Signal Transducer And Activator Of Transcription 3 Dimerization Research Articles

Combined therapeutic effect of YHO-1701 with PD-1 blockade is dependent on natural killer cell activity in syngeneic mouse models.

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival, and invasion. We discovered YHO-1701 as a small molecule inhibitor of STAT3 dimerization and demonstrated its potent anti-tumor activity using xenograft mouse models as monotherapy and combination therapy with molecular targeted drugs. STAT3 is also associated with cancer immune tolerance; therefore, we used the female CT26 syngeneic mouse model to examine the effect of combining YHO-1701 administration with PD-1/PD-L1 blockade. Pretreatment of the mice with YHO-1701 before starting anti-PD-1 antibody administration resulted in a significant therapeutic effect. In addition, the effect of monotherapy and combination treatment with YHO-1701 was significantly abolished by depleting natural killer (NK) cell activity. YHO-1701 was also found to restore the activity of mouse NK cells under inhibitory conditions in vitro. Furthermore, this combination therapy significantly inhibited tumor growth in an immunotherapy-resistant model of murine CMS5a fibrosarcoma. These results suggest that the combination of YHO-1701 with PD-1/PD-L1 blockade might be a new candidate for cancer immunotherapy involving the enhancement of NK cell activity in the tumor microenvironment.

Synthesis and evaluation of naphthalene derivatives as potent STAT3 inhibitors and agents against triple-negative breast cancer growth and metastasis.

Triple-negative breast cancer (TNBC) represents the worst prognostic subtype of breast cancer and lacks targeted therapeutic drugs. Signal transducer and activator of transcription 3 (STAT3) is overexpressed and constitutively activated in TNBCs and associated with poor patient outcomes. However, no agents targeting STAT3 have been successfully developed and marketed. Selective Estrogen Receptor Modulators (SERMs) have been reported as potential inhibitors of the IL-6/STAT3 signaling pathway. Naphthalene compounds have good pharmacological activity and significant anti-cancer activity. In this study, we synthesized a new series of naphthalene derivatives with the general structure of SERM and evaluated their effects on TNBC and STAT3 signals. A new series of compounds based on the scaffold of SERMs and an amino group were designed and screened based on the structure-activity relationship by MTT assay. The binding activity of SMY002 to STAT3 was predicted and validated by docking and SPR. The STAT3 signaling target and anti-cancer effects of SMY002 were evaluated with three TNBC cell lines and the mice transplanted tumor model. Among the compounds, SMY002 displayed the most potent activity, which could directly interact with STAT3 SH2-domain, and strongly inhibit the phosphorylation, dimerization, nuclear distribution, transcriptional activity, and target genes expression of STAT3. Furthermore, SMY002 markedly suppressed migration, invasion, survival, growth, and metastasis of TNBC cells in vitro and in vivo via down-regulating the expression of Cyclin D1 and MMP9. SMY002 can significantly inhibit the growth and metastasis of TNBC cells by targeting the STAT3 signal.

Inhibiting STAT3 signaling pathway by natural products for cancer prevention and therapy: In vitro and in vivo activity and mechanisms of action

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in signal transmission from the plasma membrane to the nucleus, regulating the expression of genes involved in essential cell functions and controlling the processes of cell cycle progression and apoptosis. Thus, STAT3 has been elucidated as a promising target for developing anticancer drugs. Many natural products have been reported to inhibit the STAT3 signaling pathway during the past two decades and have exhibited significant anticancer activities in vitro and in vivo. However, there is no FDA-approved STAT3 inhibitor yet. The major mechanisms of these natural product inhibitors of the STAT3 signaling pathway include targeting the upstream regulators of STAT3, directly binding to the STAT3 SH2 domain and inhibiting its activation, inhibiting STAT3 phosphorylation and/or dimerization, and others. In the present review, we have systematically discussed the development of these natural product inhibitors of STAT3 signaling pathway as well as their in vitro and in vivo anticancer activity and mechanisms of action. Outlooks and perspectives on the associated challenges are provided as well.

SHF Acts as a Novel Tumor Suppressor in Glioblastoma Multiforme by Disrupting STAT3 Dimerization.

