STAT3 Serine 727 Phosphorylation: A Relevant Target to Radiosensitize Human Glioblastoma

Radiotherapy is an essential component of glioma standard treatment. Glioblastomas (GBM), however, display an important radioresistance leading to tumor recurrence. To improve patient prognosis, there is a need to radiosensitize GBM cells and to circumvent the mechanisms of resistance caused by interactions between tumor cells and their microenvironment. STAT3 has been identified as a therapeutic target in glioma because of its involvement in mechanisms sustaining tumor escape to both standard treatment and immune control. Here, we studied the role of STAT3 activation on tyrosine 705 (Y705) and serine 727 (S727) in glioma radioresistance. This study explored STAT3 phosphorylation on Y705 (pSTAT3‐Y705) and S727 (pSTAT3‐S727) in glioma cell lines and in clinical samples. Radiosensitizing effect of STAT3 activation down‐modulation by Gö6976 was explored. In a panel of 15 human glioma cell lines, we found that the level of pSTAT3‐S727 was correlated to intrinsic radioresistance. Moreover, treating GBM cells with Gö6976 resulted in a highly significant radiosensitization associated to a concomitant pSTAT3‐S727 down‐modulation only in GBM cell lines that exhibited no or weak pSTAT3‐Y705. We report the constitutive activation of STAT3‐S727 in all GBM clinical samples. Targeting pSTAT3‐S727 mainly in pSTAT3‐Y705‐negative GBM could be a relevant approach to improve radiation therapy.     

Aberrant cell signalling in PBMCs upon IFN‐α stimulation in primary Sjögren's syndrome patients associates with type I interferon signature

Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease with heterogeneous disease manifestations. Genetic predisposition, hormonal and environmental factors are all thought to contribute to disease etiology and pathogenesis. A better understanding of the disease pathogenesis is required in order to establish new targeted therapies. We analysed MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMCs) upon stimulation with interferon alpha 2b (IFN‐α2b) by flow cytometry to define potentially dysfunctional intracellular signalling pathways involved in disease pathogenesis. Cells derived from pSS patients displayed small but significant increases in basal phosphorylation levels of numerous signalling proteins compared to cells from healthy donors. The phosphorylation profiles following stimulation with IFNα2b differed significantly between pSS patients and healthy donors, especially regarding STAT1 Y701. PCA further grouped patients according to clinical characteristics. Type I IFN induced gene expression was found to negatively correlate with the IFN‐α2b induced phosphorylation of STAT3 S727 in T cells and positively with pSTAT1 Y701 in B cells. Increases in pSTAT1 Y701 were associated with the presence of autoantibodies. Our results indicate involvement of both STAT3 S727 and STAT1 Y701 pathways in pSS patients. Therapies targeting these pathways might therefore be beneficial for certain subgroups of patients.     

STAT3 serine 727 phosphorylation influences clinical outcome in glioblastoma

Besides STAT3 tyrosine 705 phosphorylation (pTyr705-STAT3), phosphorylation of STAT3 at serine 727 (pSer727-STAT3) is shown to contribute to tumorigenesis and be closely related with resistance to radiotherapy and chemotherapy in glioma, but there is currently no study regarding its relevance to prognosis in glioblastoma (GBM). Here, the expression of phosphorylated STAT3 was detected in tumor specimens from 88 patients with newly diagnosed GBM by immunohistochemistry, the Kaplan-Meier survival curve and COX proportional hazards regression model were applied to estimate its influences on progression-free survival (PFS) and overall survival (OS). Immunohistochemical assay showed elevated expression of pSer727-STAT3 in GBM compared with normal brain tissue. Univariate analysis indicated significant correlations of high percentage of pSer727-STAT3 positive tumor cells with shorter PFS (P = 0.006) and OS (P = 0.002). In multivariate analysis, high pSer727-STAT3 expression was demonstrated as an independent unfavorable prognostic indicator for PFS (HR 1.830, P = 0.022) and OS (HR 1.797, P = 0.040). And patients with high expression of both pTyr705-STAT3 and pSer727-STAT3 had a poorer prognosis compared with the remainder (P < 0.005). In conclusion, the high proportion of pSer727-STAT3 positive neoplastic cells in GBM is an independent unfavorable prognostic factor, and increased expression of both pTyr705-STAT3 and pSer727-STAT3 is predictive of poorer clinical outcome, thereby adding to the growing evidence that STAT3 inhibition may be a potential therapeutic strategy in glioblastoma.     

Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats

Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer's disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer's-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.     

Interleukin-4 and interleukin-13 inhibit the expression of leukemia inhibitory factor and interleukin-11 in fibroblasts

Cytokines produced by inflammatory or resident mesenchymal cells play important modulatory roles in the pathogenesis of inflammation induced bone loss. In the present study, the effects of IL-4 and IL-13 on the expression of three osteotropic cytokines in the IL-6 family expressed in human gingival fibroblasts were studied. IL-4Rα and IL-13Rα1 mRNA were constitutively expressed in human gingival fibroblasts. The inflammatory cytokines IL-1β and TNF-α increased expression of IL-6, LIF, and IL-11 mRNA and protein in the gingival fibroblasts. Addition of IL-4 or IL-13 had no effect on IL-6 expression, but significantly inhibited LIF and IL-11 mRNA and protein stimulated by IL-1β and TNF-α. No involvement of NF-κB or STAT1 was observed in the inhibition. STAT6 was phosphorylated at Y641 by treatment with IL-4 and knockdown of STAT6 with siRNA decreased the inhibition of IL-11 and LIF expression by IL-4 in IL-1β and TNF-α stimulated cells. This study suggests that activation of STAT6 by IL-4 and IL-13, through type 2 IL-4 receptors, inhibits production of IL-11 and LIF stimulated by IL-1β and TNF-α in human gingival fibroblasts. A negative modulatory role of IL-4 and IL-13 in osteotropic cytokine production could be a mechanism playing an important inhibitory role in inflammation induced periodontitis.     

Rapamycin inhibits both motility through down-regulation of p-STAT3 (S727) by disrupting the mTORC2 assembly and peritoneal dissemination in sarcomatoid cholangiocarcinoma

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.