greater clonogenic and organoid-forming capacity compared with ALDH(lo) cells. Notably, the number of ALDH(hi) cells in tumor cell lines and primary tumors inversely correlated with differentiation grade. Expression analysis revealed upregulation of IL6 receptor subunits and signal transducers CD126 and GP130 in ALDH(hi) endometrial cancer cells. Accordingly, targeted inhibition of the IL6 receptor and its downstream effectors JAK1 and STAT3 dramatically reduced tumor cell growth. Overall, our results provide a preclinical rationale to target IL6 or its effector functions as a novel therapeutic option in endometrial cancer.

T cell receptor stimulation impairs IL-7 receptor signaling by inducing expression of the microRNA miR-17 to target Janus kinase 1.

T cell receptor (TCR)-mediated inhibition of interleukin-7 (IL-7) signaling is important for lineage fate determination in the thymus and for T cell survival in the periphery because uninterrupted IL-7 signaling results in T cell death. The initial event in IL-7 signaling is the transactivation of Janus kinases 1 and 3 (Jak1 and Jak3), which are associated with the cytosolic tails of the IL-7 receptor α chain (IL-7Rα) and the γc subunit, the two cell surface proteins that constitute IL-7R. We found that Jak1 is a highly unstable protein with a half-life of only 1.5 hours, so that continuous Jak1 protein synthesis is required to maintain Jak1 protein in sufficient abundance to support IL-7 signaling. However, we also found that Jak1 protein synthesis was acutely reduced by TCR-responsive microRNAs in the miR-17 family, which targeted Jak1 mRNA (messenger RNA) to inhibit its translation. Thus, this study identifies a molecular mechanism by which TCR engagement acutely disrupts IL-7 signaling.

Myeloid-specific disruption of tyrosine phosphatase Shp2 promotes alternative activation of macrophages and predisposes mice to pulmonary fibrosis.

The alternative activation of M2 macrophages in the lungs has been implicated as a causative agent in pulmonary fibrosis; however, the mechanisms underlying M2 polarization are poorly characterized. In this study, we investigated the role of the ubiquitously expressed Src homology domain-containing tyrosine phosphatase Shp2 in this process. Shp2 inactivation augmented IL-4-mediated M2 polarization in vitro, suggesting that Shp2 regulates macrophage skewing and prevents a bias toward the M2 phenotype. Conditional removal of Shp2 in monocytes/macrophages with lysozyme M promoter-driven Cre recombinase caused an IL-4-mediated shift toward M2 polarization. Additionally, an increase in arginase activity was detected in Shp2(∆/∆) mice after i.p. injection of chitin, whereas Shp2-deficient macrophages showed enhanced M2 polarization and protection against schistosome egg-induced schistosomiasis. Furthermore, mutants were more sensitive than control mice to bleomycin-induced inflammation and pulmonary fibrosis. Shp2 was associated with IL-4Rα and inhibited JAK1/STAT6 signaling through its phosphatase activity; loss of Shp2 promoted the association of JAK1 with IL-4Rα, which enhanced IL-4-mediated JAK1/STAT6 activation that resulted in M2 skewing. Taken together, these findings define a role for Shp2 in alveolar macrophages and reveal that Shp2 is required to inhibit the progression of M2-associated pulmonary fibrosis.

The Janus kinases inhibitor AZD1480 attenuates growth of small cell lung cancers in vitro and in vivo.

PURPOSE: The prognosis of small cell lung cancer (SCLC) is poor, and there has been very little progress in the medical treatment of SCLC in the past two decades. We investigated the potential of Janus-activated kinases (JAK) inhibitor, AZD1480, for treatment of SCLC in vitro and in vivo. EXPERIMENTAL DESIGN: JAK1 and JAK2 were inhibited by AZD1480 or siRNAs, and the effect of inhibition of JAK gene family on SCLC cell viability was evaluated. The effect of AZD1480 on cell-cycle distribution and apoptosis induction was studied. Antitumor effects of AZD1480 in tumor xenografts were assessed. RESULTS: AZD1480 significantly inhibited growth of six out of 13 SCLC cells with IC50s ranging from 0.73 to 3.08 µmol/L. Knocking down of JAK2 and JAK1 inhibited proliferation of Jak2-positive/Jak1-negative H82 cells and Jak1-positive/Jak2-negative GLC4 cells, respectively. Treatment of SCLC cells with AZD1480 for 24 hours resulted in an increase of 4N DNA content and histone 3 serine 10 phosphorylation, indicative of G2-M phase arrest. Moreover, SCLCs underwent apoptosis after AZD1480 treatment as exemplified by the downregulation of MCL1, the accumulation of cleaved caspase 3, cleaved PARP, and increase of annexin-V-positive cells. Finally, xenograft experiments showed that AZD1480 attenuated the growth of H82 and GLC4 tumors in mice, and we observed stronger apoptosis as well as decreased CD31-positive endothelial cells in H82 and GLC4 xenografts upon AZD1480 treatment. CONCLUSIONS: JAK inhibitor AZD1480 attenuated growth of SCLC cells in vitro and in vivo. Clinical development of anti-JAKs therapies in SCLC warrants further investigation.

Overcoming cancer cell resistance to VSV oncolysis with JAK1/2 inhibitors.

Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity but some cancer cells are resistant to VSV killing, either constitutively or due to type I interferon (IFN) inducing an antiviral state in the cells. Here, we evaluated VSV oncolysis of a panel of human head and neck cancer cells and showed that VSV resistance in SCC25 and SCC15 cells could be reversed with Janus kinase (JAK) 1/2 inhibitors (JAK inhibitor I and ruxolitinib). Pre-treatment of cells with JAK1/2 inhibitors before or in conjunction with VSV enhanced viral infection, spread and progeny yield (100- to 1000-fold increase). In contrast, inhibitors of histone deacetylase (LBH589), phosphatidylinositol 3-kinase (GDC-0941, LY294002), mammalian target of rapamycin (rapamycin) or signal transducer and activator of transcription 3 (STAT3 inhibitor VII) were ineffective. Compared with VSV-sensitive SW579 cells, IFNα/ß responsive antiviral genes (IRF-9, IRF-7, OAS1 but not MxA) are constitutively expressed in SCC25 cells. Pretreatment with JAK inhibitors reduced mRNA levels of these genes, increasing VSV expression in the cells. Interestingly, 1 h of drug exposure was sufficient to reverse SCC25 resistance to VSV and was still effective if virus was added 24 h later. Overall, we show here that JAK inhibitor I and ruxolitinib (Jakafi) can reverse resistance to VSV, supporting the rationale to incorporate JAK1/2 inhibitors in future VSV virotherapy trials.

Aspirin enhances IFN-α-induced growth inhibition and apoptosis of hepatocellular carcinoma via JAK1/STAT1 pathway.

STAT1 has a key role in exerting the antiproliferative and proapoptotic effects of interferon (IFN)-α on tumors, and its defects in expression is associated with IFN-α resistance. In this study we want to investigate whether aspirin can improve the antitumor efficiency of IFN-α on hepatocellular carcinoma (HCC) through the activation of STAT1. We found that aspirin not only significantly enhanced IFN-α-induced antiproliferation and apoptosis of HCC in vitro study but also enhanced tumor growth inhibition in nude mice. Although IFN-α alone resulted in significant phosphorylation of both STAT1 and STAT3, aspirin only prompted the IFN-α-induced phosphorylation of STAT1. Further study revealed that aspirin-prompted phosphorylation of STAT1 was activated through phosphorylation of JAK1. Furthermore, aspirin-activated STAT1 upregulated the transcription of proapoptotic IFN-stimulated gene (ISG) of X-linked inhibitor of apoptosis-associated factor-1 and downregulated the transcription of antiapoptotic ISG of G1P3, which in turn promoted the expression of Bax and activation of caspase-9 and caspase-3, thereby sensitizing HCC cells to IFN-α-induced apoptosis. Taken together, our findings suggest a novel strategy of using aspirin to overcome tumor resistance and enhance the effectiveness of IFN-α in HCC treatment through activating STAT1 gene, and have potential implications for improving future IFN-α protein and gene therapy.

Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy.

The identification of somatic activating mutations in JAK2 (refs 1­4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK­STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

Quercetin abrogates IL-6/STAT3 signaling and inhibits glioblastoma cell line growth and migration.

Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.

Correlation analysis of JAK-STAT pathway components on prognosis of patients with prostate cancer.

