P roteins of the STAT (signal transducer and activator of transcription) family are activated in response to cytokines and growth factors and promote proliferation, survival, and other biological processes (1-3). STATs are activated by phosphorylation of a critical tyrosine residue, which is mediated by growth factor receptor tyrosine kinases, Janus kinases, or the Src family kinases. Upon tyrosine phosphorylation, dimers of STATs formed between two phosphorylated monomers translocate to the nucleus, bind to specific DNA-response elements in the promoters of target genes, and induce gene expression. Aberrant activity of one of the family members, Stat3, contributes to carcinogenesis and tumor progression by up-regulating gene expression and promoting dysregulated growth, survival, and angiogenesis and modulating immune responses (2-9).As a critical step in STAT activation (10), the dimerization between two STAT monomers presents an attractive target to abolish Stat3 DNA-binding and transcriptional activity and to inhibit Stat3 biological functions (11, 12). Stat3 dimerization relies on the reciprocal binding of the SH2 domain of one monomer to the Pro-pTyr-Leu-Lys-Thr-Lys sequence of the other Stat3 monomer. To pursue the development of inhibitors of Stat3 signaling, key structural information gleaned from the x-ray crystal structure of the Stat3␤ homodimer (13) was used in the computational modeling and automated docking of small molecules into the SH2 domain of a Stat3 monomer, relative to the bound native pTyr peptide, to identify binders of the Stat3 SH2 domain, and potentially disruptors of Stat3⅐Stat3 dimers (14, 15).Structure-based high-throughput virtual screening of the National Cancer Institute (NCI) chemical libraries identified the high-scoring compound NSC 74859 (resynthesized as a pure sample and named S3I-201), which selectively inhibits Stat3 DNA-binding activity in vitro with an IC 50 value of 86 Ϯ 33 M. Furthermore, S3I-201 induces growth inhibition and apoptosis of malignant cells in part by constitutively inhibiting active Stat3 and induces human breast tumor regression in xenograft models. ResultsComputational Modeling and Virtual Screening. Our computational modeling and virtual screening study used the GLIDE (Grid-based Ligand Docking from Energetics) software (16, 17) (available from Schrödinger, Portland, OR) for the docking simulations and relied on the x-ray crystal structure of the Stat3␤ homodimer bound to DNA (13) determined at 2.25-Å resolution (1BG1 in the Protein Data Bank). For the virtual screening, DNA was removed and only one of the two monomers was used (see Fig. 1). To validate the docking approach, the native pTyr (pY) peptide, APpYLKT, was extracted from the crystal structure of one of the monomers and docked to the other monomer, whereby GLIDE produced a docking mode that closely resembled the x-ray crystal structure (data not shown). Three-dimensional structures of compounds from the NCI's chemical libraries were downloaded from the NCI Developmental Therapeutics Progra...

Studies in the past few years have provided compelling evidence for the critical role of aberrant Signal Transducer and Activator of Transcription 3 (STAT3) in malignant transformation and tumorigenesis. Thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design. This review focuses on aberrant STAT3 and its role in promoting tumor cell survival and supporting the malignant phenotype. A brief evaluation of the current strategies targeting STAT3 for the development of novel anticancer agents against human tumors harboring constitutively active STAT3 will also be presented.

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 G 0 -G 1 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-x L 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.