Oncogenic Signal Transducer And Activator Of Transcription 3 Research Articles

TMET-03. ONCOGENIC STAT3 PROMOTES TUMOR GROWTH VIA STIMULATION THE SCAP- AND SREBP-1-REGULATED LIPOGENIC PATHWAY

Abstract Augmented lipogenesis is a hallmark of cancer. In addition, signal transducer and activator of transcription 3 (STAT3) is aberrantly activated and associated with poor prognosis in multiple cancers. Whether oncogenic STAT3 has an intrinsic and causal link to lipogenesis in cancer remains unclear. Here, we show that inhibition of STAT3 significantly alters the lipid profile of tumor cells, including free fatty acids and derived membrane structural phospholipids (phosphatidic acid, phosphatidylinositol, phosphatidylserine and sphingomyelin), this change results in cell death which could be rescued by lipids (oleic acid and palmitoleic acid) supplementation, while suppressing the gene and protein expression of major mediators of the lipogenic pathway including sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), SREBP-1, fatty acid synthase and stearoyl-CoA desaturase 1. Furthermore, we show that STAT3 binds the promoter of SCAP, SREBP-1a and SREBP-1c, to promote their transcription in tumor cells. Finally, targeting STAT3 in a xenograft mouse model blocked glioblastoma growth and lipogenic enzyme expression. Our study reveals a novel regulatory pathway of STAT3-SCAP/SREBP-1 involved in tumor growth in vitro and in vivo, connecting STAT3 signaling with SCAP- and SREBP-1-mediated lipogenesis.

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.

Fangchinoline diminishes STAT3 activation by stimulating oxidative stress and targeting SHP-1 protein in multiple myeloma model

IntroductionThe development of cancer generally occurs as a result of various deregulated molecular mechanisms affecting the genes that can control normal cellular growth. Signal transducer and activator of transcription 3 (STAT3) pathway, once aberrantly activated can promote carcinogenesis by regulating the transcription of a number of oncogenic genes. ObjectivesHere, we evaluated the impact of fangchinoline (FCN) to attenuate tumor growth and survival through modulation of oncogenic STAT3 signaling pathway using diverse tumor cell lines and a xenograft mouse model. MethodsTo evaluate the action of FCN on STAT3 cascade, protein levels were analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). Translocation of STAT3 was detected by immunocytochemistry. Thereafter, FCN-induced ROS was measured by GSH/GSSG assay and H2DCF-DA. FCN-induced apoptosis was analyzed using Western blot analysis and flow cytometry for various assays. Finally, anti-cancer effects of FCN in vivo was evaluated in a myeloma model. ResultsWe noted that FCN abrogated protein expression levels of STAT3 and upstream signals (JAK1/2 and Src). In addition, FCN also attenuated DNA binding ability of STAT3 and its translocation into the nucleus. It altered the levels of upstream signaling proteins, increased SHP-1 levels, and induced substantial apoptosis in U266 cells. FCN also promoted an increased production of reactive oxygen species (ROS) and altered GSSG/GSH ratio in tumor cells. Moreover, FCN effectively abrogated tumor progression and STAT3 activation in a preclinical myeloma model. ConclusionOverall, this study suggests that FCN may have a tremendous potential to alter abnormal STAT3 activation and induce cell death in malignant cells along with causing the suppression of pathogenesis and growth of cancer through a pro-oxidant dependent molecular mechanism.

Codelivery of STAT3 and PD-L1 siRNA by hyaluronate-TAT trimethyl/thiolated chitosan nanoparticles suppresses cancer progression in tumor-bearing mice

Immunotherapy methods using potential tumor microenvironment modulators have elicited durable therapeutic responses in cancer treatment. Immune checkpoint molecule programmed cell death-ligand 1 (PD-L1) and oncogenic transcription factor STAT3 (signal transducer and activator of transcription-3) assigned as inhibitory targets of our study and particular delivery system designed to deliver small interfering RNAs (siRNAs) to silence the targeted genes. Generated trimethyl chitosan (TMC) and thiolated chitosan (TC) nanoparticles (NPs) conjugated with HIV-1-derived TAT peptide and HA (hyaluronic acid) exhibited eligible physicochemical characteristics, notable siRNA encapsulation, serum stability, non-toxicity, controlled siRNA release, and extensive cellular uptake by cancer cells. Dual inhibition with STAT3/PD-L1 siRNA-loaded HA-TAT-TMC-TC NPs led to promising results, including significant downregulation of PD-L1 and STAT3 genes, striking suppressive effects on proliferation, migration, and angiogenesis of breast and melanoma cancer cell lines, and restrained tumor growth in vivo. These findings infer the capability of HA-TAT-TMC-TC NPs containing STAT3/PD-L1 siRNAs as a novel tumor-suppressive candidate in cancer treatment.

The pleiotropic role of transcription factor STAT3 in oncogenesis and its targeting through natural products for cancer prevention and therapy.

Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors, responsible for regulating cellular proliferation, cellular differentiation, migration, programmed cell death, inflammatory response, angiogenesis, and immune activation. In this review, we have discussed the classical regulation of STAT3 via diverse growth factors, cytokines, G-protein-coupled receptors, as well as toll-like receptors. We have also highlighted the potential role of noncoding RNAs in regulating STAT3 signaling. However, the deregulation of STAT3 signaling has been found to be associated with the initiation and progression of both solid and hematological malignancies. Additionally, hyperactivation of STAT3 signaling can maintain the cancer stem cell phenotype by modulating the tumor microenvironment, cellular metabolism, and immune responses to favor drug resistance and metastasis. Finally, we have also discussed several plausible ways to target oncogenic STAT3 signaling using various small molecules derived from natural products.

JAB1-STAT3 activation loop is associated with recurrence following 5-fluorouracil-based adjuvant chemotherapy in human colorectal cancer.

Jun activation domain-binding protein 1 (JAB1) has been shown to have multiple roles in tumorigenesis, including the degradation of tumor suppressor proteins such as p53, Smad7, Runx3 and the cyclin-dependent kinase inhibitor p27Kip1, and the activation of oncogenic transcription factors, such as c-Jun and hypoxia-inducible factor-1α. In addition, our previous study revealed that JAB1 positively regulates signal transducer and activator of transcription 3 (STAT3) DNA-binding activity in human colon cancer cells. In turn, the oncogenic transcription factor STAT3 positively regulates JAB1 expression, indicative of a positive feedback loop. Furthermore, high JAB1 expression is associated with a poor prognosis in numerous malignant carcinomas. However, the association between JAB1 expression and prognosis in colorectal cancer remains unclear. The aim of the present study was to elucidate the association between JAB1 and STAT3 expression and recurrence in colorectal cancer. In the present study, it was found that high JAB1 expression in primary colorectal cancer tissues is an independent predictor of recurrence following 5-fluorouracil (5-FU)-based adjuvant chemotherapy in colorectal cancer patients, and that high expression of both JAB1 and STAT3 in primary colorectal cancer tissues is associated with a lower recurrence-free survival rate following 5-FU-based adjuvant chemotherapy compared to high expression of only JAB1 or STAT3. Overall, these results suggest that JAB1 is a novel predictive marker of recurrence following 5-FU-based adjuvant chemotherapy in colorectal cancer patients, and that the JAB1-STAT3 activation loop may be a potential therapeutic target in recurrent colorectal cancer following 5-FU-based adjuvant chemotherapy.

Abstract 2231: 15-Keto prostaglandin E2 inhibits STAT3 signaling in H-Ras transformed mammary epithelial cells

