Zangbéwendé Guy Ouédraogo

MD-PhD Julian Biau

MD-MSc Jean-Louis Kemeny

MD-PhD Laurent Morel

PhD Pierre Verrelle

MD-PhD Emmanuel Chautard
email: emmanuel.chautard@clermont.unicancer.fr

Jean Centre

Perrin

Role of STAT3 in genesis and progression of human malignant gliomas

Keywords: STAT3, Glioma, Tyrosine 705, Serine 727

Signal Transducer and Activator of Transcription 3 (STAT3) is aberrantly activated in glioblastoma and has been identified as a relevant therapeutic target in this disease and many other human cancers. After two decades of intensive research, there is not yet any approved STAT3-based glioma therapy. In addition to the canonical activation by tyrosine 705 phosphorylation, concordant reports described a potential therapeutic relevance of other post-translational modifications including mainly serine 727 phosphorylation. Such reports reinforce the need to refine the strategy of targeting STAT3 in each concerned disease. This review focuses on the role of serine 727 and tyrosine 705 phosphorylation of STAT3 in glioma. It explores their contribution to glial cell transformation and to the mechanisms that make glioma escape to both immune control and standard treatment.

INTRODUCTION

Glioma are the most frequent primary adult brain tumors. In 2016, the WHO published a new classification of glioma integrating both histological and biomolecular features [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. They are classified by histological pattern as astrocytic and oligodendroglial tumors according to the World Health Organization (WHO) classification. The most of adults' glioma are infiltrative or diffuse tumors which make resection almost always incomplete. Diffuse glioma are categorized into low-grade glioma (WHO grade II), with slow growth, and high-grade or anaplastic glioma (WHO grades III and IV) that are fast-growing. Glioblastoma (GBM) is the most frequent and malignant type of glioma (grade IV), the most common primary tumors of the central nervous system. Primary GBMs arise de novo while secondary GBMs develop from grade II and III glioma. Primary and secondary GBMs are indistinguishable histologically, but they differ in their genetic and epigenetic profiles [START_REF] Ricard | Primary brain tumours in adults[END_REF]. Isocitrate dehydrogenase 1 (IDH1) and less frequently IDH2 mutations are major molecular markers of secondary GBMs [START_REF] Ohgaki | The definition of primary and secondary glioblastoma[END_REF]. The first line setting of GBM patient treatment encompasses surgery followed by tumor bed irradiation and concomitant and then adjuvant chemotherapy with temozolomide [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. Despite such a complex therapy, recurrence is unavoidable, due to GBM cell radiochemoresistance [START_REF] Chan | Survival and Failure Patterns of High-Grade Gliomas After Three-Dimensional Conformal Radiotherapy[END_REF] and the median survival is nowadays around 14 months. Management of GBM patients at recurrent disease state is not unanimously codified because of an enhanced aggressiveness [START_REF] Yin | The treatment of glioblastomas: A systematic update on clinical Phase III trials[END_REF]. Glioma are one of human cancers in which existence of cancer-initiating cells also called cancer stem-like cells, has been established [START_REF] Galli | Isolation and Characterization of Tumorigenic, Stem-like Neural Precursors from Human Glioblastoma[END_REF][START_REF] Singh | Identification of a Cancer Stem Cell in Human Brain Tumors[END_REF]. Cancer stem cells have self-renewal and tumorigenic capacities and reproduce some but not all stem cells behaviors [START_REF] Lee | Epigenetic-Mediated Dysfunction of the Bone Morphogenetic Protein Developmental Pathway Inhibits Differentiation of Human Glioblastoma Tumor Initiating Cells[END_REF]. Genetic studies using murine glioma models revealed that glioma stemlike cells may arise from neural stem cells that are localized mainly at the ependymal surface of the ventricles [START_REF] Wang | Expression of mutant p53 proteins implicates a lineage relationship between neural stem cells and malignant astrocytic glioma in a murine model[END_REF]. Furthermore, it has been reported that GBM in close proximity to the ependymal surface of the ventricles convey a worse prognosis [START_REF] Young | Longitudinal MRI evidence for decreased survival among periventricular glioblastoma[END_REF][START_REF] Jafri | Relationship of glioblastoma multiforme to the subventricular zone is associated with survival[END_REF]. Therefore, glioma stem-like cells and their specific features are thought to be involved in glioma genesis, recurrence and aggressiveness.

Several pathways have been reported as involved in the pathogenesis of GBM [START_REF] Network | Comprehensive genomic characterization defines human glioblastoma genes and core pathways[END_REF]. Some of them have been reported as prognostic markers or therapeutic targets. Signal Transducer and Activator of Transcription 3 (STAT3) has been identified as a therapeutic target [START_REF] Brantley | STAT-3: A Molecular Hub for Signaling Pathways in Gliomas[END_REF] in glioma because (i) several signalling pathways altered in glioma converge to STAT3 and (ii) it is involved in several hallmarks of glioma aggressiveness, through modulation of genes notably implicated in cell proliferation, growth, apoptosis, migration, invasion and neoangiogenesis. STAT3 belongs to a protein family encompassing seven members sharing a common structure but largely differing in their functions. It is well established that STAT3 is an intracellular cell signalling protein activated by canonical phosphorylation [START_REF] Wegenka | Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level[END_REF] on its tyrosine 705 residue (pSTAT3-Y705), which induces its transfer to the nucleus for target gene regulation [START_REF] Darnell | Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins[END_REF][START_REF] Hervas-Stubbs | Direct Effects of Type I Interferons on Cells of the Immune System[END_REF][START_REF] Vallières | L'interleukine-6 dans le système nerveux central[END_REF]. In addition to glioma, many other human cancers display activated STAT3. Therefore, studying the role STAT3 in cancer provided rational for design of STAT3 inhibitors to be used as anticancer targeted therapy [START_REF] Miklossy | Therapeutic modulators of STAT signalling for human diseases[END_REF][START_REF] Szeląg | Comparative screening and validation as a novel tool to identify STAT-specific inhibitors[END_REF]. However such inhibitors have not yet been approved for treatment of glioma patients because of toxicity or inefficiency. This strengthens the need to refine the strategy of targeting STAT3 in glioma. Furthermore, recent concordant evidences attributed biological functions to posttranslational STAT3 modifications other than Y705 phosphorylation, including lysine methylation [START_REF] Yang | Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes[END_REF] and acetylation [START_REF] Shea | Stat Acetylation--A Key Facet of Cytokine Signaling?[END_REF][START_REF] Ray | STAT3 NH2-Terminal Acetylation Is Activated by the Hepatic Acute-Phase Response and Required for IL-6 Induction of Angiotensinogen[END_REF][START_REF] Yuan | Stat3 Dimerization Regulated by Reversible Acetylation of a Single Lysine Residue[END_REF] as well as serine 727 phosphorylation (pSTAT3-S727). All these modifications cooperate with or act independently from the canonical pSTAT3-Y705 in regulating STAT3 activity [START_REF] Fan | NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts[END_REF][START_REF] Qin | Activation of Stat3 through a Phosphomimetic Serine727 Promotes Prostate Tumorigenesis Independent of Tyrosine705 phosphorylation[END_REF][START_REF] Villalva | STAT3 is essential for the maintenance of neurosphere-initiating tumor cells in patients with glioblastomas: A potential for targeted therapy?[END_REF]. Existence of such post-translational STAT3 modifications should be taken in account in STAT3 based drug designing. However, investigators did not always assess STAT3 activation through detection of all activated forms, including mainly pSTAT3-Y705 and pSTAT3-S727 together. Understanding the fine regulation of STAT3 in glioma and the role of each of its post-translational modifications in the disease aggressiveness may help improve the therapeutic targeting of STAT3.

After a general description of STAT3 signalling, this review focuses on S727 and Y705 phosphorylation of STAT3 and their involvement in the biological processes that lead to glial cell transformation as well as tumor growth and escape to both immune control and standard treatment.

