Inflammatory mediators, including cytokines, histamine, bradykinin, prostaglandins, and leukotrienes, impact the immune system, usually as proinflammatory factors. Other mediators act as regulatory components to establish homeostasis after injury or prevent the inflammatory process. Histamine, a biogenic vasoactive amine, causes symptoms such as allergies and has a pleiotropic effect that is dependent on its interaction with its four histamine receptors. In this review, we discuss the dualistic effects of histamine: how histamine affects inflammation of the immune system through the activation of intracellular pathways that induce the production of inflammatory mediators and cytokines in different immune cells and how histamine exerts regulatory functions in innate and adaptive immune responses. We also evaluate the interactions between these effects. Histamine and its receptors represent a complex system of immunoregulation with distinct effects mediated by four GPCRs (G protein-coupled receptors HRs 1–4) and their differential expression, which changes according to the stage of cell differentiation and microenvironmental influences. Several host factors, in addition to genetic factors, may influence histamine/receptor effects, including the microbiota, gender, ageing, autoimmune diseases, inflammatory skin, cancer, gut, and pulmonary diseases.Inflammatory conditions  have long been thought to be mainly mediated by the activation of histamine receptor 1 (H1R). However, in the treatment of diseases such as chronic pruritus, asthma, and allergic rhinitis, the use of selective H4R ligands and/or modulation of H1 and H4 receptor synergism may be more effective for such pathophysiological conditions. Recent evidence strongly suggests that H4R ligands might be exploited as potential therapeutics in allergy, inflammation, autoimmune disorders, and possibly cancer. Overall, exploiting the impact of histamine on innate and adaptive immune responses may be helpful for understanding receptor signaling and trends during inflammation or regulation.Histamine shows a dichotomous nature, whereby it is able to promote inflammatory and regulatory responses that contribute to pathological processes, such as allergy induction, as well as homeostatic functions, such as intestinal regulation. In this review, we summarize recent findings about the regulation of the immune response by histamine. A general overview of the immune cascades triggered by histamine receptor activation is provided.Histamine (2-[3H-imidazol-4-yl]ethanamine) is an important chemical mediator that causes vasodilation and increased vascular permeability and may even contribute to anaphylactic reactions . It alsoThe pleiotropic effects of histamine are mediated by 4 histamine receptors (HRs), H1R, H2R, H3R, and H4R, which are G protein-coupled receptors. The active and inactive conformations of these receptors coexist in equilibrium. Agonists of these receptors stabilize the active conformation, whereas antagonists stabilize the inactive conformation. Curiously, the ageing process impairs expression or activity of HRs, and the enzymes HDC and DAO may contribute to the progression of allergic reactions and various neurodegenerative disorders . ChronicH1R is expressed in various cell types, such as neurons, endothelial cells, adrenal medulla, muscle cells, hepatocytes, chondrocytes, monocytes, neutrophils, eosinophils, DCs, T cells, and B cells. H1R signaling results in the following: synthesis of prostacyclins; activation of platelet factor; synthesis of nitric oxide, arachidonic acid and its metabolites, and thromboxane; and contraction of smooth muscle cells. In addition, H1R activation leads to increased chemotaxis of eosinophils and neutrophils at the site of inflammation, higher functional capacity of antigen-presenting cells (APCs), activation of Th1 lymphocytes, and decreased humoral immunity but the promotion of IgE production . As expe2+. Recently, H1R and H4R signaling was implicated in MAPK signaling and cAMP accumulation, leading to increased proinflammatory gene expression [Signaling via H1R leads to the activation of intracellular transcription factors, such as IP3 (inositol triphosphate), PLC (phospholipase C), PKC (protein kinase C), DAG (diacylglycerol), and Capression . In addipression . In addipression .H2R is expressed by parietal cells of the gastric mucosa, muscle, epithelial, endothelial, neuronal, hepatocyte, and immune cells. H2R antagonizes some of the effects mediated by H1R and leads to the relaxation of smooth muscle cells, causing vasodilation. H2R activation regulates several of the functions mediated by histamine, including cardiac contraction, gastric acid secretion, cell proliferation, and differentiation . It alsoα stimulation of IL-10, which is likely associated with Th2 polarization [κB and AP-1 pathways occurs due to cAMP activation downstream of H2R activation [In monocyte-derived DCs from healthy adult subjects, H2R activation counterbalances the Toll-like receptor (TLR) response, leading to inhibition of CXCL10, IL-12, and TNF-rization . Mechanitivation .2+ accumulation [While the activation H1R and H2R mainly accounts for mast cell- and basophil-mediated allergic disorders , H3R funmulation . H3R knomulation . H3R expmulation .A receptors acting on histamine neurons in the posterior hypothalamus induce a natural NREM-like sleep [Neuromodulation and the waking state are related to histaminergic neurons. The waking state is maintained by continual activation of aminergics . Three subtypes of HRs are widely distributed in the brain, not only on neurons but also on astrocytes and blood vessels. Positive-allosteric modulators of GABAke sleep . Targetike sleep . In factke sleep .α, IL-6, IL-10, and IL-13, leading to inhibition of cAMP accumulation and activation of MAPK (mitogen-activated protein kinases) by H4R. Activation of this receptor causes chemotaxis in mast cells and eosinophils, leading to an accumulation of inflammatory cells and control of cytokine secretion by DC and T cells. H4R is also involved in increased secretion of IL-31 by Th2 cells [H4R is preferentially expressed in the intestine, spleen, thymus, bone marrow, peripheral haematopoietic cells, and cells of the innate and adaptive immune systems. Expression of H4R is regulated by stimulation with IFN, TNF-h2 cells . Treatmeh2 cells . The useh2 cells .HRs are present on tumour cells, making them sensitive to variations in histamine. High levels of histamine are associated with bivalent behaviour in the regulation of several tumours  by promoting or inhibiting their growth . The preγ by those cells, which demonstrates regulation between these factors [2+ mobilization, promoting F-actin polymerization in immature DCs derived from monocytes [γ or LPS-driven IL-12 production in a H1R/H2R dependent manner [In HDC-deficient mice, a decrease in H4R expression on iNKT cells is associated with lower production of IL-4 and IFN- factors . Several factors , such asonocytes , and prot manner . H4R hast manner , 30.Asthma is prevalent in males during childhood but is more frequent in females during adolescence and adulthood. Furthermore, allergic diseases are common in women of childbearing age. Both asthma and atopic conditions may worsen, improve, or remain the same during pregnancy. Female hormones, such as estrogen, can modulate the inflammatory response, and histamine receptors can differ between males and females, which might explain the different incidence of allergy between the sexes . For exaThe cascades and effects of different histamine receptors are summarized in Inflammatory mediators are molecules produced by activated cells that intensify and prolong the inflammatory response. Histamine is a potent inflammatory mediator, commonly associated with allergic reactions, promoting vascular and tissue changes and possessing high chemoattractant activity. The binding of histamine to eosinophil H4R induces increased expression of macrophage-1 antigen (Mac1) and ICAM-1 adhesion molecules, in addition to promoting actin filament rearrangement , 34. Theβ-glucuronidase [α and IL-12 when stimulated with LPS [Histamine also modulates the inflammatory response by acting on other cellular populations, in human lung macrophages, binding of histamine to H1R induces production of the proinflammatory cytokine IL-6 and ronidase , 38, a mronidase . Togethewith LPS .κB activation. Treatment with H1R antagonists reduces expression of phosphorylated p38 and NF-κB and, consequently, IL-6 production.In an animal model of allergic airway inflammation, H4R-knockout mice present lower inflammation, reduced pulmonary infiltrate of eosinophils and lymphocytes and an attenuated Th2 response . The conβ (profibrotic cytokine), and collagen deposition [κB phosphorylation and IL-8 secretion, indicating an increased response of epithelial cells to microbial ligands [Blocking H4R in a model of pulmonary fibrosis alleviates the inflammatory response, reducing COX2 expression and activity, leukocyte infiltration, production of TGF-position . Histami ligands .α [In the nervous system, microglial activation is regulated by histamine in a dose-dependent manner, which leads to the production of proinflammatory cytokines, such as IL-6 and TNF-α . This acα , 47. Oveβ, CCL5, and CCL4. However, the H2R pathway promotes IL-10 production and inhibits IL-12 synthesis by immature DCs, favouring the development of a Th2 response profile [Histamine also modulates the response of DCs. Stimulation of immature DCs induces expression of CD86, CD80, and MHC class II, increasing the efficiency of T cell activation . In the  profile . This mo profile .