Down-regulation Of Signal Transducer And Activator Of Transcription 3 Research Articles

Abstract LB037: E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333

Abstract To elucidate the underlying mechanisms of STAT3 degradation and the suitability of VHL as an E3 ligase partner for targeting STAT3 in cancer using our clinical degrader, KT-333. Signal transducer and activator of transcription 3 (STAT3) is an undrugged oncogenic transcription factor and its role as cancer driver and tumor microenvironment modulator has been validated in a multitude of studies. Based on it’s potential as a target for cancer therapeutics and limitations of prior approaches, we developed KT-333, a first-in-class, potent, highly selective, heterobifunctional STAT3 degrader currently in Phase 1 clinical trials. Here, based on STAT3 degradation by multiple E3s based degraders, structure of STAT3-KT333-VHL and a lysine site-resolved target ubiquitination model, we provide evidence for VHL as the ideal partner E3 for targeting STAT3 in cancer. We successfully identified potent and selective STAT3 degraders using either CRBN or VHL as the E3 ligase, but ultimately preferred VHL based degraders for their increased potency, and consistent STAT3 degradation across multiple cancer lines. KT-333, our VHL based clinical STAT3 degrader, induces a strong ternary complex between STAT3 and VHL. We present a high resolution cryo-electron microscopy based ternary complex structure of STAT3-KT333-VHL which provides mechanistic insights further validating VHL as the E3 of choice for deep, selective and fast STAT3 degradation. Specifically, KT-333 enables favorable protein-protein interactions between STAT3 and VHL, burying a large protein interface in the ternary complex to the extent typically observed only in native protein complexes. Additionally, the residues forming the STAT3-VHL interface are not conserved in other STAT family members corroborating with degradation selectivity observed with KT-333. Next, we leveraged this structure and generated a ubiquitination super-complex model by deploying a combination of biochemical and proteomics techniques and reveal that KT-333-induced ubiquitination occurs by precise targeting of specific lysine resides on STAT3. Furthermore, to decode the mechanism for KT-333 activity against SUDHL1 cell line both in vitro and in a mouse xenograft model, we measured temporal changes at protein level by discovery proteomics. Reduced expression of canonical STAT3 targets and down-regulation of cytokine-mediated signaling and cell cycle signature genes indicated that cell cycle arrest and subsequent apoptosis are the main drivers of efficacy in vitro and in vivo. In summary, we show that our clinical STAT3 degrader, KT-333, is designed and optimally paired with VHL resulting in a very stable ternary complex exhibiting properties of native protein complexes. Our data provides precise structural and molecular mechanisms behind potent, selective, consistent, and fast degradation of STAT3, and downstream mechanism of action observed in vitro and in vivo. Citation Format: Kirti Sharma, Xue Fei, Yatao Shi, Christopher Browne, Dirk Walther, Caroline Daigle, Anand Ramanathan, Richard Miller, Karen Yuan, Kiran Mahasenan, Sean Zhu, Xin Huang, Bin Yang. E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB037.

Pyrimethamine, a STAT3 Inhibitor, Synergizes with Venetoclax and Shows Efficacy in Hypomethylating Agent Resistant MDS and AML

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arise after accumulation of multiple genetic and epigenetic alterations in hematopoietic stem cells (HSCs). Alterations in HSCs promote malignant transformation and give rise to leukemic stem cells (LSCs). Current therapies for MDS and AML, including recently FDA approved agents such as venetoclax (Ven), a Bcl-2 inhibitor, have failed to eliminate LSCs, therefore, molecular targeting of aberrant LSCs may be a potential strategy for developing targeted drugs capable of eliminating LSCs. Studies have shown that aberrant HSCs exhibit multiple disrupted cellular pathways, including overexpression of the signal transducer and activator of transcription 3 (STAT3) (Shastri, 2017). Elevated levels of STAT3 are observed in MDS and AML patient samples. Interestingly, an increased level of STAT3 in MDS and AML patients is predictive of a significantly adverse prognosis, thus supporting a critical role of STAT3 signaling in MDS and AML. We have previously shown that an antisense oligonucleotide inhibitor of STAT3, gets rapidly incorporated into HSCs and induces apoptosis and downregulation of STAT3 when compared to health controls (Shastri, JCI 2018). An initial screen to identify compounds that could target STAT3 revealed that pyrimethamine (PYR), an FDA approved anti-parasitic drug, inhibits STAT3 transcriptional activity at concentrations readily achieved in patients (Nelson, 2011). Therefore, we are further evaluating the efficacy of PYR for treatment of MDS and AML. HMA and Ven resistance is a critical issue in MDS/AML and can occur due to the persistence of relapse associated aberrant stem and progenitor cells that overexpress STAT3. Hence, we are evaluating the efficacy of PYR in a hypomethylating agent (HMA) and venetoclax (Ven) resistant model. Our in vitro studies show that PYR can inhibit viability by inducing apoptosis in multiple leukemic cells within its therapeutic index. Western blot analysis reveals that STAT3 protein is overexpressed in a Ven resistant MOLM13 AML line, and in a HMA resistant cell THP1 AML cell line. In HMA and Ven resistant leukemia cell lines, PYR can significantly decrease proliferation when combined with azacytidine (P= 0.0109) (Fig 1A) or venetoclax (P= 0.0271) treatments. Remarkably, PYR and Ven drug combination show a strong synergistic interaction in HMA resistant cell line (Z score = 14.523) and Ven resistant cell line (Z score = 11.065). An in vivo study to evaluate the efficacy of PYR shows that PYR treatment leads to a significant decrease of blasts counts in a cell-line derived xenograft (CDX) leukemic mouse model (Fig 1B) compared to a PBS control. In addition, primary MDS patient samples treated with PYR show increased myeloid differentiation compared to healthy controls. Our results show that MDS/AML cell lines, including HMA and Ven resistant leukemia cell lines, are sensitive to PYR treatment, in a dose dependent manner within the therapeutic index of STAT3 inhibition (5-10 uM). Interestingly, PYR with Ven drug combination shows significant reduction in cell proliferation and likely synergy. Our current findings support PYR synergy with Ven and the combination as a possible therapeutic option for patients with MDS/AML after prior treatment with hypomethylating agents for MDS and AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetics and Functional Mechanisms of STAT3 Polymorphisms in Human Tuberculosis.

Signal transducer and activator of transcription-3 (STAT3) plays an important role in biological balance. Our and others previous studies implied that STAT3 had a great effect on fast-acting innate immunity against tuberculosis (TB). We hypothesized that stat3 SNP down-regulation of STAT3 leads to a change in susceptibility to TB in humans. To test this hypothesis, we investigated STAT3 SNPs using SNP scan™ technique in a case-control study of TB patients (n = 470) and HC subjects (n = 356), and then conducted functional studies of them using cellular models. We found that SNPs in STAT3 3`-UTR of rs1053004 TT and rs1053005 AA genotypes or T-A haplotype were associated with susceptibility to TB or TB severity. While the TT/AA genotype correlated with the low constitutive expression of stat3 and IL-17A in PBMC, the variant stat3 of rs1053004-rs1053005 T-A haplotype indeed reduced stat3 expression in reporter assays. Interestingly, host PBMC expressing the rs1053005 AA genotype and low constitutive stat3 exhibited the reduced ability to mount fast-acting innate immunity against mycobacterial infection in cellular models. Finally, mechanistic experiments showed that the STAT3 down-regulation broadly depressed STAT3 downstream anti-mycobacterial activities involving VDR-related CAMP pathway as well as IL-32, iNOS and autophagy mechanisms, leading to an enhanced mycobacterial infection. The findings of this study suggest that low constitutive stat3 derived from the TT/AA genotype/T-A haplotype acts to down-regulate STAT3, depressing multiple anti-mycobacterial pathways/mechanisms downstream, which leads to an enhanced mycobacterial infection or TB in high-risk individuals.

Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C–X–C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.

The inhibitory effect of curcumin via fascin suppression through JAK/STAT3 pathway on metastasis and recurrence of ovary cancer cells