STAT3 IN THE STAT PROTEIN FAMILY

The STAT proteins constitute a family of signal transducers and transcriptional regulators. This family encompasses STAT1, 2, 3, 4, 5A, 5B and 6 [28]. Each of them is encoded by a unique different gene. The STAT proteins are characterized by a common structure (Figure 1) that typically consists of an N-terminal domain, a coiled-coil domain, a DNA-binding domain, a linker, an SH2 domain, and a transactivation (TA) domain [START_REF] Ren | Crystal structure of unphosphorylated STAT3 core fragment[END_REF]. The TA domain contains tyrosine and serine phosphorylation sites that are involved in STAT activation. There are truncated isoforms which are generated by alternative mRNA splicing of the single gene transcript of STAT1 [START_REF] Shuai | A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma[END_REF], STAT3 [START_REF] Caldenhoven | STAT3β, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription[END_REF][START_REF] Chakraborty | Granulocyte colony-stimulating factor activation of Stat3 alpha and Stat3 beta in immature normal and leukemic human myeloid cells[END_REF][START_REF] Schaefer | Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3[END_REF], STAT4 [START_REF] Hoey | Distinct requirements for the naturally occurring splice forms Stat4α and Stat4β in IL-12 responses[END_REF], STAT5A [START_REF] Wang | Naturally occurring dominant negative variants of Stat5[END_REF], STAT5B [START_REF] Ripperger | Transcription Factors Stat3 and Stat5b Are Present in Rat Liver Nuclei Late in an Acute Phase Response and Bind Interleukin-6 Response Elements[END_REF] and STAT6 [START_REF] Patel | Regulation of interleukin 4-mediated signaling by naturally occurring dominant negative and attenuated forms of human Stat6[END_REF]. In addition, isoforms resulting from proteolytic cleavage of STAT3 [START_REF] Chakraborty | Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils[END_REF], STAT5A [START_REF] Azam | Functionally Distinct Isoforms of STAT5 Are Generated by Protein Processing[END_REF][START_REF] Caldenhoven | Activation of a functionally distinct 80-kDa STAT5 isoform by IL-5 and GM-CSF in human eosinophils and neutrophils[END_REF], STAT5B and STAT6 [START_REF] Sherman | IL-4 Induces the Proteolytic Processing of Mast Cell STAT6[END_REF] have been identified. Most truncated isoforms act as dominant-negative of the corresponding full-length proteins [START_REF] Chakraborty | Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils[END_REF][START_REF] Ilaria | Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation[END_REF][START_REF] Piazza | Myeloid differentiation of FdCP1 cells is dependent on Stat5 processing[END_REF]. STAT2, 4 and 6 are mainly involved in immune system regulation whereas STAT1, 3 and 5 play immunological and many other biological functions. STAT3 and STAT5 are hyper-activated in many human cancers where they contribute to cell transformation [START_REF] Furqan | STAT inhibitors for cancer therapy[END_REF][START_REF] Lavecchia | STAT-3 inhibitors: state of the art and new horizons for cancer treatment[END_REF][START_REF] Yu | The STATs of cancernew molecular targets come of age[END_REF] and to survival and proliferation of transformed cells [START_REF] Catlett-Falcone | Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells[END_REF]. Therefore, they have been identified as relevant targets for the development of anticancer therapies [START_REF] Furqan | STAT inhibitors for cancer therapy[END_REF][START_REF] Kim | STAT3 Activation in Glioblastoma: Biochemical and Therapeutic Implications[END_REF].

The gene encoding STAT3 is located in the human 17q21.31 genomic region and is expressed in several cell types [START_REF] Zhong | Stat3 and Stat4: members of the family of signal transducers and activators of transcription[END_REF]. Alternative mRNA splicing and proteolytic cleavage give rise to STAT3α (full-length) isoform consisting in 770 aminoacids and to STAT3β, a 722 aminoacids dominant-negative variant [START_REF] Chakraborty | Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils[END_REF]. STAT3 expression is crucial for the embryogenesis, attested by mouse models, since homozygous knock-out of STAT3 resulted in lethal embryonic degeneration at day 6.5-7.5 [START_REF] Takeda | Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality[END_REF]. Consistently, there is no reported homozygous inactivation of STAT3 in human, strengthening the requirement of STAT3 in embryonic development.

Heterozygous inactivating mutations by transition, transversion or punctual deletions in STAT3, mainly located in the DNA-binding domain and the SH2 domain, have been associated to Autosomal Dominant Hyper-Immunoglobulin E Syndrome (AD-HIES) previously called Job's syndrome [START_REF] Buckley | Extreme hyperimmunoglobulinemia E and undue susceptibility to infection[END_REF][START_REF] Davis | Job's Syndrome. Recurrent, "cold", staphylococcal abscesses[END_REF][START_REF] Holland | STAT3 Mutations in the Hyper-IgE Syndrome[END_REF][START_REF] Kane | STAT3 is a central regulator of lymphocyte differentiation and function[END_REF][START_REF] Minegishi | Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome[END_REF]. AD-HIES patients have an elevated IgE level in serum, with an immune deficiency that involves lymphocytes, mainly Th17, Tfh, memory B cells and TCD8+ cells. They also have a susceptibility to develop B type lymphomas and are exposed to diverse infectious diseases.

Furthermore, gain-of-function mutations in the DNA binding domain or the dimerization domain of STAT3 have been associated to auto-immune disorders that confirmed the involvement of STAT3 in the immune system functional maturation [START_REF] Haapaniemi | Autoimmunity, hypogammaglobulinemia, lymphoproliferation and mycobacterial disease in patients with dominant activating mutations in STAT3[END_REF][START_REF] Milner | Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations[END_REF]. Functional activation of STAT3 due to punctual mutations in SH2 domain has been observed in chronic lymphoproliferative disorders of NK cells and in T-cell large granular lymphocyte leukemia [START_REF] Jerez | STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia[END_REF][START_REF] Koskela | Somatic STAT3 Mutations in Large Granular Lymphocytic Leukemia[END_REF].

GENERAL DESCRIPTION OF STAT3 PATHWAY SIGNALLING

The pool of STAT3 in cells is submitted to a fine regulation consisting in several mechanisms. This includes a synthesis-degradation cycle, controlled by proteasomedependent pathways, that regulates total STAT3 availability [START_REF] Malek | Ciliary neurotrophic factor and phorbol ester each decrease selected STAT3 pools in neuroblastoma cells by proteasome-dependent mechanisms[END_REF][START_REF] Nie | Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin-proteasome pathway[END_REF]. Another mechanism is an activation-inactivation cycle that makes STAT3 activation transient as a rule. Phosphorylation/dephosphorylation is the best known STAT3 pathway modulating mechanism. This is due to cooperation between finely regulated enzymes which are tyrosine kinases, serine/threonine kinases and many phosphatases [START_REF] Mandal | Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 -Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells[END_REF]. Phosphorylation is prior to nuclear translocation and subsequent gene expression modulation. The main conserved signalling pathways driving STAT3 phosphorylation are summarized in Figure 2.

From STAT3-Y705 phosphorylation to dimerization and transcription activation

At its discovery in 1994, STAT3 was described as an acute-phase response factor activated in response to cytokines that induce signal through the gp130 transmembrane protein. Those cytokines include interleukin-6 (IL-6), leukemia inhibitory factor (LIF), oncostatin M (OSM), and the ciliary neurotrophic factor (CNTF) [START_REF] Akira | Molecular cloning of APRF, a novel IFNstimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway[END_REF][START_REF] Zhong | Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6[END_REF]. Upon phosphorylation of gp130, STAT3 is recruited to the gp130 cytoplasmic region by interaction with its SH2 domain. To phosphorylate the Y705 residue of STAT3, the receptor complex recruits a member of Janus Kinase (JAK) family of cytoplasmic kinases including JAK1, JAK2, JAK3 and TYK2. Among them, JAK2 is the main STAT3-Y705 activator (Figure 2).

Plasma membrane receptors carrying an intrinsic intracellular protein-tyrosine kinase activity may also trigger STAT3 phosphorylation of the Y705 residue. These include growth factor receptors such as the EGFR [START_REF] Grandis | Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor-mediated cell growth In vitro[END_REF][START_REF] Sartor | Role of Epidermal Growth Factor Receptor and STAT-3 Activation in Autonomous Proliferation of SUM-102PT Human Breast Cancer Cells[END_REF] and PDGFR [START_REF] Vignais | Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins[END_REF].