γ production [The Th1/Th2 balance is directly regulated by histamine, as described above. Th1 cells display higher H1R expression, and their binding to histamine promotes activation of Th1 responses, potentiating IFN-oduction . In contoduction . In addioduction .Other evidence, indicates that histamine plays an important role in inflammatory skin diseases. When stimulated via H4R, the NK cells present in skin lesions, increase expression of the chemokines CCL3 and CCL4, favouring cell recruitment to injured tissue . As H4R Within the context of vascular inflammatory diseases, histamine produced by the tunica intima stimulates the monocytes present in atherosclerotic plaques to express CCL2 and its receptor CCR2, via activation of H2R . FurtherAs discussed above, the pleiotropic effects of histamine are a consequence of the existence of four different receptors that belong to the same family of G-coupled proteins and trigger different signaling cascades; these receptors are differentially distributed across tissues and cells . Therefoβ in an H1R-dependent manner [Although histamine is commonly associated with skin inflammation processes, for example, allergic dermatitis , it may t manner .The contribution of histamine to inflammatory neurological diseases, such as multiple sclerosis, is controversial: although H1R and H2R appear to favour the inflammatory response in brain lesions , H3R dam−/− mice were prone to develop inflammation and had a higher tumour burden at mucosal sites (intestine and skin) in models of chemically induced carcinogenesis but that their phenotype was attenuated by histamine administration [Regardless, the role of histamine in gut homeostasis maintenance is clearly essential, as exemplified in a murine model of HDC deficiency. Yang et al. showed that HDCstration . Most HDstration .Expression levels of HDC and H2R are positively associated with increased survival in colorectal cancer patients , and som−/− mice), whereby histamine worsened dextran sodium sulfate- (DSS-) induced colitis and pharmacological inhibition of H4R ameliorated the health of the mice [In addition to tumour biology, histamine has a regulatory function in gut inflammatory diseases, such as in a murine trinitrobenzene sulfonic acid- (TNBS-) induced model of colonic inflammation, which promotes a delayed hypersensitivity reaction that resembles the phenotype . By emplthe mice . DiffereRecently, the microbiota was revealed to be very important for gut pathophysiology. Indeed, the microbial community in the intestine shapes the health status of the host and its susceptibility to a broad range of local and systemic diseases . The micEscherichia coli, Lactobacillus vaginalis, and Morganella morganii) are found at higher frequency in faecal samples from asthmatic patients, with a possible contribution to their atopic phenotype [For example, histamine-secreting bacteria . H1R leads to the production of inflammatory mediators by triggering PKC-dependent NF-κB activation [L. reuteri-derived DGK interrupts H1R signaling and blocks the inflammatory effects of histamine but preserves H2R-associated immunomodulation [In addition to H2R agonism, tivation . PKC actdulation .L. reuteri administration protects HDC-deficient mice from colon tumours in a model of intestinal carcinogenesis with azoxymethane/dextran sodium sulfate (AOS/DSS) administration. L. reuteri produces or restores the histamine pool in the intestine, lowering recruitment of immature myeloid cells and production of inflammatory cytokines, which blocks tumour development [L. reuteri only produces histamine; therefore, it exerts regulatory activity regardless of whether the precursor (histidine) is exogenously provided, for example, by food intake [elopment . Importad intake . This hiL. reuteri, other bacteria have probiotic potential. Another lactobacilli, Lactobacillus rhamnosus, is a source of histamine that promotes a regulatory Foxp3-T cell response profile in intestinal Peyer patches while dampening Th1 polarization in an H2R-dependent fashion [In addition to  fashion .γ by T cells [The lung is a classical mucosal site under histamine control. Although histamine is associated with deleterious inflammation in asthmatic patients, triggering airway hyperresponsiveness and remodelling , 85, rec T cells .Although the regulatory properties of histamine reveal a promising therapeutic potential, histamine may also exert deleterious effects in some clinical conditions. For example, histamine immunomodulation might contribute to higher susceptibility to sepsis in diabetic mice. Mast cell-derived histamine is increased in diabetic settings and reduces neutrophil recruitment due to repression of CXCR2 . In addiHistamine research is an attractive perspective for the potential therapeutic exploitation of new drug targets. The main actions of histamine in controlling the immune response are summarized in


Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family of signaling molecules. ERK is predominantly found in two forms, ERK1 (p44) and ERK2 (p42), respectively. There are also several atypical forms of ERK, including ERK3, ERK4, ERK5 and ERK7. The ERK1/2 signaling pathway has been implicated in many and diverse cellular events, including proliferation, growth, differentiation, cell migration, cell survival, metabolism and transcription. ERK1/2 is activated  in the cytosol and subsequently translocated to the nucleus, where it activates transcription factors including, but not limited to, ETS, c-Jun, and Fos. It is not surprising that the ERK1/2 signaling cascade has been implicated in many pathological conditions, namely, cancer, arthritis, chronic inflammation, and osteoporosis. This narrative review examines many of the cellular events in which the ERK1/2 signaling cascade plays a critical role. It is anticipated that agents designed to inhibit ERK1/2 activation or p-ERK1/2 activity will be developed for the treatment of those diseases characterized by dysregulated gene expression through ERK1/2 activation. Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the “generic” mitogen-activated protein kinase (MAPK) signaling pathway  and NF-κmapk) and ERK2 (p42mapk), respectively, on SDS-PAGE, when the migration rates of ERK1 and ERK2 were compared alongside molecular weight standards.MAPK signaling regulates many cellular events such as cell proliferation, differentiation, cell migration, controlled cell death (apoptosis), and senescence , as well204/187 and then at Thr202/185 , the target gene of mIR-340-5p [In addition to the classic epigenetic regulators of ERK activation exhibited by HDAC, recently published results have also implicated microRNAs (miR) as a crucial component for the “fine-tuning” of various signaling pathways, including those of chondrocytes  and osteof TNF-α . This fiR-340-5p . FibromoR-340-5p . It is aR-340-5p .It has become increasingly evident that new drug development for the treatment of various forms of cancer and arthritides will be based on our increased understanding of the role of ERK1/2 signaling, which lags far behind the therapies developed against other targets that also play a role in diseases associated with chronic inflammation. Perhaps one can argue that the increased focus on other signaling pathways besides the ERK1/2 pathway, including PI-3K/Akt/mTOR  and JAK/STAT3 gene expression. Obviously, further study is required to sort this all out.However, a complication has arisen regarding the choices that must be made in any decision to further develop new drugs for specific diseases. This will entail making advances in our general understanding of how many of the pro-inflammatory cytokines activate more than one signaling pathway. This will also inform the MMPs, ADAMTS, and ADAMs that drive the progression of cell proliferation and tissue destruction, as well as also activating various transcription factors that play a role in the progression of these diseases . In addiClinical inhibitors of the ERK cascade have been developed over the past decade or so ,156,157.It is also anticipated that some of these anti-MEK/ERK1/2 cascade agents could eventually be employed for evaluation in patients diagnosed with RA, PsA, as well as bone and inflammatory bowel diseases, going forward.


Glioblastoma (GBM) is the most common malignant tumor arising from brain parenchyma. Although many efforts have been made to develop therapies for GBM, the prognosis still remains poor, mainly because of the difficulty in total resection of the tumor mass from brain tissue and the resistance of the residual tumor against standard chemoradiotherapy. Therefore, novel adjuvant therapies are urgently needed. Recent genome-wide analyses of GBM cases have clarified molecular signaling mechanisms underlying GBM biology. However, results of clinical trials targeting phosphorylation-mediated signaling have been unsatisfactory to date. Protein phosphatases are enzymes that antagonize phosphorylation signaling by dephosphorylating phosphorylated signaling molecules. Recently, the critical roles of phosphatases in the regulation of oncogenic signaling in malignant tumor cells have been reported, and tumorigenic roles of deregulated phosphatases have been demonstrated in GBM. However, a detailed mechanism underlying phosphatase-mediated signaling transduction in the regulation of GBM has not been elucidated, and such information is necessary to apply phosphatases as a therapeutic target for GBM. This review highlights and summarizes the phosphatases that have crucial roles in the regulation of oncogenic signaling in GBM cells. Malignant gliomas are the most common primary intracranial malignant neoplasm arising from brain parenchyma. And, Glioblastoma (GBM) is the most malignant and aggressive form of gliomas (WHO grade Ⅳ) with the highly infiltrative phenotype, refractoriness, and genetical complexity ,2. DespiRegulation of molecular signaling, including signaling related to tumor biology, generally involves post-translational modification of the signaling molecules. One of the most common modifications is phosphorylation. As a result of upstream signaling activation, kinases are activated and attach phosphate groups to certain amino-acid residues of their specific substrate molecules, resulting in downstream signaling activation. In GBM, kinases are often hyperactivated as a result of genetic alteration or enhanced upstream signaling activation, and, therefore, kinase hyperactivation has been focused on as a therapeutic target ,18,19,20Protein phosphatases (PPPs), as negative regulators of phosphorylation-dependent signaling, remove phosphate groups of certain phosphorylated amino-acid residues of specific substrate molecules ,26,27,28PPPs are classified as classical or atypical PPPs  21,23,2,223,27. To develop clinical GBM therapies targeting PPPs, it is important to understand the detailed molecular mechanisms underlying both tumor-suppressive and oncogenic PPPs in GBM cells. In what follows, we discuss PPPs that are suggested to have major roles in GBM biology in detail.2+/calmodulin-dependent protein kinase inhibitor (H7), PI3K inhibitor (LY29644), and multi-RTKs inhibitor (sunitinib). These knowledges suggest not only expressional but also enzymatic inhibition of PP2A in GBM cells would be important for the maintenance of GBM malignancy, and the possible role of PP2A reactivation as the therapeutic strategy of GBM would also be considered  is one of the most major PSPs. PP2A is a heterotrimeric protein phosphatase complex which consists of the alpha (PPP2R1A) or beta (PPP2R1B) isoform of the structural A subunit, the alpha (PPP2CA) or beta (PPP2CB) isoform of the catalytic C subunit, and the regulatory B subunit. The A subunit and C subunit forms core heterodimer, and association of one of the multiple B subunits with the core dimer directs various substrate specificity (more than 60 combinations) of PP2A . PP2A reBM cells . TreatmeBM cells . TreatmeBM cells . PharmacBM cells ,59,60. ABM cells ,61,62,63PPP4C gene, which encodes the catalytic subunit of PP4 (PP4C), in the TCGA GBM