Peibin Yue

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.

Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts

Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity (KD) of 504 nM, blocks Stat3–phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4–6.8 μM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102–mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3–NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.

A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes

The molecular modeling of the phosphotyrosine (pTyr)-SH2 domain interaction in the Stat3:Stat3 dimerization, combined with in silico structural analysis of the Stat3 dimerization disruptor, S3I-201, has furnished a diverse set of analogs. We present evidence from in vitro biochemical and biophysical studies that the structural analog, S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity (K(D)) of 2.74microM, and disrupts the binding of Stat3 to the cognate pTyr-peptide, GpYLPQTV-NH(2), with an IC(50) of 23microM. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2(MAPK) pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 of malignant cells harboring aberrantly active Stat3 down-regulated the expression of c-Myc, Bcl-xL, Survivin, the matrix metalloproteinase 9, and VEGF. The in vivo administration of S3I-201.1066-induced significant antitumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Our studies identify a novel small-molecule that binds with a high affinity to Stat3, blocks Stat3 activation and function, and thereby induces antitumor response in human breast tumor xenografts harboring persistently active Stat3.

A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

Evidence is presented for the nuclear presence of a functional heteromeric complex of epidermal growth factor (EGFR), Src and the Signal Transducer and Activator of Transcription (Stat)3 proteins in pancreatic cancer cells. Stat3 remains nuclear and associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src, demonstrating the resistance of the complex to the modulation of EGFR or Src alone. Significantly, chromatin immunoprecipitation (ChIP) analyses reveal the nuclear EGFR, Src and Stat3 complex is bound to the c-Myc promoter. The siRNA knockdown of EGFR or Src, or the pharmacological inhibition of Stat3 activity only marginally suppressed c-Myc expression. By contrast, the concurrent modulation of Stat3 and EGFR, or Stat3 and Src, or EGFR and Src strongly suppressed c-Myc expression, demonstrating that the novel nuclear heteromeric complex intricately regulates the c-Myc gene. The prevalence of the transcriptionally functional EGFR, Src, and Stat3 nuclear complex provides an additional and novel mechanism for supporting the pancreatic cancer phenotype and explains in part the insensitivity of pancreatic cancer cells to the inhibition of EGFR, Src or Stat3 alone.

Disruption of Transcriptionally Active Stat3 Dimers with Non‐phosphorylated, Salicylic Acid‐Based Small Molecules: Potent in vitro and Tumor Cell Activities

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that relays signals from receptors in the plasma membrane directly to the nucleus, and is routinely hyperactivated in many human cancers and diseases.[1] Regarded as an oncogene, Stat3 is well-recognized as a master regulator of cellular events that lead to the cancer phenotype, making this protein viable target for molecular therapeutic design.[2] Stat3 inhibitors have included peptides,[3–4] peptidomimetics,[5–9] small molecules[10–14] and metal complexes.[15] Despite significant advances in Stat3 inhibition,[1] truly potent (in vivo), isoform-selective, small molecule Stat3 agents have not been readily forthcoming; this is likely due in part to the challenge of disrupting protein–protein interactions.[16]

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.

Small Molecule STAT5-SH2 Domain Inhibitors Exhibit Potent Antileukemia Activity

A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i) < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.

Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that is aberrantly activated in numerous human cancers. Inhibitors of activated Stat3-Stat3 protein complexes have been shown to hold therapeutic promise for the treatment of human cancers harboring activated Stat3. Herein, we report the design and synthesis of a focused library of salicylic acid containing Stat3 SH2 domain binders. The most potent inhibitor, 17o, effectively disrupted Stat3-phosphopeptide complexes (K(i)=13 μM), inhibited Stat3-Stat3 protein interactions (IC(50)=19 μM) and silenced intracellular Stat3 phosphorylation and Stat3-target gene expression profiles. Inhibition of Stat3 function in both breast and multiple myeloma (MM) tumor cells correlated with induced cell death (EC(50)=10 and 16 μM, respectively).

Antagonism of the Stat3–Stat3 Protein Dimer with Salicylic Acid Based Small Molecules

More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure–activity relationship (SAR) study based on the previously identified inhibitor S3I‐201 (IC50=86 μM, Ki>300 μM). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC50 range of 18.7–51.9 μM, and disruption of Stat3–pTyr peptide interactions with Ki values in the 15.5–41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.

Enhanced sensitivity of pancreatic cancer cells to concurrent inhibition of aberrant signal transducer and activator of transcription 3 and epidermal growth factor receptor or Src.

Many molecular aberrations occur in pancreatic cancer. Although aberrant epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) are implicated in pancreatic cancer, therapies that target only one of these entities are undermined by signaling cross-talk. In the human pancreatic cancer lines, Panc-1 and Colo-357, pY845EGFR, pY1068EGFR, pY1086EGFR, and pY1173EGFR levels and pY416c-Src are concurrently elevated with aberrantly active Stat3 in a complex signaling cross-talk. Thus, understanding the signaling integration would facilitate the design of effective multiple-targeted therapeutic modalities. In Panc-1 and Colo-357 lines, pY845EGFR, pY1068EGFR, and pY1086EGFR levels are responsive to c-Src inhibition in contrast to pY1173EGFR, which is EGFR kinase-dependent. Constitutively active Stat3 is sensitive to both EGFR and Src inhibition, but the early suppression of aberrantly active Stat3 in response to the inhibition of EGFR and Src is countered by a Janus kinase (Jaks)-dependent reactivation, suggesting that Jaks activity is a compensatory mechanism for Stat3 induction. The inhibition of EGFR, Src, or Stat3 alone induced weak biological responses. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis and decreased the migration/invasion of pancreatic cancer cells in vitro. Significantly, the concurrent inhibition, compared with monotargeting modality, induced stronger human pancreatic tumor growth inhibition in xenografts. We infer that the tumor growth inhibition in vivo is caused by the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer.