Inhibition Of Signal Transducer And Activator Of Transcription 3 Activation Research Articles

Abstract B037: Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer known for its high rate of recurrence and metastatic ability. Patients face a poor prognosis and the five-year survival rate for those diagnosed with metastatic TNBC is very low. The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively active in TNBC and functions to promote proliferation and metastasis. Therefore, it is a potential therapeutic target for TNBC. We used CLUE, an online bioinformatics tool from the Broad Institute, to identify potential STAT3 inhibitors. To do this, a STAT3 gene signature generated from TNBC cells was used to query the gene signatures from drug treatments in the CLUE dataset for compounds that caused a similar signature. The hypothesis is that a drug with a similar signature to the STAT3 inhibition signature would be a potential STAT3 inhibitor. Drugs were chosen based on high similarity rating and known function. One of the top drugs we chose to pursue was TWS-119, a GSK3β inhibitor. GSK3β is a serine/threonine kinase that regulates proliferation and cell survival. TWS-119 was found to decrease the viability of MDA-MB-231, MDA-MB-468, and Hs587T TNBC cell lines. To assess the ability of TWS-119 to inhibit STAT3 activity, we used a STAT3 responsive reporter and found that TWS-119 inhibited STAT3 activity in MDA-MB-468 cells. To determine the mechanism of inhibition of STAT3 activity, we analyzed the effects of TWS-119 on STAT3 tyrosine phosphorylation. Preliminary results suggest that TWS-119 inhibits STAT3 activity via inhibition of tyrosine phosphorylation. Having determined that TWS-119 inhibited STAT3 activity, we wanted to determine if TWS-119 had any effect on TNBC migration. We found that TWS-119 reduced the migratory ability of MDA-MB-231 cells. An additional drug identified from CLUE was Cilengitide, an inhibitor of integrin ⍺vβ3 which plays an important role in proliferation and migration. While Cilengitide only slightly decreased the viability of the three different TNBC cell lines (MDA-MB-231, MDA-MB-468, Hs587T), we found that combinations of Cilengitide and TWS-119 had synergistic effects on cell viability in the TNBC cells. Preliminary western blot analysis demonstrated that the combination of both drugs reduced the level of pY-STAT3. This suggests that this combination targeting STAT3 may be a viable treatment strategy for TNBC. However, the mechanism through which these drugs interact is unknown, and the cell signaling relationship between their known targets (GSK3β and integrin ⍺vβ3) and STAT3 is unclear. We are currently working to define the mechanism of action of these combination effects in TNBC cells. Using TWS-119 and Cilengitide, we can determine the connection between integrins, GSK3β, and STAT3 to better understand proliferation and metastasis and to identify new therapeutic options for TNBC patients. Citation Format: Emily A Pratt, Sarah R Walker. Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B037.

Dienogest attenuates STAT3 activation in ovarian endometriotic cysts

ObjectiveRecent studies have suggested that endometriosis could be the result of excessive activation of signal transducer and activator of transcription 3 (STAT3), which is associated with the regulation of essential cellular mechanisms such as proliferation, invasion, and apoptosis. That finding implies that regulating STAT3 activation could play a key role in treating endometriosis. In the present study, we aimed to evaluate whether the anti-endometriotic effects of dienogest is mediated by the regulation of STAT3 activation. Study designSTAT3 activation was evaluated in normal endometrial and ovarian endometriotic tissues obtained from patients with/without preoperative dienogest treatment. A subsequent in vitro analysis with endometriotic cyst stromal cells (ECSCs) was used to confirm the direct influence of dienogest in STAT3 activation. ResultSTAT3 activation is significantly higher in endometriotic tissues from non-treated patients than in normal endometrial tissues, and that difference is reversed by preoperative administration of dienogest. Similarly, the inhibitory effects of dienogest on STAT3 activation are demonstrated by in vitro results showing that dienogest treatment significantly inhibits IL-6-stimulated STAT3 activation in cultured ECSCs. That inhibition was accompanied by decreased expression of proliferative (PCNA), invasive (MMP-2), and anti-apoptotic (BCL-2) proteins. Furthermore, downregulating STAT3 activity with siRNA decreased PCNA, MMP-2, and BCL-2 expression in IL-6-treated ECSCs. ConclusionDienogest inhibits STAT3 activation in ECSCs, which affects their proliferation, invasiveness, and apoptosis.

Quercetin, a key active ingredient of Jianpi Zishen Xiehuo Formula, suppresses M1 macrophage polarization and platelet phagocytosis by inhibiting STAT3 activation based on network pharmacology.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease, and abnormal M1 macrophage polarization participates in the pathogenesis of ITP. Jianpi Zishen Xiehuo (JZX) Formula has a good therapeutic effect on ITP. However, its key active ingredients and molecular mechanisms remain unclear. In this study, we explored the key active ingredients and potential targets of JZX in treating ITP using network pharmacology combined with in vitro experimental verification. A total of 157 active ingredients of JZX were identified from public databases, and quercetin was the most important one. One hundred sixty-five intersection targets of active ingredients in JZX, ITP, and macrophage polarization were obtained by Venn diagram. The top three potential targets were signal transducer and activator of transcription 3 (STAT3), protein kinase B (PKB/AKT) 1, and c-JUN through protein-protein interaction analysis. Molecular docking showed that quercetin had strong binding affinities with them all. In vitro experiment, CD16+ monocytes increased in ITP patients compared with healthy controls, which indicated a M1/M2 polarization imbalance in ITP. The expression levels of M1 polarization markers, CD86, CD80, and inducible nitric oxide synthase (iNOS), M1 polarization-associated cytokines, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), and antibody-opsonized platelet phagocytosis significantly increased in THP-1 macrophages stimulated with lipopolysaccharide (LPS). Quercetin markedly inhibited the expressions of M1 markers, decreased the levels of TNF-α and IL-6, and down-regulated the phosphorylated STAT3 (p-STAT3) protein, which confirmed the prediction by network pharmacology and molecular docking. Importantly, quercetin significantly reduced the phagocytosis of antibody opsonised platelet. In conclusion, quercetin suppressed platelet phagocytosis in M1 macrophages via its anti-inflammatory effects and may serve as a potential drug forthe treatment of ITP. Quercetin could be a key ingredient for JZX against ITP.

(E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol alleviates inflammatory responses in LPS-induced mice liver sepsis through inhibition of STAT3 phosphorylation

Sepsis is a life-threatening disease with limited treatment options, and the inflammatory process represents an important factor affecting its progression. Many studies have demonstrated the critical roles of signal transducer and activator of transcription 3 (STAT3) in sepsis pathophysiology and pro-inflammatory responses. Inhibition of STAT3 activity may therefore represent a promising treatment option for sepsis. We here used a mouse model to demonstrate that (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) treatment prevented the liver sepsis-related mortality induced by 30 mg/kg lipopolysaccharide (LPS) treatment and reduced LPS-induced increase in alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels, all of which are markers of liver sepsis progression. These recovery effects were associated with decreased LPS-induced STAT3, p65, and JAK1 phosphorylation and proinflammatory cytokine (interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha) level; expression of cyclooxygenase-2 and induced nitric oxide synthase were also reduced by MMPP. In an in vitro study using the normal liver cell line THLE-2, MMPP treatment prevented the LPS-induced increase of STAT3, p65, and JAK1 phosphorylation and inflammatory protein expression in a dose-dependent manner, and this effect was enhanced by combination treatment with MMPP and STAT3 inhibitor. The results clearly indicate that MMPP treatment prevents LPS-induced mortality by inhibiting the inflammatory response via STAT3 activity inhibition. Thus, MMPP represents a novel agent for alleviating LPS-induced liver sepsis.

Brevilin A exerts anti-colorectal cancer effects and potently inhibits STAT3 signaling invitro