dataset was not confirmed [Protein phosphatase 4 (PP4) is a member of the type 2A PSP family, which includes PP2A, PP4, and protein phosphatase 6 (PP6) ,65. Thesonfirmed . In addionfirmed . AlthougPPP5C and highly expressed in central nervous system and neurons [Protein phosphatase 5 (PP5) is one of the classical PSPs encoded by  neurons ,72. Phys neurons . On the  neurons ,75. Howe neurons . TherefoLike PP4, Protein phosphatase 6 (PP6) is a member of the type 2A PSP family. It regulates various physiological processes, such as anti-inflammation, cell-cycle regulation, DNA-damage repair, and lymphocyte development. Furthermore, it is reportedly involved in the regulation of tumorigenesis. In melanomas, PP6 has been shown to act as a tumor suppressor . On the Tyrosine-protein phosphatase non-receptor type 6  is a PTP primarily expressed in hematopoietic cells in normal tissue and regulates hematopoietic signaling, such as tyrosine-protein kinase Lyn-mediated pathway. In a series of glioma cases, high expression of SHP1 in glioma tissue was associated with poor prognosis . FurtherTyrosine-protein phosphatase non-receptor type 11  is a PTP activated mainly by RTKs and is one of the representative PTPs that have oncogenic roles in various cancers ,83,84,85Tyrosine-protein phosphatase non-receptor type 13 (PTPN13 or PTPL1) is a PTP that regulates various cellular functions, such as proliferation and differentiation, via the Ras-ERK cascade or the Rho-associated protein kinase (ROCK) pathway ,93,94,95PTPRD gene and is inactivated in about 2 to 3% of GBM cases in the TCGA dataset [PTPRD triggers craniosynostosis, hearing loss, or intellectual disability [PTPRD is frequently observed [Receptor-type tyrosine-protein phosphatase delta (PTPRD) is one of the receptor-type tyrosine kinases (RTPTPs). PTPRD is encoded by the  dataset . Physiol dataset , and mutsability ,104. In observed ,106. Forobserved ,107. TheReceptor-type tyrosine-protein phosphatase mu (PTPRM or RTPTPμ) is RPTPT expressed in endothelial, glial, and neuronal cells. PTPRM regulates various biological process, such as cell growth, differentiation, and mitosis. And importantly, PTPRM positively regulates cell–cell adhesion by association with another PTPRM expressed at adjacent cells via homophilic binding . The rolPTPRZ1 [Receptor-type tyrosine-protein phosphatase zeta (PTPRZ) is an RTPTP encoded by PTPRZ1 ,114. In PTPRZ1 ,116. PhoPTPRZ1 . In GBM PTPRZ1 ,118. ImpPTPRZ1 . In addiPTPRZ1 ,120,121.PTPRZ1 . The bioPTEN promoter methylation-mediated PTEN silencing has also been detected in GBMs [Phosphatase and tensin homolog (PTEN) is one of the best known DUSPs in gliomas, because inactivation of PTEN by deletion or mutation was discovered in 41% of the TCGA GBM dataset . In phys in GBMs . Because in GBMs ,127,128, in GBMs , and exo in GBMs , suggestDUSP1, and DUSP1 expression is transcriptionally regulated by tumor suppressor p53 [Most DUSP family members regulate the phosphorylation (activation) status and subcellular localization of MAPKs ,28,131. ssor p53 , which issor p53 . Thus, sssor p53 . Knockdossor p53 . These fDUSP4 and is widely expressed in the nucleus in various tissues. Like other DUSPs, DUSP4 dephosphorylates MAPKs, such as ERK1, ERK2, and JNK, and regulates proliferation and differentiation [DUSP4 promoter methylation [DUSP4 promoter methylation is reported to be associated with alteration of the IDH1 gene encoding isocitrate dehydrogenase 1 (IDH1) [IDH1 mutation is one of the most important genetic alteration in gliomas, which is not only necessary for molecular diagnosis of glioma cases, but is also essential for understanding glioma biology [IDH1 mutations in gliomas are gain-of-function mutations that lead to the generation of the oncometabolite 2-hydroxyglutarate instead of 2-oxoglutarate from isocitrate, which induces a wide range of epigenetic dysregulation collectively termed “glioma CpG island methylator phenotype” [IDH1 mutation is one of the most important determining factors for molecular diagnosis in the WHO 2016 classification of gliomas [IDH1 mutation and to contribute to glioma tumorigenesis through enhancement of MAPK signaling. Accordingly, the DUSP4 expression level might serve as a predictor of IDH1 mutation, which currently is crucial for the molecular diagnosis of gliomas.Dual-specificity phosphatase 4 (DUSP4)/mitogen-activated protein kinase phosphatase 2 (MKP-2) is encoded by ntiation ,131. In ntiation ,138,139,ntiation . This dohylation ,141. Imp1 (IDH1) , which c1 (IDH1) . IDH1 mu biology . IDH1 muenotype” . The pre gliomas ,146. TheDUSP26 and dephosphorylates MAPKs, including ERK, JNK, and p38; however, its physiological function remains unclear. In tumor cells, DUSP26 is known to act as both oncogene and tumor suppressor. A recent study revealed that the oncogenic activity of DUSP26 depends on dephosphorylation and inactivation of tumor suppressor p53 [Dual-specificity phosphatase 26 (DUSP26) is encoded by ssor p53 . In GBMsssor p53 . Anotherssor p53 . CollectPhosphatase of regenerating liver 3 (PRL-3) is a DUSP that positively regulates various cellular signaling factors, such as PI3K, Src, and Rho ,151,152.CDKN3 gene, which encodes KAP, in the TCGA GBM dataset was not confirmed [Cyclin-dependent kinase inhibitor 3  is a DUSP that physiologically dephosphorylates cyclin-dependent kinase 2 (CDK2) and induces mitotic G1-S arrest ,160. In onfirmed , KAP exponfirmed ; howeveronfirmed . An in vonfirmed . In addionfirmed . TherefoThe cell division cycle 25 (CDC25) family of DUSPs is commonly involved in mitotic entry in the cell cycle through dephosphorylation and activation of cyclin-dependent kinases (CDKs). In many cancers, the upregulated expression of CDC25s and its role in tumor progression by accelerating proliferation has been confirmed. All three CDC25 variants  are involved in the regulation of GBM biology. Although genetic alterations of the genes encoding CDC25s are not observed in the TCGA GBM dataset, in a certain series of human glioma samples, expression of the cell-cycle marker Ki67 is increased in parallel with increased CDC25A expression, and dephosphorylation of PKM2 by CDC25A induces glycolytic metabolism, resulting in the Warburg effect and tumorigenicity of GBM cells ,164. On EYA2 is observed in around 0.7% of the total cases [The eyes absent (EYA) family (EYA1–4) is a group of atypical PPPs that function as transcriptional cofactors as well as phosphatases. As for the physiological functions of the EYA family, they are known to be involved in organ development by regulating RTK, transforming growth factor (TGF) and Hedgehog-mediated signaling ,175,176.al cases . Thus, aRecently, to establish the therapy targeting phosphatases based on the biological features of each phosphatase upon neoplasms, the small molecule inhibitors and activators against some phosphatases have been developed as the antitumor agents. The summary of these agents is shown in https://clinicaltrials.gov/ct2/show/NCT03027388). However, okadaic acid-induced dormancy of GBM stem-like cells was also reported [As noted above, PP2A works as either oncogenic protein or tumor suppressor which is dependent on the type harboring tumor, and the inhibitor and activator have been the focused as novel anticancer drug against various malignancies previously. LB-100, a water-soluble small molecule inhibitor of PP2A, and its lipid-soluble derivative LB-102 have been shown antitumor effects against various tumors . And impreported . This mereported . OP440, reported . Anticanreported . In GBMsreported . In addireported . Currentreported . Efficacreported ,188; howhttps://clinicaltrials.gov/ct2/show/NCT03114319); however, the efficacy of these allosteric inhibitors of SHP2 in GBM treatment has not yet been well-proven. On the other hand, the activator of SHP2, geranylnaringenin (CG902), is reported to inhibit proliferation of the prostate cell line via SHP2-dependent STAT3 suppression [As mentioned above, not only tumor-suppressive roles but tumorigenic functions of SHP2 have been suggested in various cancer systems. Therefore, SHP2 inhibitors, as well as SHP2 activators, have been developed as the anticancer drugs. The classical SHP2 inhibitors, such as NSC-87877 or PHPS1 which targets the catalytic domain of SHP2 ,212, dempression . Althougpression .As mentioned above, suppression of PTPRZ expression resulted in inhibition of GBM cell oncogenicity. SCB4380, a cell-impermeable small molecule PTPRZ inhibitor which also potentially blocks PTPRG activity, suppressed migration, proliferation, and tumorigenicity of the Rat GBM cell line by liposome-mediated intracellular loading . NAZ2329The various inhibitors targeting certain DUSPs  have been developed for cancer therapy . Among tInhibitors targeting PRL-3 have been developed as anti-cancer agents for years. Thienopyridone, a classical PRL-3 inhibitor targeting active site of PRL-3, blocks PRLs activity and has demonstrated anticancer effects . In addiCDC25s play fundamental roles in regulation of GBM biology, and pharmacological inhibition of CDC25B or CDC25C expression resulted in growth arrest of GBM cells ,172,173.As noted above, EYA2 is overexpressed and regulates growth and invasion of high-grade gliomas including GBMs . MLS0005https://clinicaltrials.gov/ct2/show/NCT03027388), and the drugs targeting PPPs with greater permeability of the BBB, such as SMAPs, have been developed recently [Given current knowledge, it still remains difficult to determine which PPPs might be appropriate as a therapeutic target for GBM. Therefore, it is necessary to verify the effect and usefulness of PPP inactivation or activation in vitro before the efficacy of targeting PPPs which can be studies in in vivo models or clinical studies. As for inhibitors or activator of PPPs, substrate specificity and blood–brain barrier (BBB) permeability might pose issues, especially in the case of GBMs . Now, asrecently ,187,188;Recent studies on glioma biology have indicated the roles of PPPs in the regulation of GBM oncogenicity, and the modulation of PPP expression or activity for clinical GBM treatment is gradually getting closer. However, further in-depth studies are needed before the application of PPP modulation in clinical GBM treatment can be contemplated, as PPPs are more difficult as therapeutic targets than certain other molecules because a single PPP regulates multiple and complex signaling pathways. However, from another angle, PPP modulation might avoid relapse and the development of therapeutic insensitivity in GBM exactly because PPPs modulate diverse intracellular signaling pathways. In any case, targeting of PPPs in GBM therapy would be challenging but is worth further development. In future, highly specific and blood–brain barrier-permeable PPP inhibitors might have potential as novel therapeutic agents against refractory GBMs.