Besides intrinsic and extrinsic activity involving cell-surface cytokine receptors, pSTAT3-Y705 can be induced directly by some cytoplasmic kinases including the Src family [START_REF] Yu | Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein[END_REF], Bone Marrow X-linked (BMX [START_REF] Saharinen | The Bmx Tyrosine Kinase Induces Activation of the Stat Signaling Pathway, Which Is Specifically Inhibited by Protein Kinase Cδ[END_REF][START_REF] Wen | Kinase Activation of the Non-receptor Tyrosine Kinase Etk/BMX Alone Is Sufficient to Transactivate STAT-mediated Gene Expression in Salivary and Lung Epithelial Cells[END_REF]) and the Bcr-Abl fusion protein [START_REF] Allen | c-Abl regulates Mcl-1 gene expression in chronic lymphocytic leukemia cells[END_REF][START_REF] Coppo | Constitutive and specific activation of STAT3 by BCR-ABL in embryonic stem cells[END_REF].

Two pSTAT3-Y705 associate to form active dimers capable of translocating to the nucleus. Therein, they interact with specific response element consisting in TTN 4-5 AA consensus DNA sequences called Interferon Gamma-activated sequences (GAS). They therefore induce transcription of specific target genes [START_REF] Darnell | Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins[END_REF][START_REF] Hervas-Stubbs | Direct Effects of Type I Interferons on Cells of the Immune System[END_REF] (Figure 3).

Serine 727 phosphorylation and other post-translational modifications of STAT3

In addition to Y705 phosphorylation, STAT3 is also activated by serine 727 phosphorylation. The functions and the kinases responsible of S727 phosphorylation of STAT3 have been discussed during the last 2 decades [START_REF] Chung | STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation[END_REF][START_REF] Lim | Serine Phosphorylation and Negative Regulation of Stat3 by JNK[END_REF][START_REF] Wen | Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3[END_REF]. S727 phosphorylation is achieved by serine-threonine kinases that are members of the Protein Kinase C (PKC) family such as PKC-δ [START_REF] Jain | Protein Kinase C δ Associates with and Phosphorylates Stat3 in an Interleukin-6-dependent Manner[END_REF], PKC-ε [START_REF] Aziz | Protein kinase C? mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression and cell invasion in various human cancer cell lines via integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)[END_REF][START_REF] Aziz | Protein Kinase Cε Interacts with Signal Transducers and Activators of Transcription 3 (Stat3), Phosphorylates Stat3Ser727, and Regulates Its Constitutive Activation in Prostate Cancer[END_REF][START_REF] Xu | Knockdown of PKCε Expression Inhibits Growth, Induces Apoptosis and Decreases Invasiveness of Human Glioma Cells Partially Through Stat3[END_REF]. Cyclin-dependent kinase 5 was recently shown to interact and phosphorylate STAT3-S727 [START_REF] Lam | Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727[END_REF]. Other kinases downstream the MAP Kinase pathway [START_REF] Chung | STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation[END_REF][START_REF] Lim | Serine Phosphorylation and Negative Regulation of Stat3 by JNK[END_REF][START_REF] Turkson | Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation[END_REF] and the NOTCH pathway [START_REF] Fan | NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts[END_REF] have also been reported as involved in STAT3-S727 phosphorylation (Figure 2).

Recent studies provided evidence that the biological function and the regulation of pSTAT3-S727 depend on cell type and cell-differentiation state [START_REF] Qin | Activation of Stat3 through a Phosphomimetic Serine727 Promotes Prostate Tumorigenesis Independent of Tyrosine705 phosphorylation[END_REF][START_REF] Villalva | STAT3 is essential for the maintenance of neurosphere-initiating tumor cells in patients with glioblastomas: A potential for targeted therapy?[END_REF][START_REF] Miyakoshi | Serine 727 phosphorylation of STAT3: An early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine[END_REF][START_REF] Sherry | STAT3 Is Required for Proliferation and Maintenance of Multipotency in Glioblastoma Stem Cells[END_REF]. Considering together the published data, it becomes clear that STAT3 plays a critical role in the control of many cellular processes (Figure 3). However the contribution of each of pSTAT3-Y705 and pSTAT3-S727 in those processes remains unclear because authors did not use methods that allow such distinguishing. In the light of the current knowledge, one can hypothesize that some genes are preferentially regulated by only one or both pSTAT3-Y705 and pSTAT3-S727. In case of involvement of both phosphorylations, their action could be consistent or not. For instance, results reported by Xu et al. provided evidence that knockdown of PKCε attenuates pSTAT3-S727 and expression of Bcl-XL but not that of Bcl-2 which is another STAT3 downstream target. The authors hypothesized that knockdown of PKCε may influence signalling pathways that may compensate each other in regulating Bcl2 expression [START_REF] Xu | Knockdown of PKCε Expression Inhibits Growth, Induces Apoptosis and Decreases Invasiveness of Human Glioma Cells Partially Through Stat3[END_REF]. We further speculate, because pSTAT3-Y705 was unaffected by PKCε inhibition, that it may have maintained Bcl-2 expression.

In addition to phosphorylation, concordant evidences attributed biological functions to other post-translational modifications of STAT3 including lysine methylation [START_REF] Yang | Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes[END_REF] and acetylation [START_REF] Shea | Stat Acetylation--A Key Facet of Cytokine Signaling?[END_REF]. Such modifications require further investigation to decipher their role in the STAT3 cascade.

STAT3 pathway down-regulation by tyrosine phosphatase activity

The identified phosphatases involved in the dephosphorylation of pSTAT3-Y705 belong to a family of receptor-type phosphatases and to a group of non-receptor (intracellular) protein-tyrosine phosphatases. The category of receptor-like phosphatases includes mainly the Receptor-type tyrosine-protein phosphatase delta (PTPRD) [START_REF] Jiang | Germline PTPRD Mutations in Ewing Sarcoma: Biologic and Clinical Implications[END_REF][START_REF] Ostman | Protein-tyrosine phosphatases and cancer[END_REF] and the Receptor-type tyrosine-protein phosphatase O (PTPRO) [START_REF] Hou | Estrogen-sensitive PTPRO expression represses hepatocellular carcinoma progression by control of STAT3[END_REF] that are enzymatic transmembrane proteins. The inhibiting pSTAT3-Y705 intracellular phosphatases are members of the Protein-tyrosine phosphatase non-receptor (PTPN) family including PTPN1 (or PTP1B) [START_REF] Lund | Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling[END_REF] , PTPN2 (or TC-PTP) [START_REF] Lee | Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation[END_REF][START_REF] Yamamoto | The nuclear isoform of proteintyrosine phosphatase TC-PTP regulates interleukin-6-mediated signaling pathway through STAT3 dephosphorylation[END_REF] , PTPN6 (or SHP1) [START_REF] Phromnoi | Dihydroxypentamethoxyflavone Down-Regulates Constitutive and Inducible Signal Transducers and Activators of Transcription-3 through the Induction of Tyrosine Phosphatase SHP-1[END_REF][START_REF] Yin | SHP-1 Arrests Mouse Early Embryo Development through Downregulation of Nanog by Dephosphorylation of STAT3[END_REF] , PTPN9 (or PTPMeg2) [START_REF] Bu | Protein tyrosine phosphatase PTPN9 regulates erythroid cell development through STAT3 dephosphorylation in zebrafish[END_REF][START_REF] Su | Protein tyrosine phosphatase Meg2 dephosphorylates signal transducer and activator of transcription 3 and suppresses tumor growth in breast cancer[END_REF], PTPN11 (or SHP2) [START_REF] Kim | Protein Tyrosine Phosphatases, TC-PTP, SHP1, and SHP2, Cooperate in Rapid Dephosphorylation of Stat3 in Keratinocytes Following UVB Irradiation[END_REF] and PTPN12 [START_REF] Su | PTPN12 inhibits oral squamous epithelial carcinoma cell proliferation and invasion and can be used as a prognostic marker[END_REF].