Colorectal cancer (CRC) is the third most common cause of cancer-related morbidity worldwide, with an estimated of 1.85 million new cases and 850,000 deaths every year. Nevertheless, the current treatment regimens for CRC have many disadvantages, including toxicities and off-targeted side effects. STAT3 (signal transducer and activator of transcription 3) has been considered as a promising molecular target for CRC therapy. Brevilin A, a sesquiterpene lactone compound rich in Centipedae Herba has potent anticancer effects in nasopharyngeal, prostate and breast cancer cells by inhibiting the STAT3 signaling. However, the anti-CRC effect of brevilin A and the underlying mechanism of action have not been fully elucidated. In this study, we aimed to investigate the involvement of STAT3 signaling in the anti-CRC action of brevilin A. Here, HCT-116 and CT26 cell models were used to investigate the anti-CRC effects of brevilin A in vitro. HCT-116 cells overespressing with STAT3 were used to evaluate the involvement of STAT3 signaling in the anti-CRC effect of brevilin A. Screening of 49 phosphorylated tyrosine kinases in the HCT-116 cells after brevilin A treatment was performed by using the human phospho-receptor tyrosine kinase (phospho-RTK) array. Results showed that brevilin A inhibited cell proliferation and cell viability, induced apoptosis, reduced cell migration and invasion, inhibited angiogenesis, lowered the protein expression levels of phospho-Src (Tyr416), phospho-JAK2 (Y1007/1008) and phospho-STAT3 (Tyr705), and inhibited STAT3 activation and nuclear localization. Brevilin A also significantly reduced the protein expression levels of STAT3 target genes, such as MMP-2, VEGF and Bcl-xL. More importantly, over-activation of STAT3 diminished brevilin A's effects on cell viability. All these results suggest that brevilin A exerts potent anti-CRC effects, at least in part, by inhibiting STAT3 signaling. Our findings provide a strong pharmacological basis for the future exploration and development of brevilin A as a novel STAT3-targeting phytotherapeutic agent for CRC treatment.

Diospyros lotus leaf extract and its main component myricitrin regulate pruritus through the inhibition of astrocyte activation.

Diospyros lotus is a deciduous plant native to Asian countries, including Korea, Japan and China, and southeast Europe. In traditional medicine, Diospyros lotus is used as an anticancer, antidiabetic and antipyretic agent. The present study aimed to evaluate the effect of Diospyros lotus leaf extract (DLE) in ameliorating histamine-independent pruritus. Activation of signal transducer and activator of transcription 3 (STAT3) in astrocytes contributes to pruritus. In this study, the effects of DLE and its main component, myricetin (MC), on the activation of STAT3, expression of glial fibrillary acidic protein (GFAP), and production of lipocalin-2 (LCN2) in IL-6-treated astrocytes and chloroquine-injected mice were investigated through western blot, reverse transcription-quantitative PCR, and immunofluorescence staining. DLE and MC inhibited STAT3 activation, GFAP expression and LCN2 release via inositol 1,4,5-trisphosphate receptor type 1 blockade in astrocytes. DLE and MC ameliorated scratching behavior, expression of GFAP, mast cell infiltration and serum IL-6 levels in chloroquine-injected mice. These results suggested that DLE and MC can be used as oral therapeutic agents for the treatment and management of pruritus.

Onionin A inhibits small-cell lung cancer proliferation through suppressing STAT3 activation induced by macrophages-derived IL-6 and cell–cell interaction with tumor-associated macrophage

Tumor-associated macrophage (TAM)-derived IL-6 is involved in small-cell lung cancer (SCLC) progression and chemoresistance via the activation of signal transducer and activator of transcription 3 (STAT3) in the tumor microenvironment. This study aimed to identify natural compounds that suppress cell–cell interactions between TAMs and SCLC cells by inhibiting STAT3 activation. We used a library of natural compounds to identify candidate agents possessing anti-SCLC effects by inhibiting macrophage-induced tumor proliferation. SBC-3 and SBC-5, human SCLC cell lines, were used for in vitro experiments. Furthermore, we assessed the efficacy of these candidate agents in a murine xenograft model of human SCLC. Among the natural compounds examined, onionin A (ONA) inhibited IL-6-induced STAT3 activation and SCLC cell proliferation. ONA also reduced the secretion of IL-6 from macrophages and interfered with the direct effect of cell–cell interactions between macrophages and SCLC cells. Furthermore, ONA administration suppressed tumor progression in a tumor-bearing mouse model. ONA was identified as the most useful candidate for targeting cell–cell interactions between cancer cells and TAMs for anti-SCLC therapy.

Curcumin analog WZ26 induces ROS and cell death via inhibition of STAT3 in cholangiocarcinoma

ABSTRACT Cholangiocarcinoma (CCA) is an aggressive biliary epithelial tumor with limited therapeutic options and poor prognosis. Curcumin is a promising active natural compound with several anti-cancer properties, though its clinical uses remain hindered due to its poor bioavailability. We recently synthesized curcumin analogs with multifunctional pharmacological and bioactivities with enhanced bioavailability. Among these novel curcumin analogs, WZ26 is a representative molecule. However, the anti-tumor effect of WZ26 against CCA is unclear. In this study, we evaluated the anti-tumor effect of WZ26 in both CCA cells and CCA xenograft mouse model. The underlying molecular anti-cancer mechanism of WZ26 was also studied. Our results show that WZ26 significantly inhibited cell growth and induced mitochondrial apoptosis in CCA cell lines, leading to significant inhibition of tumor growth in xenograft tumor mouse model. Treatment of WZ26 increased reactive oxygen species (ROS) generation, subsequently decreased mitochondrial membrane potential and inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inducing G2/M cell cycle arrest and cell apoptosis. Pretreatment of N-acetyl cysteine (NAC), an antioxidant agent, could fully reverse the WZ26-induced ROS-mediated changes in CCA cells. Our findings provide experimental evidence that curcumin analog WZ26 could be a potential candidate against CCA via enhancing ROS induction and inhibition of STAT3 activation.

IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis: IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression.

Is interleukin-10 (IL-10) anti-fibrotic in endometriosis? IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis, because IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression. We previously showed that persistent activation of signal transducer and activator of transcription 3 (STAT3) via IL-6 trans-signaling promotes fibrosis of endometriosis. Studies showed marked anti-fibrotic effects of IL-10 via the STAT3 signaling pathway, which is generally considered to be anti-inflammatory, in various organs. Endometrial and/or endometriotic samples of 54 patients who had histological evidence of deep endometriosis, and endometrial samples from 30 healthy fertile women were analyzed. The effects of IL-10/STAT3 signaling as well as inhibition of STAT3 activation by knockdown of STAT3 gene on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Then, the effects of various time points of IL-10 treatment in combination with transforming growth factor (TGF)-β1 and/or IL-6/soluble IL-6 receptor (sIL-6R) on the profibrotic phenotype of endometrial and endometriotic stromal cells were investigated. IL-10 induced pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers and collagen gel contraction) of endometriotic stromal cells. Knockdown of STAT3 gene decreased the IL-10 induced pro-fibrotic phenotype of endometriotic stromal cells. In contrast, IL-10 had no significant effects on pro-fibrotic phenotype of endometrial stromal cells of healthy women. Sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R induced persistent activation of STAT3 and significantly increased proliferation of myofibroblasts (cells with α-smooth muscle actin positive stress fibers) and protein expression of collagen type I in endometriotic stromal cells. TGF-β1 and/or IL-6/sIL6RIL-6/sIL6R treatment significantly increased tissue inhibitor of metalloproteinase 1 (TIMP1) protein expression, whereas IL-10 had no significant effects. Knockdown of STAT3 gene significantly decreased the TGF-β1 and/or IL-6/sIL6R induced TIMP1 protein expression. In contrast, pre-treatment with IL-10 before TGF-β1 and/or IL-6/sIL-6R treatment and sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R significantly decreased proliferation of fibroblasts (cells without α-smooth muscle actin positive stress fibers) and collagen type I protein expression in endometrial stromal cells of healthy women. N/A. Given the large number of complex interactions and signaling pathways of pro- and anti-inflammatory mediators that are involved in the pathophysiology of endometriosis, the present study investigated only a very small portion of the whole. Further in vivo studies are required to validate the present findings. Inflammatory mediators in the pathophysiology of endometriosis have been extensively investigated as potential therapeutic targets. However, the present study showed that anti-inflammatory signals of IL-10 and IL-6 through persistent STAT3 activation may promote endometriosis fibrosis. Therapeutic strategies, such as suppression of 'inflammation', might dysregulate the cross-regulation of 'pro- and anti-inflammatory mediators', leading to detrimental effects in patients with endometriosis, such as fibrosis. To develop new, but not deleterious, therapeutic strategies, studies are required to investigate whether, how and what 'anti-inflammatory mediators' along with pro-inflammatory mediators are involved in individual patients with endometriosis. This study was supported in part by KARL STORZ SE & Co. KG (Tuttlingen, Germany). The authors have no conflict of interest to disclose.

HUC-MSCs Attenuate Acute Graft-Versus-Host Disease through Chi3l1 Repression of Th17 Differentiation.