Specifically, elevated SCG3 levels were found in subjects with high fasting plasma glucose (FPG) levels, central obesity, or hypertriglyceridemia. Additionally, MetS was an independent factor of serum SCG3 levels in multivariate linear regression analyses. Moreover, FPG, free fatty acids, and waist circumference were positively associated with serum SCG3 concentrations after adjusting for insulin levels, high-sensitivity C-reactive protein, and cardiovascular risk factors. In conclusion, serum SCG3 concentrations were higher in subjects with MetS and were independently associated with FPG levels.Secretogranin III (SCG3) plays a crucial role in the biogenesis of secretory granules in endocrine cells, and thus affects glucose homeostasis by regulating insulin secretion by pancreatic beta cells. Insulin resistance and compensatory hyperinsulinemia are hallmarks of metabolic syndrome (MetS). However, the role of SCG3 in MetS remains unclear. Therefore, we investigated the relationship between serum SCG3 levels and metabolic parameters in subjects with and without MetS. This was a case control study, and 295 subjects were recruited. Serum SCG3 concentrations were compared between groups. Associations between SCG3 levels and clinico-metabolic parameters were also examined. We found serum SCG3 levels were higher in the MetS group than non-MetS group (122.6 ± 79.2 vs. 90.6 ± 58.5 nmol/L,  Metabolic syndrome (MetS) is a cluster of interrelated risk factors which include an elevated waist circumference (WC), hypertriglyceridemia, reduced high-density lipoprotein cholesterol (HDL-C), high blood pressure, and dysglycemia . Given oSecretogranin III (SCG3) is a member of the granin family of proteins, which consists of chromogranins, secretogranins, and other related proteins . The graThe present study was a case-control study. All protocols of the study were approved by the Human Experiment and Ethics Committee of National Cheng Kung University Hospital (NCKUH B-ER-102-418), and written informed consent was obtained from all participants. All subjects aged 20~80 years who were admitted to the Preventive Health Center of National Cheng Kung University Hospital for a health checkup from June 2007 to July 2009 were screened. Individuals with the following conditions or diseases were excluded: (1) Type 1 diabetes mellitus; (2) receiving insulin therapy, glucagon like peptide-1, or oral antidiabetic drugs; (3) taking drugs that affect glucose homeostasis, including corticosteroids, thiazides, atypical antipsychotic drugs, or sympathomimetic agents; (4) being pregnant; (5) having an acute infection, such as pneumonia, urinary tract infection, soft-tissue infection, or sepsis; (6) having experienced a recent event of acute coronary syndrome, cerebrovascular accident, or pancreatitis during the past 3 months; (7) having any advanced malignant diseases or other major diseases with generalized inflammation contraindicating this study; (8) taking lipid-lowering medications; and (9) taking antihypertensive drugs.2) was calculated as weight (in kilograms) divided by body height (in meters) squared. The WC was measured on bare skin midway between the lower rib margin and the iliac crest at the end of normal expiration. Additionally, after a 15-minute rest, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of participants were checked twice with an interval of at least 5 minutes in a supine position in a quiet environment.With the help of a trained assistant, body height (to the nearest 0.1 cm) and weight (to the nearest 0.1 kg) measurements of each subject were conducted. The body-mass index (BMI) , glycated hemoglobin A1c (HbA1C), renal function (creatinine), liver enzymes (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)), and lipid profiles , triglycerides (TGs), and high- (HDL-C) and low-density lipoprotein cholesterol (LDL-C)). In addition, using commercial enzyme-linked immunosorbent assay (ELISA) kits, serum concentrations of SCG3  of 8% and interassay CV of 10%), insulin , high-sensitivity C-reactive protein (hsCRP) , and free fatty acid (FFA) levels  were determined. Moreover, after the fasting blood test was obtained, a subsequent 75-g oral glucose tolerance test (OGTT), which collected data on the plasma glucose level at 120 minutes, was carried out.2), the modification of diet in renal disease (MDRD) equation was used. Furthermore, the homeostasis model assessment of IR (HOMA-IR) was applied to investigate IR with the formula HOMA-IR = fasting insulin in mU/L × FPG in mmol/L/405 [To calculate the estimated glomerular filtration rate  t-tests were respectively used to analyze differences in categorical and continuous variables between the two groups. In addition, Pearson’s correlation coefficients were analyzed to assess the strength of associations between serum SCG3 levels and clinical parameters of all participants. Furthermore, we conducted multiple linear regression analyses to identify independent factors related to SCG3 concentrations, using stepwise and backward variable selection strategies. A p value of <0.05 was considered statistically significant.The Windows version of the Statistical Package for the Social Sciences  was used for all statistical analyses. All normally distributed continuous variables are expressed as the mean ± standard deviation (SD), and categorical variables as percentages. All participants were divided into a MetS group and non-MetS group. Chi-square tests and Student’s n = 54) were grouped into the MetS group, and 81.7% of participants (n = 241) belonged to the non-MetS group. In addition, 90, 165, and 40 subjects were diagnosed with normal glucose tolerance, prediabetes (impaired fasting glucose or impaired glucose tolerance), and diabetes, respectively, according to the results of FPG and the OGTT.In total, 295 subjects, including 129 women and 166 men, were enrolled in the study. Among them, 18.3% of participants (p < 0.001), BMI , SBP , DBP , FPG , HDL-C , TGs , insulin , HOMA-IR , glucose level at two hours during the OGTT , hsCRP , and FFAs  between the MetS group and non-MetS group. Moreover, there were higher serum SCG3 levels in the MetS group than in the non-MetS group  (p < 0.001), elevated WC , or hypertriglyceridemia , but not elevated blood pressure  or reduced HDL-C levels   . Of note = 0.47) .p = 0.001) after adjusting for age and sex (model 1). In addition, this independent association was still found  after further adjusting for FFAs, hsCRP, insulin, the eGFR, and GPT (model 2). It is particularly noteworthy that serum FFA levels were also an independent factor associated with serum SCG3 levels in model 2 . Moreover, when we further took every single component of MetS into consideration, FPG  and WC , but not TGs, HDL-C, or SBP, were proven to be independently associated with serum SCG3 levels (model 3).Correlations between serum SCG3 levels and clinical parameters of all participants are shown in To the extent of our knowledge, this is the first report providing evidence that serum SCG3 levels are higher in subjects with MetS. Specifically, there were higher serum SCG3 levels in subjects with high FPG levels, larger WCs, or hypertriglyceridemia. Additionally, the presence of MetS, FPG, FFAs, and WC were independently associated with serum SCG3 levels after adjusting for the insulin level, hsCRP, and other cardiovascular risk factors.Secretory granules are organelles that contain bioactive peptide hormones in endocrine cells. Their formation and further secretion from cells involve sophisticated processes, in which SCG3 is indispensable. First, SCG3 binds to prohormone-chromogranin A aggregates. Then SCG3 acts as a bridge of the core hormone aggregates and the cholesterol component of the trans-Golgi network, thus sorting the peptide prohormones into secretory granules ,17. In pInterestingly, several loci on chromosomes were linked with MetS, diabetes, and obesity, including regions on chromosome 15q ,27.The gThere are some limitations in this work. First, as previous studies suggested that SCG3 may derive from hypothalamus-pituitary-adrenal axis, dysregulation in this axis might affect circulating SCG3 levels. Regarding this, we did not measure and adjust plasma cortisol levels in the present study to exclude this possibility. However, we excluded subjects with conditions of acute or chronic inflammation/infection in our study, and also adjusted hsCRP in the regression analysis. The resulting independent association between MetS and SCG3 levels implies the HPA axis had little effect. Second, to minimize the effects of external factors, the recruited population did not take oral or injectable antidiabetic drugs, lipid-lowering medications, or antihypertensive agents. Therefore, our findings have not been verified in subjects with MetS who were taking such medications. Third, all study subjects were Taiwanese, and thus our results might not be able to be applied to other ethnicities.Serum SCG3 levels were higher in subjects with MetS. In addition, FPG, FFAs, and WC were important factors independently associated with serum SCG3 concentrations. These findings support the possibility of SCG3 being a promising target for controlling dysglycemia. However, further work is still needed to investigate the exact mechanisms involved in the elevated serum SCG3 levels and their clinical implications in MetS.