Janus kinases (JAK)/signal transducers and activator of transcription (STAT) pathway is activated constitutively in prostate cancer (PCa). Despite previous reports implying a role of this pathway in the development of clinical hormone-refractory PCa, the correlation of pathway members with the clinicopathologic features and prognosis of patients with PCa has not been elucidated. To address this problem, pJAK-1(Tyr1022/1023) and pSTAT-3(Tyr705) were evaluated by immunostaining in needle biopsies of the prostate from 202 PCa patients treated by definitive therapy (105 cases) or hormonal therapy (97 cases). The correlation of two protein expression with the clinicopathologic features and the prognosis of PCa were subsequently assessed. The expression levels of pJAK-1(Tyr1022/1023) and pSTAT-3(Tyr705) were both positively correlated with Gleason score and clinical stage of patients with PCa. Their expression was also significantly higher in patients with biochemical (prostate-specific antigen, PSA) failure than that in those with no PSA failure (both P < 0.001). In all patients, the recurrence-free survival (RFS) rates were significantly higher in those with low pJAK-1(Tyr1022/1023) and pSTAT-3(Tyr705) expression than that in those with high expression (both P < 0.001). Moreover, for patients treated by definitive or hormonal therapy, the RFS rates in those with lower pJAK-1(Tyr1022/1023) (P < 0.001 and 0.012, respectively) and pSTAT-3(Tyr705) expression (P < 0.001 and 0.015, respectively) were significantly higher than in those with higher expression. Cox multivariate analysis showed that the expression levels of pJAK-1(Tyr1022/1023) (P = 0.002) and pSTAT-3(Tyr705) (P = 0.005) were prognostic factors for PCa in addition to extraprostatic extension (P = 0.026) and Gleason score (P = 0.018). The results of pJAK-1(Tyr1022/1023) and pSTAT-3(Tyr705) immunostainings in needle-biopsy specimens are prognostic factors for PCa.

Interferon-gamma enhances the apoptosis of macrophages under trophic stress through activation of p53 and the JAK1 pathway.

Apoptosis has been implicated as an important mediator of immunosuppression associated with a depleted nutritional state. Since a number of cytokines are likely to influence the rate of apoptosis, we determined the effect of interferon-gamma (IFN-gamma) on apoptosis of macrophages in serum/amino acid deprived cell culture conditions. IFN-gamma further increased the rate of apoptosis compared to trophically stressed macrophages. In addition, we showed that enhancement of apoptosis by IFN-gamma is caused by a decrease in mitochondrial membrane potential due to upregulation of p53 and Bax and down-regulation of Bcl-xL. Moreover, we found that IFN-gamma increases caspase-3 activity that had been induced by serum/amino acid depletion, and that JAK1 signaling is involved in capase-3 activation and in the enhancement of apoptosis by IFN-gamma.

Oncostatin M enhances the antiviral effects of type I interferon and activates immunostimulatory functions in liver epithelial cells.

Oncostatin M (OSM) is released together with type I interferon (IFN) by activated dendritic cells, suggesting a concerted action of these cytokines in the biological response against infection. We found that OSM increases the antiviral effect of IFN-alpha in Huh7 hepatoma cells infected with hepatitis A or hepatitis C virus and synergizes with IFN-alpha in the induction of antiviral genes. The combination of OSM and IFN-alpha led to upregulation of both STAT1 and STAT3 together with intense and prolonged activation of STAT1, STAT3, and Jak1. OSM with or without IFN-alpha also activated p38 mitogen-activated protein kinase, which is known to enhance transcription of IFN-alpha-inducible genes. Interestingly, OSM combined with IFN-alpha strongly induced immunoproteasome genes and other genes involved in antigen processing and presentation. Moreover, OSM, alone or in combination with IFN-alpha, upregulated relevant innate immunity molecules and increased the expression of intracellular adhesion molecule 1 and interleukin-15 receptor alpha (IL-15Ralpha) in liver cells. Hepatoma cells transfected with a plasmid encoding a viral antigen were able to activate effector T cells when pretreated with IFN-alpha plus OSM but not with each cytokine separately. Also, OSM, more than IFN-alpha, augmented the ability of Huh7 cells to transpresent IL-15 to responding lymphocytes and increased the immunostimulatory activity of liver epithelial cells by presenting a short viral peptide to sensitized cytotoxic T cells. In conclusion, OSM enhances the antiviral effects of type I interferon and cooperates with it in the induction of adaptive immune responses to pathogens. These findings may have therapeutic implications.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in hepatitis C virus replicon.

BACKGROUND: Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. RESULTS: HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. CONCLUSION: This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.