It was reported that pSTAT3-S727 activates TC45, a nuclear PTPN2 isoform to dephosphorylate pSTAT3-Y705 [START_REF] Wakahara | Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45[END_REF]. Therefore, TC45, may provide an explanation to a kind of a STAT3 autoregulation system described in early study of STAT3 showing that pSTAT3-S727 negatively regulates pSTAT3-Y705 [START_REF] Chung | STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation[END_REF]. More recent investigations demonstrated that PASD1 protein competed with TC45 to associate with STAT3, blocking thus TC45-mediated dephosphorylation of STAT3. PADS1 serves therefore as a critical nuclear positive regulator of pSTAT3-Y705 [START_REF] Xu | PASD1 promotes STAT3 activity and tumor growth by inhibiting TC45-mediated dephosphorylation of STAT3 in the nucleus[END_REF].

STAT3 pathway down-regulation by serine/threonine phosphatase activity

Besides protein-tyrosine phosphatases, STAT3 dephosphorylation also involves protein serine/threonine phosphatases, mainly the Phosphoprotein phosphatase 2A (PP2A) [START_REF] Mandal | Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 -Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells[END_REF][START_REF] Seshacharyulu | Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer[END_REF][START_REF] Zhang | 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells[END_REF]. Activity of such phosphatases on STAT3 is regulated by other STAT3 interacting proteins like the STAT3-Interacting Protein As a Repressor (SIPAR) that promotes dephosphorylation of pSTAT3-Y705 [100] and like 14-3-3ζ, a protein that protects pSTAT3-S727 from dephosphorylation by PP2A [START_REF] Zhang | 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells[END_REF]. It has also been reported that a non-coding long RNA called Inc-DC interacts with STAT3 in dendritic cells and prevents fixation of SHP1 protein-tyrosine phosphatase [101].

Other mechanisms modulating STAT3 pathway

The fine regulation of STAT3 activity also involves the Protein Inhibitor of Activated STAT3 (PIAS3) that is an E3 SUMO-protein ligase [102,103] STAT3 is also submitted to a cytokine-induced acetylation and deacetylation process mediated on its SH2 domain lysine K685 respectively by p300 and type I HDAC family members [START_REF] Yuan | Stat3 Dimerization Regulated by Reversible Acetylation of a Single Lysine Residue[END_REF]. K685 acetylation induces stable dimer formation required for cytokinestimulated DNA binding and gene transcription regulation. K685 acetylation is also critical for functional regulation of STAT3 lacking Y705 phosphorylation [106]. Acetylation of the STAT3 Coiled-coil domain on the K49 and K87 lysines has been reported. These modifications are also controlled by p300 and type I HDAC family members. However, functional analysis showed that unlike K685 acetylation, they are not involved in STAT3 dimerization and translocation to the nucleus, but they are required for IL-6-induced gene transcription [START_REF] Ray | STAT3 NH2-Terminal Acetylation Is Activated by the Hepatic Acute-Phase Response and Required for IL-6 Induction of Angiotensinogen[END_REF].

Activated STAT3 is also regulated by methylation on K140 by the histone methyltransferase SET9 and by demethylation mediated by LSD1 [START_REF] Yang | Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes[END_REF]. K140 methylation of STAT3 is a negative regulator of transcription activation by promoter bound STAT3.

THE MECHANISMS LEADING TO STAT3 ACTIVATION IN HUMAN GLIOMA CELLS Activation of protein tyrosine kinases

There is not any identified gain-of-function mutation of human STAT3 in glioma; the constitutive activation of STAT3 in this disease is therefore due to an aberrant signal from upstream regulators. This includes, on one hand, any gain-of-function mutation or increase in activation of an upstream activator, and, on the other hand, any loss-of-function mutation or any decrease in activation of an upstream repressor. The first group involves mainly alterations in the EGFR signalling pathway and those in the IL-6 signalling axis. Both pathways are induced by receptor-associated tyrosine kinase activities. Gene amplification and/or rearrangements that result in the EGFRvIII truncated variant of EGFR, or the EGFR-SEPT 14 fusion mutant of EGFR, induce an aberrant, excessive signal leading to a hyperactive pSTAT3-Y705 [115,116]. Insulin-like growth factor binding protein 2 (IGFBP2) augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signalling pathway [117].

In addition to or independently of EGFR activation, IL-6 participates in complex genetic and epigenetic regulatory circuits that orchestrate STAT3 constitutive activation in glioma [118]. We have reported IL-6 overexpression in GBM patients and cell lines [119,120]. IL-6 induces STAT3 activation by a signal transduced through an hexameric transmembrane receptor complex that implicates the IL-6Rα (gp80) and the gp130 subunits and that is shared with receptors of other cytokines such as IL-11, LIF, CT-1, CTNF, OSM and CLC [121]. Among them, OSM plays, as IL-6, a critical role in glioma [122,123]. OSM mainly contributes to aggressiveness of mesenchymal subtype of glioma [124]. OSM receptor (OSMR) was reported to form a complex with EGFRvIII. This complex activates STAT3 and upregulates OSMR expression. The result is a feed-forward signalling mechanism that drives oncogenesis [125]. IL-6 induced STAT3 activation was usually assessed by monitoring pSTAT3-Y705 [126]. But Liu et al. reported a dose-dependent increasing accumulation of pSTAT3-S727 in T98G and U251 human GBM cell lines treated by various concentrations of . In that assay, variation of pSTAT3-Y705 has not been evidenced. To our knowledge, such demonstration has not yet been reproduced. We used IL-6 blocking antibodies to challenge GBM cells and it resulted in a decrease in pSTAT3-Y705 while pSTAT3-S727 remained stable (unpublished data). Apart from gp130 signalling chemokines, IL-22, hypothetically secreted by stromal cells was reported to activate pSTAT3-Y705 in glioma cells [128]. Moreover, it has been shown that STAT3 activation increased transactivation of VEGF promoter and VEGF secretion by GBM cells [129,130]. In addition to those pathways, pSTAT3-Y705 can be induced through signal induced by many other cytokines or growth factor receptors such as PDGFR, depending on cell type or experimental conditions [131].

Besides its induction by receptor-associated tyrosine kinase activity, activation of STAT3 in glioma can also be triggered by non-receptor tyrosine kinases. This implicates intracellular kinases such as BMX which is expressed by some GBM stem-like cell subpopulation [132]. Since knocking down BMX impaired pSTAT3-Y705 accumulation, it has been described as an upstream activator of STAT3 in GBM stem-like cells.

Activation of protein serine/threonine kinases

Serine/threonine kinases are intracellular proteins that can mediate STAT3 activation by phosphorylating its serine 727 residue. The serine/threonine kinase protein kinase C epsilon (PKCε) is overexpressed in human anaplastic astrocytoma, GBM and gliosarcoma and is associated to a worse disease prognosis [133]. This overexpression of PKCε has been shown to take part in the constitutive activation of STAT3 through serine 727 phosphorylation in several human glioma cell lines [START_REF] Aziz | Protein kinase C? mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression and cell invasion in various human cancer cell lines via integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)[END_REF][START_REF] Xu | Knockdown of PKCε Expression Inhibits Growth, Induces Apoptosis and Decreases Invasiveness of Human Glioma Cells Partially Through Stat3[END_REF].

The NOTCH pathway has been implicated in STAT3 activation since it has been shown that NOTCH signalling stimulation by Delta-like 4 (Dll4) and Jagged 1 (Jag1) NOTCH ligands promotes human embryonic stem cell survival through STAT3 serine 727 phosphorylation [134]. This NOTCH-mediated effect on normal stem cell fate can be blocked by the NOTCH pathway inhibitor DAPT, a gamma-secretase inhibitor that impairs activating cleavage of NOTCH by Presenilin 1. Moreover, the preponderant role of NOTCH in activating STAT3-S727 in glioma became evident when Fan et al. demonstrated that blocking activated NOTCH pathway in GBM stem-like cells by Gamma-Secretase Inhibitor 18 , reversed accumulation of pSTAT3-S727 and furthermore selectively impeded cell proliferation [START_REF] Fan | NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts[END_REF]. Those findings suggest that there is a regulation loop involving activated STAT3 and NOTCH signalling because NOTCH pathway regulates pSTAT3-S727 and pSTAT3-Y705 participates with NFκB in regulating activated NOTCH signalling in GBM stem-like cells [135]. More studies should help decipher this regulatory loop in GBM stemlike cells.