The normalized expression (log2 ratio) of GmWRKY49 was 2.75- and 1.90-fold in salt-tolerant and salt-susceptible soybean genotypes, respectively. The transcripts of GmWRKY49 could be detected in roots, stems, leaves, flowers, and almost no expression in pod tissues. The salinity-tolerance response of this gene was studied through overexpression in soybean composite seedlings and transgenic Arabidopsis. The effect of GmWRKY49 overexpression on root length of transgenic Arabidopsis was also investigated. Under salt stress, several parameters including germination rate, survival rate, root length, rosette diameter, relative electrolyte leakage, and proline content were significantly higher in composite seedlings and transgenic Arabidopsis than those in wild-type. Moreover, GmWRKY49 enhanced salinity tolerance in soybean mosaic seedlings and transgenic Arabidopsis. These results suggest that GmWRKY49 is a positive regulator of salinity tolerance in soybean and has high potential utilization for crop improvement.Plant WRKY transcription factors (TFs) are active guardians against pathogens’ insurgency, key components in developmental processes, contributors in signal transduction pathways, and regulators of diverse biotic and abiotic stress responses. In this research, we isolated, cloned, and functionally characterized a new WRKY TF GmWRKY49 from soybean. GmWRKY49 is a nuclear protein which contains two highly conserved WRKY domains and a C Being sessile, plants are forced to cope with their immediate environment and encounter multiple biotic and abiotic stresses simultaneously. Extreme environmental conditions impact detrimental effects on the growth and development of economically important crops. Soil salinity is a major abiotic stress worldwide , and concis-acting elements in the promoter region , and signal-related protein kinases-encoding genes . Severalr region  and modur region .2H2-type (Cx4-5Cx22-23HxH) or C2HC-type (Cx7Cx23HxC). Additionally, they contain a nuclear localization signal (NLS), a kinase domain, a TIR-NBS-LRR domain, leucine zippers, Ser/Thr-rich stretches, and Gln- and Pro-rich regions (1–β4). β1 and β2 are highly conserved, whereas β3 and β4 show discrepancy in terms of amino acid numbers and conservation. Depending on the number of WRKY domains and type of zinc-finger motifs, all WRKY TFs are categorized into three groups. Group I proteins contain two WRKY domains and a C2H2-type zinc-finger structure. Groups II and III are marked by only one WRKY domain with C2H2 and C2HC-type zinc-finger structures, respectively. Group II proteins are further categorized into five subgroups (IIa–IIe)   . WRKY prIIa–IIe) . WRKY TFIIa–IIe) , are theIIa–IIe) , in signIIa–IIe) , and resIIa–IIe) . GmWRKY2id (ABA) .Glycine soja (STGoGS) and a salt-sensitive genotype of Glycine max (SSGoGM) in response to salinity stress. Three STGoGS specific and two SSGoGM specific WRKY TFs were expressed and upregulated. Overall, seven common genes were expressed and upregulated in both species  data from a salt tolerant genotype of  species . Of thesy to ABA . Based oGmWRKY49, was further functionally characterized to explore its role in salinity tolerance. The expression pattern of GmWRKY49 in soybean seedlings at different time intervals and in specific tissues was inspected under varying salt stress levels. Furthermore, the functional analysis of soybean composite seedlings and transgenic Arabidopsis overexpressing candidate gene was also investigated.In this study, salt-responsive WRKY TFs were cloned and analyzed in response to NaCl stress. One salt-responsive candidate gene, Arabidopsis and soybean WRKY TFs, comprising 75 AtWRKYs and 187 GmWRKYs, classified WRKY genes into three major groups . The second group was further categorized into five subgroups, named IIa, IIb, IIc, IId, and IIe . The conserved domains and motifs of each group, as based on AtWRKYs. Group I contains two WRKY domains with a C2H2-type zinc-finger motif. Groups II and III contain a single WRKY domain with C2H2 and C2HC-type zinc-finger motifs, respectively, whereas five subgroups (IIa–IIe) differ from each other with respect to amino acids and their conservation. GmWRKY49 contains two WRKY domains, a C2H2-type Zn-finger motif and is most similar to AtWRKY33 and both belong to group I . The same group also contains 12 Arabidopsis and 35 soybean WRKY genes. AtWRKY33 and AtWRKY25 are NaCl-inducible TFs which respond to multiple abiotic stresses and induction of AtWRKY33 is dependent upon ABA signaling . To detect subcellular localization, a GmWRKY49-green fluorescent protein (GFP) fusion construct and a control construct containing only GFP were created. In both constructs, cauliflower mosaic virus-35S (CaMV 35S)-mediated transcription was driven. By following the PEG-4000-mediated method, constructs were introduced into Arabidopsis protoplasts and cells were inspected with a confocal laser scanning microscope. Fluorescence of GmWRKY49-GFP was specifically detected in the nucleus; fluorescence of control construct was distributed throughout the cell . These results indicate that GmWRKY49 likely functions in the nucleus.A comparative phylogenetic analysis of full-length ignaling . The NLSGmWRKY49 and cDNAs from both species were amplified and sequenced. Sequence alignment results indicated that cultivated soybean gene, GmWRKY49, has a mutation of two bases in the translated gene sequence . The base C at 303 position substitutes with T (303C > T); similarly, base A at 942 position substitutes with G (942A > G). Despite changes in the coding sequence, no changes in amino acid residues were observed. Both mutated codons code for the same amino acids, AAT and AAC code aspartic acid  and AGA and AGG code arginine , due to the degeneracy of genetic code. So, the two mutations are silent mutations, and the structure and functions of GmWRKY49 protein are conserved between cultivated and wild species.For the confirmation of nucleotide sequence similarity, full-length gene primers were designed for GmWRKY49 expression distribution in soybean plant tissues, in silico expression of genome-wide GmWRKYs performed, following 14 samples: young leaf, flower, 1 cm pod, pod shells at 10 and 14 days after flowering (DAF), seeds at 10, 14, 21, 25, 28, 35, and 42 DAF, roots, and nodules, and showing that most of WRKY genes expressed in roots, some expressed in flower and young pod, and no expression in seeds, but GmWRKY49 mainly expressed in roots and flower (Supplementary Table S1).In order to figure out the GmWRKY49 transcripts in leaf tissues at the V1 and R2 stages: a decreasing response with growth advance . The expression patterns in roots altered significantly, as minimum transcription observed at the V1, maximum at the R2, and intermediate at the R4 stages. A similar expression pattern was observed in stem tissues. However, GmWRKY49 was expressed only at the R2 stage in flowers and almost no expression in pod tissues.To clarify transcripts’ abundance in specific tissues, the total RNA was extracted from roots, leaves, stems, flowers, and pods of soybean plants at first trifoliate (V1), full bloom (R2), and full pod (R4) stages. RNA was reverse transcribed to cDNA, and real time-quantitative polymerase chain reaction (RT-qPCR) was performed. Transcription profiles showed relative abundance of GmWRKYs was altered in salt-tolerant and salt-sensitive genotypes (Supplementary Table S1). Two genes were upregulated and one gene was downregulated specifically in STGoGS. Two genes, GmWRKY49 and GmWRKY111, were upregulated in both species. In comparison with control treatment, GmWRKY49 was differentially upregulated by 2.75- and 1.90-fold  in G. soja and G. max, respectively.Previously, DGEP data were generated from four cDNA libraries of control and NaCl-treated samples of STGoGS and SSGoGM to identify differentially expressed genes (DEGs) . ExpressGmWRKY49, root samples were collected from STGoGS and SSGoGM at different time intervals after salt stress application. The time-course qPCR results showed that transcription of GmWRKY49 was initially increased at 2 h, reached a maximum at 4 h and then gradually decreased at 8, 12, and 24 h after salt stress, respectively . The normalized expression of GmWRKY49 increased by approximately 17- and 9-fold in the roots of G. max and G. soja after 4 and 8 h of NaCl treatments, respectively.To study the expression of GmWRKY49 carrying transgenic Arabidopsis lines  and WT, survival rate, rosette diameter, relative electrolyte leakage, and proline contents were determined. Under normal conditions, both transgenic lines showed no significant survival rate difference with WT plants, one exception that, transgenic line GmWRKY49-1 seedlings showed a weaker growth. As GmWRKY49 expression was induced by higher salt concentrations, 4-week-old transgenic Arabidopsis seedlings were treated with 200 mM sodium chloride for 1 week and wild-type (WT) seedlings were used as a control. Seedlings of GmWRKY49-1 and GmWRKY49-3 showed improved growth compared to WT seedlings . Both transgenic lines showed significantly higher survival rate, rosette diameter, relative electrolyte leakage, and proline content values than those of WT . Electrolyte leakage from cell and proline accumulation in the cytoplasm is plant responses to salt stress . The majority of the seedlings carrying the salt-responsive gene, GmWRKY49, were survived with some wilted leaves . As expected, more than 91% transgenic seedlings carrying empty vector could not survive and eventually died.To investigate whether overexpression of GmWRKY49 overexpression on germination and root length, transgenic Arabidopsis seedlings were grown on 1/2 strength MS medium supplemented with 0, 50, 100, 150, and 200 mM NaCl treatments. In the absence of salt stress, WT and GmWRKY49 carrying Arabidopsis seeds showed non-significant phenotypic and germination rate differences . The germination rate of GmWRKY49 carrying seeds started to decline after 4 days in 50 and 100 mM NaCl treatments; increase in germination rate was observed after 2 and 3 days under 150 and 200 mM treatments, respectively. These results indicate that GmWRKY49 is induced by high salt concentrations. A reduction in root length was observed compared with WT when GmWRKY49 overexpressing seedlings were under no salt  and 50 mM NaCl stress . However, a significant reduction (P < 0.05) in root length was noted when seedlings were under 50 mM stress. At higher salt treatments, 100, 150, and 200 mM, a highly significant (P < 0.01) increase in root length was recorded