Mesenchymal stem cells (MSCs) have already demonstrated definitive evidence of their clinical benefits in acute graft-versus-host disease (aGvHD) and other inflammatory diseases. However, the comprehensive mechanism of MSCs' immunomodulation properties has not been elucidated. To reveal their potential immunosuppressive molecules, we used RNA sequencing to analyze gene expression in different tissue-derived MSCs, including human bone marrow, umbilical cord, amniotic membrane, and placenta, and found that chitinase-3-like protein 1 (Chi3l1) was highly expressed in human umbilical cord mesenchymal stem cells (hUC-MSCs). We found that hUC-MSCs treated with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) exhibited increased expression of Chi3l1 and concurrently repressed T-helper 17 cell (Th17) differentiation through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, Chi3l1 knockdown hUC-MSCs exhibited impaired therapeutic efficacy in aGvHD mice with an increased inflammatory response by promoting Th17 cell differentiation, including an increase in IL-17A in the spleen, intestine, and serum. Collectively, these results reveal a new immunosuppressive molecule, Chi3l1, in hUC-MSCs in the treatment of aGvHD that regulates Th17 differentiation and inhibits STAT3 activation. These novel insights into the mechanisms of hUC-MSC immunoregulation may lead to the consistent production of hUC-MSCs with strong immunosuppressive functions and thus improved clinical utility.

STAT3/miR-130b-3p/MBNL1 feedback loop regulated by mTORC1 signaling promotes angiogenesis and tumor growth

BackgroundAberrantly activated mammalian target of rapamycin complex 1 (mTORC1) plays a vital role in tumor angiogenesis, but its precise mechanisms are still unclear.MethodsMicro-RNA-130b-3p (miR-130b-3p) expression in mTORC1-activated and control cells was examined by quantitative real-time PCR (qRT-PCR). MiR-130b-3p levels and their correlation with mTORC1 activity were evaluated by analyzing publicly available databases and in-house head and neck squamous cell carcinoma (HNSCC) tissues. The role of miR-130b-3p in mTORC1-mediated angiogenesis and tumor growth was examined using tube formation assay, chicken chorioallantoic membrane assay, cell line − derived xenograft models, and an HNSCC patient-derived xenograft (PDX) model. The regulatory mechanisms among signal transducer and activator of transcription 3 (STAT3), miR-130b-3p, and muscleblind-like protein 1 (MBNL1) were investigated via bioinformatics analyses, qRT-PCR, western blot, RNA immunoprecipitation, immunofluorescence, luciferase reporter assay, and chromatin immunoprecipitation assay.ResultsElevated miR-130b-3p enhanced the angiogenic and tumorigenic abilities of mTORC1-activated cells both in vitro and in vivo. STAT3, a downstream effector of mTORC1, transactivated miR-130b-3p by direct binding promoter of the miR-130b gene. MBNL1 was identified as a direct target of miR-130b-3p. MBNL1 depletion rescued the compromised angiogenesis and tumor growth caused by miR-130b-3p inhibition. MiR-130b-3p levels were significantly upregulated and positively correlated with mTORC1 signaling in multiple cancers. MiR-130b-3p inhibition attenuated tumor angiogenesis and growth in an HNSCC PDX model. MBNL1 feedback inhibited STAT3 activation in mTORC1-activated cells.ConclusionsThe STAT3/miR-130b-3p/MBNL1 feedback loop plays a vital role in mTORC1-mediated angiogenesis and tumor progression. This pathway could be targeted for therapeutic intervention of mTORC1-related cancers.

Persistent activation of signal transducer and activator of transcription 3 via interleukin-6 trans-signaling is involved in fibrosis of endometriosis.