Deficiency in upstream repression of STAT3

Apart from an increased activity of an upstream activator, any loss-of-function mutation or any decrease in activation of an upstream repressor may explain the constitutive activation of STAT3 in glioma. This second group of deregulating mechanisms involves the PIAS3, the SOCS, and PTPRD.

Concordantly with pSTAT3-Y705 and pSTAT3-S727 overexpression, the PIAS3 is less expressed in GBM in comparison with non-neoplastic brain tissue [136]. Furthermore, inhibition of PIAS3 in U87-MG human glioma cell line by RNA interference boosted cell proliferation despite no or little change in the level of phosphorylated STAT3. Conversely, overexpression of PIAS3 in U251-MG human glioma cell line dumped OSM-enhanced expression of the STAT3 targeted genes Survivin, Bcl-xL as well as SOCS3 and resulted in 80% slow-down of cell proliferation [136].

The SOCS protein family includes cytokine inducible signal transducers that encompass several members involved in negatively regulating cytokine and growth factorinduced signal [137]. Among them, SOCS3 is not only a STAT3 target-gene but also an upstream repressor of STAT3 via its inhibitory action on JAK kinases [138]. Thus, SOCS3 functions in a negative feedback loop to regulate STAT3 activity. GBM tumors and cell lines overexpress SOCS3 whereas they repress SOCS1 [136,139,140]. This aberrant expression of SOCS3, concomitant with a constitutive activation of STAT3, suggests that SOCS3 is induced by STAT3 pathway [140] even if SOCS3 might fail in inhibiting STAT3 in GBM. Further study should help understand the link between the constitutive silencing of SOCS1 and activated STAT3.

PTPRD belongs to a family of protein-tyrosine phosphatases which are involved in the regulation of many normal and cancer cell processes such as adhesion, proliferation and migration, through the regulation of multiple cell-signalling pathways [START_REF] Ostman | Protein-tyrosine phosphatases and cancer[END_REF]. PTPRD function is frequently inactivated by genetic and epigenetic alterations in GBM as well as in other cancers, and is associated to a worse patient prognosis [141,142]. Functional analysis in mice, recently published by Ortiz et al. showed that loss of heterozygoty of PTPRD resulted in accumulation of pSTAT3-Y705 and cooperated with CDKN2A/p16 IN4KA to promote glioma tumorigenesis [143].

Other STAT3 activating mechanisms

Apart from deregulation of upstream regulators, other factors cooperate in inducing hyperactivity of STAT3 in glioma. Some of them have been identified but more studies are required to well characterize the mechanisms of their action. This is the case of the mutation in the GDNF Family Receptor Alpha 4 (GFRA4) gene exon 2 that induces mislocated weaklyinteracting GFRA4-persephin (PSPN) complexes. These complexes aberrantly dissociate from the RET proto-oncogene product (RET) at the plasma membrane and relocate to the endoplasmic reticulum [144]. Moreover, GFRA4 silencing or artificial redirection of GFRA4 to the plasma membrane reduced pSTAT3-Y705 and resulted in slowed glioma cell proliferation.

EVIDENCE AND PROGNOSTIC VALUE OF STAT3 ACTIVATION IN GLIOMA SPECIMENS Phosphorylation of STAT3-Y705

Phosphorylated STAT3 has been detected by immunohistochemistry assays performed on frozen or paraffin-embedded glial tumour sections and by immunoblotting of tumour tissue samples. The early studies reported positive detection of pSTAT3-Y705 in a variable proportion of glioma of the studied cohorts [116,[145][146][147][START_REF] Wang | Analysis of the activation status of Akt, NFκB, and Stat3 in human diffuse gliomas[END_REF]. This variability might be due to the cohort size and to the limits of sensitivity and specificity of the diagnosis and of the detection methods. Moreover, comparison of the published results should consider that the cut-off point of positive detection was different. The detected pSTAT3-Y705 was located in the nucleus of a variable number of glial tumour cells or tumour-associated lymphocytes or vascular endothelial cells [116,146,[START_REF] Wang | Analysis of the activation status of Akt, NFκB, and Stat3 in human diffuse gliomas[END_REF]. Normal brain tissue was negative for pSTAT3-Y705 staining except in one over 5 specimens reported by Lo et al. [116]. Considering the group of specimens in which the section showed more than 20% pSTAT3-Y705 positively stained cells, Lo et al. found a positive correlation of pSTAT3-Y705 and glioma grade. However, in a cohort of 111 GBM (WHO grade IV glioma) cases and 25 WHO grade III gliomas, Birner et al. did not find any correlation with the disease grade among cases that showed more than 5% positive cells [149]. Using other methods such as electrophoretic mobility shift assay and western blotting, greater proportions of pSTAT3-Y705 expressing tumours or higher levels of pSTAT3-Y705 have been positively correlated to glioma tumour grade [136,150].

Constitutive phosphorylation of STAT3-S727

The detection of serine pSTAT3-S727 in U87-MG and U373-MG glioma cell lines has been achieved by western blot showing STAT3 inhibition that accompanied cell death induced by telomere 3' overhang-specific DNA oligonucleotides [151]. The first description of serine pSTAT3-S727 in glioma tissues has been reported by Brantley et al. [136]. Two years later, this constitutive activation of STAT3-S727 has been confirmed in patient-and cell line-derived GBM stem(-like) cells [START_REF] Fan | NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts[END_REF][START_REF] Villalva | STAT3 is essential for the maintenance of neurosphere-initiating tumor cells in patients with glioblastomas: A potential for targeted therapy?[END_REF]. This study of Villalva et al. evidenced that STAT3 was activated in GBM stem-like cells mainly through pSTAT3-S727. Moreover, disruption of STAT3 activity impaired proliferation and neurosphere formation. Recently pSTAT3-S727 has been described by immunohistochemistry in 70.5% of 88 GBM cases [152]. Even though pSTAT3-S727 was found in the cytoplasm, the main subcellular location was the nucleus of the neoplastic cells and some vascular endothelial cells. Normal brain tissue did not show any expression of pSTAT3-S727. Moreover, this study of Lin et al evidenced a statistically significant association of pSTAT3-S727 and pSTAT3-Y705 [152]. In a cohort of 30 GBM clinical specimens and a panel of 15 GBM cell lines, we showed that pSTAT3-S727 is present in all studied human malignant glioma cell lines and all the examined GBM specimens [153]. However, pSTAT3-Y705 was not detected in some GBM cell lines or specimens and was moreover restricted to a variable number of GBM cells in the positive clinical samples. These results are consistent with an involvement of pSTAT3-S727 in the process of oncogenesis as a driving mechanism as suggested for mouse hepatocarcinomas [START_REF] Miyakoshi | Serine 727 phosphorylation of STAT3: An early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine[END_REF] or as a consequence of cell transformation. Analysis of these two phosphorylations in relation with tumor grade might help to confirm or infirm this hypothesis.

STAT3 AND PROGNOSIS

Many studies have examined the prognostic value of STAT3 expression and activation in glioma. Early studies of the signalling pathways activated downstream EGFR, including MAPK, Akt and STAT3, established that pSTAT3-Y705 is correlated to the presence of the EGFRVIII muted variant of EGFR in anaplastic astrocytoma and GBM [147]. Among those downstream effectors of EGFR pathway, MAPK and Akt but not STAT3 have been correlated to the progression of grade III astrocytoma to GBM (grade IV). In accordance, activated Akt but not pSTAT3-Y705 was predictive of a worse glioma prognosis.