Sustained activation of signal transducer and activator of transcription 3 (STAT3) is a critical contributor in tumorigenesis and chemoresistance, thus making it an attractive cancer therapeutic target. Here, SH2 domain‐containing adapter protein F (SHF) is identified as a tumor suppressor in glioblastoma Multiforme (GBM) and its negative regulation of STAT3 activity is characterized. Mechanically, SHF selectively binds and inhibits acetylated STAT3 dimerization without affecting STAT3 phosphorylation or acetylation. Additionally, by blocking STAT3‐DNMT1 (DNA Methyltransferase 1) interaction, SHF relieves methylation of tumor suppressor genes. The SH2 domain is documented to be essential for SHF's actions on STAT3, and almost entirely replaces the functions of SHF on STAT3 independently. Moreover, the peptide C16 a peptide derived from the STAT3‐binding sites of SHF inhibits STAT3 dimerization and STAT3/DNMT1 interaction, and achieves remarkable growth inhibition in GBM cells in vitro and in vivo. These findings strongly identify targeting of the SHF/STAT3 interaction as a promising strategy for developing an optimal STAT3 inhibitor and provide early evidence of the potential clinical efficacy of STAT3 inhibitors such as C16 in GBM.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

Targeting STAT3 prevents bile reflux-induced oncogenic molecular events linked to hypopharyngeal carcinogenesis.

The signal transducer and activator of transcription 3 (STAT3) oncogene is a transcription factor with a central role in head and neck cancer. Hypopharyngeal cells (HCs) exposed to acidic bile present aberrant activation of STAT3, possibly contributing to its oncogenic effect. We hypothesized that STAT3 contributes substantially to the bile reflux‐induced molecular oncogenic profile, which can be suppressed by STAT3 silencing or pharmacological inhibition. To explore our hypothesis, we targeted the STAT3 pathway, by knocking down STAT3 (STAT3 siRNA), and inhibiting STAT3 phosphorylation (Nifuroxazide) or dimerization (SI3‐201; STA‐21), in acidic bile (pH 4.0)‐exposed human HCs. Immunofluorescence, luciferase assay, Western blot, enzyme‐linked immunosorbent assay and qPCR analyses revealed that STAT3 knockdown or pharmacologic inhibition significantly suppressed acidic bile‐induced STAT3 activation and its transcriptional activity, Bcl‐2 overexpression, transcriptional activation of IL6, TNF‐α, BCL2, EGFR, STAT3, RELA(p65), REL and WNT5A, and cell survival. Our novel findings document the important role of STAT3 in bile reflux‐related molecular oncogenic events, which can be dramatically prevented by STAT3 silencing. STA‐21, SI3‐201 or Nifuroxazide effectively inhibited STAT3 and cancer‐related inflammatory phenotype, encouraging their single or combined application in preventive or therapeutic strategies of bile reflux‐related hypopharyngeal carcinogenesis.

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.

Targeting Canonical and Non-Canonical STAT Signaling Pathways in Renal Diseases.

Signal transducer and activator of transcription (STAT) plays an essential role in the inflammatory reaction and immune response of numerous renal diseases. STATs can transmit the signals of cytokines, chemokines, and growth factors from the cell membrane to the nucleus. In the canonical STAT signaling pathways, upon binding with their cognate receptors, cytokines lead to a caspase of Janus kinases (JAKs) and STATs tyrosine phosphorylation and activation. Besides receptor-associated tyrosine kinases JAKs, receptors with intrinsic tyrosine kinase activities, G-protein coupled receptors, and non-receptor tyrosine kinases can also activate STATs through tyrosine phosphorylation or, alternatively, other post-translational modifications. Activated STATs translocate into the nucleus and mediate the transcription of specific genes, thus mediating the progression of various renal diseases. Non-canonical STAT pathways consist of preassembled receptor complexes, preformed STAT dimers, unphosphorylated STATs (U-STATs), and non-canonical functions including mitochondria modulation, microtubule regulation and heterochromatin stabilization. Most studies targeting STAT signaling pathways have focused on canonical pathways, but research extending into non-canonical STAT pathways would provide novel strategies for treating renal diseases. In this review, we will introduce both canonical and non-canonical STAT pathways and their roles in a variety of renal diseases.

15-Deoxy-Δ12,14 -prostaglandin J2 binds and inactivates STAT3 via covalent modification of cysteine 259 in H-Ras-transformed human breast epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) functions as an allosteric inhibitor of STAT3. 15d-PGJ2 inhibits phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3 in H-Ras-transformed human mammary epithelial cells (MCF10A-Ras) through the Michael addition reaction at cysteine 259 of STAT3. Comparative studies with 15d-PGJ2 analogues reveal that both C12-C13 and C9-C10 double bonds conjugated to the carbonyl group in the cyclopentenone ring of 15d-PGJ2 are essential for STAT3 binding. Antiproliferative and pro-apoptotic activities of 15d-PGJ2 in MCF10A-Ras cells are attributable to covalent modification of STAT3 on Cys259, and mimic the effects induced by mutation of this amino acid.