compared to WT. Interestingly, highest gains in root length were observed under 200 mM treatment, confirming that high salt concentrations induce salinity-tolerance response of GmWRKY49.To investigate the effect of G. max WRKY TF, GmWRKY49. Phylogenetic analysis clustered 187 soybean and 75 Arabidopsis WRKY family genes into three groups  and five sub-groups (IIa–IIe) . This clustering of WRKY TFs is similar to those of GmWRKY49 was clustered with AtWRKY25, AtWRKY26, and AtWRKY33; these genes belong to WRKY group I. OsWRKY30 from rice and TaWRKY2 from wheat also belong to the same WRKY group I . Sequence alignment of GmWRKY49 from wild and cultivated species indicated substitution of two base pairs in the coding region; however, no change in amino acid sequence was observed  indicating that it was a silent mutation. Nevertheless, the expression levels of GmWRKY49 across wild and cultivated species were different . This discrepancy in expression might arise due to the presence/absence of some cis-acting elements in the promoter region, as these elements are reported to regulate the gene expression . The highest expression of GmWRKY49 was detected in leaves, roots, and nodule tissues  which are similar to the transcripts’ abundance of GmbZIP110, a NaCl-responsive bZIP TF  in our study, which is consistent with AtWRKY25 and AtWRKY33 in Arabidopsis  which are in accordance with other TFs from WRKY group I: AtWRKY25, AtWRKY33, DgWRKY5, and TaWRKY2. Based on these studies, it is quite possible that TFs of WRKY group I have some analogous effects on abiotic stress tolerance.The majority of the  bZIP TF . GmWRKY4bidopsis . But in t stress ; we infeanthemum  and TaWRin wheat . ComposiChrysanthemum plants compared to WT under control and salt stress conditions. They suggested that DgWRKY5, a Chrysanthemum salinity-tolerant TF, can regulate the expression of ROS scavengers and weaken the damage of plasma membrane to enhance salinity-tolerance. To withstand salinity stress, plant cells often tend to synthesize and accumulate higher proline content and demonstrate higher relative electrolyte leakage to reduce salinity-induced toxic effects. Proline accumulation is a protective measure taken by the plants to maintain a high osmotic pressure and insurance for moisture absorption from the surrounding environment. It has been reported that plant cells with higher accumulation of organic molecules, especially proline and soluble sugars, had better tolerance to salt stress . Previously, GmWRKY49-overexpressing plants may withstand higher salinity levels by the efficient accumulation of osmotic adjustment substances, enhancing the cation conductance of the plasma membrane and cell membrane protection through the possible regulation of ROS scavenging enzymes.High salinity leads to the accumulation of osmotic adjustment substances, such as proline, reactive oxygen species (ROS) and higher electrolyte leakage . In plant stress . Similart stress  which isnditions . In our Arabidopsis seeds demonstrated a higher germination rate under 150 and 200 mM NaCl treatments; however, under 50 and 100 mM NaCl treatments, the germination rate of transgenic seeds was lower than WT seeds . This suggests that GmWRKY49 is induced by high salt concentrations. Furthermore, GmWRKY49 carrying transgenic seedlings showed significantly higher survival rate and rosette diameter compared to WT after 1 week of 200 mM NaCl stress . Similarly, roots’ length of transgenic Arabidopsis seedlings was significantly higher than WT at all NaCl treatments . Triticum aestivum TFs, TaWRKY1, TaWRKY10, and TaWRKY33, also exhibited an increase in germination and survival rates and roots’ length in transgenic Arabidopsis and tobacco plants and their overexpression improved drought and salinity-tolerance , full bloom (R2), and full pod (R4) stages. For total RNA extraction, root samples of both accessions were harvested after 0, 2, 4, 8, 12, and 24 h of NaCl treatment, promptly frozen in liquid nitrogen, and stored at -80°C. RNA was reverse transcribed into first-strand cDNA for further use as a template in PCR. Each experiment contained three biological replicates.2 ratio  either > + 1 or < -1, were identified among control and NaCl-treated samples of STGoGS and SSGoGM by generating DGEP data  < 0.05  and foldGEP data . Two WRKAtWRKYs  were retrieved from Phytozome v12.1. Forward (5′-ATGGCATCTTCTTCTGGTAGT-3′) and reverse primers (5′-GCATAGTAGAGACTCAAGGAAC-3′) were designed and synthesized by InvitrogenTM. cDNA templates from G. max and G. soja were used for PCR amplification of GmWRKY49. Amplicons were cloned and sequenced by GenScript.CDS and peptide sequences of 3. NLS was predicted using online tools at http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi.Domain analyses were performed using MEME online serverGmWRKY49 (1728 bp) was fused to pJIT166-GFP vector driven by CaMV 35S to generate the GmWRKY49-GFP construct. Arabidopsis protoplasts were isolated by following the previously published protocol (Arabidopsis protoplast by the PEG4000-mediated method as described by The coding sequence of protocol . Both fu® reagent (Invitrogen & Co.) according to the manufacturer’s protocol. Full-length gene-specific forward (5′-TTCCTGCAGCCCGTGCAGT-3′) and reverse (5′-GGCAAGAGCCTTACGGAAGTGGC-3′) primers were used for qPCR and RT-PCR analyses. A soybean actin gene  was used as the internal control. Sample preparation, qPCR, and RT-PCR analyses were performed as For qPCR analysis, total RNAs were extracted from STGoGS and SSGoGM roots under control and 200 mM NaCl treatments. RNA was extracted using the TRIzolAgrobacterium-mediated genetic transformation.Seeds of soybean variety “Dongnong 690” were sown in pots containing vermiculite. After cotyledons, emergency, healthy, and robust seedlings were selected for GmWRKY49 full-length cDNA was cloned into plant expression vector pCXSN under CaMV 35S promoter. The obtained pCXSN-GmWRKY49 construct was transformed into young soybean seedlings at the cotyledonary node and/or hypocotyl via Agrobacterium rhizogenes. The seedlings were grown under controlled conditions at 25°C and 60% humidity with 12 h of light/dark cycle. Water was applied as required. After 3 weeks of Agrobacterium infection when induced hairy roots could support the plants  to further transform Arabidopsis Columbia-0 plants via floral dip method as Arabidopsis seeds were employed for subsequent experimentation.The coding sequence of GmWRKY49 transgenic Arabidopsis and WT were surface sterilized and placed on Petri plates containing 1/2 strength Murashige and Skoog (MS) basal medium supplemented with 0, 50, 100, 150, and 200 mM NaCl, respectively. The Petri plates were kept in a growth chamber at 25°C under a 16-h photoperiod for 7 days. For root length measurements, germinated seeds were supplied with the same nutritional and stress treatments for one additional week. The germination rates were recorded everyday after sow for 7 days, whereas the root length was measured after 14 days, separately from each experiment.For germination assay, 100 seeds of Arabidopsis were sown in pots containing vermiculite and nutritional soil (3:1). Pots were placed into the growth chamber set at 22°C/12 h light and 20°C/12 h dark cycle. Water was applied as required. For salt treatment, Arabidopsis seedlings after 4 weeks of growth were supplied with 10 ml of 200 mM NaCl every day for 1 week. Untreated seedlings were used as a control. After 1 week of treatment, survival rate, rosette diameter, proline content, and relative electrolyte leakage were measured. The experiment contained three biological repeats.For morphological and physiological assays, seeds of transgenic and WT The relative electrolyte leakage was assayed as described by t-test and P-values < 0.05 were considered to be statistically significant. All the error bars were SD (Standard Deviation) value.All data were processed using the SPSS version 15.0 . The significance of data was computed by using Student’s HM, ZA, and JY designed the experiments. ZX, QR, LX, XH, YH, and DZ finished the experiments. ZX and QR analyzed the data. ZX and QR finished the manuscript. H-BS revised the paper. All authors approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer. Human exonuclease 1 (EXO1) contributes to checkpoint progression and to several DNA repair pathways involved in reducing DNA replication stress, for example, in mismatch repair (MMR), translesion DNA synthesis (TLS), nucleotide excision repair (NER), double-strand break repair (DSBR), and checkpoint activation to restart stalled DNA forks ,3,4,5,6.N-terminal region of the protein , EXO1, and polymerase δ are the main factors in primer removal and Okazaki fragment maturation at the lagging strand in the process of strand displacement during replication ,17,18,19ut cells . This suut cells ,15. Furtut cells ,15,21. Iut cells ,23. Bothut cells ,23, whicut cells ,26,27,28ut cells ,30. In vut cells . It is pSchizosaccharomyces pombe) after it was co-purified with mismatch repair factor MSH2 [High-fidelity DNA replication is required to maintain an unaltered genetic code during cell division. The MMR pathway is a post-replicative DNA repair system, which mainly corrects DNA polymerase slippage and damaged bases, such as chemically-induced base adducts; base mismatches; and base insertions, deletions, and loops. The MMR pathway consists of several steps, which are detailed below. The initial recognition step of eukaryotic MMR utilizes the MutSα complex made up of mutS homolog 2 (MSH2) and mutS homolog 6 (MSH6) or MutSβ complex (MSH2 and mutS homolog 3 (MSH3)). The MutSα mainly recognizes single base mismatches, while the MutSβ complex detects larger lesions, insertion/deletions, or loops ,32,33. Ttor MSH2 ,40,41,42tor MSH2 . The reptor MSH2 . Malfunctor MSH2 ,46,47. −/− and WRN−/− that show no defective MMR [More recently, it was shown that MMR occurs in the absence of EXO1 ,49, suggtive MMR ,63. Altotive MMR . The contive MMR . A recentive MMR . In vitrtive MMR . It has tive MMR ; howeverTranslesion DNA synthesis (TLS) describes the process by which a DNA polymerase can synthesize a DNA strand across a lesion on the template strand. This process is critical to maintaining functional DNA replication in the face of genotoxic stress and may act as a pathway to cope with ultra violet (UV) induced DNA damage . Indeed,rad2 (XPG homolog in human) and exo1 are knocked out [UV radiation from sunlight mainly damages DNA by causing cyclobutane pyrimidine dimers, and 6–4 photoproducts, lesions typically repaired