In opposition to Mizoguchi et al. who evaluated STAT3 activity by quantifying pSTAT3-Y705 in tumours, Alvarez et al., assessed functional activation of STAT3 through expression of a subset of 12 genes admitted as defining the STAT3 signature [154]. This study found a positive correlation linking high expression level of STAT3 target genes with a poorer prognosis in glioma. However, they did not correlate phosphorylated STAT3 with gene expression in tumour samples. Another study established that pSTAT3-Y705 was a negative prognostic marker in anaplastic astrocytoma [145]. Furthermore, some other studies reaffirmed the bad prognostic value of the constitutive activation of pSTAT3-Y705 alone or in association with others mesenchymal markers such as C/EBP-β and C/EBP-δ in GBM [155,156].

The bad clinical meaning of STAT3 activation has recently been attested by the studies carried out by Lin et al., which established that high number of pSTAT3-Y705 and pSTAT3-S727 positive cells in GBM specimens, independently each other impact negatively the patients' progression-free survival and overall survival [152,157]. Moreover, increased expression of the both makes worse the clinical outcome [152]. Since, pSTAT3-Y705 is not detected is some glioma specimens and does not accumulate in all glioma neoplastic cells [153], we speculate that it is not a major driver of GBM aggressiveness. In contrast, pSTAT3-S727 detected is all neoplastic cells of all GBM specimens may be a best prognostic marker, provided that a discriminative detection assay is performed.

ROLE OF STAT3 IN GLIOMA TUMORIGENESIS AND AGGRESSIVENESS

After phosphorylation and dimerization, STAT3-dimers translocate into the nucleus where they modulate expression of targeted genes harboring GAS sequences. STAT3 is involved in cell transformation but could also act in tumor cell including GBM stem-like cells to promote aggressiveness and resistance to current therapies. Thus, STAT3 signalling modulates expression of several genes implicated in transformation, survival, proliferation, immunosuppression, migration, invasion and neoangiogenesis (Figure 3).

STAT3 in glioma genesis

The first demonstration of STAT3 involvement in cell transformation rose from the observation that it was activated through pSTAT3-Y705 in fibroblasts and epithelial cells, which have been stably transformed by the Src oncogene tyrosine kinase [START_REF] Yu | Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein[END_REF]. Moreover, transgenic mice expressing v-src under control of GFAP gene regulatory elements developed transplantable astrocytoma that progress to GBM, thus providing evidence of the involvement of v-src and probably the consecutive activated STAT3 in initiating and promoting glioma genesis in a multistep way [158,159]. Consistent with the implication of activated STAT3 in the tumorigenesis of glioma, another analysis have evidenced that malignant transformation by v-src and maintenance of transformed phenotype require STAT3 that is however dispensable for normal cell growth [160]. The genuine oncogenic function of STAT3 by its own became clear with functional analysis using interfering negative or positive STAT3 dominant proteins. Negative dominant mutants rendered cells refractory to v-src transformation [START_REF] Turkson | Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation[END_REF]161] whereas positive dominants induced by themselves carcinogenesis [162,163]. The involvement of STAT3 in tumorigenesis is further explained by studies showing that pSTAT3-S727 localizes in mitochondria of diverse cell-types and sustain cellular functions including mitochondrial gene regulation [164], viability [165] and cell transformation [166]. However, a report demonstrated that STAT3 activation in glioma can also play a tumor suppressor function depending on tumor genetic background mainly on the mutational status of PTEN [167]. Rat orthotopic glioma models generated with the STAT3 S727A mutant (inactivated STAT3-S727) cell line, resulted in more aggressive tumors in comparison to the STAT3 WT expressing stable cell line tumors [START_REF] Mandal | Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 -Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells[END_REF]. However, interpretation of such results should take in account an obvious concomitant increase in pSTAT3-Y705 level in the STAT3 S727A tumor cells. This increased pSTAT3-Y705 may be sufficient to boost tumor aggressiveness in the STAT3-S727A mutant. Concurrently modulating S727 and Y705 is essential in deciphering the contribution of both in tumorigenesis.

STAT3 function in cell survival and proliferation

Inhibition of STAT3 pathway by various strategies resulted in a decreased accumulation of anti-apoptotic gene products such as Survivin, , (Figure 3). In addition, inhibiting activated STAT3 reduced expression of cell cycle regulators: c-myc, cyclin D1 and cyclin E [170]. Those experiments have been conducted in vitro [169] as well as in vivo [170,171]. Through control of regulators of cell cycle and apoptosis, inhibiting STAT3 activity resulted in increased cell death and decreased cell or tumor growth. Since STAT3 activity has been modulated by targeting pSTAT3-Y705 (pharmacological inhibitors, blocking antibodies, and dominant negative proteins) or total STAT3 protein accumulation (RNAi) or STAT3 dimers formation or binding to DNA (decoy oligonucleotides), the role of pSTAT3-S727 remained misunderstood in the cell fate control by STAT3. Interestingly, inhibiting STAT3 dimer formation and DNA-binding by the mean of STA-21, S3I-201 [START_REF] Sherry | STAT3 Is Required for Proliferation and Maintenance of Multipotency in Glioblastoma Stem Cells[END_REF], hydroxamic acid-based (SH5-07) and benzoic acid-based inhibitors (SH4-54) [172] STAT3 inhibitors which target the STAT3 SH2 domain resulted in apoptosis or decreased cell proliferation (assessed by low BrdU incorporation). This depended on the state of cell differentiation, on the cell culture conditions and on the profile of STAT3 phosphorylation (pSTAT3-Y705 or pSTAT3-S727). MicroRNA-506 (miR-506) and MicroRNA-519a (miR-519a) have been reported to act as tumor suppressors and inhibit cell proliferation in glioma by targeting STAT3 mRNA and decreasing expression of STAT3 target genes [173,174]. Any specific pSTAT3-S727 inhibitor should help in understanding the interference of pSTAT3-S727 and pSTAT3-Y705 in the control of cell survival and proliferation.

Regulation of cell survival by STAT3 is tightly linked to blocking of both apoptosis and autophagy since inhibiting active STAT3 by several means induced autophagy and concomitantly sensitized glioma cells to induction of apoptosis [175,176]. These findings suggested that autophagy contributes to glioma escape to anticancer therapy.

STAT3 activation role in glioma cell migration and invasion

The use of inhibitors of STAT3 upstream regulators pointed that STAT3 might be involved in glioma cell migration and invasion [173,174,177,178]. This inhibition of STAT3 has been linked to down-regulation of matrix metalloproteinase gene expression such as MMP2 [179], MMP9 and to epithelial-mesenchymal transition regulating genes like Snail [180] (Figure 3). STAT3 has also been shown to cooperate with other transcription factors such as NF-κB (p65) [181] and Nuclear factor I-X3 [182] in up-regulating respectively ICAM-1 and YKL-40, resulting in promotion of glioma cell migration and invasion.

Role of STAT3 activation in local immune suppression

STAT3 activation has been shown in several models to promote cancer cell immune escape by disturbing the processes involved in normal functioning of both innate and adaptive immune system components. Through plasma membrane interactions [183] and released cytokines such as IL-6, IL-8, IL-10, IP-10 (CXCL10), RANTES, TNFα [184], TGFβ, and the PGE-2 prostaglandin [185], STAT3 activation in glioma cells including cell lines, primary cultured cells and cancer stem cells, has been shown in inducing immune tolerance. Through a mechanism that is not fully understood, STAT3 activation in glioma cells blocks differentiation, maturation and function of dendritic cells, inhibits T-cell proliferation [186] (Figure 3), induces T-cell anergy, T-regulatory cells, and immunosuppressive microglia [187]. An explicative mechanism is provided by the demonstration that activated STAT3 in glioma stem-like cells makes them secrete cytokines that induce paracrine activation of STAT3 in immune cells, thus contributing to their functional inactivation [188]. Furthermore, a very recent work established that glioma stem-like cells produce IL-6 and IL-10, which activate B7-H4 expression in tumor infiltrating macrophages through STAT3 signalling. B7-H4 expression in the tumor microenvironment, surrounding stroma and invasive edge, may contribute to the neoplastic cell escape from immune surveillance by blocking T-cell function [189]. Glioma-induced immunosuppression is potentiated by hypoxia [190]. It can be hampered or reversed by STAT3 inhibition [186,191], suggesting the relevance of using STAT3 inhibitors in glioma immunotherapy. However inhibition of STAT3 did not prevent IL-6 induced suppression of Langerhans cell differentiation [192], indicating that STAT3 activation is not the only one mechanism of glioma-induced immune tolerance. Combined inhibition of STAT3 and the p38 MAPK pathways is therefore proposed as a synergic anticancer immunotherapeutic approach [185]. The role of STAT3 in tumor mediated immune suppression has been recently reviewed by Fergusson et al. [193].