Is activation of signal transducer and activator of transcription 3 (STAT3) via interleukin-6 (IL-6) trans-signaling involved in fibrosis of endometriosis? Persistent activation of STAT3 via IL-6 trans-signaling is involved in fibrosis of endometriosis. Our previous study showed that sustained low-grade inflammation promotes a fibrotic phenotype in endometriotic stromal cells. However, the underlying mechanisms of the establishment of non-resolving, low-grade inflammation in endometriosis remain to be clarified. Endometrial and/or endometriotic samples of 60 patients who had histological evidence of deep endometriosis and endometrial samples from 32 healthy fertile women were analyzed. The effects of priming with ligands of Toll-like receptors (TLRs) 2, 3 and 4 on secretion of inflammatory mediators (tumor necrosis factor-α, C-X-C motif chemokine ligand-10 [CXCL-10], IL6 and IL-10) after a second challenge with TLR ligands in endometrial and endometriotic stromal cells were investigated. Then, the effects of IL-6/soluble (s) IL-6 receptor (R)/STAT3 signaling, as well as inhibition of STAT3 activation by knockdown of STAT3 or pharmacological inhibition (S3I-201), on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Priming with TLR ligands for 4 h had no significant effects, whereas 24 h of priming significantly decreased secretion of IL-6, after a second challenge in endometrial stromal cells of healthy women. In endometriotic stromal cells, whereas 24 h of priming had no significant effects, priming with TLR ligands for 4 h significantly increased secretion of IL-6 after a second challenge. IL-6/soluble IL-6 receptor (sIL-6R) induced a pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers, cell migration and collagen gel contraction) as well as nuclear factor-kappa B (NF-κB) activation of endometriotic stromal cells. In contrast, IL-6/sIL-6R had no significant effects on either a pro-fibrotic phenotype or NF-κB activation of endometrial stromal cells of healthy women. Stimulation with transforming growth factor (TGF)-β1 and/or IL-6/sIL-6R for 1 h and 48 h activated STAT3, but induced very low or no suppressor of cytokine signaling (SOCS) 1 and 3 protein expression in endometriotic stromal cells. In endometrial stromal cells of healthy women, IL-6/sIL-6R-induced STAT3 and SOCS1/3 expression at 1 h, whereas no STAT3 activation was detected at 48 h. Knockdown of STAT3 gene or S3I-201 (a STAT3 inhibitor) decreased the IL-6/sIL-6R-induced pro-fibrotic phenotype as well as NF-κB activation and TGF-β1-induced cell proliferation of endometriotic stromal cells. N/A. In vivo studies are required to confirm the present in vitro results. However, it remains challenging to mimic non-resolving chronic inflammation in animal models, as active inflammation can resolve spontaneously. Dysfunction of negative regulators of IL-6/sIL-6R/STAT3 signaling may cause persistent activation of STAT3 in endometriosis. Since STAT3 activation in the endometrium is essential for successful embryo implantation, treatment with STAT3 inhibitors would not be appropriate for women wishing to conceive. However, targeting impaired negative regulation of IL-6/sIL-6R/STAT3 signaling may still represent a promising avenue for the treatment of endometriosis. This study was supported in part by the KARL STORZ SE & Co. KG (Tuttlingen, Germany). There are no conflicts of interest.

Acacetin Inhibits the Growth of STAT3-Activated DU145 Prostate Cancer Cells by Directly Binding to Signal Transducer and Activator of Transcription 3 (STAT3).

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the formation and growth of human cancer. Therefore, STAT3 is a therapeutic target for cancer drug discovery. Acacetin, a flavone present in various plants, inhibits constitutive and inducible STAT3 activation in STAT3-activated DU145 prostate cancer cells. Acacetin inhibits STAT3 activity by directly binding to STAT3, which we confirmed by a pull-down assay with a biotinylated compound and two level-free methods, namely, a drug affinity responsive target stability (DARTS) experiment and a cellular thermal shift assay (CETSA). Acacetin inhibits STAT3 phosphorylation at the tyrosine 705 residue and nuclear translocation in DU145 cells, which leads to the downregulation of STAT3 target genes. Acacetin then induces apoptosis in a time-dependent manner. Interestingly, acacetin induces the production of reactive oxygen species (ROS) that are not involved in the acacetin-induced inhibition of STAT3 activation because the suppressed p-STAT3 level is not rescued by treatment with GSH or NAC, which are general ROS inhibitors. We also found that acacetin inhibits tumor growth in xenografted nude mice. These results suggest that acacetin, as a STAT3 inhibitor, could be a possible drug candidate for targeting STAT3 for the treatment of cancer in humans.

The novel STAT3 inhibitor WZ-2-033 causes regression of human triple-negative breast cancer and gastric cancer xenografts.

Hyperactive signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in human triple-negative breast cancer (TNBC) and gastric cancer, leading to uncontrolled tumor growth, resistance to chemotherapy, and poor prognosis. Thus, inhibition of STAT3 signaling is a promising therapeutic approach for both TNBC and gastric cancer, which have high incidences and mortality and limited effective therapeutic approaches. Here, we report a small molecule, WZ-2-033, capable of inhibiting STAT3 activation and dimerization and STAT3-related malignant transformation. We present in vitro evidence from surface plasmon resonance analysis that WZ-2-033 interacts with the STAT3 protein and from confocal imaging that WZ-2-033 disrupts HA-STAT3 and Flag-STAT3 dimerization in intact cells. WZ-2-033 suppresses STAT3-DNA-binding activity but has no effect on STAT5-DNA binding. WZ-2-033 inhibits the phosphorylation and nuclear accumulation of pY705-STAT3 and consequently suppresses STAT3-dependent transcriptional activity and the expression of STAT3 downstream genes. Moreover, WZ-2-033 significantly inhibited the proliferation, colony survival, migration, and invasion of TNBC cells and gastric cancer cells with aberrant STAT3 activation. Furthermore, administration of WZ-2-033 in vivo induced a significant antitumor response in mouse models of TNBC and gastric cancer that correlated with the inhibition of constitutively active STAT3 and the suppression of known STAT3 downstream genes. Thus, our study provides a novel STAT3 inhibitor with significant antitumor activity in human TNBC and gastric cancer harboring persistently active STAT3.

