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NF-κB (RelA) is constitutively active in many cancers, where it upregulates antiapoptotic and other oncogenic genes. While proinflammatory stimulus-induced NF-κB activation involves IKK-dependent nuclear translocation, mechanisms for maintaining constitutive NF-κB activity in tumors have not been elucidated. We show here that maintenance of NF-κB activity in tumors requires Stat3, which is also frequently constitutively activated in cancer. Stat3 prolongs NF-κB nuclear retention through acetyltransferase p300-mediated RelA acetylation, thereby interfering with NF-κB nuclear export. Stat3-mediated maintenance of NF-κB activity occurs in both cancer cells and tumor-associated hematopoietic cells. Both murine and human cancers display highly acetylated RelA, which is associated with Stat3 activity. This Stat3/NF-κB interaction is thus central to both the transformed and nontransformed elements in tumors.

Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its alpha subunit (HIF-1alpha), which is tightly regulated in normal tissues, is elevated in tumors due to hypoxia and overactive growth signaling pathways. Although much is known about HIF-1alpha regulation in cancer cells, crucial molecular targets that affect HIF-1alpha levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1alpha accumulation is unclear. This study shows a novel mechanism by which HIF-1alpha availability is regulated in both cancer cells and in myeloid cells in the tumor microenvironment. We show a requirement of signal transducer and activator of transcription 3 (Stat3) for HIF-1alpha RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1alpha mRNA relative to their counterparts from normal tissues in a Stat3-dependent manner. Additionally, Stat3 activity in the nontransformed cells in the tumor milieu affects HIF-1alpha RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1alpha RNA transcription, elevated Stat3 activity increases HIF-1alpha promoter activity, and Stat3 protein binds to the HIF-1alpha promoter in both transformed cells and in growing tumors. Taken together, these findings show a novel mode by which HIF-1alpha is regulated not only in cancer cells but also in the tumor-associated inflammatory cells, suggesting Stat3 as an important molecular target for inhibiting the oncogenic potential of HIF-1 induced by both hypoxia and overactive growth signaling pathways prevalent in cancer.

Endothelial integrity is dependent on intracellular adherens junctions formed by complexes of vascular endothelial (VE)-cadherin and catenins. We have previously demonstrated that exposing endothelial cells (EC) to breast cancer cell-conditioned media (CM) for 24h results in a reduction in VE-cadherin protein and mRNA levels. Herein, we examined the mechanism(s) involved in the downregulation of VE-cadherin by CM. Human dermal microvascular EC exposed to CM showed a downregulation in VE-cadherin promoter activity and upregulation of Twist, Slug, and Snail expression. Reporter gene analysis demonstrated a direct repression of the VE-cadherin promoter by Slug, Snail, and Twist expression plasmids. At least two E-box motifs appear to be involved in this regulatory process as shown by electrophoretic mobility shift assays. These results suggest that factors released by breast cancer cells are able to upregulate Twist, Slug, and Snail expression in EC, which in turn downregulate the activity of the VE-cadherin promoter.

Abnormal activation of human epidermal growth factor receptor 2 (HER2; ErbB-2) in breast tumors results in increased metastasis and angiogenesis, as well as reduced survival. Here, we show that angiopoietin-2 (Ang-2) expression correlates with HER2 activity in human breast cancer cell lines. Inhibiting HER2 activity with anti-HER2 monoclonal antibody trastuzumab (Herceptin) or HER2 short interfering RNA in tumor cells down-regulates Ang-2 expression. Consistent with the important roles of AKT and mitogen-activated protein kinase in the HER2 signaling pathway, AKT and ERK mitogen-activated protein kinase (MAPK) kinase activity is necessary for Ang-2 up-regulation by HER2. Moreover, overexpression of HER2 protein up-regulates Ang-2 expression. Heregulin-beta1-induced Ang-2 up-regulation is abrogated when AKT and ERK kinase activity are blocked. Immunohistochemical analysis of HER2 and Ang-2 proteins in human breast carcinomas shows that Ang-2 expression in breast cancer correlates with HER2 expression. These studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.

