Aberrant Activation Of STAT3 Research Articles

Targeting STAT3 in cancer: how successful are we?

Background: Aberrant activation of the signal transducer and activator of transcription (STAT)3 occurs in many human tumors. Moreover, studies utilizing genetic and pharmacological approaches to modulate constitutive STAT3 activity have provided compelling evidence for the critical role of aberrant STAT3 activity in malignant transformation and tumor progression, and thereby validated STAT3 as a novel cancer drug target. Objective: This review is intended to be a full coverage of the efforts to develop direct STAT3 inhibitors and will provide a discussion on the inhibitory modalities developed to date. Methods: Review of the literature focused on the modalities and mechanisms that directly target and inhibit the STAT protein or its functions. Results/conclusion: While a variety of STAT3 inhibitors have been identified that induce antitumor cell effects in vitro and in vivo, the landscape remains murky. With a few exceptions, most of the STAT3 inhibitors reported to date have not undergone an in vivo efficacy, pharmacology or toxicity testing. Also, there is no evidence, per the published literature of an impending clinical development for the few agents that were reported to exhibit in vivo efficacy. Overall, there is the need for a reassessment of the ongoing strategies to target STAT3 intended not only for refinement, but also for incorporating some new technologies to strengthen our efforts and ensure the success – sooner, rather than later – of identifying suitable anti-STAT3 agents for development into clinically useful anticancer therapeutics.

SOCS3 Negatively Regulates the gp130–STAT3 Pathway in Mouse Skin Wound Healing

Proliferation and differentiation of keratinocytes during wound healing are regulated by cytokines and chemokines, which are secreted by resident and inflammatory cells and activate the transcription factor signal transducer and activator of transcription (STAT)3. However, it is not clear to what extent STAT3 in keratinocytes is activated by gp130-containing receptors. We addressed this question genetically by deleting the suppressor of cytokine signaling (SOCS)3, a negative regulator of gp130-mediated STAT3 activation. Socs3 alleles flanked by loxP sites were deleted in mice with either an MMTV-Cre or K5-Cre transgene. While both transgenes are active in keratinocytes, the MMTV-Cre deletes floxed genes also in immune cells. Deletion of Socs3 using the MMTV-Cre transgene resulted in aberrant STAT3 activation, impaired wound healing, prolonged secretion of chemokines, a hyperproliferative epidermis, and neutrophil infiltration into wounds. Simultaneous deletion of the Socs3 and gp130 genes restored normal wound healing. Moreover, deletion of Socs3 only in keratinocytes caused impaired wound healing. These results demonstrate that wound healing is controlled in keratinocytes by the gp130-SOCS3-STAT3 pathway and an imbalance of this pathway results in delayed wound healing.

STAT3 inhibition in prostate and pancreatic cancer lines by STAT3 binding sequence oligonucleotides: differential activity between 5′ and 3′ ends

Signal transducers and activators of transcription (STAT) were originally discovered as components of signal transduction pathways. Persistent aberrant activation of STAT3 is a feature of many malignancies including prostate cancer and pancreatic cancer. One consequence of persistently activated STAT3 in malignant cells is that they depend on it for survival; thus, STAT3 is an excellent molecular target for therapy. Previously, we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. Here, we report that authentic STAT3 binding sequences, identified from published literature, were more effective for inducing apoptosis in prostate cancer cells and pancreatic cancer cells than was oligonucleotide 13410. Moreover, the authentic STAT3 binding sequences showed differing efficacies in the malignant cell lines depending on whether the canonical STAT3 binding sequence was truncated at the 5' or the 3' end. Finally, expression of one STAT3-regulated gene was decreased following treatment, suggesting that STAT3 may regulate the same set of genes in the two types of cancer. We conclude that truncating the 5' end left intact enough of the canonical STAT3 binding site for effective hybridization to the genome, whereas truncation of the 3' end, which is outside the canonical binding site, may have affected binding of required cofactors essential for STAT3 activity, thereby reducing the capacity of this modified oligonucleotide to induce apoptosis. Additional experiments to answer this hypothesis are under way.

A signal network involving coactivated NF‐κB and STAT3 and altered p53 modulates BAX/BCL‐XL expression and promotes cell survival of head and neck squamous cell carcinomas

Abrogation of apoptosis to sustain cell survival is an essential step in development of cancer. Aberrant activation of signal transcription factors NF-kappaB or STAT3, alterations in p53 status, or BCL/BAX family expression have each been reported to affect cell survival in cancer, including head and neck squamous cell carcinomas (HNSCC). However, molecular targeting of these alterations individually has yielded disappointing results. In our study, we examined the hypothesis that alterations in a signal network involving NF-kappaB, STAT3 and p53 modulates expression of proapoptotic BAX and antiapoptotic BCL-XL proteins, and promotes cell survival of HNSCC. We found that NF-kappaB and STAT3 are coactivated together, and with cytokine stimulation or siRNA knock-down, both modulate BAX/BCL-XL. Greater modulation among HNSCC lines expressing low wt p53 than those over-expressing mt p53 protein suggested that decreased p53 expression might enhance activation of NF-kappaB, STAT3 and BCL-XL. Reexpression of wt p53 suppressed NF-kappaB and STAT3 nuclear binding activity, and BCL-XL expression, while inducing p21 and BAX. Over-expression of p53 together with inhibition of NF-kappaB or STAT3 induced greater increase in the BAX/BCL-XL ratio and apoptosis than modulation of these transcription factors individually. Conversely, NF-kappaB or STAT3 inducing cytokines decreased the BAX/BCL-XL ratio. Thus, a network involving signal coactivation of NF-kappaB and STAT3, differentially modified by p53 inactivation or mutation, promotes altered BAX/BCL-XL expression and cell survival in HNSCC. Inhibition of signal activation of both NF-kappaB and STAT3 together with reexpression of p53 could be the most effective strategy to restore BAX/BCL-XL regulation and for cytotoxic therapy of HNSCC.

Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma

A variety of cancers, including malignant gliomas, show aberrant activation of STAT3, which plays a pivotal role in negative regulation of antitumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T cell adoptive transfer therapy by reversal of STAT3-induced immunosuppression in a murine GL261 intracranial glioma model. In vitro treatment of GL261 cells with JSI-124, a STAT3 inhibitor, reversed highly phosphorylated status of STAT3. Systemic i.p. administration of JSI-124 in glioma-bearing immunocompetent mice, but not athymic mice, resulted in prolonged survival, suggesting a role of adaptive immunity in the antitumor effect. Furthermore, JSI-124 promoted maturation of tumor-infiltrating CD11c(+) dendritic cells and activation of tumor-conditioned cytotoxic T cells, enhanced dendritic cells and GL261 production of CXCL-10, a critical chemokine for attraction of Tc1 cells. When i.p. JSI-124 administration was combined with i.v. transfer of Pmel-I mouse-derived type-1 CTLs (Tc1), glioma-bearing mice exhibited prolonged survival compared with i.p. JSI-124 or i.v. Tc1 therapy alone. Flow cytometric analyses of brain infiltrating lymphocytes revealed that JSI-124-treatment enhanced the tumor-homing of i.v. transferred Tc1 cells in a CXCL-10-dependent fashion. Systemic JSI-124 administration also up-regulated serum IL-15 levels, and promoted the persistence of transferred Tc1 in the host. These data suggest that systemic inhibition of STAT3 signaling can reverse the suppressive immunological environment of intracranial tumor bearing mice both systemically and locally, thereby promoting the efficacy of adoptive transfer therapy with Tc1.

Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity

S3I-201 (NSC 74859) is a chemical probe inhibitor of Stat3 activity, which was identified from the National Cancer Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3beta homodimer. S3I-201 inhibits Stat3.Stat3 complex formation and Stat3 DNA-binding and transcriptional activities. Furthermore, S3I-201 inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3. Constitutively dimerized and active Stat3C and Stat3 SH2 domain rescue tumor cells from S3I-201-induced apoptosis. Finally, S3I-201 inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin and inhibits the growth of human breast tumors in vivo. These findings strongly suggest that the antitumor activity of S3I-201 is mediated in part through inhibition of aberrant Stat3 activation and provide the proof-of-concept for the potential clinical use of Stat3 inhibitors such as S3I-201 in tumors harboring aberrant Stat3.

SD-1029 Inhibits Signal Transducer and Activator of Transcription 3 Nuclear Translocation

Signal transducer and activator of transcription 3 (Stat3) proteins have important roles in cancer cell survival and proliferation. Recent studies show that aberrant Stat3 activation promotes tumor growth and survival in several human cancers, and thus, presents an attractive pathway for the development of targeted anticancer therapy. Stat3 is a DNA-binding transcription factor, and thus, its function depends on cytoplasmic to nuclear translocation. To discover novel inhibitors of the Stat3 signaling pathway, we designed a cell-based screening assay capable of identifying compounds that inhibit Stat3 nuclear translocation and activity. Cell-based fluorescence microscope screening and quantitative measurement of enhanced green fluorescent protein-Stat3 nuclear translocation assays were used to identify novel Stat3 inhibitors. The effects of identified Stat3 inhibitors on Janus kinase (Jak), Stat3 expression, and activation were determined by Western blotting and kinase in vitro autophosphorylation assay. The effects of identified Stat3 inhibitors on cell growth was evaluated by cell proliferation assay and apoptosis assay. Among the National Cancer Institute Diversity set, a 2,000-member library of bioactive small molecules, we identified SD-1029 as a micromolar inhibitor of IL-6 or oncostatin-induced Stat3 nuclear translocation. Biochemical analysis shows that SD-1029 inhibits tyrosyl phosphorylation of Stat3 implicating SD-1029 as an inhibitor of Jak. Further analysis shows that this compound inhibits tyrosyl phosphorylation of the Jak2 isoenzyme. The antiapoptotic proteins Bcl-X(L) and survivin, target proteins of activated Stat3, are down-regulated by SD-1029 resulting in the induction of apoptosis in several human breast and ovarian cancer cell lines. SD-1029 also enhances apoptosis induced by paclitaxel in ovarian cancer cells. These results show that SD-1029 directly abrogates the Jak-Stat3 signaling pathway in human cancer cells expressing constitutively active Stat, and add to the growing literature that validates this pathway as a viable target for further drug development. Finally, SD-1029 may represent a suitable prototype for structural optimization and exploration as a therapeutic lead.

Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein

Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G0-G1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-xL and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.

Unique Structural Determinants for Stat3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor at Phosphotyrosine Ligands 704 and 744

G-CSFR cytoplasmic tyrosine (Y) residues (Y704, Y729, Y744, and Y764) become phosphorylated upon ligand binding and recruit specific Src homology 2 domain-containing proteins that link to distinct yet overlapping programs for myeloid cell survival, differentiation, proliferation, and activation. The structural basis for recruitment specificity is poorly understood but could be exploited to selectively target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation demonstrated in a subset of acute myeloid leukemia patients with poor prognosis. Recombinant Stat3 bound to G-CSFR phosphotyrosine peptide ligands pY704VLQ and pY744LRC with similar kinetics. Testing of three models for Stat3 Src homology 2-pY ligand binding in vitro and in vivo revealed unique determinants for Stat3 recruitment and activation by the G-CSFR, the side chain of Stat3 R609, which interacts with the pY ligand phosphate group, and the peptide amide hydrogen of E638, which bonds with oxygen/sulfur within the + 3 Q/C side chain of the pY ligand when it assumes a beta turn. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of this interaction that can be exploited to target Stat3 activation for the treatment of a subset of acute myeloid leukemia patients.

Aberrant Stat3 Signaling by Interleukin-4 in Malignant Glioma Cells: Involvement of IL-13Rα2

Interleukin (IL)-4 exhibits antitumor activity in rodent experimental gliomas, which is likely mediated by the actions of IL-4 on a variety of immune cells present in and around the tumor masses. Here, we show that IL-4, which activates Stat6 in normal human astrocytes and in a variety of other cells, induces an aberrant activation of Stat3 in glioblastoma multiforme (GBM) cells but not in normal human astrocytes. Previously, we have shown that autocrine IL-6 signaling induces a persistent activation of Stat3. Now, we show that Stat3 is further activated by IL-4 stimulation of GBM cells. Expression of IL-13Ralpha2, a decoy receptor for IL-13 that partly blocks IL-4-mediated activation of Stat6 in GBM cells, up-regulates the activation of Stat3 as shown by a small interfering RNA-mediated inhibition of IL-13Ralpha2 expression. In addition, transient expression of the IL-13Ralpha2 transgene in 293T cells increases the IL-4-mediated activation of Stat3 and subsequent expression of Stat3-targeted gene. Coimmunoprecipitation results reveal that IL-13Ralpha2-mediated activation of Stat3 does not require a direct physical interaction between Stat3 and IL-13Ralpha2. Chromatin immunoprecipitation assay employing anti-Stat3 antibody confirms the in vivo binding of activated Stat3 to the promoters of genes that encode antiapoptotic proteins Bcl-2, Bcl-x(L), and Mcl-1. IL-4 significantly up-regulates of the steady-state levels of Bcl-2, Bcl-x(L), and Mcl-1 in GBM cells. These results indicate that IL-4/IL-13 receptor-mediated Stat3 signaling may contribute to the pathogenesis of GBM cells by modulating the expression of the Bcl-2 family of antiapoptotic proteins.

A SOCS-1 peptide mimetic inhibits both constitutive and IL-6 induced activation of STAT3 in prostate cancer cells

Prostate cancer is the second highest cause of cancer-related deaths of men in the US. Signal transducers and activators of transcription (STATs) proteins are a small family of latent cytoplasmic transcription factors that act downstream of Janus kinase (JAK) activation and mediate intracellular signaling from a wide variety of cytokines, growth factors, and hormones. Aberrant activation of STAT3 has been implicated in the progression of many human carcinomas, including prostate cancer. Previously, we have characterized a novel tyrosine kinase inhibitor peptide, Tkip, that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1). Similar to SOCS-1, Tkip binds to the autophosphorylation site of JAK2 and inhibits phosphorylation of STAT1alpha. In this study, we determined the inhibitory effects of Tkip on the human prostate cancer cell lines DU145 and LNCaP. Tkip inhibited cellular proliferation of both DU145 and LNCaP cells, with a slightly greater antiproliferative effect on DU145 cells. Cell cycle analysis using flow cytometry showed Tkip blockage of progression into the S phase of the cell cycle. Tkip also inhibited constitutive (DU145) and IL-6-induced (LNCaP) activation of STAT3, consistent with the fact that STAT3 activation is mediated by JAK2. Tkip also slightly reduced the levels of cyclin D1, an important regulator of cell cycle progression into S phase, in DU145 and LNCaP cancer cell lines. These data describe a potentially important therapeutic that targets both constitutive and IL-6-induced STAT3 activation in human prostate cancer cell lines.

Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent antitumor activity

DNA-alkylating agents that are platinum complexes induce apoptotic responses and have wide application in cancer therapy. The potential for platinum compounds to modulate signal transduction events that contribute to their therapeutic outcome has not been extensively examined. Among the signal transducer and activator of transcription (STAT) proteins, Stat3 activity is frequently up-regulated in many human tumors. Various lines of evidence have established a causal role for aberrant Stat3 activity in malignant transformation and provided validation for its targeting in the development of small-molecule inhibitors as novel cancer therapeutics. We report here that platinum-containing compounds disrupt Stat3 signaling and suppress its biological functions. The novel platinum (IV) compounds, CPA-1, CPA-7, and platinum (IV) tetrachloride block Stat3 activity in vitro at low micromolar concentrations. In malignant cells that harbor constitutively activated Stat3, CPA-1, CPA-7, and platinum (IV) tetrachloride inhibit cell growth and induce apoptosis in a manner that reflects the attenuation of persistent Stat3 activity. By contrast, cells that do not contain persistent Stat3 activity are marginally affected or are not affected by these compounds. Moreover, CPA-7 induces the regression of mouse CT26 colon tumor, which correlates with the abrogation of persistent Stat3 activity in tumors. Thus, the modulation of oncogenic signal transduction pathways, such as Stat3, may be one of the key molecular mechanisms for the antitumor effects of platinum (IV)-containing complexes.

Structural Determinants for Signal Transducer and Activator of Transcription (STAT) 3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) at Phosphotyrosine Ligands 704 and 744.

Four tyrosine (Y) residues within the cytoplasmic domain of the G-CSFR (Y704, Y228, Y744 and Y764 in the human receptor; Y703, Y227, Y743 and Y763 in the murine receptor) become phosphorylated by Jak kinases upon ligand binding leading to recruitment of Src homology (SH) 2 domain-containing proteins that link to programs for myeloid cell survival and differentiation (Stat3 recruitment to Y704 and Y744) and proliferation (SHP-2 and PI3K recruitment to Y04; Grb2, Shc and SHP-2 recruitment to Y764). While the preference of SH2 domain binding to specific phospho (p) Y peptide ligands was shown to map to the three residues immediately C-terminal to the pY (+1, +2, +3 residues), the structural basis for these preferences is poorly understood but could be exploited to specifically target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation, which has been demonstrated in a subset of acute myelogenous leukemia (AML) patients whose cells contain Flt3 internal tandem duplications and who suffer relapse following initial chemotherapy. To establish the structural basis for Stat3 recruitment and activation by the G-CSFR at Y704 and Y744, we generated purified recombinant full-length Stat3 and phosphododecapeptides based on the sequence surrounding each Y within the G-CSFR. In peptide pull-down assays, recombinant Stat3 bound only to Y704 and Y744 phosphododecapeptide, which contain core pY motifs consisting of pYVLQ and pYLRC, respectively. In mirror resonance affinity assays employed to obtain quantitative binding information, Stat3 bound to each phosphododecapeptide with similar kinetics (e.g. KDs = 0.703 and 0.95 μM, respectively). We tested three models for Stat3 SH2-pY ligand binding proposed by us and others using wild type and mutant recombinant Stat3 proteins in peptide pull-down and mirror resonance affinity assays along with computer modeling of this interaction using the known structures of Statβ SH2 and EGFR pY ligand (EpY1068INQ). Our results revealed loss of binding of Stat3 to Y704 and Y744 phosphododecapeptides only in Stat3 mutated within the SH2 domain at K591 or R609, whose side chains interacted with the pY phosphate group, and in Stat3 mutated within the SH2 domain at E638, whose amide hydrogen bonded with oxygen within the +3 Q side chain (or with sulfur within the +3 C side chain) when the pY ligand assumes a β turn. G-CSF stimulation of cells co-expressing full-length G-CSFR and either wild type or mutant Stat3 constructs confirmed the requirements for the side chain of R609 and the amide hydrogen of E638 within the Stat3 SH2 domain for binding to the G-CSFR and subsequent phosphorylation of Stat3 on Y705. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of their interaction at Y704 and Y744 i.e. a β turn within the receptor pY motif and a key hydrogen bond formed between the polar side chain of the +3 residue and the amide hydrogen of E638 within the Stat3 SH2 domain. These features explain the preference of the Stat3 SH2 domain for pY peptide ligands with +3 Q or C as well as +3 T (pY705LKT within Stat3) and +3 S (pY743IRS within the murine G-CSFR) and can be exploited using a structure-assisted drug design strategy to develop new therapies for a subset of AML patients with poor prognosis whose cells demonstrate aberrant activation of Stat3.