Sanguinarine combats hypoxia-induced activation of EphB4 and HIF-1α pathways in breast cancer

BackgroundBreast cancer is the most common female cancer worldwide. Large hypoxic area is one of the features of tumor microenvironment. Highly activated hypoxia-induced pathways positively correlate with poor clinical response to chemo- and radiotherapy and high mortality in breast cancer patients. PurposeWe explore the effect of sanguinarine on hypoxia-induced activation of Ephrin type-B receptor 4 (EphB4) and hypoxia inducible factor-1α (HIF-1α) pathways in breast cancer. ResultsHypoxia-induced expression of a receptor tyrosine kinase EphB4 was observed in hypoxic breast cancer cell models. Sanguinarine, a natural alkaloid, could effectively combat hypoxia-induced EphB4 and HIF-1α expression. Sanguinarine inhibited the activation of downstream protein signal transducer and activator of transcription-3 (STAT3), thereby blocking hypoxia-induced HIF-1α/STAT3 interaction and downregulating the mRNA levels of their target genes. Mechanically, sanguinarine attenuated HIF-1α protein levels via inhibition of MAPK/ERK pathways and promotion of HIF-1α proteasome degradation. Sanguinarine inhibited STAT3 activation through targeting its upstream EphB4 and accelerating STAT3 dephosphorylation. Correspondingly, xenograft models confirmed that sanguinarine treatment disrupted hypoxia-induced pathways and inhibited tumor growth in vivo. ConclusionsOur results may bring insights to the hypoxia-induced pathways in breast cancers, and suggest sanguinarine as a promising candidate for EphB4 and HIF-1α-targeted inhibition.

The sesquiterpene lactone eupatolide induces apoptosis in non-small cell lung cancer cells by suppressing STAT3 signaling

We aimed to evaluate the role of a natural sesquiterpene lactone, eupatolide, in non-small-cell lung cancer (NSCLC) and further explore its underlying mechanism on regulating the activation of signal transducer and activator of transcription 3 (STAT3), which is thought to have carcinogenic function in a variety of malignancies including lung cancer. Cell survival was measured by Cell Counting Kit-8 assay. in vivo experiments were performed by inoculating NSCLC cells into nude mice. Western blot and qRT-PCR were applied to detect the activation level of STAT3 and the mRNA levels of anti-apoptotic markers. The cell apoptosis was measured by Annexin V-FITC/PI Apoptosis Detection Kit. Our results showed that eupatolide suppressed the survival of NSCLC cells in a dose and time dependent manner. Furthermore, eupatolide increased the anti-tumor activity of the chemotherapeutic drugs cisplatin and 5-Fluoracil (5-FU). The xenograft study revealed that eupatolide suppressed tumor growth of NSCLC cells in vivo. Furthermore, eupatolide induced apoptosis by suppressing the activation of STAT3 in NSCLC cells. Sustained activation or knockdown of STAT3 suppressed and enhanced the activity of eupatolide, respectively. This paper is the first to report that eupatolide could effectively inhibit NSCLC progression, suggesting that eupatolide might be utilized as a novel STAT3 inhibitor for treating NSCLC.

An Electrophilic Deguelin Analogue Inhibits STAT3 Signaling in H-Ras-Transformed Human Mammary Epithelial Cells: The Cysteine 259 Residue as a Potential Target.

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,β-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

10,11-dehydrocurvularin exerts antitumor effect against human breast cancer by suppressing STAT3 activation.