BACKGROUND:HER2 overexpression imparts a metastatic advantage in breast cancer. We have shown that HER2 signaling in breast cancer cells induces adjacent endothelial cell (EC) retraction, disrupting endothelial integrity. Because endothelial integrity is dependent on the adherens junctions, we postulated that the mechanism of tumor cell-induced EC retraction involves dissociation of catenin proteins from vascular endothelial (VE) cadherin. In this study, we report a loss of VE-cadherin in tumor-associated EC. We also tested for a change of catenin dissociation from VE-cadherin by manipulating HER2 signaling in tumor cells.METHODS:We tested confluent monolayers of human EC for downregulation of VE cadherin and dissociation of catenins from VE cadherin after exposure to breast cancer cells or conditioned media. Using immunoprecipitation, we quantitated the remaining complexed catenins to VE-cadherin in tumor-associated EC after different treatments to manipulate HER2 signaling.RESULTS:Treatment of EC with conditioned media from MCF-7 cells expressing HER2 induced a loss of VE-cadherin expression, and time-dependent dissociation of catenins from VE cadherin. Catenin dissociation from VE-cadherin was enhanced by Heregulin beta1 (P < .05) stimulation and decreased by trastuzumab (P < .05) blockade of HER2 signaling in cancer cells. An increase in EC phosphoSrc (Tyr 416) was seen by 8 hours.CONCLUSIONS:Our data suggest that HER2 induction of EC retraction involves both down-regulation of VE-cadherin and dissociation of catenins. HER2 signaling appears to regulate this potential metastatic mechanism. Further, Src phosphorylation suggests that this pathway may be involved in this mechanism.

Infiltration of immune elfector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.

Vascular endothelial growth factor (VEGF) upregulation is induced by many receptor and intracellular oncogenic proteins commonly activated in cancer, rendering molecular targeting of VEGF expression a complex challenge. While VEGF inducers abound, only two major transcription activators have been identified for its promoter: hypoxia inducible factor-1 (HIF-1) and signal transducer and activator of transcription (Stat3). Both HIF-1 expression and Stat3 activity are upregulated in diverse cancers. Here, we provide evidence that Stat3 is required for both basal and growth signal-induced expression of HIF-1. Moreover, induction of VEGF by diverse oncogenic growth stimuli, including IL-6R, c-Src, Her2/Neu, is attenuated in cells without Stat3 signaling. We further demonstrate that Stat3 regulates expression of Akt, which is required for growth signal-induced HIF-1 upregulation. Targeting Stat3 with a small-molecule inhibitor blocks HIF-1 and VEGF expression in vitro and inhibits tumor growth and angiogenesis in vivo. Furthermore, tumor cells' in vivo angiogenic capacity induced by IL-6R, which simultaneously activates Jak/STAT and PI3K/Akt pathways, is abrogated when Stat3 is inhibited. Activation of Stat3 signaling by various growth signaling is prevalent in diverse cancers. Results presented here demonstrate that Stat3 is an effective target for inhibiting tumor VEGF expression and angiogenesis.

To investigate the role of Stat3 (signal transducer and activator of transcription-3) in neoplastic transformation by the Large Tumor antigen of Simian Virus 40 (TAg), murine fibroblasts were rendered deficient in Stat3 activity through expression of a Stat3-specific siRNA or a Cre-loxP recombination system. The results demonstrate that growth rate, formation of foci overgrowing a monolayer of normal cells and colony formation in soft agar were dramatically reduced in Stat3-deficient cells. In addition, TAg expression led to increased Stat3 tyrosine phosphorylation, DNA binding, and transcriptional activity, suggesting that Stat3 is required for TAg-mediated neoplasia. Stat3 activation was prevented by blocking the binding of TAg to pRb (retinoblastoma-susceptibility gene product), whereas genetic ablation of pRb increased Stat3 activity, suggesting that pRb inactivation by TAg might be responsible for the observed Stat3 activation. Stat3 activation by TAg was suppressed after inhibition of c-Src, JAKs or the insulin-like growth factor receptor. On the other hand, targeted disruption of the Fer kinase or pharmacological inhibition of Abl had no effect. Inhibition of Src activity led to Stat3 down-regulation as well as apoptosis of sparsely growing, TAg-transformed cells. However, Src inhibition was relatively ineffective in confluent cells, consistent with previous results indicating that cell to cell adhesion activates Stat3 by a Src-independent mechanism. Direct Stat3 inhibition on the other hand induced apoptosis very effectively in confluent cells, which could have significant therapeutic implications. Taken together, our results suggest that Stat3 is an important component of a pathway emanating from TAg and leading to neoplastic conversion.

The immune system can act as an extrinsic suppressor of tumors. Therefore, tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here, we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells, and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells, T cells, natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3 −/− hematopoietic cells, and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell– and NK cell–dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity.

L'invention concerne des compositions et des procedes permettant de traiter le cancer et des angiopathies proliferantes. Une composition de cette invention peut comprendre un inhibiteur du trajet de signalisation Jak2/Stat3 et un inhibiteur du trajet de signalisation PI3k/Akt. Dans certains cas, les deux inhibiteurs sont capables d'agir en synergie, par opposition a chaque inhibiteur seul.

Loss of p53 function by mutation is common in cancer. However, most natural p53 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p53 expression but no p53 mutations. It remains to be fully determined what mechanisms disable p53 during malignant initiation and in cancers without mutations that directly affect p53. We show here that oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Site-specific mutation of a Stat3 DNA-binding site in the p53 promoter partially abrogates Stat3-induced inhibition. Stat3 activity also influences p53 response genes and affects UV-induced cell growth arrest in normal cells. Furthermore, blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. As a point of convergence for many oncogenic signaling pathways, Stat3 is constitutively activated at high frequency in a wide diversity of cancers and is a promising molecular target for cancer therapy. Thus, repression of p53 expression by Stat3 is likely to have an important role in development of tumors, and targeting Stat3 represents a novel therapeutic approach for p53 reactivation in many cancers lacking p53 mutations.

Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. The mechanisms that block initiation of immune responses during cancer development are poorly understood. We report here that constitutive activation of Stat-3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators. Blocking Stat-3 in tumor cells increases expression of proinflammatory cytokines and chemokines that activate innate immunity and dendritic cells, leading to tumor-specific T-cell responses. In addition, constitutive Stat-3 activity induces production of pleiotropic factors that inhibit dendritic cell functional maturation. Tumor-derived factors inhibit dendritic cell maturation through Stat-3 activation in progenitor cells. Thus, inhibition of antitumor immunity involves a cascade of Stat-3 activation propagating from tumor to dendritic cells. We propose that tumor Stat-3 activity can mediate immune evasion by blocking both the production and sensing of inflammatory signals by multiple components of the immune system.

Activation of protein tyrosine kinases is prevalent in human cancers and previous studies have demonstrated that Stat3 signaling is a point of convergence for many of these tyrosine kinases. Moreover, a critical role for constitutive activation of Stat3 in tumor cell proliferation and survival has been established in diverse cancers. However, the oncogenic signaling pathways in melanoma cells remain to be fully defined. In this study, we demonstrate that Stat3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens examined. Blocking Src tyrosine kinase activity, but not EGF receptor or JAK family kinases, leads to inhibition of Stat3 signaling in melanoma cell lines. Consistent with a role of Src in the pathogenesis of melanoma, we show that c-Src tyrosine kinase is activated in melanoma cell lines. Significantly, melanoma cells undergo apoptosis when either Src kinase activity or Stat3 signaling is inhibited. Blockade of Src or Stat3 is also accompanied by down-regulation of expression of the anti-apoptotic genes, Bcl-xL and Mcl-1. These findings demonstrate that Src-activated Stat3 signaling is important for the growth and survival of melanoma tumor cells.

Non-receptor and receptor tyrosine kinases, such as Src and EGF receptor (EGFR), are major inducers of vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis. While tyrosine kinases signal through multiple pathways, signal transducer and activation of transcription 3 (Stat3) is a point of convergence for many of these and is constitutively activated with high frequency in a wide range of cancer cells. Here, we show that VEGF expression correlates with Stat3 activity in diverse human cancer cell lines. An activated Stat3 mutant (Stat3C) up-regulates VEGF expression and stimulates tumor angiogenesis. Stat3C-induced VEGF up-regulation is abrogated when a Stat3-binding site in the VEGF promoter is mutated. Furthermore, interrupting Stat3 signaling with dominant-negative Stat3 protein or Stat3 antisense oligonucleotide in tumor cells down-regulates VEGF expression. Consistent with an important role of Stat3 in VEGF up-regulation induced by various oncogenic tyrosine kinases, v-Src-mediated VEGF expression is inhibited when Stat3 signaling is blocked. Moreover, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in vivo and mutation of a Stat3-binding site in the VEGF promoter abrogates v-Src-induced VEGF promoter activity. These studies provide evidence that the VEGF gene is regulated directly by Stat3 protein, and indicate that Stat3 represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in human cancers.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR–ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr–Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr–Abl kinase. In this study, we show that blocking Bcr–Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC 50 of 5 n M . Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr–Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr–Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr–Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr–Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.