by the nucleotide excision repair pathway (NER) independent of replication . Howevercked out . In addicked out . The connull/nullExo1 mice show an increase in chromosomal breaks and base substitution, and predominately develop lymphomas [-/-exo1 has no significant defect in recombinational repair, with only minor defects in DNA end processing [null/nullExo1 cells showed a defect in the DNA damage response [null/nullExo1 cells with the topoisomerase inhibitor camptothecin, which creates single strand breaks (SSB) that ultimately lead to DSB during the S-phase, results in a reduction in phosphorylated RPA (pRPA) foci at the DSBs [Mre11, Rad50, or Nbs1 genes has been shown to be lethal in mice [Nbs1 (ΔB/ΔBNbs1) are viable, but show severe developmental impairment, embryonic death, and chromosomal instability when Exo1 is lost [ΔB/ΔBNbs1 MEFs depleted in EXO1 strongly influenced DNA replication, DNA repair, checkpoint signaling, and the DNA damage response [Homologous recombination (HR) is an essential process involved in the repair of double strand DNA breaks, mainly in the S and G2-phases of the cell cycle. A possible piece of evidence suggesting the involvement of EXO1 in double strand DNA repair is the observation that ymphomas . In addiymphomas . This processing ,18,19,72ocessing . The 5′→ocessing . In mousresponse . Treatmethe DSBs . Recruitthe DSBs . PARP1, the DSBs ,76 and sthe DSBs . Poly  (the yeast homolog is SAE2) [C-terminal region of WRN. Biochemical assays suggest that WRN and EXO1 function in replication stress, where WRN enhances EXO1 in processing stalled forks or regressed replication forks [The single-stranded DNA binding protein RPA has a central role in DNA replication, DNA repair, recombination, and DNA resection . DNA respathways , suggestis SAE2) ,84,85,86is SAE2) ,88. Theris SAE2) ,90,91. Eon forks . More reon forks . Human con forks . In summSeveral lines of evidence suggest that EXO1 may be a central regulator of the cell cycle. For example, in S-phase, EXO1 co-localizes with MMR protein MSH2 and cell cycle regulator PCNA . In humaFurther evidence for a regulatory function of EXO1 in the cell cycle comes from yeast, where the absence of cell cycle regulator 14-3-3 leads to checkpoint defects . In huma-/- and MLH1-/- cells is the presence of MSI, leading to increased chromosomal instability, which is believed to be the underlying molecular driver of tumor formation in Lynch syndrome [Exo1 leads to an increased incidence of lymphomas, but interestingly not to increased MSI [EXO1 has been associated with different types of cancers, including Lynch Syndrome, breast, ovarian, lung, pancreatic, and gastric tract cancer see  101,102,116,117.syndrome ,45,118. syndrome ,116. Notsyndrome ,101. Howsyndrome ,120. Thesyndrome . The cliased MSI . Pathogeased MSI . Neverthased MSI ,123,124.ased MSI ,126,127.Evidently, EXO1 is a central player in DNA metabolic processes. As elucidated herein, EXO1 contributes to several DNA repair pathways, which safeguard DNA replication, including MMR, TLS, HR, and cell cycle regulation . ReplicaNonetheless, several questions remain to be answered. For example, given the putative central role of EXO1, it remains a mystery why the knockout of EXO1 in mice, as well as loss of function, leads to a relatively mild phenotype. Further, the mechanism of EXO1-independent MMR is still unclear, particularly regarding at what point this specific pathway is active. Given the biochemical activity of EXO1, it is possible that an unknown helicase or exonuclease can contribute to MMR repair in the absence of EXO1. DNA polymerase δ is a strong candidate, as well as the helicases BLM and/or WRN with minor contributions ,57,58,65EXO1 gene variants have been associated with different types of cancers. Interestingly, large GWAS analyses support that specific mutations in domains required for interaction with other proteins in EXO1 are more commonly occurring in particular types of cancer, as summarized in


TP53 mutation and the frequently accompanied by a large number of gene copy-number alterations, including the amplification of important oncogenes, such as CCNE1 and c-MYC. However, a proportion of cases (20%) probably represent the progression of tumors initially belonging to the copy-number low endometrioid-like molecular subtype , after the acquisition of the TP53 mutations. Only a few ECS belong to the microsatellite-unstable hypermutated molecular type and the POLE-mutated, ultramutated molecular type. A common characteristic of all ECSs is the modulation of genes involved in the epithelial to mesenchymal process. Thus, the acquisition of a mesenchymal phenotype is associated with a switch from E- to N-cadherin, the up-regulation of transcriptional repressors of E-cadherin, such as Snail Family Transcriptional Repressor 1 and 2 (SNAI1 and SNAI2), Zinc Finger E-Box Binding Homeobox 1 and 2 (ZEB1 and ZEB2), and the down-regulation, among others, of members of the miR-200 family involved in the maintenance of an epithelial phenotype. Subsequent differentiation to different types of mesenchymal tissues increases tumor heterogeneity and probably modulates clinical behavior and therapy response.Endometrial carcinosarcoma (ECS) represents one of the most extreme examples of tumor heterogeneity among human cancers. ECS is a clinically aggressive, high-grade, metaplastic carcinoma. At the morphological level, intratumor heterogeneity in ECS is due to an admixture of epithelial (carcinoma) and mesenchymal (sarcoma) components that can include heterologous tissues, such as skeletal muscle, cartilage, or bone. Most ECSs belong to the copy-number high serous-like molecular subtype of endometrial carcinoma, characterized by the  Endometrial carcinosarcoma (ECS), also known as malignant mixed Müllerian tumor (MMMT), is a high-grade tumor characterized by a biphasic growth of malignant epithelial (carcinomatous) and mesenchymal (sarcomatous) components  1]. ECS. ECS1]. Matsuo et al.  analyzedSeveral studies have demonstrated that tamoxifen use may be associated with an increased incidence of ECS. In women with breast cancer, the incidence of ECS is 6.35-fold higher in those treated with tamoxifen . Matsuo From a morphological point of view, the epithelial component of ECS could be endometrioid (most common in most series) or non-endometrioid  ,13,14,15Regarding other pathological features, 55% to 60% of ECS show less than 50% of myometrial invasion at diagnosis. Lymphovascular invasion (LVI) prevalence in ECS seems to be higher than in other types of endometrial cancer (60.4–62% vs. 26–52%) . Matsuo Also, the pattern of metastasis differs between the epithelial and mesenchymal parts of the ECS. Thus, for example, Matsuo et al.  analyzedECS follows an aggressive clinical course. Patients with International Federation of Gynecology and Obstetrics (FIGO) stage 1–2 disease have a five-year disease-specific survival of 59%, while those with stage 3 and 4 disease have a five-year disease-specific survival of 22% and 9%, respectively ,13,15,22Molecular studies have demonstrated similar genetic alterations in both the carcinomatous and sarcomatous components of ECS . Thus, iPOLE mutated), the copy-number low, and the copy-number high groups. These groups not only have different molecular alterations but also different prognoses; patients from the ultramutated group show the best prognosis, whereas patients in the copy-number high group have the highest risk of recurrence [Four molecular groups have been defined for ECs: the hypermutated (mismatch repair deficiency), the ultramutated , while part of the tumors displayed an endometrioid carcinoma-like mutation profile characterized by the presence of ARID1A, CTNNB1, KRAS, and PTEN mutations. Based on both combined genetic and immunohistochemical profiles in their cohort, 18 tumors presented serous-like and 11 tumors presented endometrioid-like molecular profiles. There was a good correlation between the histological subtyping  and the molecular subtyping in 27 of 29 uterine carcinosarcomas (93%) [POLE mutation, while another had no mutation in the genes used for classification. Interestingly, all 10 stage IV tumors were serous-like [Considering the mutational profile  [Previous studies combining aberrant expression of p53 and mutational analysis estimated a f 50–60% ,33,34,35 and 91% ,38,39,40s in ECS . The lacih.gov/) . TP53 gene mutation, which points to a monoclonal origin of both components  than in endometrial endometrioid carcinoma (EEC). Accordingly, mutations of FBXW7 and PPP2R1A have been reported in 19% to 39% and 1% to 38%, respectively, in different series [The carcinomatous and sarcomatous components show a concordance of 85% for the p53 protein overexpression and 96% for the mponents . p16 ovet series ,38,39,40BRCA1 and BRCA2 genes, the frequency of ECS in patients carrying germinal BRCA1/2 mutations has been analyzed in different studies. The estimated relative risk for mutation carriers is approximately 2% per year, most importantly among serous carcinoma [BRCA1/2 were found mutated in 18% and 27%, respectively of ECS [BRCA2 mutations were detected and at a lower frequency (5%) [BRCA1/2 germline mutations [Regarding the arcinoma ,46,47. Ay of ECS , althougncy (5%) . Carcinoutations ,49,50.histone H2A and H2B, as well as a significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. Thus, mutations in histone H2A/H2B genes were significantly enriched in carcinosarcomas (CSs) compared with carcinomas . These findings implicate mutations in histone H2A/H2B genes in ECS. Zhao et al.  found anFOXA2) mutations in 15.1% of ECS. FOXA2 had not previously been implicated in ECSs and was predominated by frameshift and nonsense mutations. Sequencing of FOXA2 in 160 primary endometrial carcinomas revealed somatic mutations in 5.7% of serous, 22.7% of clear cell, 9% of endometrioid, and 11.1% of mixed endometrial carcinomas, the majority of which were frameshift mutations. Collectively, the findings of the study of Le Gallo et al. [FOXA2 is a pathogenic driver gene in the etiology of primary uterine cancers, including ECSs.Le Gallo et al.  have repo et al.  provide Similarly to ESC, ECS is characterized by aneuploidy and a high frequency of copy number variations (CNVs). Analysis of ploidy and whole-genome doubling has established a median ploidy of 3.3 and that 90% of ECS had undergone at least one whole-genome-doubling event. This percentage is significantly higher than in serous ovarian tumors, the tumor type with the next highest frequency of genomic doubling in the TCGA . TERC (3q26.2), FGFR3 (4p16.3), MYC (8q24.21), KAT6A (10q22.2), MDM2 (12q15), ERBB2 (17q12), CCND1 (11q13), CCNE1 (19q12), BCL2L1 (20q11.21), and RIT1 (1q22)  .CCNE1) is the most frequently amplified gene in ECS, 41% according to data derived from TCGA  [c-MYC in only 21% of ECSs [Cyclin D1 (rom TCGA . In othen (FISH) . However of ECSs .ERBB2 amplification in ECS ranged from 3–20% [ERBB2 amplification could benefit from anti-HER2  therapies, such as Trastuzumab. For patients unresponsive to chemotherapy and Trastuzumab, T-DM1 (Trastuzumab emtansine) may offer an alternative treatment option, as recent studies show how ECS cell lines and derived xenografts with ERBB2 amplification respond well to T-DM1 [PIK3CA is amplified in 11% of ECS, further highlighting the importance of the phosphatidylinositol 3-kinase (PIK3) pathway  [EGFR, and URI , have not been found in the TCGA data set. Biscuola et al. reported EGFR amplification by FISH in 19% of tumors [URI1 amplification has been reported in 40% of ECS [URI1 amplification was also associated with poor survival and reduced response to adjuvant treatment. Likewise, in a cultured cell model, overexpression of URI1 induced ATM (ATM Serine/Threonine Kinase) expression and resistance to cisplatin [GPC5 (Glypican 5) gain/amplification has been detected in a subset of ECS, mostly in the sarcoma component, and the authors linked the involvement of GPC5 with sarcomatous transformation [Schipf et al. detected gical CS . Similarwer (9%) ,38. Two f tumors , while if tumors ,58,59. U% of ECS . URI1 amisplatin . Recurriormation .PIK3CA mutations have been found in 11% to 40% [TP53, with mutations concentrated on HotSpot regions, the mutations in PIK3CA are found scattered in the different functional domains. In addition to the traditional PIK3CA hotspots in exons 9 and 20, a smaller portion of ECS has mutations in exon 1, in the adaptor binding domain, helical domain, and C2 domain which increase kinase enzymatic activity [Mutations in genes encoding for the kinase or regulatory proteins of the PI3K/AKT (phosphatidylinositol 3-kinase/(Protein Kinase B) pathway have been detected in up to 67% of ECS . Moreove% to 40% ,57,62,63activity ,57.PI3KCA mutations have been detected in both the carcinoma and sarcoma components of the primary tumor and also in the metastatic tumor. This implies that they are important early events in the tumorigenesis of carcinosarcoma and thus could be targeted with PIK3CA/mTOR  inhibitors [The importance of mutations in this pathway comes from the fact that hibitors ,38. PIK3hibitors .PTEN) mutations are not as frequent as PIK3CA, but they are present in approximately 20% of ECS: 17% and 19% in the series reported by McConechy et al. [PTEN mutation, but their series included only 17 cases [PTEN and PIK3CA mutations frequently coexist in the same ECS [Phosphatase And Tensin Homolog  (10–17%), PIK3R2, AKT1, AKT2, and AKT3 (less than 5% for each gene) [Other genes with less frequency of mutations in the PI3K/AKT pathway in ECSs include Phosphatidylinositol 3-Kinase Regulatory Subunit 1 (ch gene) ,36,38,57ARID1A) and Catenin Beta 1 (CTNNB1) are commonly mutated in EEC, and ARID1A mutations occur also in 10% to 15% of ECS, leading usually to loss of of protein expression, while mutations in CTNNB1 are infrequent in ECS [KRAS mutations were found in 12% and Cadherin 4 (CDH4) mutations in 18% [AT-Rich Interaction Domain 1A , MutS Homolog 2 (MSH2), MutS Homolog 6 (MSH6), and Mismatch Repair Endonuclease PMS2 (PMS2). In sporadic EC, MMR-def is detected in 15–30% of cases [MLH1 promotor methylation. In addition, between 2–6% of endometrial carcinoma occurs in the context of Lynch syndrome due to germline mutations [MLH1 promoter methylation is probably the major cause for MMR-def in most tumors [MLH1 was epigenetically silenced in the two samples with MMR-def in the TCGA series [Mismatch repair deficiency (MMR-def) and of cases , althougof cases , most frutations . The freutations ,67, whilutations ,68. MLH1A series . (POLE) are present in some ECS, both of the most common HotSpot-mutations (P286R and V114L) have been identified in individual cases of ECS [Mutations in DNA Polymerase Epsilon, Catalytic Subunit s of ECS ,69,70. TSeveral studies have analyzed mRNA and microRNA (miRNA) expression profiles in ECS in comparison to other histological types of EC . RegardiThere are over 200 CTAs, which are classified into different families according to their sequence homology. In general, CTA genes are expressed only in normal testis and cancerous tissue. In many instances, CTA families are formed by clusters of nearly identical genes that are frequently located on the X-chromosome. A shared regulatory mechanism for related CTA clusters has been suggested as whole families of CTAs are often co-expressed together in tumors ,73.MAGEA) members , XAGE2, CTCFL, and CTAG1A (cancer/testis antigen 1A) has been reported in ECS [CTCF, also known as the brother of the regulator of imprinted sites (BORIS), is an oncogene that deregulates the cancer epigenome, which is a common event in ECS [CTCFL) probably mediates the demethylation of another CTA gene, thus resulting in activation via repression [NY-ESO-1) and Preferentially Expressed Antigen In Melanoma (PRAME) [Overexpression of many members of the CTA family, such as melanoma antigen family A  ,76. Cons (PRAME) .The miRNA signature of carcinosarcomas differs from endometrioid and serous carcinomas . The funIn addition to miRNAs related to EMT , when compared to the normal endometrium ,82. RegaKLF4, NDN, WT1, PROX1, among others. Promoter hypermethylation of these genes is also common in other types of EC [PTEN mutations was higher in the first group. A number of tumor suppressor genes with recurrent hypermethylated promoters has also been reported in ECS, es of EC ,82. Intees of EC  reportedA constant characteristic of ECS is the aberrant DNA methylation of miR-200 genes  epithelial cells, endowing these cells with migratory and invasive properties, promoting cancer progression, preventing cell death and senescence, and inducing resistance to chemotherapy . EMT hasSPARC, SNAIL, ZEB1, ZEB2, TWIST-1, TCF4, TGFβ1, and TGFβ2 between the epithelial and mesenchymal components of 23 ECSs. Also, we used immunohistochemistry to evaluate the expression of E-, P- and N-cadherin, cadherin-11, p120, vimentin, SPARC, fascin, and caveolin-1 in 76 ECS. In the mesenchymal component, a ‘‘cadherin switch’’ from E-cadherin to N-cadherin and cadherin 11 was observed. In addition, upregulation of all of E-cadherin repressors together with overexpression of all mesenchymal markers tested was demonstrated.We have previously suggested that EMT is activated in ECS ,73,84,85Also, High Mobility Group AT-Hook 2 (HMGA2) has a role in EMT as a regulator of SNAI1 expression and of other transcription factors downstream of SNAI1, such as Slug, ZEB1, and ZEB2. HMGA2 has been proposed to be regulated by the let-7/Lin28B pathway. Accordingly, we have previously demonstrated that an increase of Lin28B expression correlated with let-7b down-regulation and HMGA2 overexpression in ECS . CTNNB1 mutation, in the sarcomatous but not in the carcinomatous component [A role of the WNT pathway in the transition from an epithelial to a mesenchymal status is demonstrated by the fact that up to 23% of ECS showed nuclear β-catenin, not associated with omponent . Nuclearomponent .Similarly to β-catenin, in another study, ALK tyrosine kinase receptor (ALK) was frequently over-expressed in the sarcomatous components of EC . The autMicroRNA signatures associated with EMT and their relationships with EMT markers in human carcinosarcomas have been studied by us and more recently by Cherniack et al. ,38,84. WIt has been demonstrated that epithelial cells undergoing EMT to acquire mesenchymal features are more likely to possess stemness. In addition, some studies suggested that stemness can be associated with cells undergoing a partial EMT and showing a hybrid Epithelial/Mesenchymal phenotype. Jolly et al. postulated that the core EMT and stemness modules, miR-200/ZEB and Lin28/let7, govern EMT decision making . AccordiMyh7) and embryonic (Myh3) myosin heavy chains and skeletal α-actin (Acta1), was also observed. Similar to our previous results, Lu et al. [Cells with an Epithelial/Mesenchymal hybrid phenotype evolve to an epithelial or a mesenchymal phenotype depending on factors acting on the EMT and stemness modules . Both rou et al.  reportedThe tumor microenvironment has an important role in cancer and immunomodulation of the microenvironment is a new focus in cancer medicine . AccumulAyers et al. have created a Tumor Inflammation Signature (TIS) using gene expression data from baseline tumor samples of pembrolizumab-treated patients. The signature includes 18 genes that reflect a suppressed adaptive immune response  and is enriched in tumors with sensitivity to Programmed cell death 1 ligand 1 (PD-L1) inhibitors . In anotSeveral studies have demonstrated that EMT contributes to evasion of immune surveillance ,104,105.Regarding carcinosarcomas, PD-L1 expression was significantly higher in lung carcinosarcoma than in conventional non–small-cell lung carcinoma , providiPTEN or PIK3CA are more prone to acquire TP53 mutations than those associated with MMR-def, POLE, or CTNNB1 mutations. Mutations in TP53 seem to be essential, but not sufficient, to ECS development, since they are as frequent in ECS as in endometrial serous carcinoma. Although it is not clear what triggers EMT in tumors with TP53 mutation, a common characteristic of all ECS is the switching of cadherins, the overexpression EMT-TF, and the down-regulation of miR-200 genes. Probably, the crosstalk of different EMT-TF and the differential regulation of miR-200 genes by transcriptional repression or by epigenetic silencing through DNA hypermethylation play a major role in fixing the mesenchymal phenotype. Subsequent activation of specific transcription programs could induce differentiation to diverse mesenchymal tissues.Carcinosarcoma is a heterogeneous aggressive endometrial carcinoma that probably represents the end-stage of the evolution of both endometrioid and serous carcinomas after triggering a stable EMT program . MoleculPOLE mutations, MMR-def, and high TMB, and also the use of Poly (ADP-ribose) polymerase (PARP) inhibitors in tumors with homologous recombination deficiency, especially due to germline or somatic BRCA mutations.At present, most patients with ECS are not stratified for treatment according to molecular alterations ,111. How