Hyperactive EGF receptor, Jaks and Stat3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells.

We present evidence that the cisplatin-resistant human ovarian cancer lines, A2780S/CP1 (S/CP1), A2780S/CP3 (S/CP3) and A2780S/CP5 (S/CP5), derived by subjecting the sensitive A2780S ovarian cancer line to multiple rounds of cisplatin treatments followed by recovery and are resistant to 1, 3 and 5 µM cisplatin, respectively, have increased colony-forming ability and altered morphology that is consistent with enhanced motility, migration and invasiveness in vitro. The malignant phenotype progresses with increasing resistance and is associated with hyperactive epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (Erk)1/2 and janus kinases (Jaks), aberrant signal transducer and activator of transcription (Stat) 3 activation promoted by EGFR and Jaks, and epithelial-mesenchymal transition (EMT) in vitro. Survivin and FLIP anti-apoptotic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase activities are also elevated in the resistant cells. Accordingly, the ectopic expression of constitutively-active Stat3C in the sensitive A2780S cells diminished cisplatin sensitivity. The inhibition of EGFR or Stat3 activity repressed Survivin, VEGF and Vimentin expression and the colony-forming potential, viability, motility and migration of the resistant cells, and sensitized them to cisplatin. Analysis of human ovarian cancer patients' tumor tissues shows aberrantly-active EGFR and Stat3 that in certain cases correlate with Vimentin over-expression. Intra-peritoneal mouse xenograft studies revealed, compared with the sensitive A2780S line that had low tumor incidence restricted to the ovary, a high tumor incidence for the resistant S/CP3 and S/CP5 lines that formed tumor nodules at several locations on the small intestine and colon, and which responded poorly to cisplatin, but were sensitive to concurrent treatment with cisplatin and EGFR or Stat3 inhibitor. Hyperactive EGFR signaling through Stat3 and the Jak-Stat3 activity together promote ovarian cancer progression to cisplatin resistance and therefore represent targets for preventing the development of cisplatin resistance and the recurrent disease during cisplatin therapy in ovarian cancer.

Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation.

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression.

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.

Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling.

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully defined. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse fibroblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the findings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These findings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse fibroblasts.

STATs in oncogenesis.

Since their discovery as key mediators of cytokine signaling, considerable progress has been made in defining the structure-function relationships of Signal Transducers and Activators of Transcription (STATs). In addition to their central roles in normal cell signaling, recent studies have demonstrated that diverse oncoproteins can activate specific STATs (particularly Stat3 and Stat5) and that constitutively-activated STAT signaling directly contributes to oncogenesis. Furthermore, extensive surveys of primary tumors and cell lines derived from tumors indicate that inappropriate activation of specific STATs occurs with surprisingly high frequency in a wide variety of human cancers. Together, these findings provide compelling evidence that aberrant STAT activation associated with oncogenesis is not merely adventitious but instead contributes to the process of malignant transformation. These studies are beginning to reveal the molecular mechanisms leading to STAT activation in the context of oncogenesis, and candidate genes regulated by STATs that may contribute to oncogenesis are being identified. Recent studies suggest that activated STAT signaling participates in oncogenesis by stimulating cell proliferation and preventing apoptosis. This review presents the evidence for critical roles of STATs in oncogenesis and discusses the potential for development of novel cancer therapies based on mechanistic understanding of STAT signaling. Oncogene (2000).

Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon activation by tyrosine phosphorylation. Several protein-tyrosine kinases can induce phosphorylation of STATs in cells, including Janus kinase (JAK) and Src family kinases. One STAT family member, Stat3, is constitutively activated in Src-transformed NIH3T3 cells and is required for cell transformation. However, it is not entirely clear whether Src kinase can phosphorylate Stat3 directly or through another pathway, such as JAK family kinases. To address this question, we investigated the phosphorylation of STATs in baculovirus-infected Sf-9 insect cells in the presence of Src. Our results show that Src can tyrosine-phosphorylate Stat1 and Stat3 but not Stat5 in this system. The phosphorylated Stat1 and Stat3 proteins are functionally activated, as measured by their abilities to specifically bind DNA oligonucleotide probes. In addition, the JAK family member Jak1 efficiently phosphorylates Stat1 but not Stat3 in Sf-9 cells. By contrast, we observe that AG490, a JAK family-selective inhibitor, and dominant negative Jak1 protein can significantly inhibit Stat3-induced DNA binding activity as well as Stat3-mediated gene activation in NIH3T3 cells. Furthermore, wild-type or kinase-inactive platelet-derived growth factor receptor enhances Stat3 activation by v-Src, consistent with the receptor serving a scaffolding function for recruitment and activation of Stat3. Our results demonstrate that Src kinase is capable of activating STATs in Sf-9 insect cells without expression of JAK family members; however, Jak1 and platelet-derived growth factor receptor are required for maximal Stat3 activation by Src kinase in mammalian cells. Based on these findings, we propose a model in which Jak1 serves to recruit Stat3 to a receptor complex with Src kinase, which in turn directly phosphorylates and activates Stat3 in Src-transformed fibroblasts.

