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

Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade.

Because of the essential role of signal transducer and activator of transcription 3 (STAT3) in proliferation, anti-apoptosis, and chemoresistance of multiple myeloma (MM), we investigated whether icariin, a prenylated flavonol glycoside, inhibits both constitutive and inducible STAT3 activation in human myeloma cell lines. We noted that icariin could block constitutive STAT3 phosphorylation as well as its nuclear translocation and DNA binding ability in U266 cells. Icariin also suppressed IL-6-induced STAT3 activation through the inhibition of upstream kinases (Janus activated kinase-1 and -2, and c-Src). We found that icariin downregulated the protein expression of STAT3 downstream target gene products such as Bcl-2, Bcl-xl, survivin, IAP-1/2, COX-2, VEGF, and matrix metallopeptidase 9 (MMP-9) in a concentration-dependent manner. Moreover, this flavonoid also exhibited the capacity to significantly induce apoptosis and suppress proliferation of MM cells. Interestingly, this agent also significantly potentiated the apoptotic effects of bortezomib through the suppression of STAT3 activation in MM cells. Altogether, our data indicates that the potential application of icariin as a STAT3 blocker in myeloma therapy.

Targeting STAT3 by HO3867 induces apoptosis in ovarian clear cell carcinoma.

Advanced ovarian clear cell carcinoma (OCCC) carries a very poor prognosis in large part secondary to the extremely high rate of resistance to standard platinum and taxane chemotherapy. Signal transducer and activator of transcription 3(STAT3) expression and activation has been shown to regulate tumor progression in various human cancers, though has not been well studied in OCCC. Preliminary work in our lab has demonstrated constitutive activation of STAT3 (pSTAT3Tyr705 or pSTAT3727) in OCCC cell lines as well as human OCCC tumor tissue samples. Significantly, pSTAT3 is expressed in the absence of other forms of activated STAT (pSTAT1, 2, 6). Therefore, this work was planned to investigate the role of STAT3 and examine the efficacy of a novel anti-cancer compound -HO-3867, which is an inhibitor of STAT3, using known OCCC cell lines. Results demonstrate that treatment with HO-3867 decreased expression of pSTAT3 Tyr705 as well pSTAT3 Ser727, while total STAT3 remained constant. STAT3 overexpression increased the migration capability in OVTOKO cells in vitro and led to an increased tumor size when injected in vivo. The inhibitory effect of HO-3867 on cell proliferation and cell survival was accompanied by increased apoptosis, within 24 h post treatment. Treatment with HO-3867 resulted in a decrease in Bcl-2 and increase of cleavage of caspase 3, caspase 7, and PARP, confirming induction of apoptosis after treatment with HO-3867. In addition, HO-3867 significantly inhibited formation of human umbilical vein endothelial cells capillary-like structures and invasion at both 5 and 10 µM concentrations. STAT3 expression plays an important role in the spread of OCCC in vitro as well as in vivo. Thus, we can exploit the STAT3 pathway for targeted drug therapy. Inhibition of pSTAT3 using HO-3867in OCCC cell lines appears to be a promising therapy. This is of utmost importance given the poor response of OCCC to standard chemotherapy regimens.

Geranylnaringenin (CG902) inhibits constitutive and inducible STAT3 activation through the activation of SHP-2 tyrosine phosphatase

The roles and significance of signal transducer and activator of transcription 3 (STAT3) in human cancers have been extensively studied and STAT3 is a promising therapeutic target for cancer drug discovery. During the screening of natural products to identify STAT3 inhibitors, we identified geranylnaringenin (CG902), which decreased luciferase activity in a dose-dependent manner. CG902 specifically inhibited STAT3 phosphorylation at Tyr-705 in DU145 prostate cancer cells and decreased the expression levels of STAT3 target genes, such as cyclin D1, cyclin A, and survivin. Notably, the knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of CG902 to inhibit STAT3 activation and CG902 activated the phosphatase activity of SHP-2 through direct interaction with SHP-2 and induced the phosphorylation of SHP-2. The interactions between CG902 and SHP-2 were confirmed by pull-down assay using biotinylated CG902. The interactions were also further validated by the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). The inhibitory effect of CG902 on cell growth was confirmed using the DU145 mouse xenograft model. We propose that CG902 inhibits STAT3 activity through a mechanism that involves the interactions between CG902 and SHP-2, and the phosphorylation of SHP-2, which leads to SHP-2 activation in DU145 cells. CG902 is the first compound to regulate STAT3 activity via the modulation of SHP-2 activity, and our results suggest that CG902 is a novel inhibitor of the STAT3 pathway and an activator of SHP-2, and may be a useful lead molecule for the development of a therapeutic STAT3 inhibitor.

