Disruption Of STAT3 Signaling Research Articles

Abstract 4880: Disruption of STAT3 signaling promotes K-Ras induced lung tumorigenesis

Abstract Lung cancer related and unrelated to smoking is still leading cause of cancer deaths worldwide with an overall survival rate of 15%. Several genetic alterations have been associated with lung cancer: loss of tumor suppressor genes such as p53, INK4a, LKB1 and mutations/amplifications in several oncogenes like K-Ras, EGFR, or c-Myc. Most frequent smoking-related mutations impair GTP hydrolysis in K-Ras, an oncogene downstream of the EGFR pathway, causing persistent cell growth and proliferation in 20-30% of lung adenocarcinomas. Another important downstream effector of EGFR signaling is the signal transducer and activator of transcription (STAT)-3. STAT-3 regulates important pathways in tumorigenesis, through upregulation of genes encoding apoptosis inhibitors (Bcl-XL, Bcl-2, Mcl-1, survivin). In patient samples and NSCLC cell lines nuclear, phosphorylated STAT3 is enhanced and correlates with subsequent suppression of apoptosis of NSCLC tumors. However, the role of STAT-3 in lung cancer in vivo has not yet been established. In order to investigate the role of STAT-3 in lung tumors, we have established a genetic mouse model that allows inducing K-RasG12D-dependent lung tumors and simultaneously genetically ablating STAT-3. Survival analysis of this model showed a significant advantage of K-RasG12D male mice harbouring STAT-3 compared to K-RasG12D mice lacking one or both alleles of Stat-3, indicating a hapoloinsufficient tumor suppressor role in oncogenic K-RasG12D tumor formation. Furthermore, animals with deleted STAT-3 and activated K-RasG12D have a significant increase in tumor burden and develop more adenomas and adenocarcinomas than age-matched animals, harbouring only oncogenic K-RasG12D. This data suggest that disruption of STAT-3 signaling promotes tumorigenesis in K-Ras induced tumors. We are currently investigating the molecular and cellular mechanisms responsible for this observation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4880. doi:1538-7445.AM2012-4880

Abstract 2399: Inhibition of murine breast cancer growth by immunization with tumor cells transfected with a dominant negative vector of Stat3

Abstract Stat3 is constitutively activated in several cancer types. Its activation in tumors promotes the transcription of genes associated with cell-cycle progression, cell survival, angiogenesis, and immune evasion. In particular in breast cancer, elevated activation levels of Stat3 are linked to breast tumorigenesis. We have already shown that progestins induce Stat3 activation in murine and human breast cancer cells. Moreover, we demonstrated that in the C4HD tumor that belongs to a progestin-dependent murine mammary tumor model, which requires medroxiprogesterone acetate (MPA) administration to proliferate, the presence of activated Stat3 is an absolute requirement for progestin stimulation of in vitro and in vivo breast cancer growth. Based on the evidence that disruption of Stat3 signaling in tumor cells can overcome tumor immune evasion, the present study focused on whether serial injection of irradiated C4HD cells expressing a dominant negative form of Stat3 (DNStat3), Stat3Y705F, could provide protection against C4HD wild-type tumor challenge, and focused as well on elucidating the mechanism mediating this effect. Our results showed that Balb/c mice immunized with DNStat3-transfected C4HD cells experienced significant inhibition of tumor growth with MPA (77%, 74% and 78% as compared with mice injected with PBS, with wild-type C4HD cells, or with empty vector-transfected C4HD cells, respectively). The lack of protection against tumor formation in nude mice suggested that T lymphocytes were involved in the antitumor response. Delayed-type hypersensitivity (DTH) assays in immunized Balb/c mice showed a strong increase in DTH reactivity in mice injected with DNStat3-transfected C4HD cells as compared to control groups, confirming that a cellular immune response was involved in the antitumoral effect. To evaluate this in vitro, we performed a 51Cr release assay to study the cytotoxic potential of the stimulated splenocytes towards C4HD cells. Only splenocytes isolated from mice injected with DNStat3-transfected C4HD cells were effective in lysing C4HD (37,9+0,6% cytotoxic activity at 50:1 effector-to-target ratio). We also assessed the capacity of splenocytes from immunized mice to proliferate in response to C4HD wild-type cells. Only splenocytes from mice injected with DNStat3-transfected C4HD cells proliferated strongly in response to mitomycin C-treated C4HD cells. Induction of the immunogenic phenotype in DNStat3 C4HD cells occurred along with a dim increase of costimulatory molecule CD86 expression and with the secretion of pro-inflammatory cytokines like TNF-α, IL-6, IFN-γ and Rantes. In conclusion, our results demonstrate for the first time that breast cancer growth can be inhibited through induction of a specific protective immune response in vivo with tumor immunogens derived from DNStat3-transfected breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2399.