A novel STAT3 inhibitor W2014-S regresses human non-small cell lung cancer xenografts and sensitizes EGFR-TKI acquired resistance.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is a common feature in human non-small cell lung cancer (NSCLC). STAT3 plays an important role in cancer progression as a driver oncogene and acquired resistance of targeted therapies as an alternatively activated pathway. W2014-S with pharmacophore structure of imidazopyridine, which was firstly reported to be utilized in STAT3 inhibitor discovery, was screened out as a potent STAT3 inhibitor from a library of small molecules. The aim of this study is to investigate the antitumor activities and mechanisms of W2014-S in NSCLC and effect on epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in vitro and in vivo.Methods: SPR analysis, Co-immunoprecipitation, confocal microscope imaging, and luciferase report gene assays were utilized to determine the mechanisms. Cell viability, colonial survival, wound healing, cell invasion assay, human cancer cell xenografts and PDX tumor xenografts were used to determine antitumor activities.Results: W2014-S disrupted STAT3 dimerization and selectively inhibited aberrant STAT3 signaling in NSCLC cell line. W2014-S strongly suppressed proliferation, survival, migration and invasion of lung cancer cells with aberrant STAT3 activation and inhibited the growth of human NSCLC cell xenografts and PDX tumor xenografts in mouse model. Furthermore, W2014-S significantly sensitized resistant NSCLC cell line to gefitinib and erlotinib in vitro and enhances the anti-tumor effect of gefitinib in TKI-resistant lung cancer xenografts in vivo.Conclusions: Our study has provided a novel STAT3 inhibitor with significant anti-tumor activities in NSCLC and suggests that combination of STAT3 inhibitor such as W2014-S with gefitinib could serve as a promising strategy to overcome EGFR-TKIs acquired resistance in NSCLC patients.

STX-0119, a novel STAT3 dimerization inhibitor, prevents fibrotic gene expression in a mouse model of kidney fibrosis by regulating Cxcr4 and Ccr1 expression.

Kidney fibrosis is a histological hallmark of chronic kidney disease (CKD) and is believed to be involved in the progression of CKD. Therefore, inhibition of kidney fibrosis is a potential strategy for slowing CKD progression. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated by interleukin‐6 and is reported to be involved in fibrosis. Previously, S3I‐201, an inhibitor of STAT3 phosphorylation, was shown to inhibit renal fibrosis in a mouse model, but its mechanism was not clarified completely. In this study, we investigated whether STX‐0119, a new inhibitor of STAT3 dimerization, suppressed kidney fibrotic gene expression using a mouse model of kidney fibrosis and examined the underlying mechanisms. Kidney fibrosis was induced by unilateral ureteral obstruction (UUO), which was accompanied by upregulation of STAT3 target genes. STX‐0119 administration suppressed the expression of fibrotic genes in UUO kidneys without affecting STAT3 phosphorylation. STX‐0119 decreased Cxcr4 mRNA in cultured rat kidney fibroblasts and Ccr1 mRNA in blood cells from UUO mice, both of which are reported to be involved in the progression of kidney fibrosis. These results suggest that STX‐0119 inhibits fibrotic gene expression in kidney by suppressing Cxcr4 and Ccr1 expression. This is the first report to indicate a part of the mechanism of the antifibrotic effects of a STAT3 inhibitor and suggests that STX‐0119 may be a lead compound for the treatment of kidney fibrosis.

Retraction Note: Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Trichothecin Inhibits Cancer-Related Features in Colorectal Cancer Development by Targeting STAT3.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that contributes to cancer progression through multiple processes of cancer development, which makes it an attractive target for cancer therapy. The IL-6/STAT3 pathway is associated with an advanced stage in colorectal cancer patients. In this study, we identified trichothecin (TCN) as a novel STAT3 inhibitor. TCN was found to bind to the SH2 domain of STAT3 and inhibit STAT3 activation and dimerization, thereby blocking STAT3 nuclear translocation and transcriptional activity. TCN did not affect phosphorylation levels of STAT1. TCN significantly inhibited cell growth, arrested cell cycle at the G0/G1 phase, and induced apoptosis in HCT 116 cells. In addition, the capacities of colony formation, migration, and invasion of HCT 116 cells were impaired upon exposure to TCN with or without IL-6 stimulation. In addition, TCN treatment abolished the tube formation of HUVEC cells in vitro. Taken together, these results highlight that TCN inhibits various cancer-related features in colorectal cancer development in vitro by targeting STAT3, indicating that TCN is a promising STAT3 inhibitor that deserves further exploration in the future.

Antitumor activity of a novel oral signal transducer and activator of transcription 3 inhibitor YHO-1701.