Contribution of STAT3 activation in glioma angiogenesis

Through pSTAT3-Y705, STAT3 upregulates VEGF expression in glioma tumor cells in vitro and in vivo [129,194]. The role of pSTAT3-Y705-induced VEGF expression in glioma angiogenesis and tumor growth is confirmed since U87 human GBM cells expressing dominant-negative STAT3 induced slowly growing tumors with impaired neoangiogenesis [171]. More recently, this was confirmed by the antiangiogenic effect of JSI-124 JAK-STAT chemical inhibitor [195]. Furthermore, it has been shown that angiogenesis in glioma is accompanied by VEGF-A-induced loss of calpastatin (CAST) in endothelial cells. This downregulation of CAST triggers µ-calpain-induced proteolysis of SOCS3 and subsequent hyperactivation of STAT3 which stimulates transcription of VEGF-C. Induced VEGF-C and VEGF-A act synergistically in angiogenesis [196]. Moreover, elevated ROS production plays a crucial role for STAT3 activation and for angiogenesis in hypoxic glioblastoma cells since blocking ROS resulted in STAT3 inhibition [197]. Clinical trials regarding anti-angiogenic therapy with Bevacizumab [198,199], an anti-VEGF monoclonal antibody approved by US Food and Drug Administration (FDA) in recurrent glioma patient treatment, and chemical inhibitors of VEGFR such as Cediranib [200,201] did not exhibit the expected benefit in terms of both safety and overall survival prolongation. Those unexpected results suggest that it might be relevant to integrate the modulation of STAT3 in the strategy of anti-angiogenic therapy because of its function, at least in part, as upstream regulator of not only angiogenesis, but also autophagy and hypoxia-induced biological effects [START_REF] Brantley | STAT-3: A Molecular Hub for Signaling Pathways in Gliomas[END_REF]175].

Role of STAT3 in glioma stem-like cell maintenance

GBM-derived stem-like cells displaying pSTAT3-Y705 and pSTAT3-S727 showed a modified pattern of STAT3 phosphorylation when grown in different conditions [START_REF] Sherry | STAT3 Is Required for Proliferation and Maintenance of Multipotency in Glioblastoma Stem Cells[END_REF]. Using Stattic, an SH2 domain inhibitor, Villalva et al. showed that disruption of STAT3 function impaired neurosphere formation by slowing cell proliferation and inducing apoptosis of GBM stem-like cells [START_REF] Villalva | STAT3 is essential for the maintenance of neurosphere-initiating tumor cells in patients with glioblastomas: A potential for targeted therapy?[END_REF]. STAT3 pathway phosphorylation on S727 or Y705 has been reported to be essential in the maintenance of not only the capacity of these stem-like cells to initiate neurospheres but also their multipotency [START_REF] Fan | NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts[END_REF][START_REF] Sherry | STAT3 Is Required for Proliferation and Maintenance of Multipotency in Glioblastoma Stem Cells[END_REF] and their immunosuppressive ability [186] (Figure 3). The carcinoembryonic antigen-related cell adhesion molecule Ceacam1L was described as a crucial factor in glioma stem-like cell maintenance and tumorigenesis by activating c-Src/STAT3 signalling [202]. In addition, Pigment Epithelium-Derived Factor (PEDF) has been identified as an EGFRvIII-STAT3 induced autocrine factor that promotes stemness of glioma stem-like cells [203]. The work of Talukdar et al. highlighted that pSTAT3-Y705-nanog signalling mediates the MDA-9/Syntenin (SDCBP) scaffold proteincontrolled stemness of GBM-stem-like cells while NOTCH1/c-Myc pathway governs the survival [204]. Mesenchymal stem cells isolated from human glioma sustain stemness of GBM-stem like cells by stimulating the IL-6/gp130/STAT3 axis in a coculture assay [205]. Therefore, GBM stem-like cell stemness generation and maintenance involve complex dialog between tumoral cells and stromal cells in which pSTAT3 isoforms are undoubtedly implicated. Such findings enhanced the need of STAT3 inhibitors because of relevance in targeting both tumoral cells including stem-like cells and stromal cells.

Role of STAT3 activation in glioma resistance to standard treatment

Treatment of malignant glioma is based on surgery, radiotherapy, and concomitant and adjuvant chemotherapy with temozolomide [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. This standard treatment has been reported to prolong patient survival and to delay disease progression [206]. Despite this gold treatment, recurrence is still unavoidable in GBM and recurrent GBM are chemoresistant, mainly due to a selection of resistant tumor clones [START_REF] Yin | The treatment of glioblastomas: A systematic update on clinical Phase III trials[END_REF]207].

The DNA-repair enzyme MGMT has been identified as conferring resistance to temozolomide. Repression of its encoding gene mainly by promoter methylation in tumors is a predicting factor of GBM response to alkylators [208,209]. GBM, the highest degree of glioma malignancy harbor high level of STAT3 activation. This suggests an involvement of STAT3 pathway in glioma progression and a possible link with resistance to treatment. This is strengthened by a report showing that recurrent astrocytic glioma displayed an increased STAT3 protein accumulation compared to primary astrocytic tumors [210].

The involvement of the canonical pSTAT3-Y705 and/or the so far less studied pSTAT3-S727 in resistance to treatment is not yet well defined. Therefore published data are often incomplete because only one of the two phosphorylations (mainly pSTAT3-Y705) is assessed by authors resulting sometimes in contradictory results. Lee et al., reported an increase in total STAT3 and pSTAT3-S727 accumulation and a concomitant decrease in pSTAT3-Y705 in selected temozolomide-resistant cells, independently of MGMT expression, compared to (isogenic) parental [211]. Kohsaka et al., found that down-modulating STAT3 activity by shRNA or chemical inhibitor of pSTAT3-Y705 resulted in attenuated resistance to temozolomide and concomitant decrease in MGMT accumulation [146]. One explanation is the fact that temozomlomide resistance may not be governed only by MGMT. Such observations lead us to hypothesize that pSTAT3-Y705 and pSTAT3-S727 are two alternative drug resistance mechanism in GBM. STAT3 could affect chemoresistance via Y705 phosphorylation and MGMT expression or via S727 phosphorylation and other unknown mechanisms. It has also been reported that impairment of temozolomide resistance by inhibition of pSTAT3-Y705 is mediated at least in part through modulation of pAkt and βcatenin [212]. Thus, increased STAT3 activity by protein accumulation and/or increased level of pSTAT3-S727 or pSTAT3-Y705 is involved in temozolomide-resistance induction through complex mechanisms including upregulation of MGMT, pAkt and β-catenin activities. Further experiments are really needed to decipher implication of STAT3 signalling in temozolomide resistance.