(-)-Epigallocatechin-3-Gallate Protects SH-SY5Y Cells Against 6-OHDA-Induced Cell Death through STAT3 Activation

As a natural product, (-)-Epigallocatechin-3-gallate (EGCG), has demonstrated remarkable neuronal protection by depressing oxidative stress in Parkinson's disease (PD). However, the molecular mechanisms underlying EGCG neuronal protection have not been clarified. Using 6-hydroxydopamine (6-OHDA)-treated human neuroblastoma SH-SY5Y cells as a PD cell model, we found that 6-OHDA can cause neuronal death by regulating the activity of STAT3. Pretreatment of SH-SY5Y cells with EGCG (0.1-10 microM) significantly attenuated cell death induced by 6-OHDA. In addition, STAT3 activity decline induced by 6-OHDA in SH-SY5Y cells can be completely prevented by the presence of 1 microM of EGCG, and neuronal cell proliferation can be stimulated by EGCG treatment. These results clearly demonstrate that the disruption of STAT3 signaling by 6-OHDA makes significant contribution to the neuronal death in PD, and the protection of EGCG on neurons against oxidative stress-induced cell death may result from the re-stimulation of STAT3 signaling pathway. Our study not only clarified the role of STAT3 signaling pathway in oxidative stress-induced neuronal cell death, but also identified its involvement in the protection mechanism of EGCG on neurons in PD. The data resulting from our study also suggest that STAT3 may serve as a potential therapeutic target for drug development in PD.

Cell Density–Dependent Increase of Constitutive Signal Transducers and Activators of Transcription 3 Activity in Melanoma Cells Is Mediated by Janus Kinases

Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and sustained activation of STATs, especially STAT3, is a trait of many different cancers and their derived cell lines. Constitutively active STAT3 has been reported to prevent programmed cell death and enhance cell proliferation, whereas the disruption of STAT3 signaling can inhibit tumor growth. The physiologic activation of STAT3 by cytokines has been well established; however, little is known about altered, stimulation-independent STAT3 activation. Here, we show that, in most but not all melanoma cell lines, STAT3 phosphorylation increased substantially with cell density and that this STAT3 was able to bind to DNA and to activate transcription. Inhibitor studies showed that the cell density-dependent STAT3 activation relies on Janus kinases (JAK) rather than Src kinases. Using a specific JAK inhibitor, sustained STAT3 activation was completely abrogated in all tested melanoma lines, whereas inhibition of Src or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 had no effect on constitutively tyrosine-phosphorylated STAT3 levels. Although STAT3 activation was completely blocked with JAK inhibitor I and to a lesser extent with the common JAK inhibitor AG490, only the latter compound markedly decreased proliferation and induced apoptosis. Taken together, variations in cell density can profoundly modify the extent of JAK-mediated persistent STAT3 phosphorylation; however, STAT3 activation was not sufficient to provide critical growth and survival signals in melanoma cell lines.

Disruption of STAT3 Signaling in Antigen Presenting Cells Represents a Novel Strategy To Overcome Tumor-Induced Antigen-Specific CD4+ T-Cell Tolerance.

Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen presenting cells (APCs) are critical in the induction of this unresponsive state. Recently, we have identified STAT3 signaling in APCs as an important regulatory pathway that determines the functional outcome of antigen-specific CD4+ T-cells in response to cognate antigen. Indeed, while disruption of this signaling pathway in APCs led to effective T cell priming, enhanced STAT3 activity resulted in the induction of T cell unresponsiveness1.Given the above results, we explored in this study whether disruption of STAT3 signaling in APCs may preserve the responsiveness of antigen-specific CD4+ T-cells during the growth of a tumor that induces antigen specific T-cell tolerance. First, mice with a genetic disruption of Stat3 in macrophages, neutrophils and a sub-population of myeloid DCs (LysMcre/Stat3flox/− mice) or control C57BL/6 mice were given subcutaneously 1x106 B16 melanoma tumor cells engineered to express Ovalbumin as a model tumor antigen (B16-OVA). Four days later, naive CD4+ T-cells (1x106) specific for a MHC class II-restricted epitope of Ovalbumin (OT-II cells) were adoptively transferred intravenously into tumor bearing mice as well as into tumor-free controls. Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses to in vitro re-stimulation with OVA-peptide were evaluated. As expected, antigen-specific T cells re-isolated from tumor-bearing C57BL/6 mice were fully tolerant (lack of HA-specific proliferation and cytokine production). In sharp contrast, anti-OVA CD4+ T-cells isolated from tumor bearing LysMcre/Stat3flox/− mice remained fully functional as determined by their capacity to proliferate and produce IL-2 and IFN-gamma in response to cognate OVA-peptide. The demonstration that tumor-induced antigen-specific CD4+ T-cell tolerance occurs in mice with an intact STAT3 signaling in APCs, but not in mice with genetic disruption of this signaling pathway, led us next to evaluate the efficacy of pharmacologic inhibitors of STAT3 in preventing and or overcoming tumor-induced T-cell tolerance. In vivo treatment of tumor bearing mice with Tyrphostin AG490 (0.5 mg/ i.p. /twice a day x 5 days), a well-known inhibitor of STAT3 signaling, also resulted in preservation of the responsiveness of tumor-antigen specific CD4+ T-cells. Furthermore, in vitro treatment of APCs with this compound led to effective priming of naive antigen-specific T cells and breaking of antigen-specific T-cell anergy. More recently, we have evaluated the efficacy of a novel STAT3 inhibitor, compound 295558, which efficiently inhibits the DNA-binding activity of STAT3. Treatment of DCs or macrophages with this specific inhibitor led to the generation of inflammatory APCs capable of restoring the responsiveness of tolerized CD4+ T-cells isolated from tumor bearing mice.Taken together, our findings establish a critical role for STAT3 signaling in the induction of tolerance to tumor antigens in vivo. Inhibition of this signaling pathway in APCs provides a novel molecular target to overcome the remarkable barrier that tolerance to tumor antigens imposes to cancer vaccination strategies.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis.

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3beta protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3beta protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF --> Src --> Stat3 --> Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.