BackgroundFascin is an actin-binding protein and highly expressed in ovarian cancer cells. It is associated with metastasis of cancer and may be a useful prognostic factor. Anticancer activity of curcumin is related to its effect on several signaling mechanisms. Although there have been many reports regarding the anticancer properties of curcumin, its inhibitory effects on migration and invasion of ovarian cancer cells, particularly in the context of fascin expression, have not been reported. The purpose of this study was to investigate the effect of curcumin on fascin expression in ovarian cancer cells and to propose a possible mechanism for the anticancer activity of curcumin through reduced fascin expression.MethodsSKOV3, human epithelial ovary cancer cell line, was cultured with curcumin at various dose and duration. The fascin was quantified using cell viability test and Western blot. To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. Attachment assay, migration assay and invasion assay were analyzed to approve the change of cellular invasiveness of ovary cancer after curcumin. To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed.ResultsMTS assays showed that cell viability was different at various concentration of curcumin, and as concentration increased, cell viability tended to decrease. Curcumin appears to suppress fascin expression, even with a minimal concentration and short exposure time. Also, curcumin may suppress fascin expression in ovarian cancer cells through STAT3 downregulation. The attachment assay, migration assay and invasion assay of the ovarian cancer cells exhibited a statistically significant decrease. Immunofluorescence revealed a change of cell shape from a typical form of uninfluenced cells to a more polygonal appearance, with a significant reduction in filopodia formation.ConclusionsCurcumin reduces fascin expression through JAK/STAT3 pathway inhibition, which interferes with the cellular interactions essential for the metastasis and recurrence of ovarian cancer cells. Higher curcumin concentrations and longer exposure times concomitantly decreased fascin expression.

STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is one of the most lethal childhood brain tumors. This tumor is unique because it is detected exclusively in the ventral pons of patients aged between 6 and 7 years, which suggests a developmental nature of its formation. Signal transducer and activator of transcription 3 (STAT3) is a critical molecule for the differentiation of neural stem cells into astrocytes during neurodevelopment. Additionally, STAT3 is associated with oncogenesis and the epithelial-mesenchymal transition (EMT) in various types of tumor. In recent years, several studies have demonstrated the oncogenic role of STAT3 in high-grade gliomas. However, the role of STAT3 in DIPG at the cellular level remains unknown. To assess the possible association between gliogenesis and DIPG, the expression levels of various molecules participating in the differentiation of neural stem cells were compared between normal brain control tissues and DIPG tissues using open public data. All of the screened genes exhibited significantly increased expression in DIPG tissues compared with normal tissues. As STAT3 expression was the most increased, the effect of STAT3 inhibition in a DIPG cell line was assessed via STAT3 short hairpin (sh)RNA transfection and treatment with AG490, a STAT3 inhibitor. Changes in viability, apoptosis, EMT and radiation therapy efficiency were also evaluated. Downregulation of STAT3 resulted in decreased cyclin D1 expression and cell viability, migration and invasion. Additionally, treatment with STAT3 shRNA or AG490 suppressed the EMT phenotype. Finally, when radiation was administered in combination with STAT3 inhibition, the therapeutic efficiency, assessed by cell viability and DNA damage repair, was increased. The present results suggest that STAT3 is a potential therapeutic target in DIPG, especially when combined with radiation therapy.

Curcumin Combined with Thalidomide Reduces Expression of STAT3 and Bcl-xL, Leading to Apoptosis in Acute Myeloid Leukemia Cell Lines.

IntroductionAcute myeloid leukemia (AML) is a type of blood disorder that exhibits uncontrolled growth and reduced ability to undergo apoptosis. Signal transducer and activator of transcription 3 (STAT3) is a family member of transcription factors which promotes carcinogenesis in most human cancers. This effect on AML is accomplished through deregulation of several critical genes, such as B cell lymphoma-extra-large (BCL-XL) which is anti-apoptotic protein. The aim of this study was to evaluate the effect of curcumin (CUR) and thalidomide (THAL) on apoptosis induction and also the alteration of the mRNA expression level of STAT3 and BCL-XL mRNA on AML cell line compounds.MethodsThe growth inhibitory effects of CUR and THAL and their combination were measured by MTT assay in U937 and KG-1 cell lines. The rates of apoptosis induction and cell cycle analysis were measured by concurrent staining with Annexin V and PI. The mRNA expression level of STAT3 and BCL-XL was evaluated by Real-Time PCR.ResultsCUR inhibited proliferation and induced apoptosis in both KG-1 and U937 cells and this effect increased by combination with THAL. The expression level of STAT3 and BCL-XL was significantly down-regulated in KG-1 cells after treatment by CUR and THAL and their combination.ConclusionOverall, our findings suggested that down-regulation of STAT3 and BCL-XL mRNA expression in response to CUR and THAL treatment lead to inhibition of cell growth and induction of apoptosis.