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival and invasion. Aberrant STAT3 has been demonstrated in various malignant cancers. YHO‐1701 is a novel quinolinecarboxamide derivative generated from STX‐0119. Here, we examined the effect of YHO‐1701 on STAT3 and evaluated antitumor activity of YHO‐1701 as a single agent and in combination. YHO‐1701 inhibited STAT3‐SH2 binding to phospho‐Tyr peptide selectively and more potently than STX‐0119 in biochemical assays. Molecular docking studies with STAT3 suggested more stable interaction of YHO‐1701 with the SH2 domain. YHO‐1701 exhibited approximately 10‐fold stronger activity than STX‐0119 in abrogating the STAT3 signaling pathway of human oral cancer cell line SAS. YHO‐1701 also blocked multi‐step events by inhibiting STAT3 dimerization and suppressed STAT3 promoter activity. As expected, YHO‐1701 exerted strong antiproliferative activity against human cancer cell lines addicted to STAT3 signaling. Orally administered YHO‐1701 showed statistically significant antitumor effects with long exposure to high levels of YHO‐1701 at tumor sites in SAS xenograft models. Moreover, combination regimen with sorafenib led to significantly stronger antitumor activity. In addition, the suppression level of survivin (a downstream target) was superior for the combination as compared with monotherapy groups within tumor tissues. Thus, YHO‐1701 had a favorable specificity for STAT3 and pharmacokinetics after oral treatment; it also contributed to the enhanced antitumor activity of sorafenib. The evidence presented here provides justification using for this approach in future clinical settings.

Can asymmetric post-translational modifications regulate the behavior of STAT3 homodimers?

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus‐STAT3 bimolecular fluorescence complementation assay that allows the visualization and study of STAT3 dimerization and protein‐protein interactions in living cells. Inactivating mutations on residues susceptible to post‐translational modifications (PTMs) (K49R, K140R, K685R, Y705F and S727A) changed significantly the intracellular distribution of unstimulated STAT3 dimers when the dimers were formed by STAT3 molecules that carried different mutations (ie they were “asymmetric”). Some of these asymmetric dimers changed the proliferation rate of HeLa cells. Our results indicate that asymmetric PTMs on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. We put forward these observations as a working hypothesis, since confirming the existence of asymmetric STAT3 homodimers in nature is extremely difficult, and our own experimental setup has technical limitations that we discuss. However, if our hypothesis is confirmed, its conceptual implications go far beyond STAT3, and could advance our understanding and control of signaling pathways.

Anticancer activity of dietary xanthone \u03b1-mangostin against hepatocellular carcinoma by inhibition of STAT3 signaling via stabilization of SHP1

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide. The dietary xanthone α-mangostin (α-MGT) exhibits potent anti-tumor effects in vitro and in vivo. However, the anti-HCC effects of α-MGT and their underlying mechanisms are still vague. Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is involved in the progression of HCC. We therefore investigated whether α-MGT inhibited the activation of STAT3 and thereby exhibits its anti-HCC effects. In this study, we found that α-MGT significantly suppressed cell proliferation, induced cell cycle arrest, and triggered apoptosis in HCC cells, including HepG2, SK-Hep-1, Huh7, and SMMC-7721 cells in vitro, as well as inhibiting tumor growth in nude mice bearing HepG2 or SK-Hep-1 xenografts. Furthermore, α-MGT potently inhibited the constitutive and inducible activation of STAT3 in HCC cells. In addition, α-MGT also suppressed IL-6-induced dimerization and nuclear translocation of STAT3, which led to inhibition of the expression of STAT3-regulated genes at both mRNA and protein levels. Mechanistically, α-MGT exhibited effective inhibition of the activation of STAT3’s upstream kinases, including JAK2, Src, ERK, and Akt. Importantly, α-MGT increased the protein level of Src homology region 2 domain-containing phosphatase-1 (SHP1), which is a key negative regulator of the STAT3 signaling pathway. Furthermore, α-MGT enhanced the stabilization of SHP1 by inhibiting its degradation mediated by the ubiquitin–proteasome pathway. Knockdown of SHP1 using siRNA obviously prevented the α-MGT-mediated inhibition of the activation of STAT3 and proliferation of HCC cells. In summary, α-MGT exhibited a potent anti-HCC effect by blocking the STAT3 signaling pathway via the suppression of the degradation of SHP1 induced by the ubiquitin–proteasome pathway. These findings also suggested the potential of dietary derived α-MGT in HCC therapy.

Structural Implications of STAT3 and STAT5 SH2 Domain Mutations.