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) plays a critical role in many types of cancers. As a result, STAT3 has been identified as a potential target for cancer therapy. In this study we identified 10,11-dehydrocurvularin (DCV), a natural-product macrolide derived from marine fungus, as a selective STAT3 inhibitor. We showed that DCV (2-8 μM) dose-dependently inhibited the proliferation, migration and invasion of human breast cancer cell lines MDA-MB-231 and MDA-MB-468, and induced cell apoptosis. In the two breast cancer cell lines, DCV selectively inhibited the phosphorylation of STAT3 Tyr-705, but did not affect the upstream components JAK1 and JAK2, as well as dephosphorylation of STAT3. Furthermore, DCV treatment strongly inhibited IFN-γ-induced STAT3 phosphorylation but had no significant effect on IFN-γ-induced STAT1 and STAT5 phosphorylation in the two breast cancer cell lines. We demonstrated that the α, β-unsaturated carbonyl moiety of DCV was essential for STAT3 inactivation. Cellular thermal shift assay (CETSA) further revealed the direct engagement of DCV with STAT3. In nude mice bearing breast cancer cell line MDA-MB-231 xenografts, treatment with DCV (30 mg·kg-1·d-1, ip, for 14 days) markedly suppressed the tumor growth via inhibition of STAT3 activation without observed toxicity. Our results demonstrate that DCV acts as a selective STAT3 inhibitor for breast cancer intervention.

Role of crosstalk between STAT3 and mTOR signaling in driving sensitivity to chemotherapy in osteosarcoma cell lines.

Osteosarcoma (OS) is a malignant bone neoplasm, mostly occurring in pediatric patients. OS is characterized by a highly aggressive and metastatically active tumor. Chemotherapy followed by surgical excision is the treatment of choice but is often associated with both chemoresistance and relapse. Hence, it is important to develop further understanding of OS pathogenesis and identify potential therapeutic targets. Both the signal transducer and activator of transcription 3 (STAT3) and mammalian target of rapamycin (mTOR) have been implicated in OS pathogenesis. Crosstalk between mTOR and STAT3 signaling has been shown to regulate hypoxia-induced angiogenesis in other diseases. In this study, we determined using OS cell lines if there is a crosstalk between these two pathways and how that impacts sensitivity to treatment with Rapamycin. OS cell lines exhibited differential sensitivity to mTOR inhibitor Rapamycin. Evaluation of phosphorylated STAT3 showed that in Rapamycin-sensitive 143B cells, the inhibitor decreased phosphorylation of STAT3 at Y705, but not at S727 whereas, in Rapamycin-resistant U2OS cells, the inhibitor decreased S727 phosphorylation but not Y705. However, knockdown of STAT3 in U2OS cells made them sensitive to Rapamycin. Immunofluorescence (IF) analysis showed that mTOR is constitutively activated in the 143B cells but is suppressed in the U2OS cells, indicating that this might be their reason for being resistant to Rapamycin. Both cell lines were sensitive to treatment with the STAT3 inhibitor Napabucasin (NP). Treatment with NP inhibited STAT3 activation at Y705 and additionally inhibited mTOR activation, indicating crosstalk between STAT3 and mTOR signaling pathways. Rapamycin could effectively prevent lung metastasis in an orthotropic OS mice model using 143B cells. However, Rapamycin could not inhibit lung metastasis in mice injected with U2OS cells. The STAT3 inhibitor NP attenuated lung metastasis with the U2OS cells. Our results thus established yet undefined crosstalk of STAT3 and mTOR signaling pathways in OS and highlight the possibility of using mTOR inhibitors for treatment in patients with OS.

A novel small molecule STAT3 inhibitor SLSI-1216 suppresses proliferation and tumor growth of triple-negative breast cancer cells through apoptotic induction

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, characterized by the lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Owing to the absence of molecular targets, there are limited treatment options, and TNBC patients exhibit high mortality rates. Signal transducer and activator of transcription 3 (STAT3) is overexpressed and aberrantly activated in TNBC cells. Therefore, inhibition of STAT3-mediated signaling provides a potential strategy for the treatment of TNBC. In this study, A series of synthetic derivatives of SLSI-1 (a STAT3 inhibitor) were designed and evaluated for antitumor activity in TNBC cells. A novel derivative (SLSI-1216) exhibited the most potent anti-proliferative activity. SLSI-1216 effectively inhibited STAT3 activity and activation of STAT3, leading to the downregulation of AXL, a downstream target of STAT3 and epithelial-mesenchymal transition (EMT) progression. The inhibition of EMT by SLSI-1216 was associated with modulation of E-cadherin and N-cadherin. Furthermore, SLSI-1216 induced apoptosis by targeting STAT3 and effectively inhibited tumor growth in vivo. These findings suggest that SLSI-1216, as a potential inhibitor of STAT3, may be a promising therapeutic agent for TNBC.