MiR-296-5p inhibits cell invasion and migration of esophageal squamous cell carcinoma by downregulating STAT3 signaling.

Many studies have emphasized the function of microRNA-296 (miR-296) that inhibits tumor formation. To some extent, the role of miR-296 in esophageal squamous cell carcinoma (ESCC) remains misleading. Therefore, the current research was designed to investigate the regulatory mechanisms of miR-296 and signal transducer and activator of transcription 3 (STAT3) in ESCC. The mRNA expression of miR-296-5p and STAT3 in ESCC tissues or cell lines was measured via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The protein level of STAT3 was measured by Western blotting assay. The Luciferase reporter assay was used to verify the binding sites between miR-296-5p and STAT3. The transwell assay was employed to identify cell migration and invasion. Down-regulation of miR-296-5p was detected in ESCC tissues and cell lines (p<0.01). Additionally, miR-296-5p was found to target STAT3 directly. Functionally, up-regulation of miR-296-5p or down-regulation of STAT3 significantly inhibited cell migration and invasion in ESCC. MiR-296-5p inhibited cell invasion and migration in ESCC by downregulating STAT3. The overexpression of miR-296-5p by targeting STAT3 suppressed tumorigenesis of ESCC cells.

Cyanidin-3-glucoside attenuates the angiogenesis of breast cancer via inhibiting STAT3/VEGF pathway.

Angiogenesis plays a pivotal role in breast cancer progression. Cyanidin-3-glucoside (C3G), one of the most widely distributed anthocyanins in edible fruits, shows antioxidative and anti-inflammatory property as well as induction of breast cancer cells apoptosis. However, the effect of C3G on breast cancer-induced angiogenesis remains unknown. In the present study, we found that C3G could attenuate breast cancer-induced angiogenesis via inhibiting VEGF, a key cytokine for angiogenesis, expression and secretion. Furthermore, signal transducer and activator of transcription 3 (STAT3) could transcriptionally activate VEGF, and C3G reduced STAT3 expression at both mRNA and protein level. Subsequently, our data showed that C3G induced miR-124 expression. Moreover, miR-124 could directly repress STAT3 expression, and miR-124-mediated STAT3 down-regulation was responsible for the inhibition of C3G on VEGF and angiogenesis. Taken together, we supplied more evidence to the anti-breast cancer property of C3G.

Induction of apoptosis in prostate cancer by ginsenoside Rh2.

The therapeutic action of ginsenoside Rh2 on several cancer models has been reported. This study aimed to evaluate its apoptotic effect on prostate cancer and the underlying mechanism. Cultured DU145 cells were treated with Rh2 (5 × 10–5 to 1 × 10–4 M), peroxisome proliferator-activated receptor-delta (PPAR-delta) antagonist GSK0660 (1 × 10–6 to 5 × 10–6 M); or small interfering RNA (siRNA) of PPAR-delta. The treatment effects were evaluated with cell viability assay, life/death staining and flow cytometry for apoptosis. Immunostaining was used for reactive oxygen species (ROS) and superoxide detection. Western blot analysis for PPAR-delta and signal transducer and activator of transcription 3 (STAT3) protein expression were performed. The results showed that Rh2 significantly decreased DU145 cell survival and increased cell apoptosis. ROS and superoxide induction, PPAR-delta up-regulation and phosphorylated STAT3 (p-STAT3) down-regulation by Rh2 were demonstrated. GSK0660 partially but significantly inhibited the Rh2-induced apoptosis and restored cell viability. Treatment with siRNA reversed the Rh2-induced apoptosis as well as changes in PPAR-delta and p-STAT3 expression. In conclusion, our findings have demonstrated that ginsenoside Rh2 induces prostate cancer DU145 cells apoptosis through up-regulation of PPAR-delta expression which is associated with p-STAT3 up-regulation and ROS/superoxide induction. Rh2 may be potentially useful in the treatment of prostate cancer.

Opposite effects of non-thermal plasma on cell migration and collagen production in keloid and normal fibroblasts.

