A cell-permeable Stat3 SH2 domain mimetic inhibits Stat3 activation and induces antitumor cell effects in vitro

Abstract

Given the role of constitutively active Signal Transducer and Activator of Transcription (Stat) 3 in human tumors, Stat3 inhibitors would be useful as novel therapeutics and as tools for probing Stat3-mediated tumor processes. We herein report that a 28-mer peptide, SPI, derived from the Stat3 SH2 domain, replicates Stat3 biochemical properties. Studies show SPI and Stat3 (or Stat3 SH2 domain) bind with similar affinities to known Stat3-binding phosphotyrosine (pY) peptide motifs, including those of the epidermal growth factor receptor (EGFR) and the high-affinity, IL-6R/gp130-derived pY-peptide, GpYLPQTV-NH(2). Consequently, SPI functions as a potent and selective inhibitor of Stat3 SH2 domain:pTyr interactions and disrupts the binding of Stat3 to the IL-6R/gp130 peptide, GpYLPQTV-NH(2). Fluorescence imaging and immunofluorescence staining/laser-scanning confocal microscopy show SPI is cell membrane-permeable, associates with the cytoplasmic tail of EGFR in NIH3T3/hEGFR, and is present in the cytoplasm, but strongly localized at the plasma membrane and in the nucleus in malignant cells harboring persistently active Stat3. Moreover, SPI specifically blocks constitutive Stat3 phosphorylation, DNA binding activity, and transcriptional function in malignant cells, with little or no effect on the induction of Stat1, Stat5, and Erk1/2(MAPK) pathways, or on general pTyr profile at the concentrations that inhibit Stat3 activity. Significantly, treatment with SPI of human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring constitutively active Stat3 induced extensive morphology changes, associated with viability loss and apoptosis. Our study identifies SPI as a novel molecular probe for interrogating Stat3 signaling and that functions as a selective inhibitor of Stat3 activation with antitumor cell effects.

FIGURE 1.

Computer modeling of Stat3 or the Stat3 SH2 domain peptide (SPI) and SPI amino acid sequence. A, the spatial presentation of the 28-mer peptide (28-mer, green overlay) within the context of the three-dimensional structure of Stat3 (red), per the x-ray crystal structure of DNA-bound Stat3β. B, structure of SPI, as modeled using ModWeb (University of California, San Francisco (UCSF)) and viewed by UCSF Chimera software. Model Score, 0.87; and C, amino acid sequence for residues 588–615 of the Stat3 SH2 domain.

FIGURE 2.

SPR analysis. SPR analysis of the binding of SPI (A), Stat3 SH2 domain (B), or full-length Stat3 (C) to GpYLTQTV-NH₂ (high affinity IL-6R/gp-130 peptide), PpYLKTK (Stat3 pTyr peptide), GpYIKTE (Stat1 pTyr peptide), GpYVKPQ (Stat5 pTyr peptide), PEpYINQS (EGFR pTyr peptide), PVpYHNQP (EGFR pTyr peptide), and S3I-201 (Stat3 dimerization inhibitor). Data are representative of three independent determinations.

FIGURE 3.

FP assay. FP assay of the binding to the 5-carboxyfluorescein-GpYLPQTV-NH₂ probe of (A) increasing concentration of (i) purified His-Stat3 or (ii) SPI; (B) a fixed amount of (i) purified His-Stat3 (200 nm) or (ii) SPI (150 μm) in the presence of increasing concentrations of S3I-201; or (C) fixed amount of Stat3 (0.8 μm) in the presence of increasing concentrations of SPI. Data are representative of four independent determinations.

FIGURE 4.

Intracellular uptake and effect of SPI on Stat3 activation. A, phase-contrast and fluorescence imaging of MDA-MB-231 cells following treatment with 30 μm fluorescently labeled (right two panels) or unlabeled (left two panels) SPI for 3 h; Image was captured using Zeiss Axiovert 200 microscope; B, cytosolic extracts of equal total protein were prepared from 24-h SPI-treated or untreated (i) NIH3T3/v-Src/pLucTKS3 fibroblasts that stably express the Stat3-dependent luciferase reporter (pLucTKS3), (ii) NIH3T3/v-Src/pLucSRE that stably express the Stat3-independent luciferase reporter (pLucSRE), or (iii) NIH3T3/hEGFR transiently transfected with β-casein promoter-driven luciferase report (β-casein-Luc) and stimulated with EGF and analyzed for luciferase activity using a luminometer; (C) nuclear extracts of equal total protein were prepared from (i) the designated malignant cells or (ii) EGF-stimulated NIH3T3/hEGFR treated for 24 h with or without SPI and subjected to in vitro DNA binding assay using the radiolabeled hSIE probe (i) and (ii, left panel) that binds Stat1 and Stat3 or MGFe probe (ii, right panel) that binds Stat1 and Stat5 and analyzed by EMSA; and (D) SDS-PAGE and Western blotting analysis of whole cell lysates of equal total protein prepared from SPI-treated or untreated NIH3T3/v-Src and MDA-MB-231 cells and probing for (i) pY705Stat3, Stat3, pErk1/2, and Erk1/2, or (ii) general pY profile. Positions of STATs:DNA complexes or proteins in gel are labeled; control lanes (0) represent cytosolic or nuclear extracts, or whole cell lysates prepared from 0.05% DMSO-treated cells. Data are representative of 3–4 independent determinations or mean and S.D. of 3 independent determinations. *, p < 0.05; **, p < 0.01, and ***, p < 0.005.

FIGURE 5.

Studies of colocalization of SPI with EGF receptor or Stat3 and the effect on Stat3 activation. A, fluorescence imaging of MDA-MB-231 cells treated with fluorescently labeled SPI (green; 30 μm, 3 h), fixed, and stained with rabbit anti-EGFR antibody (red) or DAPI nuclear staining (blue), or (B) immunofluorescence with laser-scanning confocal microscopy of unstimulated (−EGF) or EGF-stimulated (+EGF, 10 ng/ml, 10 min) NIH3T3/hEGFR fibroblasts growing in culture and pretreated with fluorescently labeled SPI (green; 30 μm, 3 h), fixed and stained with (i) rabbit anti-EGFR antibody (red) or (ii) rabbit anti-Stat3 antibody (red), or DAPI nuclear staining (blue). Images were captured using Zeiss Axiovert 200 fluorescent microscope or Leica TCS SP5 laser-scanning confocal microscope; or (C) Nuclear extracts were prepared from MDA-MB-231 cells pretreated with or without SPI prior to treatment with or without sodium orthovanadate and subjected to in vitro DNA binding assay using the radiolabeled hSIE probe and analyzed by EMSA. Positions of STATs:DNA complexes in gel are labeled; control (−), no treatment. Data are representative of three independent studies.

FIGURE 6.

SPI induces morphology change, suppresses viability, and induces apoptosis of malignant cells that harbor persistently active Stat3. A, photomicrographs of human breast (MDA-MB-231, MDA-MB-435, and MCF-7), prostate (DU145 and LNCaP), non-small cell lung (A549), and pancreatic (Colo-357) cancer cells, murine thymus stromal epithelial (TE-71) cells, and normal mouse fibroblasts (NIH3T3) in culture were treated once or untreated with 50 μm SPI for 24 h; (B) indicated cells were untreated or treated once with increasing concentration of SPI for 24 h and assayed for viability using CyQuant cell proliferation kit; or (C) human breast cancer cells (MDA-MB-231) or normal mouse fibroblasts (NIH3T3) in culture were untreated (control) or treated once with SPI (40 μm, 24 h) and subjected to Annexin V and 7-AAD binding and analyzed by flow cytometry for apoptosis. Data are representative of 3–4 independent determinations.