Abstract Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. The intracellular level of PGE2 is regulated by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This enzyme catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2. Down-regulation of 15-PGDH was observed in various malignancies and overexpression of 15-PGDH has been known to suppress the carcinogenic processes. However, mechanisms underlying anti-carcinogenic effects and tumor suppressive functions of 15-keto PGE2 are poorly understood in various cancer. H-Ras transformed mammary epithelial MCF10A-ras cells show enhanced expression of cyclooxygenase-2 and activation of signal transducer and activator of transcription 3 (STAT3) and down-regulation of 15-PGDH compared to normal mammary epithelial MCF10A cells. STAT 3 plays a key role in growth of cancer cells as well as inflammation-associated carcinogenesis. This prompted us to investigate the effect of 15-keto PGE2 on STAT3 activation in MCF10A-ras cells. 15-Keto PGE2 suppressed the phosphorylation of STAT3 in MCF10A-ras cells in concentration- and time-dependent manners as determined by Western blot and immunocytochemical analyses. 15-Keto PGE2 also inhibited the dimerization of STAT3 which led to suppression of nuclear translocation and transcriptional activity of STAT3 as determined by Western blot analysis and the luciferase assay, respectively. 15-keto PGE2 suppressed the expression of cyclin D1. In addition, 15-keto PGE2 inhibited the growth of MCF10A-ras cells as determined by anchorage-independent growth and colony forming assays. However, a non-electrophilic analogue 13,14-dihydro-15-keto PGE2 which lacks the α,β-unsaturated carbonyl moiety failed to inhibit the phosphorylation, dimerization, and nuclear translocation of STAT3. Moreover, 13,14-dihydro-15-keto PGE2 did not affect the growth of MCF10A ras cells. These findings suggest that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. STAT3 has several cysteine residues in its activation sites. We observed that reducing agents N-acetyl-L-cysteine and dithiothreitol abrogated the suppressive effect of 15-keto PGE2 on STAT3 phosphorylation, suggesting that the involvement of the thiol modification. Biotinylated 15-keto PGE2 covalently bound to STAT3 as determined by immunoprecipitation with STAT3 followed by immunoblotted with streptavidin. Molecular docking analysis predicted Cys251 and Cys259 residues of STAT3 as putative binding sites of 15-keto PGE2. In conclusion, 15-keto PGE2 inhibits STAT3 signaling through cysteine thiol modification, thereby suppressing MCF10A-ras cell growth and proliferation. Thus, tumor suppressor function of 15-PGDH is attributable, at least in part, to inactivation of oncogenic STAT3 signaling by its catalytic product 15-keto PGE2. Citation Format: Eun-Ji Lee, Young-Il Hahn, Su-Jung Kim, Young-Joon Surh, Hye-Kyung Na. 15-Keto prostaglandin E2 inhibits STAT3 signaling in H-Ras transformed mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2017-2231

B1, a novel HDAC inhibitor, induces apoptosis through the regulation of STAT3 and NF-κB.

We previously demonstrated that B1 induced significant cytotoxic effects, cell cycle G1 arrest and apoptosis in human lung cancer A549 cells through the inhibition of DNA topoisomerase II activity. In the present study, we focused on the histone deacetylase (HDAC) modulation of B1 in A549 cells. HDACs, important enzymes affecting epigenetic regulation, play a crucial role in human carcinogenesis. Our findings showed that B1 could suppress the growth of A549 cells in vitro through the inhibition of HDAC activity. Additionally, B1 caused disruption of the mitochondrial membrane potential and induced DNA double-strand breaks (DSBs) in a dose- and time-dependent manner, which consequently led to cell apoptosis. We also observed that B1 inhibited cancer cell migration and angiogenesis-related signal expression, including vascular endothelial growth factor (VEGF) and pro-matrix metalloproteinases-2 and -9 (pro-MMP-2/9). Gelatin zymography suggested that B1 decreased pro-MMP-2 and pro-MMP-9 activity. Transcription factors, signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NF-κB), are vital players in the many steps of carcinogenesis. B1 showed significant dose-response inhibitory effects on cytoplasmic expression and nuclear translocation of both phosphorylated STAT3 (pSTAT3) and NF-κB. It has been well documented that reactivated telomerase confers cancer cells the ability to repair DNA. Real-time PCR results indicated that B1 inhibited STAT3 and NF-κB mRNA expression and telomerase activity. Taken together, our results demonstrated that B1 exerted significant inhibitory effects on HDAC, telomerase activities, oncogenic STAT3 and NF-κB expression. The inhibition of the intricate crosstalk between STAT3 and NF-κB may be a major factor in the molecular action mechanism of B1. The multiple targeting effects of B1 render it a potential new drug for lung cancer therapy.

HO-3867, STAT3 inhibitor: effects on cellular proliferation in hepatocellular carcinoma

Objective: Signal transducer and activator of transcription 3 (STAT3) plays an important role in cancer cell proliferation. HO-3867, a STAT3 inhibitor, has shown preclinical efficacy in ovarian cancer, but there is limited data in Hepatocellular carcinoma (HCC), a lethal disease with limited systemic therapy options. This project hypothesizes HO-3867 inhibits HCC cellular proliferation through STAT3 inhibition. Methods: Effects of increasing concentrations of HO-3867 on three HCC cell lines were assessed via calorimetric assay. Cell lysates were examined via Western blot for cell cycle arrest, pro- and anti-apoptotic markers, and traditional oncogenic pathways Notch, STAT3, and Phosphatidylinositol-3 Kinase (PI3-K)/Akt. Results: Concentrations of 2 μM HO-3867 led to a significant reduction in cellular proliferation (p=0.001). Upon Western analysis, HO-3867 was associated with increased expression of cell cycle markers p21 and p27 and reduction in cyclin D1. Furthermore, an increase in pro-apoptotic cleaved poly ADP ribose polymerase (PARP) and reduction in anti-apoptotic Bcl2, BclXL, XIAP, and Survivin was observed. HO-3867 also demonstrated a reduction in pSTAT3 and concomitant inhibition of Notch and PI3-K/AKT pathways as evidenced by reduction in Notch1-3, Hes1, and pAKT. However, higher concentrations of HO-3867 were associated with increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a cell survival protein. Conclusion: HO-3867, STAT3 inhibitor, mitigates HCC cell proliferation through cell cycle arrest and apoptosis through STAT3 inhibition. HO-3867 also reduces oncogenic targets Notch and Akt but at higher concentration increases phospho ERK1/2. This is the first study demonstrating HO-3867 effectiveness in HCC cells, providing a strong rationale for further preclinical analysis of HO-3867.

JNK1/2 expression and modulation of STAT3 signaling in oral cancer.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that link extracellular stimuli with intracellular responses and participate in numerous cellular processes such as growth, proliferation, differentiation, inflammation and apoptosis. Persistent activation of signal transducer and activator of transcription 3 (STAT3), which is accompanied by increases in STAT3 tyrosine phosphorylation, is associated with cell proliferation, differentiation and apoptosis in oral squamous cell carcinoma (OSCC). The role and significance of the activation of MAPKs, particularly of c-Jun N-terminal kinase (JNK), on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examines the effects of JNK1/2 modulation on STAT3 signaling and cellular activities in OSCC cells. The expression levels of STAT3 [total, tyrosine phosphorylated (p-Tyr) and serine phosphorylated (p-Ser)], JNK, c-Jun and cyclin D1 were assessed in the OSCC cell lines SCC25 and SCC9. Inhibition of JNK1/2 was achieved by pharmacological agents (SP600125) and by small interfering RNA (siRNA) silencing, while JNK1/2 was induced by active MAPK kinase 7. Cell proliferation and viability rates were also evaluated. Inhibition of JNK1/2 with either SP600125 treatment or specific siRNA silencing resulted in decreased levels of p-Ser STAT3 and increased levels of p-Tyr STAT3 and cyclin D1 in both cell lines. Furthermore, JNK1/2 inhibition resulted in a dose-dependent increase in cell growth and viability in both cell lines. Opposite results were observed with JNK1/2 induction in both cell lines. The present results are supportive of a potential tumor suppressive role of JNK1/2 signaling in OSCC, which may be mediated through negative crosstalk with the oncogenic STAT3 signaling pathway. The possible therapeutic implications of JNK1/2 inhibition for patients with OSCC require to be investigated.

MiR-519a functions as a tumor suppressor in glioma by targeting the oncogenic STAT3 pathway.

Glioblastoma (GBM) is among the most aggressive primary brain tumors, with a median survival rate of 12-15months. MicroRNAs have been implicated in GBM development as oncogenes or tumor suppressors. In this study, we demonstrated that miR-519a expression was frequently downregulated in GBM specimens and cell lines, and that low-levels miR-519a expression significantly correlated with poor outcomes associated with GBM. Analysis of The Cancer Genome Atlas also demonstrated that low miR-519a expression can predict poor clinical outcomes in classical and proneural GBM subtypes. Functionally, re-expression of miR-519a effectively reduced GBM cell proliferation, migration, and invasion. Mechanistically, we confirmed that the signal transducer and activator of transcription 3 (STAT3) 3'-UTR was a putative target of miR-519a, and that re-expression of STAT3 abrogated miR-519a function in GBM cells. Furthermore, we found that STAT3 expression negatively correlated with that of miR-519a in human GBM tissues. These results elucidated the prognostic value and tumor-suppressor role of miR-519a in GBM and further suggested it as a potential therapeutic target for GBM treatment.

Novel thiosemicarbazones regulate the signal transducer and activator of transcription 3 (STAT3) pathway: inhibition of constitutive and interleukin 6-induced activation by iron depletion.

Pharmacologic manipulation of metal pools in tumor cells is a promising strategy for cancer treatment. Here, we reveal how the iron-binding ligands desferrioxamine (DFO), di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibit constitutive and interleukin 6-induced activation of signal transducer and activator of transcription 3 (STAT3) signaling, which promotes proliferation, survival, and metastasis of cancer cells. We demonstrate that DFO, Dp44mT, and DpC significantly decrease constitutive phosphorylation of the STAT3 transcription factor at Tyr705 in the pancreatic cancer cell lines PANC-1 and MIAPaCa-2 as well as the prostate cancer cell line DU145. These compounds also significantly decrease the dimerized STAT3 levels, the binding of nuclear STAT3 to its target DNA, and the expression of downstream targets of STAT3, including cyclin D1, c-myc, and Bcl-2. Examination of upstream mediators of STAT3 in response to these ligands has revealed that Dp44mT and DpC could significantly decrease activation of the nonreceptor tyrosine kinase Src and activation of cAbl in DU145 and MIAPaCa-2 cells. In contrast to the effects of Dp44mT, DpC, or DFO on inhibiting STAT3 activation, the negative control compound di-2-pyridylketone 2-methyl-3-thiosemicarbazone, or the DFO:Fe complex, which cannot bind cellular iron, had no effect. This demonstrates the role of iron-binding in the activity observed. Immunohistochemical staining of PANC-1 tumor xenografts showed a marked decrease in STAT3 in the tumors of mice treated with Dp44mT or DpC compared with the vehicle. Collectively, these studies demonstrate suppression of STAT3 activity by iron depletion in vitro and in vivo, and reveal insights into regulation of the critical oncogenic STAT3 pathway.

NF-κB and STAT3 cooperatively induce IL6 in starved cancer cells

A number of genes involved in tumorigenesis have been known to be controlled by signal transducer and activator of transcription 3 (STAT3) and NF-κB, either synergistically or individually. In starved cancer cells, we found that NF-κB was activated through endoplasmic reticulum stress signals, which depend on reactive oxygen species, cytosolic calcium and preserved translation of NF-κB p65 subunit, but independent of IκBα serine phosphorylation, thereby resulting in IL6 induction. STAT3 was required for proper induction of IL6 by NF-κB. They existed as identical nuclear complexes in proximal IL6 promoters, and STAT3 had critical roles in binding to IL6 promoters as well as nuclear retention of NF-κB. The conditioned media from starved cancer cells contained various secretory factors, such as IL6, IL9, VWF (von Willebrand factor), FREM1 (FRAS1 related extracellular matrix 1), SAA1 (serum amyloid A1), SDK1 (sidekick homolog 1) and ADAM12 (ADAM metallopeptidase domain 12), induced by NF-κB and STAT3 and promoted clonogenic capacities of cancer cells, and proliferation and migration of human umbilical vein endothelial cells. These results suggest novel survival strategies of cancer cells by which two oncogenic transcriptional factors, NF-κB and STAT3, are activated simultaneously by an intrinsic mechanism during stressful conditions of cancer cells, and they cooperatively induce various survival factors.

M1635 Helicobacter pylori CagA Cytotoxin Stimulates Inappropriate STAT3 Activation in Gastric Epithelial Cells By a Phosphorylation Dependent Mechanism

Helicobacter pylori has a profound influence upon the development of gastric adenocarcinoma, however the molecular mechanisms are not clearly understood. Much attention has been devoted to the H. pylori Cytotoxin-associated antigen A (CagA), a 120-145kDa protein that is injected into gastric epithelial cells and disrupts intracellular signaling pathways including those utilised by IL-6 family cytokines. Current evidence shows that unbalancing of the two major IL-6 cytokine signaling cascades, resulting in the constitutive activation of the signal transducer and activator of transcription 3 (STAT3) and disruption of the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK pathway, emanating from the common gp130 coreceptor leads to gastric tumour development and establishes co-ordinated IL-6 family cytokine signalling as a paradigm for gastric homeostasis. This is intriguing because translocated CagA binds SHP-2 in a phosphorylation dependent manner and alters SHP-2-ras-ERK signalling leading to characteristic changes in cell morphology, motility and proliferation. We hypothesized that sequestration of intracellular SHP-2 by CagA may imbalance coordinated signalling downstream of gp130 leading to the inappropriate activation of oncogenic STAT3, as implicated by our recent work showing increased STAT3 phosphorylation in CagA-positive compared with CagA-negative H. pylori infections in precancerous lesions of humans. To clarify the mechanism by which CagA interferes with STAT3 signalling we have developed transient and inducible CagA expression systems in gastric epithelial cell lines. Overexpression of CagA in AGS cells led to a ~50% reduction in available phosphorylated SHP-2 as a consequence of its interaction with CagA, yet consistent with our expectations, we also observed a concomitant two-fold increase in STAT3 phosphorylation by western blotting. Overexpression of a phosphorylation-resistant CagA mutant had no discernable effect on either SHP-2 or STAT3 phosphorylation indicating that STAT3 activation in this context is dependent upon CagA tyrosine phosphorylation. These results reveal a novel mechanism by which CagA might provoke cellular transformation by the inappropriate activation of oncogenic STAT3 and thus constitute an early event in H. pylori mediated gastric cancer progression.

Crif1 is a novel transcriptional coactivator of STAT3

Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that performs a broad spectrum of biological functions in response to various stimuli. However, no specific coactivator that regulates the transcriptional activity of STAT3 has been identified. Here we report that CR6-interacting factor 1 (Crif1) is a specific transcriptional coactivator of STAT3, but not of STAT1 or STAT5a. Crif1 interacts with STAT3 and positively regulates its transcriptional activity. Crif1-/- embryos were lethal around embryonic day 6.5, and manifested developmental arrest accompanied with defective proliferation and massive apoptosis. The expression of STAT3 target genes was markedly reduced in a Crif1-/- blastocyst culture and in Oncostatin M-stimulated Crif1-deficient MEFs. Importantly, the key activities of constitutively active STAT3-C, such as transcription, DNA binding, and cellular transformation, were abolished in the Crif1-null MEFs, suggesting the essential role of Crif1 in the transcriptional activity of STAT3. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of STAT3 that modulates its DNA binding ability, and shed light on the regulation of oncogenic STAT3.

Tumor Suppressor LKB1 Inhibits Activation of Signal Transducer and Activator of Transcription 3 (STAT3) by Thyroid Oncogenic Tyrosine Kinase Rearranged in Transformation (RET)/Papillary Thyroid Carcinoma (PTC)

The tumor suppressor LKB1 (STK11) is a cytoplasmic/nuclear serine/threonine kinase, defects in which cause Peutz-Jeghers syndrome (PJS) in humans and animals. Recent studies showed that loss of function of LKB1 is associated with sporadic forms of lung, pancreatic, and ovarian cancer. In cancer cells, LKB1 is inactivated by two mechanisms: mutations in its central kinase domain or complete loss of LKB1 expression. Inactivation of LKB1 is associated with progression of PJS and transformation of benign polyps into malignant tumors. This study examines the effect of LKB1 on regulation of STAT3 and expression of transcriptional targets of STAT3. The results show that LKB1 inhibits rearranged in transformation (RET)/papillary thyroid carcinoma (PTC)-dependent activation of signal transducer and activator of transcription 3 (STAT3), which is mediated by phosphorylation of STAT3 tyrosine 705 by RET/PTC. Suppression of STAT3 transactivation by LKB1 requires the kinase domain but not the kinase activity of LKB1. The centrally located kinase domain of LKB1 is an approximately 260-amino-acid region that binds to the linker domain of STAT3. Chromatin immunoprecipitation studies indicate that expression of LKB1 reduces the binding of STAT3 to its target promoters and suppresses STAT3-mediated expression of Cyclin D1, VEGF, and Bcl-xL. Knockdown of LKB1 by specific small interfering RNA led to an increase in STAT3 transactivation activity and promoted cell proliferation in the presence of RET/PTC. Thus, this study suggests that LKB1 suppresses tumor growth by inhibiting RET/PTC-dependent activation of oncogenic STAT3.