Recent progress in the understanding non-thermal plasma (NTP) properties prompted its application in the treatment of various diseases. However, therapeutic effect of NTP on keloid cells has not been reported previously. We sought to investigate the effect of NTP treatment on keloid by comparing cell migration and collagen production of keloid (KFs) and normal fibroblasts (NFs) and determined the regulatory pathways involved. We assessed NTP effects on cell migration in KFs and NFs by the wound healing assay and measured the expression of the epidermal growth factor receptor (EGFR), signal transducer and activator of transcription-3 (STAT3), and collagen by western blot. Expression of the transforming growth factor-β and Type I collagen following NTP treatment was determined by reverse transcription-polymerase chain reaction, immunofluorescence staining, and the Sircol collagen assay. NTP treatment increased cell migration and collagen production of NFs. However, it reduced these parameters in KFs. NTP reduced the expression of EGFR, STAT3, and Type I collagen in KFs but increased their levels in NFs. We revealed that NTP suppressed KF cell migration via down-regulation of EGFR and STAT3 and reduced collagen production via supressing transforming growth factor-β. Our data suggest that NTP may be a new therapeutic strategy for keloids.

Effects of daphnetin combined with Bcl2‑siRNA on antiapoptotic genes in synovial fibroblasts of rats with collagen‑induced arthritis.

The aim of the present study was to investigate the effects of daphnetin combined with B cell lymphoma 2 (Bcl2)‑targeted small interfering (si)RNA (si‑Bcl2) on antiapoptotic genes in fibroblast‑like synoviocytes (FLS) in rats with collagen II‑induced arthritis (CIA). The roles of si‑Bcl2 and daphnetin were determined by measuring the expression levels of Bcl2. Protein and mRNA expression levels of Bcl2 in FLS were determined by flow cytometry and reverse transcription‑quantitative polymerase chain reaction. Apoptosis of FLS was also determined by flow cytometry. It was revealed that treatment with si‑Bcl2 or daphnetin alone resulted in downregulation of Bcl2 mRNA and protein expression. In addition, the mRNA expression levels of the signal transducer and activator of transcription 3 (STAT3), which transcriptionally regulates the activity of mitochondria, were reduced. The combination of si‑Bcl2 and daphnetin exhibited an enhanced effect on rheumatoid arthritis FLS (RAFLS), in which the apoptotic rate was significantly higher than either treatment alone. The results suggested that si‑Bcl combined with daphnetin may have an enhanced effect in promoting apoptosis of RAFLS derived from CIA rats, and a possible underlying molecular mechanism may function through the downregulation of Bcl2 expression and STAT3, which is located upstream of Bcl2 in the mitochondrial apoptotic pathway. The results of the present study are expected to provide theoretical and experimental basis for the treatment of RA and the medicinal development of daphnetin combined siRNA.

Preparation and characterization of lipid nanoparticle/pDNA complexes for STAT3 downregulation and overcoming chemotherapy resistance in lung cancer cells

Developments in the field of molecular oncology have revealed that resistance to chemotherapeutics is acqured through several mechanisms including overexpression of common oncogenic proteins. Signal Transducer and Activator of Transcription 3 (STAT3) is one of these oncogenes that is overexpressed in many cancer types. RNA interference (RNAi) is proven powerful tool for downregulating STAT3, allowing re-sensitization of resistant cancer cells. However, delivery of RNA interference-mediating molecules for STAT3 downregulation in lung cancer cells is limited to a small number of studies most of which employ commercially available transfection kits. The aim of this study was to develop and evaluate cationic solid lipid nanoparticles for delivery of RNAi-mediating plasmid DNA in order to down regulate STAT3 in cisplatin resistant lung cancer cells. We focused on obtaining cSLN:plasmid DNA complexes with size below or equal to 100nm, and a positive zeta potential. Two successful candidate cSLN:plasmid DNA complexes (K2 and K3) were selected for in vitro tests and cell culture studies. These formulations have particle sizes of 98 and 93nm, and zeta potential values of 10.5 and 8.9mV, respectively. Plasmid DNA in these complexes was protected against DNaseI and serum-mediated degradation. Substantial part of DNA retained its supercoiled and circular conformation. TEM images showed nearly spherical complex structure. Both formulations reduced STAT3 expression by approx. 5-fold in cisplatin resistant Calu1 cell line and increased the sensitivity of cells to cisplatin.

The promotion of the transformation of quiescent gastric cancer stem cells by IL-17 and the underlying mechanisms

Postoperative recurrence and metastasis have crucial roles in the poor prognosis of gastric cancer patients. Previous studies have indicated that gastric cancer originates from cancer stem cells (CSCs), and some investigators have found that a particular subset of CSCs possesses higher metastatic capacity. However, the specific mechanism remains uncertain. In the present study, we aimed to explore the biological functions of the inflammatory cytokine interleukin-17 (IL-17) in gastric cancer metastasis and the distinct IL-17-induced transformation of quiescent gastric CSCs. Our results showed that invasive gastric CSCs were CD26+ and CXCR4+ and were closely associated with increased metastatic ability. The quiescent gastric CSCs, which were CD26− and CXCR4−, were exposed to appropriate concentrations of IL-17; this resulted in the decreased expression of E-cadherin and the increased expression of vimentin and N-cadherin. In addition, the upregulation of IL-17 both in vitro and in vivo resulted in a significant induction of invasion, migration and tumor formation ability in gastric CSCs compared with the control group, which was not treated with IL-17. Further experiments indicated that the activation of the downstream phosphorylated signal transducer and activator of transcription 3 (STAT3) transcription factor pathway was facilitated by IL-17. On the contrary, the downregulation of STAT3 by the specific inhibitor Stattic significantly reversed the IL-17-induced epithelial–mesenchymal transition (EMT)-associated properties of quiescent gastric CSCs. Moreover, tumorigenesis and metastasis were suppressed. Taken together, we suggest that IL-17 is positively correlated with the transformation of quiescent gastric CSCs into invasive gastric CSCs and that targeting IL-17 may emerge as a possible novel therapeutic strategy for gastric cancer.

Investigation of JAKs/STAT-3 in lipopolysaccharide-induced intestinal epithelial cells.

Janus-activated kinase (JAKs)-signal transducer and activator of transcription 3 (STAT-3) signalling play critical roles in immunoregulation and immunopathology, which involve inflammatory responses and enteritis. JAK phosphorylates STAT-3 in response to stimulation by cytokines or growth factors, and then activates or represses the gene expression. STAT-3 is activated persistently in cancer cells and contributes to the malignant progression of various types of cancer and inflammation. To elucidate the different roles of JAKs in the activation of STAT-3, the lipopolysaccharide-induced primary intestinal epithelial cell (IEC) acute inflammatory model was established. Small interference RNAs (siRNAs) were then employed to attenuate the expression levels of JAKs. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qRT-PCR) revealed that JAK mRNA levels were reduced efficiently by JAK-specific siRNAs. Under the IEC inflammatory model transfected with si-JAK, which equates to effective silencing, qRT-PCR and Western blot assays, suggested that knockdowns of JAK attenuated the JAK-induced down-regulation of STAT-3 at the mRNA or protein levels. In particular, JAK1 played a key role, which was consistent with the RNA-Seq results. Subsequently, the expression levels of proinflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α were down-regulated in the IEC inflammatory model transfected with si-JAK1. JAK1 appears as a direct activator for STAT-3, whereas treatments targeting JAK1 repressed STAT-3 sufficiently pathways in the IEC inflammatory model. Therefore, the control of JAK1 using siRNAs has the potential to be an effective strategy against enteritis.

Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization.

Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo.

Blockage of STAT3 Signaling Pathway by Morusin Induces Apoptosis and Inhibits Invasion in Human Pancreatic Tumor Cells.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis. Here, we investigated the role of morusin, the major prenylflavonoid, isolated from Chinese herbal medicine in abrogating the constitutive STAT3 activation in human pancreatic tumor cells. The effect of morusin on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was examined. Morusin specifically inhibited constitutive STAT3 activation both at tyrosine residue 705 and serine residue 727 in 4 pancreatic tumor cells. The inhibition of STAT3 was mediated through the suppression of activation of upstream JAK1, JAK2, and c-Src kinases. Morusin led to the accumulation of the cells in different phases of the cell cycle and caused induction of apoptosis and loss of mitochondrial membrane potential. Morusin downregulated the expression of various STAT3-regulated gene products; this correlated with induction of caspase-3 activation and anti-invasive effects. Treatment with the protein tyrosine phosphatase inhibitor pervanadate reversed the morusin-induced downregulation of STAT3, thereby suggesting the involvement of a protein tyrosine phosphatase. Morusin is a novel blocker of STAT3 activation and thus may have potential in negative regulation of growth and metastasis of pancreatic tumor cells.

Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin-proteasome pathway.

We investigated the underlying mechanism for the potent proapoptotic effect of paeoniflorin (PF) on human glioma cells in vitro, focusing on signal transducer and activator of transcription 3 (STAT3) signaling. Significant time- and dose-dependent apoptosis and inhibition of proliferation were observed in PF-treated U87 and U251 glioma cells. Expression of STAT3, its active form phosphorylated STAT3 (p-STAT3), and several downstream molecules, including HIAP, Bcl-2, cyclin D1, and Survivin, were significantly downregulated upon PF treatment. Overexpression of STAT3 induced resistance to PF, suggesting that STAT3 was a critical target of PF. Interestingly, rapid downregulation of STAT3 was consistent with its accelerated degradation, but not with its dephosphorylation or transcriptional modulation. Using specific inhibitors, we demonstrated that the prodegradation effect of PF on STAT3 was mainly through the ubiquitin–proteasome pathway rather than via lysosomal degradation. These findings indicated that PF-induced growth suppression and apoptosis in human glioma cells through the proteasome-dependent degradation of STAT3.

Gallstone and alcoholic pancreatitis? Evaluation of Atlanta and determinant based classification scores

We have previously demonstrated that pterostilbene, a compound in blueberries, exerts antiproliferative effects against pancreatic adenocarcinoma. However, little is known about the anti-inflammatory effects of pterostilbene in pancreatitis. Therefore, the aim of this study was to evaluate the effect of pterostilbene on inflammatory markers in an in vitro pancreatitis model. We hypothesized that pterostilbene would ameliorate the immediate inflammatory response in tumor necrosis factor alpha (TNF-α)–induced pancreatitis through downregulation of signal transducer and activator of transcription 3 (STAT3) and inhibition of TNF-α–induced secretion of lipase and proinflammatory cytokines interleukins (ILs) 1β and 6.AR42J acinar cells were pretreated with TNF-α to induce pancreatitis followed by 25 and 50 μM pterostilbene for 15 and 30 min. Secretion of lipase was quantified using a lipase assay and used as a marker of TNF-α–induced pancreatitis. Detection of STAT3, IL-1β, and IL-6 was performed using enzyme-linked immunosorbent assay. Analysis of variance and Tukey post hoc analysis were used for statistical analysis.TNF-α increased the secretion of lipase, IL-1β, and IL-6. Pterostilbene treatment inhibited TNF-α–induced secretion of lipase (P < 0.01 and P < 0.001), IL-1β (P < 0.05), and IL-6 (P < 0.05 and P < 0.01). Inhibition of STAT3 by pterostilbene occurred with treatment doses of 25 and 50 μM (P < 0.001 and P < 0.01).The dietary compound pterostilbene exerts an anti-inflammatory effect in pancreatitis through downregulation of STAT3 and decreased the secretion of lipase, IL-1β, and IL-6. Pterostilbene's amelioration of pancreatitis in vitro makes it an advantageous anti-inflammatory agent. Further studies are necessary to determine pterostilbene's role as a protective or therapeutic agent in pancreatitis.

Farnesol inhibits tumor growth and enhances the anticancer effects of bortezomib in multiple myeloma xenograft mouse model through the modulation of STAT3 signaling pathway

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is frequently observed in multiple myeloma (MM) cancer and can upregulate the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. The effect of farnesol (FOH) on STAT3 activation, associated protein kinases, its regulated gene products, cellular proliferation, and apoptosis was examined. The in vivo effect of FOH on the growth of human MM xenograft tumors alone and in combination with bortezomib (Bor) in athymic nu/nu female mice was also investigated. We found that FOH suppressed both constitutive and inducible STAT3 activation at Tyr705 in MM cells. The suppression of STAT3 was mediated through the inhibition of activation of upstream JAK1, JAK2, and c-Src kinases. Also, treatment with the protein tyrosine phosphatase (PTP) inhibitor, pervanadate treatment reversed the FOH-induced down-regulation of STAT3, possibly indicating the involvement of a PTP. Indeed, we found that FOH treatment induces the increased expression of SHP-2 protein and knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of FOH to inhibit STAT3 activation. FOH inhibited proliferation and significantly potentiated the apoptotic effects of bortezomib (Bor) in U266 cells. When administered intraperitoneally, FOH enhanced Bor-induced growth suppression of human MM xenograft tumors in athymic nu/nu female mice. Our results suggest that FOH is a novel blocker of STAT3 signaling pathway and exerts both anti-proliferative and apoptotic activities in MM in vitro and in vivo.