Ginkgolic Acid C 17:1, Derived from Ginkgo biloba Leaves, Suppresses Constitutive and Inducible STAT3 Activation through Induction of PTEN and SHP-1 Tyrosine Phosphatase.

Ginkgolic acid C 17:1 (GAC 17:1) extracted from Ginkgo biloba leaves, has been previously reported to exhibit diverse antitumor effect(s) through modulation of several molecular targets in tumor cells, however the detailed mechanism(s) of its actions still remains to be elucidated. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that regulates various critical functions involved in progression of diverse hematological malignancies, including multiple myeloma, therefore attenuating STAT3 activation may have a potential in cancer therapy. We determined the anti-tumor mechanism of GAC 17:1 with respect to its effect on STAT3 signaling pathway in multiple myeloma cell lines. We found that GAC 17:1 can inhibit constitutive activation of STAT3 through the abrogation of upstream JAK2, Src but not of JAK1 kinases in U266 cells and also found that GAC can suppress IL-6-induced STAT3 phosphorylation in MM.1S cells. Treatment of protein tyrosine phosphatase (PTP) inhibitor blocked suppression of STAT3 phosphorylation by GAC 17:1, thereby indicating a critical role for a PTP. We also demonstrate that GAC 17:1 can induce the substantial expression of PTEN and SHP-1 at both protein and mRNA level. Further, deletion of PTEN and SHP-1 genes by siRNA can repress the induction of PTEN and SHP-1, as well as abolished the inhibitory effect of drug on STAT3 phosphorylation. GAC 17:1 down-regulated the expression of STAT3 regulated gene products and induced apoptosis of tumor cells. Overall, GAC 17:1 was found to abrogate STAT3 signaling pathway and thus exert its anticancer effects against multiple myeloma cells.

Simvastatin-induced cell cycle arrest through inhibition of STAT3/SKP2 axis and activation of AMPK to promote p27 and p21 accumulation in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is characterized by a poor prognosis and is one of the leading causes of cancer-related death worldwide. Simvastatin, an HMG-CoA reductase inhibitor, which decreases cholesterol synthesis by inhibiting mevalonate pathways and is widely used to treat cardiovascular diseases. Simvastatin exhibits anticancer effects against several malignancies. However, the molecular mechanisms underlying the anticancer effects of simvastatin on HCC are still not well understood. In this study, we demonstrated simvastatin-induced G0/G1 arrest by inducing p21 and p27 accumulation in HepG2 and Hep3B cells. Simvastatin also promoted AMP-activated protein kinase (AMPK) activation, which induced p21 upregulation by increasing its transcription. Consistent with this finding, we found genetic silencing of AMPK reduced p21 expression; however, AMPK silencing had no effect on p27 expression in HCC cells. Simvastatin decreased Skp2 expression at the transcriptional level, which resulted in p27 accumulation by preventing proteasomal degradation, an effect mediated by signal transducer and activator of transcription 3 (STAT3) inhibition. Constitutive STAT3 activation maintained high-level Skp2 expression and lower level p27 expression and significantly prevented G0/G1 arrest in simvastatin-treated HCC cells. Mevalonate decreased simvastatin-induced AMPK activation and rescued phospho-STAT3 and Skp2 expression in HCC cells, which resulted in the prevention of G0/G1 arrest through inhibition of p21 and p27 accumulation. Moreover, simvastatin significantly decreased tumor growth in HepG2 xenograft mice. Consistently, we found that simvastatin also increased p21 and p27 expression in tumor sections by reducing Skp2 expression and inducing AMPK activation and STAT3 suppression in the same tumor tissues. Taken together, these findings are demonstrative of the existence of a novel pathway in which simvastatin induces G0/G1 arrest by upregulating p21 and p27 by activating AMPK and inhibiting the STAT3–Skp2 axis, respectively. The results identify novel targets that explain the beneficial anticancer effects of simvastatin treatment on HCC in vitro and in vivo.

Abstract A12: Quercetin inhibits multiple pathways involved in interleukin 6 secretion from human lung fibroblasts and activity in bronchial epithelial cell transformation induced by benzo[a]pyrene diol epoxide

Abstract The interaction between epithelial and stromal cells through soluble factors such as cytokines plays an important role in carcinogenesis. Breaking this cancer-promoting interaction poses an opportunity for cancer prevention. The tumor-promoting function of interleukin 6 (IL-6) has been documented; however, the underlying mechanisms of this function in lung carcinogenesis are not well elucidated. Here, we show that benzo[a]pyrene diol epoxide (BPDE, the active metabolite of cigarette smoke carcinogen benzo[a]pyrene)-induced human bronchial epithelial cell (HBEC) transformation was enhanced by IL-6 in vitro. The carcinogen/IL-6-transformed cells exhibited higher expression of STAT3 (signal transducer and activator of transcription 3) when compared with cells transformed by BPDE alone. Constitutive STAT3 activation drove cell proliferation and survival through anti-apoptosis gene expression. We further show that quercetin, a dietary compound having preventive properties for lung cancer, decreased BPDE-stimulated IL-6 secretion from human lung fibroblasts through inhibition of the NF-κB and ERK pathways. The inhibition was accomplished at clinically achievable concentrations of the compound. Finally, quercetin blocked IL-6-induced STAT3 activation in HBECs, and IL-6 enhancement of HBEC transformation by BPDE was abolished by quercetin treatment. Altogether, our data reveal novel mechanisms for IL-6 in lung carcinogenesis and for the preventive role of quercetin in the process. Citation Format: Wenshu Chen, Mabel T. Padilla, Xuling Xu, Dhimant Desai, Jacek Krzeminski, Shantu Amin, Yong Lin. Quercetin inhibits multiple pathways involved in interleukin 6 secretion from human lung fibroblasts and activity in bronchial epithelial cell transformation induced by benzo[a]pyrene diol epoxide. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A12.

A novel synthetic Asiatic acid derivative induces apoptosis and inhibits proliferation and mobility of gastric cancer cells by suppressing STAT3 signaling pathway.

Activation of the transcription factor, signal transducers and activators of transcription 3 (STAT3), has been linked to the proliferation and migration of a variety of human cancer cells. These actions occur via the upregulation or downregulation of cell survival and tumor suppressor genes, respectively. Importantly, agents that can suppress STAT3 activation have the potential for use in the prevention and treatment of various cancers. In this study, an Asiatic acid (AA) derivative, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe), is reported to dose dependently suppress constitutive STAT3 activation in gastric cancer cells. This inhibition was mediated by blockade of Janus-activated kinase 2. Additionally, AA-PMe regulated the expression of STAT3-modulated gene products, including cyclin D1, Bax, Bcl-2, c-Myc, and matrix metalloproteinase (MMP)-2 and MMP-9. Finally, transfection with both a STAT3 mimic and an inhibitor reversed the AA-PMe-driven modulation of STAT3 downstream gene products. Overall, these results suggest that AA-PMe is a novel blocker of STAT3 activation and has the potential for the prevention and treatment of gastric cancer.

Signal transducer and activator of transcription 3 as a therapeutic target for cancer and the tumor microenvironment.

Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that modulates the transcription of a variety of genes to regulate important biological functions, including cell proliferation, differentiation, survival, angiogenesis, and immune response. Constitutive activation of STAT3 is important in oncogenic signaling and occurs at high frequency in human cancers, including diverse solid tumors and hematologic malignancies. Moreover, it is associated with a poor prognosis. The tumor microenvironment has recently been recognized as a key condition for cancer progression, invasion, angiogenesis, metastasis, and drug resistance by activation of STAT3 signaling. Therefore, understanding the biology associated with STAT3-mediated signaling cascades in the tumor microenvironment may offer the therapeutic potential to treat human cancers. This review presents an overview of the critical roles of STAT3 in the tumor microenvironment related to cancer biology and discusses recent advancements in the development of anticancer drugs that therapeutically inhibit STAT3 signaling cascades.

Abstract 996: STAT3 modulates HOTAIR transcription to influence HNSCC growth in vitro and in vivo

Abstract Background Squamous cell carcinoma is the most common cancer type of head and neck (HNSCC) with poor prognosis. Signal transducer and activator of transcription 3 (STAT3) activation is involved in HNSCC carcinogenesis as a transcript factor. A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. HOTAIR, lncRNA Hox transcript antisense intergenic RNA, has been characterized as a novel hall marker to predict poor prognosis in HNSCC. HOTAIR regulates downstream target gene transcription by recruiting polycomb repressive complex 2 (PRC2) to the cell nucleus and triggers triple methylation of H3K27. Methods RNA sequencing, qPCR and CHIP assay were used to determine HOTAIR is a down-stream gene of STAT3. MTT and western blot were employed to examine the regulatory mechanism between STAT3 and HOTAIR in HNSCC cancers in vitro. Additionally, a cell derived xenograft tumor model was used to further validate the anti tumor effect of targeting STAT3/HOTAIR axis in vivo. Results STAT3 depleted HNSCC cell showed decreased HOTAIR expression by qPCR assay. CHIP assay indicated STAT3 bind to the promoter region of HOTAIR encoding gene. Combined inhibition of STAT3 and HOTAIR significantly inhibited cell proliferation, sensitive to cisplatin in HNSCC. Up-regulating HOTAIR partially compensated anti-tumor effect of targeting STAT3 in HNSCC. Conclusion Our data suggested that HOTAIR overexpression is depended on constitutive STAT3 activation in HNSCC and targeting STAT3/HOTAIR axis showed significant therapeutic potential in vitro and in vivo. Citation Format: Xuan Zhou. STAT3 modulates HOTAIR transcription to influence HNSCC growth in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 996.

Abstract 4732: A novel analog of Leonurine inhibits melanoma growth and survival through STAT3 signaling pathways

Abstract Previous studies have shown that a major alkaloid of Leonurus japonicus Houtt, Leonurine, displays a variety of biological effects including inhibition of HMG-CoA reductase, kidney fibrosis, and osteoporosis. However, the full potential of this alkaloid as a cancer therapeutics has not been explored, primarily due to the lack of potency. A limited reports however, do suggest that Leonurine induces apoptosis in lung and liver cancer cells. We hypothesized that this natural product's structure can be optimized to create a more potent and drug-like molecule. A recent structure-activity relationship (SAR) study in our laboratory based on Leonurine structure has led to the identification of three novel analogs which were >100 times potent than Leonurine in killing solid cancer cells. The aim of the current study is to determine whether the novel analogs of Leonurine exhibit anti-proliferative effects against tumor cells through suppression of the signal transducer and activator of transcription 3 (STAT3) activation pathway. We investigated the effects of Leonurine analogs on constitutive STAT3 activation, modulation of tyrosine kinases and phosphatases in STAT3 activation, STAT3-regulated gene products, and growth modulation of tumor cells. We found that these compounds inhibited constitutive STAT3 activation in metastatic melanoma cells. The suppression was mediated through the inhibition of activation of the upstream kinases Janus-like kinase (JAK) 1, and JAK2. Leonurine derivatives down-regulated the expression of STAT3-regulated gene products such as survivin, Bcl-xL, Bcl-2, cyclin D1, and Mcl-1 leading to the suppression of proliferation and induction of apoptosis. Moreover, we found that these Leonurine analogs significantly inhibited the proliferation of variety of cells derived from solid tumors including breast, pancreatic, prostate, and colon cancer. Overall, these results suggest that these new compounds are novel blockers of STAT3 activation and thus may have potential to suppress melanoma cell proliferation and chemoresistance. Citation Format: Manoj K. Pandey, Jacek Krzeminski, Deepkamal Karelia, Arun K. Sharma, Shantu G. Amin. A novel analog of Leonurine inhibits melanoma growth and survival through STAT3 signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4732.

3-Formylchromone inhibits proliferation and induces apoptosis of multiple myeloma cells by abrogating STAT3 signaling through the induction of PIAS3

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently observed and closely linked with proliferation, survival, metastasis and angiogenesis of various cancer cells, and thus its inhibition can be considered a potential therapeutic strategy. We found that 3-formylchromone (3FC) inhibited both constitutive and inducible STAT3 activation in multiple myeloma (MM) cells. Besides the inhibition of STAT3 phosphorylation, 3FC also abrogated constitutive activity and nuclear translocation of STAT3. This suppression was mediated through the inhibition of phosphorylation of Janus-activated kinase (JAK) 1/2 and Src. Furthermore, 3FC induced the expression of the protein inhibitors of activated STAT3 (PIAS3), and gene silencing of the PIAS3 by small interfering RNA abolished the ability of 3FC to inhibit STAT3 activation, suggesting a critical role for PIAS3 in the action of 3FC. 3FC also downregulated the expression of STAT3-regulated gene products such as Bcl-2, Bcl-xl, Mcl-1, Survivin, inhibitor of apoptosis protein-1 (IAP-1), Cyclin D1, cyclooxygenase-2 (COX-2), and matrix metalloproteinases-9 (MMP-9) in MM cells. This correlated with induction of substantial apoptosis as indicated by an increase in the sub-G1 cell population and caspase-3 induced poly ADP ribose polymerase (PARP) cleavage. Overall, these results suggest that 3FC is a novel blocker of STAT3 activation pathway thus may have a potential in therapy of MM and other cancers.

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.

Preferential expression of functional IL-17R in glioma stem cells: potential role in self-renewal.

Gliomas are the most common primary brain tumor and one of the most lethal solid tumors. Mechanistic studies into identification of novel biomarkers are needed to develop new therapeutic strategies for this deadly disease. The objective for this study was to explore the potential direct impact of IL-17−IL-17R interaction in gliomas. Immunohistochemistry and flow cytometry analysis of 12 tumor samples obtained from patients with high grade gliomas revealed that a considerable population (2–19%) of cells in all malignant gliomas expressed IL-17RA, with remarkable co-expression of the glioma stem cell (GSC) markers CD133, Nestin, and Sox2. IL-17 enhanced the self-renewal of GSCs as determined by proliferation and Matrigel® colony assays. IL-17 also induced cytokine/chemokine (IL-6, IL-8, interferon-γ-inducible protein [IP-10], and monocyte chemoattractant protein-1 [MCP-1]) secretion in GSCs, which were differentially blocked by antibodies against IL-17R and IL-6R. Western blot analysis showed that IL-17 modulated the activity of signal transducer and activator of transcription 3 (STAT3), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), glycogen synthase kinase-3β (GSK-3β) and β-catenin in GSCs. While IL-17R-mediated secretion of IL-6 and IL-8 were significantly blocked by inhibitors of NF-κB and STAT3; NF-κB inhibitor was more potent than STAT3 inhibitor in blocking IL-17-induced MCP-1 secretion. Overall, our results suggest that IL-17–IL-17R interaction in GSCs induces an autocrine/paracrine cytokine feedback loop, which may provide an important signaling component for maintenance/self-renewal of GSCs via constitutive activation of both NF-κB and STAT3. The results also strongly implicate IL-17R as an important functional biomarker for therapeutic targeting of GSCs.

Quercetin inhibits multiple pathways involved in interleukin 6 secretion from human lung fibroblasts and activity in bronchial epithelial cell transformation induced by benzo[a]pyrene diol epoxide.

The interaction between epithelial and stromal cells through soluble factors such as cytokines plays an important role in carcinogenesis. Breaking this cancer-promoting interaction poses an opportunity for cancer prevention. The tumor-promoting function of interleukin 6 (IL-6) has been documented; however, the underlying mechanisms of this function in lung carcinogenesis are not well elucidated. Here, we show that benzo[a]pyrene diol epoxide (BPDE, the active metabolite of cigarette smoke carcinogen benzo[a]pyrene)-induced human bronchial epithelial cell (HBEC) transformation was enhanced by IL-6 in vitro. The carcinogen/IL-6-transformed cells exhibited higher expression of STAT3 (signal transducer and activator of transcription 3) when compared with cells transformed by BPDE alone. Constitutive STAT3 activation drove cell proliferation and survival through anti-apoptosis gene expression. We further show that quercetin, a dietary compound having preventive properties for lung cancer, decreased BPDE-stimulated IL-6 secretion from human lung fibroblasts through inhibition of the NF-κB and ERK pathways. The inhibition was accomplished at clinically achievable concentrations of the compound. Finally, quercetin blocked IL-6-induced STAT3 activation in HBECs, and IL-6 enhancement of HBEC transformation by BPDE was abolished by quercetin treatment. Altogether, our data reveal novel mechanisms for IL-6 in lung carcinogenesis and for the preventive role of quercetin in the process. © 2015 Wiley Periodicals, Inc.

Abstract P5-07-01: Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity

Abstract Since the neoplastic phenotype of a cell is largely driven by its gene expression patterns, increasing attention is focused on transcription factors that regulate critical mediators of tumor formation and metastatic progression like the oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). Whereas normal cells have transient activation of STAT3 due to tight control by negative regulators, cancer cells frequently have inappropriate constitutive activation of STAT3 which drives increased expression of genes involved in tumorigenesis. However, little is known about proteins that interact with STAT3 to modulate its function. To identify novel STAT3-interacting proteins, we performed liquid chromatography tandem mass spectrometry-based profiling of STAT3-containing complexes immunoprecipitated from the triple-negative breast cancer cell lines MDA-MB-468 and SUM159PT, which have constitutively active STAT3. We identified granulin (GRN) as a novel STAT3-interacting protein and validated the STAT3-GRN interaction in breast cancer cells by co-immunoprecipitation. To investigate the functional effect of GRN on STAT3 activity, we silenced GRN using small interfering RNA. We found that GRN was necessary for constitutive and maximal cytokine-induced STAT3 transcriptional activity in breast cancer cells. GRN modulated cytokine-induced STAT3 function by enhancing STAT3 DNA binding and increasing the time-integrated amount of STAT3 activation and nuclear translocation. Silencing GRN mirrored the effect of silencing STAT3 on reducing the viability, clonogenesis, and migratory capacity of triple-negative breast cancer cells. Furthermore, GRN mRNA levels were significantly and positively correlated with STAT3 gene expression signatures indicative of STAT3 activation as well as with reduced overall survival in breast cancer patients. These studies used a proteomics approach to identify GRN as a novel STAT3 interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. Citation Format: Jennifer E Yeh, Simion Kreimer, Sarah R Walker, Andrea Richardson, Alexander R Ivanov, David A Frank. Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-01.

Antitumorigenic effect of plumbagin by induction of SH2-containing protein tyrosine phosphatase 1 in human gastric cancer cells.

A recent study reported that plumbagin downregulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various antitumor effects in multiple myeloma cells. We aimed in this in vitro study to demonstrate the inhibition of JAK2/STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in the MKN-28 gastric cancer cell line. We performed western blot analysis to measure SHP1, phosphor-JAK2/STAT3 level, and observed that plumbagin induced SHP1 expression and simultaneously downregulated phosphor-JAK2/STAT3 in MKN-28 cells, with negative SHP1 expression. This effect was consistent when JAK2/STAT3 signaling was activated by interleukin-6 (IL-6), and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, vascular endothelial growth factor (VEGF)-1, Bcl-xL, survivin and matrix metalloproteinase-9 (MMP-9), known target products of STAT3 activation in gastric carcinogenesis by reverse transcription-polymerase chain reaction (RT-PCR). Several functional studies such as water soluble tetrazolium salt-1 (WST-1) assay, wound closure assay, Matrigel invasion assay and Annexin V assay were also performed, and we validated the functional effect of plumbagin for inhibition of cell proliferation, migration and invasion, and induction of apoptosis. Collectively, our findings suggest that plumbagin is a potential regulator of cellular growth, migration, invasion and apoptosis by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

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.

Antitumorigenic effect of plumbagin by induction of SHP1 in human gastric carcinoma cell lines.

74 Background: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-drived natural agent extracted from the root of Plumbago zeylanic. A recent study reported that plumbagin down-regulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various anti-tumor effects. We aimed in this in vitrostudy to demonstrate the inhibition of JAK2-STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in gastric cancer cell line. Methods: We performed Wetern blot to measure SHP1, phospho-JAK2/STAT3 level, and reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate target gene expression of STAT3. Several functional studies such as water soluble tetrazolium-1 (WST-1) assay, wound closure assay and matrigel invasion assay were also performed. Results: Plumbagin induced SHP1 expression and simultaneously down-regulated phospho-JAK2/STAT3 level via dose-and time-dependant manner in MKN28 cell, a gastric carcinoma cell line which has negative SHP1 expression. This effect was consistent when JAK2-STAT3 signaling was activated by interleukin-6, and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, VEGF1, survivin, MMP9, known target products of STAT3 activation in gastric cancinogenesis. The functional effect of plumbagin could be validated as inhibition of cell proliferation, migration and invasion, which are the results of activation of JAK2-STAT3 pathway in gastric cancer cells. Conclusions: Plumbagin is a potential negative regulator of cellular growth, migration and invasion by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

The hydrolysed products of iridoid glycosides can enhance imatinib mesylate-induced apoptosis in human myeloid leukaemia cells.

Several studies have demonstrated that deregulated activation of signal transducer and activator of transcription 3 (STAT3) has been associated with survival, proliferation, chemoresistance and angiogenesis of tumour cells. Thus, inhibition of STAT3 expression could be a potent therapeutic approach for cancer treatment. Using several leukaemia cell lines, the effect of the hydrolysed-catalpol (H-catalpol) and hydrolysed-aucubin (H-aucubin) products on the STAT3 signalling pathway, inhibition of BCR-ABL activation, cellular proliferation and potentiation of imatinib mesylate-induced apoptosis was investigated. We found that iridoid glycosides (catalpol and aucubin) did not exert any cytotoxicity in the tumour cells, whereas both H-catalpol and H-aucubin exhibited significant cytotoxic effects on K562 human myeloid leukaemia cells. Indeed, H-catalpol and H-aucubin down-regulated BCR-ABL phosphorylation and inhibited constitutive STAT3 activation through abrogating upstream JAK2 and c-Src and constitutive STAT5 activation leading to apoptosis through caspase-3 activation. Hydrolysed-catalpol enhanced the apoptosis induced by imatinib mesylate and this correlated with down-regulation of gene products that mediate cell proliferation (cyclin D1), and cell survival (Bcl-2, Bcl-xL and survivin); all known to be regulated by the STAT3. Overall, our results provide novel insight into the role of hydrolysed iridoids in potentially treating leukaemia through the modulation of STAT3 signalling pathway.

The role of STAT3 signaling in mediating tumor resistance to cancer therapy.

Signal transducer and activator of transcription 3 (STAT3) is activated in many cancer types and can regulate pathways involving tumorigenesis, cell proliferation, cell survival and angiogenesis. Upstream cytokine signaling through multiple trans-membrane receptors can enhance the activation of STAT3 and promote tumor progression. Importantly, STAT3 activation can also be induced via the Janus-activated kinase 1/2 (JAK1/2) and Src family kinases. Target-specific drug therapies have been developed to inhibit many of the upstream receptor and non-receptor activators of STAT3 and are now approved for clinical use. Recently, resistance to standard-of-care therapies has been linked to constitutive or unabated STAT3 activation, suggesting that combination therapy with STAT3 inhibitors may be of clinical benefit. Furthermore, STAT3 activity has also been shown to regulate self-renewal of cancer stem cells that are often refractory to chemotherapy treatment. This review will focus on STAT3 mediated resistance to cancer therapy and discuss strategies to overcome this resistance.