Src Homology 2 (SH2) domains arose within metazoan signaling pathways and are involved in protein regulation of multiple pleiotropic cascades. In signal transducer and activator of transcription (STAT) proteins, SH2 domain interactions are critical for molecular activation and nuclear accumulation of phosphorylated STAT dimers to drive transcription. Sequencing analysis of patient samples has revealed the SH2 domain as a hotspot in the mutational landscape of STAT proteins although the functional impact for the vast majority of these mutations remains poorly characterized. Despite several well resolved structures for SH2 domain-containing proteins, structural data regarding the distinctive STAT-type SH2 domain is limited. Here, we review the unique features of STAT-type SH2 domains in the context of all currently reported STAT3 and STAT5 SH2 domain clinical mutations. The genetic volatility of specific regions in the SH2 domain can result in either activating or deactivating mutations at the same site in the domain, underscoring the delicate evolutionary balance of wild type STAT structural motifs in maintaining precise levels of cellular activity. Understanding the molecular and biophysical impact of these disease-associated mutations can uncover convergent mechanisms of action for mutations localized within the STAT SH2 domain to facilitate the development of targeted therapeutic interventions.

A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) occurs in ∼70% of human cancers, and STAT3 is regarded as one of the most promising targets for cancer therapy. However, specific direct STAT3 inhibitors remain to be developed. Oridonin is an ent-kaurane plant-derived diterpenoid with anti-cancer and anti-inflammatory activities. Here, using an array of cell-based and biochemical approaches, including cell proliferation and apoptosis assays, pulldown and reporter gene assays, site-directed mutagenesis, and molecular dynamics analyses, we report that a thiazole-derived oridonin analogue, CYD0618, potently and directly inhibits STAT3. We found that CYD0618 covalently binds to Cys-542 in STAT3 and suppresses its activity through an allosteric effect, effectively reducing STAT3 dimerization and nuclear translocation, as well as decreasing expression of STAT3-targeted oncogenes. Remarkably, CYD0618 not only strongly inhibited growth of multiple cancer cell lines that harbor constitutive STAT3 activation, but it also suppressed in vivo tumor growth via STAT3 inhibition. Taken together, our findings suggest Cys-542 as a druggable site for selectively inhibiting STAT3 and indicate that CYD0618 represents a promising lead compound for developing therapeutic agents against STAT3-driven diseases.

Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors could promote STAT3 activation through different mechanisms. Here we show that STAT3 is posttranslationally S-palmitoylated at the Src Homology 2 (SH2) domain, promoting its dimerization and transcriptional activation. Fatty acids could directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase (PAT) regulating STAT3. Cytokine stimulation enhances STAT3 palmitoylation by promoting ZDHHC19–STAT3 association mediated by Grb2 SH3 domain. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, impairing cytokine and fatty acid-induced STAT3 activation. Importantly, ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas (LSCCs). High ZDHHC19 levels correlate with high nuclear STAT3 in patient samples. In addition, ZDHHC19 knockout in LSCC cells significantly blocks STAT3 activity, and inhibits fatty acid-induced tumorsphere formation and high-fat diet (HFD)-induced tumorigenesis in vivo. Taken together, we reveal that fatty acid and ZDHHC19-mediated palmitoylation are additional signals regulating STAT3, linking deregulation of palmitoylation to inflammation and cancer.

Development of an HPLC-UV Method for Quantification of Stattic

Background: Signal transducer and activator of transcription 3 (STAT3), an oncogenic protein found constitutively active in many types of human malignancies, is considered to be a promising target for cancer therapy. Objective: In this study for the first time, a simple and accurate method has been developed for the determination of a STAT3 dimerization inhibitor called stattic in aqueous and plasma samples. Methods: A reverse-phase high-performance liquid chromatography (RP-HPLC) composed of C18 column as stationary phase, and the mixture of acetonitrile (60%) and water (40%) as mobile phase with a UV detection at 215 nm were applied for quantification of stattic. The developed method was validated by Food and Drug Administration (FDA) guideline. Results: The method provided a linear range between 1-40 and 2.5-40 µg mL-1 for aqueous and plasma samples, respectively, with a correlation coefficient of 0.999. The accuracy (as recovery) of the developed method was found to be between 95-105% for aqueous medium and 85-115% for plasma samples. The precision (as relative standard deviation) for aqueous and plasma samples was less than 6% and 15%, respectively. The sensitivity of the developed method based on FDA guideline was 1 µg mL-1 for aqueous and 2.5 µg mL-1 for plasma samples. Conclusion: These results show that the established method is a fast and accurate quantification for stattic in aqueous and plasma samples.