Targeting STAT3 in Cancer with Nucleotide Therapeutics.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in promoting the proliferation and survival of tumor cells. As a ubiquitously-expressed transcription factor, STAT3 has commonly been considered an "undruggable" target for therapy; thus, much research has focused on targeting upstream pathways to reduce the expression or phosphorylation/activation of STAT3 in tumor cells. Recently, however, novel approaches have been developed to directly inhibit STAT3 in human cancers, in the hope of reducing the survival and proliferation of tumor cells. Several of these agents are nucleic acid-based, including the antisense molecule AZD9150, CpG-coupled STAT3 siRNA, G-quartet oligodeoxynucleotides (GQ-ODNs), and STAT3 decoys. While the AZD9150 and CpG-STAT3 siRNA interfere with STAT3 expression, STAT3 decoys and GQ-ODNs target constitutively activated STAT3 and modulate its ability to bind to target genes. Both STAT3 decoy and AZD9150 have advanced to clinical testing in humans. Here we will review the current understanding of the structures, mechanisms, and potential clinical utilities of the nucleic acid-based STAT3 inhibitors

Erythropoietin-induced serine 727 phosphorylation of STAT3 in erythroid cells is mediated by a MEK-, ERK-, and MSK1-dependent pathway

Objective. Erythropoietin (EPO) is a key regulator of erythropoiesis, playing a role in both the proliferation and differentiation of erythroid cells. One of the signal transduction molecules activated upon EPO stimulation is signal transducer and activator of transcription (STAT) 3. Besides tyrosine 705 phosphorylation of STAT3, serine 727 phosphorylation has been described upon EPO stimulation. In the present study, we investigated which molecular pathways mediate the STAT3 serine 727 phosphorylation and the functional implications of this phosphorylation. Methods. The EPO-dependent erythroid cell line ASE2 was used to investigate which signaling routes were involved in the STAT3 serine 727 phosphorylation. Western blotting using phosphospecific antibodies was used to assess the phosphorylation status of STAT3 molecules. Transfection analysis was performed to investigate the transactivational potential of STAT3, and quantitative RT-PCR was used to study the in vivo gene expression of STAT3-responsive genes. Results. Western blotting of extracts of cells exposed to various chemical inhibitors revealed that the MEK inhibitors PD98059 and U0126 abrogated the EPO-mediated STAT3 serine 727 phosphorylation without an effect on tyrosine phosphorylation. Further analysis showed that MSK1 is activated downstream of ERK, and retroviral transductions with kinase-inactive MSK1 revealed that MSK1 is necessary for STAT3 serine phosphorylation. Furthermore, the STAT3-mediated transactivation was reduced by blocking the STAT3 serine phosphorylation with the MEK inhibitor U0126 or by expression of kinase-inactive MSK1. Conclusions. The EPO-induced STAT3 serine 727 phosphorylation is mediated by a pathway involving MEK, ERK, and MSK1. Furthermore, serine phosphorylation of STAT3 augments the transactivational potential of STAT3.

TYK2-induced phosphorylation of Y640 suppresses STAT3 transcriptional activity

STAT3 is a pleiotropic transcription factor involved in homeostatic and host defense processes in the human body. It is activated by numerous cytokines and growth factors and generates a series of cellular effects. Of the STAT-mediated signal transduction pathways, STAT3 transcriptional control is best understood. Jak kinase dependent activation of STAT3 relies on Y705 phosphorylation triggering a conformational switch that is stabilized by intermolecular interactions between SH2 domains and the pY705 motif. We here show that a second tyrosine phosphorylation within the SH2 domain at position Y640, induced by Tyk2, negatively controls STAT3 activity. The Y640F mutation leads to stabilization of activated STAT3 homodimers, accelerated nuclear translocation and superior transcriptional activity following IL-6 and LIF stimulation. Moreover, it unlocks type I IFN-dependent STAT3 signalling in cells that are normally refractory to STAT3 transcriptional activation

STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn\u27s Disease

Crohn\u27s disease (CD) results from dysregulated immune responses to gut microbiota in genetically susceptible individuals, affecting multiple areas of the gastrointestinal tract. Innate lymphoid cells (ILCs) are tissue-resident innate effector lymphocytes which play crucial roles in mucosal immune defense, tissue repair, and maintenance of homeostasis. The accumulation of IFN-γ-producing ILC1s and increased level of proinflammatory cytokines produced by ILCs has been observed in the inflamed terminal ileum of CD patients. To date, the precise mechanisms of ILC plasticity and gene regulatory pathways in ILCs remain unclear. Signal transducer and activator of transcription 3 (STAT3) regulates gene expression in a cell-specific, cytokine-dependent manner, involving multiple immune responses. This study proposes the positive correlation between the prevalence of STAT3 rs744166 risky allele A with the severity of disease in a cohort of 94 CD patients. In addition, the results suggest an increased STAT3 activity in the inflamed ileum of CD patients, compared to unaffected ileum sections. Notably, IL-23 triggers the differentiation of CD117+NKp44- ILC3s and induces the activation of STAT3 in both CD117+NKp44- and CD117-NKp44- ILC subsets, implying the involvement of STAT3 in the initiation of ILC plasticity. Moreover, carriage of STAT3 A risk allele exhibited a higher basal level of STAT3 tyrosine phosphorylation, and an increased IL-23 triggered the pSTAT3 level. We also demonstrated that there was no delayed dephosphorylation of STAT3 in ILCs of both A/A and G/G donors. Overall, the results of this study suggest that IL-23-induced activation of STAT3 in the CD117-NKp44- ILC1s involves in ILC1-to-ILC3 plasticity and a potential regulatory role of ILC1 function. Those genetically susceptible individuals carried STAT3 rs744166 risky allele appear to have higher basal and cytokine-stimulated activation of STAT3 signal, leading to prolonged inflammation and chronic relapse

Signal transduction and activator of transcription-3 (STAT3) in patients with colorectal cancer: associations with the phenotypic features of the tumour and host

Purpose: In patients with colorectal cancer (CRC), a high-density local inflammatory infiltrate response is associated with improved survival, whereas elevated systemic inflammatory responses are associated with poor survival. One potential unifying mechanism is the IL-6/JAK/STAT3 pathway. The present study examines the relationship between tumour total STAT3 and phosphorylated STAT3Tyr705 (pSTAT3) expression, host inflammatory responses and survival in patients undergoing resection of stage I-III CRC. Experimental Design: Immunohistochemical assessment of STAT3/pSTAT3 expression was performed using a tissue microarray and tumour cell expression divided into tertiles using the weighted histoscore. The relationship between STAT3/pSTAT3 expression and local inflammatory (CD3+, CD8+, CD45R0+, FOXP3+ T-cell density and Klintrup-Mäkinen grade) and systemic inflammatory responses and cancer-specific survival were examined. Results: 196 patients were included in the analysis. Cytoplasmic and nuclear STAT3 expression strongly correlated (r=0.363, P<0.001); nuclear STAT3 and pSTAT3 expression weakly correlated (r=0.130, P=0.068). Cytoplasmic STAT3 was inversely associated with the density of CD3+ (P=0.012), CD8+ (P=0.003) and FOXP3+ T-lymphocytes (P=0.002) within the cancer cell nests and was associated with an elevated systemic inflammatory response as measured by modified Glasgow Prognostic Score (mGPS2: 19% vs. 4%, P=0.004). The combination of nuclear STAT3/pSTAT3 stratified five-year survival from 81% to 62% (P=0.012), however was not associated with survival independent of venous invasion, tumour perforation or tumour budding. Conclusion In patients undergoing CRC resection, STAT3 expression was associated with adverse host inflammatory responses and reduced survival. Up-regulation of tumour STAT3 may be an important mechanism whereby the tumour deregulates local and systemic inflammatory responses

Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation

Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease

Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway.

BackgroundThe activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.MethodsExpression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.ResultsStat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.ConclusionsStat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance

Target-specific glioma therapy in an immunocompetent mouse model : meeting abstract

Objective: Establishment of an immunocompetent mouse model representing the typical progressive stages observed in malignant human gliomas for the in vivo evaluation of novel target-specific regimens. Methods: Isolated clones from tumours that arose spontaneously in GFAP-v-src transgenic mice were used to develop a transplantable brain tumour model in syngeneic B6C3F1 mice. STAT3 protein was knocked down by infection of tumour cells with replication-defective lentivirus encoding STAT3-siRNA. Apoptosis is designed to be induced by soluble recombinant TRAIL + chemical Bcl-2/Bcl-xL inhibitors. Results: Striatal implantation of 105 mouse tumour cells resulted in the robust development of microscopically (2 – 3 mm) infiltrating malignant gliomas. Immunohistochemically, the gliomas displayed the astroglial marker GFAP and the oncogenic form of STAT3 (Tyr-705-phosphorylated) which is found in many malignancies including gliomas. Phosphorylated STAT3 was particularly prominent in the nucleus but was also found at the plasma membrane of peripherally infiltrating glioma cells. To evaluate the role of STAT3 in tumour progression, we stably expressed siRNA against STAT3 in several murine glioma cell lines. The effect of STAT3 depletion on proliferation, invasion and survival will be first assessed in vitro and subsequently after transplantation in vivo. Upstream and downstream components of the STAT3 signalling pathway as well as possible non-specific side effects of STAT3-siRNA expression after lentiviral infection will be examined, too. Conclusions: Its high rate of engraftment, its similarity to the malignant glioma of origin, and its rapid locally invasive growth should make this murine model useful in testing novel therapies for malignant gliomas

Shmt2: a stat3 signaling new player in prostate cancer energy metabolism

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotyp