Naijie Jing

Targeting Stat3 in cancer therapy.

Stat3 is constitutively activated in many human cancers where it functions as a critical mediator of oncogenic signaling through transcriptional activation of genes encoding apoptosis inhibitors (e.g. Bcl-x(L), Mcl-1 and survivin), cell-cycle regulators (e.g. cyclin D1 and c-Myc) and inducers of angiogenesis (e.g. vascular endothelial growth factor). This article reviews several approaches that have been pursued for targeting Stat3 in cancer therapy including antisense strategies, tyrosine kinase inhibition, decoy phosphopeptides, decoy duplex oligonucleotides and G-quartet oligodeoxynucleotides (GQ-ODN). The GQ-ODN strategy is reviewed in somewhat greater detail than the others because it includes a novel system that effectively delivers drug into cells and tissues, addresses successfully the issue of specificity of targeting Stat3 versus Stat1, and has demonstrated efficacy in vivo.

G-Quartet Oligonucleotides A New Class of Signal Transducer and Activator of Transcription 3 Inhibitors That Suppresses Growth of Prostate and Breast Tumors through Induction of Apoptosis

Stat3 is a signaling molecular and oncogene activated frequently in many human malignancies including the majority of prostate, breast, and head and neck cancers; yet, no current chemotherapeutic approach has been implemented clinically that specifically targets Stat3. We recently developed G-rich oligodeoxynucleotides, which form intramolecular G-quartet structures (GQ-ODN), as a new class of Stat3 inhibitor. GQ-ODN targeted Stat3 protein directly inhibiting its ability to bind DNA. When delivered into cells using polyethyleneimine as vehicle, GQ-ODN blocked ligand-induced Stat3 activation and Stat3-mediated transcription of antiapoptotic genes. To establish the effectiveness of GQ-ODN as a potential new chemotherapeutic agent, we systemically administered GQ-ODN (T40214 or T40231) plus polyethyleneimine or polyethyleneimine alone (placebo) by tail-vein injection into nude mice with prostate and breast tumor xenografts. Whereas the mean volume of breast tumor xenografts in placebo-treated mice increased >7-fold over 18 days, xenografts in the GQ-ODN-treated mice remained unchanged. Similarly, whereas the mean volume of prostate tumor xenografts in placebo-treated mice increased 9-fold over 10 days, xenografts in GQ-ODN-treated mice increased by only 2-fold. Biochemical examination of tumors from GQ-ODN-treated mice demonstrated a significant reduction in Stat3 activation, levels of the antiapoptotic proteins Bcl-2 and Bcl-xL, and an 8-fold increase in the number of apoptotic cells compared with the tumors of placebo-treated mice. Thus, GQ-ODN targeting Stat3 induces tumor cell apoptosis when delivered into tumor xenografts and represents a novel class of chemotherapeutic agents that holds promise for the systemic treatment of many forms of metastatic cancer.

Targeting Stat3 with G-Quartet Oligodeoxynucleotides in Human Cancer Cells

Stat3 is an oncogene that is activated in many human cancer cells. Genetic approaches that disrupt Stat3 activity result in inhibition of cancer cell growth and enhanced cell apoptosis supporting the development of novel drugs targeting Stat3 for cancer therapy. G-quartet oligodeoxynucleotides (ODNs) were demonstrated to be potent inhibitors of Stat3 DNA binding activity in vitro with the G-quartet ODN, T40214, having an IC(50) of 7 microM. Computer-simulated docking studies indicated that G-quartet ODNs mainly interacted with the SH2 domain of Stat3 and were capable of inserting between the SH2 domains of Stat3 dimers bound to DNA. We demonstrated that the G-rich ODN T40214, which forms a G-quartet structure at intracellular but not extracellular K+ ion concentrations, is delivered efficiently into the cytoplasm and nucleus of cancer cells where it inhibited IL-6-stimulated Stat3 activation and suppressed Stat3-mediated upregulation of bcl-x and mcl-1 gene expression. Thus, G-quartet represents a new class of drug for targeting of Stat3 within cancer cells.