Besides temozolomide-resistance, STAT3 activation is involved in resistance to radiation since suppressing pSTAT3-Y705 activity by Resveratrol weakened radioresistance and tumorigenic capacity of GBM cancer stem-like cells [213]. This is strengthened by the recent demonstration by Yuan et al. that suppression of autophagy augments radiosensitizing effect of STAT3 inhibition in U251 GBM cells [175]. Irradiation-induced proneural-tomesenchymal transition was also associated with activation of the STAT3 transcription factor, and the combination of pSTAT3-Y705 blockade using JAK2 inhibitors with radiation abrogated the mesenchymal transition and extended survival of in vivo glioma models [214]. We had however previously reported that inhibition of pSTAT3-Y705 by the mean of anti-gp130 neutralizing antibodies or by JSI-124 JAK-STAT chemical inhibitor did not circumvent SF763 GBM cell line radioresistance [215]. Conversely, Gö6976, a multiple kinase inhibitor, down-regulated pSTAT3-S727 and significantly reduced intrinsic radioresistance of some human GBM cell lines in which STAT3-Y705 was not activated [153]. Taken together, our results and the recent data suggest that the contribution of activated STAT3 (Y705 and/or S727) in GBM cell radioreresistance depends on cell type and/or on cell differentiation state. Specific inhibitor of pSTAT3-S727 may help highlight the contribution of each of STAT3-S727 and STAT3-Y705 phosphorylation in GBM response to radiotherapy. A relevant hypothesis is that pSTAT3-S727 might drive intrinsic radioresistance whereas pSTAT3-Y705 might sustain resistance due to tumor cell interaction with their microenvironment.

FUTURE DIRECTIONS TO IMPROVE GLIOBLASTOMAS PATIENTS TREATMENT BY STAT3 TARGETING

Understanding the mechanisms involved in STAT3 pathway regulation is a current interest in the research for GBM therapy. Because pSTAT3-Y705 and pSTAT3-S727 have been reported as GBM markers that sustain some pathological processes of the genesis and of the aggressiveness of this disease, STAT3 inhibitors have been suggested as anti-cancer targeted therapeutic agents. Many pSTAT3-Y705 inhibitors have been engaged in clinical trials but there is not yet a proposed therapeutic pSTAT3-S727 inhibitor. However, recent data suggested that pSTAT3-S727 but not pSTAT3-Y705 is a constant pathological feature of activated STAT3 involved in the GBM resistance to gold standard therapy. These results underline the relevance of a combined inhibition of both pSTAT3-S727 and pSTAT3-Y705 in GBM therapy to counteract STAT3 pathological activation in glioma cells, glioma stem-like cells and stromal cells.

Ongoing clinical trials targeting somewhat STAT3 involve (i) drugs blocking cell membrane cytokine signalling mainly IL-6 or EGF axis, (ii) drugs inhibiting JAK1/2 intracellular upstream activators of pSTAT3-Y705 and (iii) specific inhibitors of STAT3. Among those drugs, only WP1066, a JAK2 inhibitor, is going to be tested in patients with glioma (NCT01904123). Consequently, it might not show effects on selective inhibition of STAT3. Such effects are expected from STAT3 decoy oligonucleotide [170], antisense oligonucleotide such as ISIS 481464 (ONIS-STAT3R3) [216] and AZD9150 [217] or direct inhibitors of STAT3-SH2 domain OBP-51602 [218, 219]. However, one of the major drawbacks of STAT3 targeting is the risk of toxicity due to the multiple cellular processes in which STAT3 play a physiological role. Targeting STAT3 for cancer treatment is one of the current challenges, chiefly in glioma. Therefore, identification and validation of new STAT3 inhibitors are needed to pursue the preclinical/clinical testing of broad inhibitors of both phosphorylations. Up to now, pSTAT3-Y05 has been easily blocked by several means but the clinical outcome was not satisfying. There is not yet any clinical trial based on pSTAT3-S727 inhibition. When waiting on the results of ongoing clinical trials (with WP1066 and with STAT3-SH2 inhibitors), it is not less relevant to set on research towards pSTAT3-S727 inhibition because of its involvement in several disease carrying mechanisms. We further recommend to examine both pSTAT3-S727 and pSTAT3-Y705 whenever studying the role of STAT3. This will highlight any prognostic or predictive value of STAT3 phosphorylation in glioma or in other diseases. It will moreover provide knowledge to refine STAT3-targeting preclinical and clinical trials. pSTAT3-Y705 is induced downstream gp130 phosphorylation and JAK2 recruitment. Activated JAK2 can be inhibited by SOCS3. Activated EGFR or its mutated versions including EGFRvIII induce pSTAT3-Y705. pSTAT3-Y705 can also be induced by intracellular kinases like BMX. PTPRD dephosphoylates pSTAT3-Y705 whereas PIAS3 directly inhibits activated STAT3. pSTAT3-S727 is directly mediated by PKCε or through an unknown mechanism (discontinuous arrow) downstream the NOTCH1 pathway activation. Activated STAT3 homodimerise, translocate to the nucleus and interact with GAS for target gene regulation. They up-regulates expression of several genes, including HIF1α, VEGF, SOC3, and IL-6. The released IL-6 contributes to auto-and paracrine activation of its pathway. pSTAT3-Y705 can interact with NFκB (p65). STAT3-NFκB oligomers bind to DNA and regulate several target genes, including Jagged 1 that encodes a NOTCH1 ligand. In absence of Notch Intracellular Domain (NICD), the DNA-bound RBPJ acts as a gene transcription repressor. It's interaction with the NICD switches it to a co-activator of gene expression. Activation of STAT3 by phosphorylation on Y705 and S727 in non-neoplastic cell contributes to their transformation. pSTAT3-Y705 and pSTAT3-S727 are involved in stem cell proliferation and survival. They participate in the maintenance of stemness in GBM stemlike cells. In GBM cells, they sustain cell proliferation and the resistance to death induction by hypoxia or chemotherapy. pSTAT3-S727 sustains GBM cell intrinsic radioresistance. To restrain the phenomenon of hypoxia, tumor cells up-regulate, an overexpression of the VEGF through pSTAT3-Y705. VEGF induces paracrine activation of endothelial cell proliferation and neoangiogenesis. Moreover, pSTAT3-Y705 in tumor cells induces the release of several factors such as MMP that promote cell migration and non-neoplastic brain tissue invasion. Tissue invasion is also supported by pSTAT3-Y705-induced glioma cell expression of adhesion molecules such as ICAM-1. pSTAT3-Y705-induced release of chemokines and other cytokines like IL-6 contributes to inactivate immune cells or to block their maturation, then inducing immune tolerance of tumor cells.

  that directly blocks activated STAT3 [102]. It moreover includes modulation of STAT3 upstream kinases such as the inhibition exerted by the Suppressor Of Cytokine Signalling (SOCS) on JAK protein family [104]. Lately, Lee et al. described that the endogenous transmembrane protein upstream-of-mTORC2 (UT2) interacts directly with gp130 and inhibits phosphorylation of STAT3-Y705[105].

  Furthermore, phosphorylated or unphosphorylated STAT3 [107] physically interact with various other transcription factors thus contributing to their functional regulation. These include STAT1 [108], the Sp1 transcription factor [109], PPAR-γ [110], Smad1 [111], the androgen receptor [112], Nuclear Factor κB (NF-κB) [113] and c-Jun [114]. Since the STAT3 pathway regulation is complex, a precise description of activated patterns and causal mechanisms in each concerned disease should be helpful in designing STAT3-based therapy.

Figure 1 .

 1 Figure 1. STAT3 in the STAT protein familyThe N-terminal (N-Ter) domain is involved in interactions between neighboring DNA sitebound STAT dimers. The DNA binding domain (DNA) interacts with specific sequence of target-gene promoter. Coiled-coil (CC) domain interacts with partner proteins. Src Homology 2 (SH2) domain is responsible of dimer formation. The C-terminal Transactivation (TA) domain carries on the gene transcription activating function. LK indicates the linker domain.

Figure 2 .

 2 Figure 2. Conserved signalling pathways driving STAT3 phosphorylation.

Figure 3 .

 3 Figure 3. Role of STAT3 in glioblastoma tumorigenesis and tumor aggressiveness.

ACKNOWLEDGEMENTS

The CREaT/ EA7283-Auvergne University research team was supported by the Ligue Nationale Contre le Cancer (Comité du Puy-De-Dôme), of the Institut National du Cancer and by the Auvergne Region. ZG.O. was the recipient of a fellowship from the Ministère des Enseignements Secondaire et Supérieur, Burkina Faso.

This work was supported by the Ligue Nationale Contre le Cancer (Comité du Puy-De-Dôme), by the Institut National du Cancer and by the Region Auvergne. ZG.O. was the recipient of a fellowship from the Ministère des Enseignements Secondaire et Supérieur, Burkina Faso.