STAT proteins: novel molecular targets for cancer drug discovery.

Signal Transducers and Activators of Transcription (STATs) are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Frequently, however, abnormal activity of certain STAT family members, particularly Stat3 and Stat5, is associated with a wide variety of human malignancies, including hematologic, breast, head and neck, and prostate cancers. Application of molecular biology and pharmacology tools in disease-relevant models has confirmed Stat3 as having a causal role in oncogenesis, and provided validation of Stat3 as a target for cancer drug discovery and therapeutic intervention. Furthermore, a constitutively-active mutant form of Stat3 is sufficient to induce oncogenic transformation of cells, which form tumors in vivo. Constitutive activation of Stat3 signaling is accompanied by upregulation of cyclin D1, c-Myc, and Bcl-x, changes consistent with subversion of normal cellular growth and survival control mechanisms. Block of constitutive Stat3 signaling results in growth inhibition and apoptosis of Stat3-positive tumor cells in vitro and in vivo. The observed dependence of certain tumors on constitutive Stat3 signaling for growth and survival has wide implications for cancer therapy, offering the potential for preferential tumor cell killing. This review evaluates constitutive Stat3 activation as a 'cancer-causing' factor, and proposes a number of molecular strategies for targeting Stat3 signaling for therapeutic intervention.

Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway.

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.

Stat3 activation by Src induces specific gene regulation and is required for cell transformation.

While signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src could induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3beta, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3beta did not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3beta does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3beta together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3beta had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3beta were generated and characterized, demonstrating that Stat3beta overexpression does not have a toxic effect on cell viability. These Stat3beta-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.

Staurosporine induces hydrolysis of phosphatidyl inositol 4,5-bisphosphate in human platelets.

Staurosporine in the micromolar range raised inositol trisphosphate in intact human platelets to levels comparable to that mediated by thrombin. This response was inhibited by neomycin, a phospholipase C antagonist. Staurosporine alone induced a weak, transient rise in cytosolic free calcium levels ([Ca2+]i) from release of internal Ca2+ stores but potentiated the effect induced by thrombin. Therefore, it is unlikely that this alkaloid suppressed inositol trisphosphate mobilization of Ca2+. Additional studies show that staurosporine, 0.5-5 microM, stimulated GTPase activity in platelet membranes while 2 microM K252a and 20 microM H7 were inactive. Present results suggest that staurosporine may activate platelet phospholipase C at the level of G proteins or receptors.

Staurosporine clamps cytosolic free Ca2+ concentrations of human neutrophils.

The present studies indicate that 50 nM-10 microM-staurosporine increased cytosolic free Ca2+ concentrations ([Ca2+]i) of fura-2-loaded neutrophils in a non-linear manner. The rise in [Ca2+]i was rapid, reaching a plateau (e.g. to 0.4 microM with 1 microM-staurosporine) within 30 s, and was maintained for more than 20 min. Pretreating cells with pertussis toxin had no effect on this reaction. The elevation of [Ca2+]i was insensitive to extracellular Ca2+ concentrations and was due entirely to mobilization of intracellular Ca2+ stores. Mn(2+)-quench studies confirmed the absence of Ca2+ influx. No Ca2+ efflux occurred in staurosporine-treated cells. In combination studies, staurosporine potentiated Ca2+ influx induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) and did not block Ca2+ efflux associated with peptide stimulation of neutrophils. Studies with permeabilized cells showed that staurosporine did not directly release intracellular Ca2+ stores, nor did it affect the sequestration of Ca2+ by a Ca2+/ATPase pump. A radioligand-binding assay failed to detect changes in the level of inositol 1,4,5-trisphosphate in neutrophils incubated with less than or equal to 1 microM-staurosporine, but in cells treated with 10 microM-staurosporine the assay recorded a transient increase in this second messenger similar to that induced by FMLP. Finally, lysozyme, but not beta-glucuronidase, was released from staurosporine-treated cells. The present results suggest that staurosporine increased [Ca2+]i by indirectly mobilizing internal Ca2+ stores. Staurosporine suppression of Ca2+ efflux and generation of a persistent signal may account for the maintained elevation of [Ca2+]i.