Targeting signal transducer and activator of transcription 3 with G-quartet oligonucleotides: a potential novel therapy for head and neck cancer

Signal transducer and activator of transcription 3 (Stat3) is a critical mediator of oncogenic signaling activated frequently in many types of human cancer where it contributes to tumor cell growth and resistance to apoptosis. Stat3 has been proposed as a promising target for anticancer drug discovery. Recently, we developed a series of G-quartet oligodeoxynucleotides (GQ-ODN) as novel and potent Stat3 inhibitors, which significantly suppressed the growth of prostate and breast tumors in nude mice. In the present study, we showed that GQ-ODN specifically inhibited DNA-binding activity of Stat3 as opposed to Stat1. Computer-based docking analysis revealed that GQ-ODN predominantly interacts with the SH2 domains of Stat3 homodimers to destabilize dimer formation and disrupt DNA-binding activity. We employed five regimens in the treatment of nude mice with tumors of head and neck squamous cell carcinoma (HNSCC): placebo, paclitaxel, GQ-ODN T40214, GQ-ODN T40231, and T40214 plus paclitaxel. The mean size of HNSCC tumors over 21 days only increased by 1.7-fold in T40214-treated mice and actually decreased by 35% in T40214 plus paclitaxel–treated mice whereas the mean size of HNSCC tumors increased 9.4-fold in placebo-treated mice in the same period. These findings show that GQ-ODN has potent activity against HNSCC tumor xenografts alone and in combination with paclitaxel. [Mol Cancer Ther 2006;5(2):279–86]

Rational Drug Design of G-Quartet DNA as Anti-Cancer Agents

The ability of certain DNA sequences to form G-quartet structures has been exploited recently to develop novel anti-cancer agents including small molecules that promote G-quartet formation within the c-MYC promoter thereby repressing c-MYC transcription and introducing G-quartet-forming oligodeoxynucleotides (GQ-ODN) into cancer cells resulting in p53-dependent cell cycle arrest and inhibition of DNA replication. GQ-ODNs also have been developed as potent inhibitors of signal transducer and activator of transcription (STAT) 3, a critical mediator of oncogenic signaling in many cancers. This review summarizes the rational design of G-quartet forming DNA drugs as Stat3 inhibitors. Topics that are reviewed include the strategy of structure-based drug design, establishment of a structure-activity relationship, development of a novel intracellular delivery system for G-quartet-forming DNA agents and in vivo drug testing to assess the anti-cancer effects of DNA drugs in tumor xenografts. Results to date with GQ-ODN targeting Stat3 are encouraging, and it is hoped that continued pursuit of the methodology outlined here may lead to development of an effective agent for treatment of metastatic cancers, such as prostate and breast, in which Stat3 is constitutively activated.

Signal Transducer and Activator of Transcription 3 (STAT3) Regulates Collagen-Induced Platelet Aggregation Independently of Its Transcription Factor Activity

Background— Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results— The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLC&ggr;2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLC&ggr;2 was detected in HEK293 cells transfected with cDNAs for Syk and PLC&ggr;2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide–induced but not thrombin receptor–activating peptide– or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions— These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.Background —Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results —The signal transducers and activators of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was also detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2, and stimulated with interleukin-6 (IL-6). Pharmacological inhibition of STAT3 blocked ~50% of collagen- and a collagen-related peptide-, but not TRAP- or ADP-induced aggregation and ~80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine IL-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions —These data demonstrate a non-transcriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for platelet hyperactivity found in conditions of inflammation.

T40214/PEI complex: A potent therapeutics for prostate cancer that targets STAT3 signaling

Prostate cancer (PC) is the most common cancer among men in American and the second leading cause of cancer death. The treatment options employed for patients with advanced and metastatic PC are limited. As a critical mediator of oncogenic signaling, STAT3 is active in 82% of patients with PC. STAT3 has become a very important molecular target for PC therapy since it upregulates the oncogenes encoding apoptosis inhibitors, cell cycle regulators, and inducers of angiogenesis. However, no anti‐tumor drug whose primary mode of action is to target STAT3 has yet reached the clinic. To this end, we have laid the initial groundwork to develop the STAT3‐inhibiting G‐quartet oligodeoxynucleotide (GQ‐ODN), T40214, for treatment of PCs.

Combined treatment targeting HIF‐1α and Stat3 is a potent strategy for prostate cancer therapy

The Stat3 pathway and the hypoxia‐sensing pathway are both up‐regulated in prostate cancer. Stat3 is a specific regulator of pro‐carcinogenic inflammation and represents a promising therapeutic target. Hypoxia‐inducible factor‐1 (HIF‐1)α, which mediates the cellular response to hypoxia, has been demonstrated to be over‐expressed in many human cancers and is associated with poor prognosis and treatment failure in clinic. To develop a potent strategy to increase therapeutic efficacy and reduce drug resistance in prostate cancer therapy, we combined two anti‐cancer agents: T40214 (a p‐Stat3 inhibitor) and JG244 (a HIF‐1α inhibitor) together to treat nude mice bearing human prostate tumor (DU145) and immunocompetent mice (C57BL/6) bearing murine prostate tumor (TRAMP‐C2).

G-rich Oligonucleotides Inhibit HIF-1α and HIF-2α and Block Tumor Growth

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.

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 β 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.