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

Abstract

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.

Figure 1

(A and B), Structures of (A) S3I-201, (B) S3I-201.1066; (C and D), GOLD docking of (C) S3I-201 (green), and (D) S3I-201 (green) and S3I-201.1066 (yellow) to the SH2 domain of Stat3; arrow denotes potential binding sub-pocket accessed by S3I-201.1066, but not S3I-201.

Figure 2. Effects of S3I-201.1066 on the activities of STATs, Src, Shc, and Erks

(A) Nuclear extracts of equal total protein containing activated Stat1, Stat3, and/or Stat5 were pre-incubated with or without S3I-201.1066 for 30 min at room temperature prior to the incubation with the radiolabeled (i) and (ii) hSIE probe that binds Stat1 and Stat3 or the (iii) MGFe probe that binds Stat5 and subjecting to EMSA analysis; (B) Nuclear extracts of equal total protein prepared from the designated malignant cells following 24-h treatment with or without S3I-201.1066 were subjected to in vitro DNA-binding assay using the radiolabeled hSIE probe and analyzed by EMSA; (C) Cytosolic extracts of equal total protein were prepared from 24 h, S3I-201.1066-treated or untreated NIH3T3/v-Src fibroblasts that stably express the Stat3-dependent luciferase reporter (pLucTKS3) or from treated or untreated NIH3T3/v-Src fibroblasts, the human pancreatic (Panc-1) and breast (MDA-MB-231) carcinoma lines that are transiently-transfected with pLucSRE or pLucTKS3 and analyzed for luciferase activity using a luminometer; and (D) SDS-PAGE and Western blotting analysis of whole-cell lysates of equal total protein prepared from S3I-201.1066-treated or untreated NIH3T3/v-Src, Panc-1 and MDA-MB-231 cells probing for pY705Stat3, Stat3, pY416Src, Src, pShc, Shc, pErk1/2 and Erk1/2. Positions of STATs:DNA complexes or proteins in gel are labeled; control lanes (0) represent nuclear extracts treated with 0.05% DMSO, or nuclear extracts or whole-cell lysates prepared from 0.05% DMSO-treated cells; luciferase activities were normalized to β-galactosidase activity. Data are representative of 3-4 independent determinations. **p - <0.05.

Figure 3. Studies of the interaction of S3I-201.1066 with Stat3 or the Stat3 SH2 domain

(A) EMSA analysis of in vitro binding activity of Stat3 to the radiolabeled hSIE probe using nuclear extracts containing activated Stat3 pre-incubated with 0 or 100 μM S3I-201.1066 in the presence or absence of 0-500 ng of purified His-tagged Stat3 SH2 domain; (B) Surface Plasmon Resonance analysis of the binding of (i) 0-5 μM GYLPQTV-NH₂ (unphosphorylated, gp-130 peptide), (ii) 0-5 μM GpYLTQTV-NH₂ (phosphorylated, high affinity gp-130 peptide), or (iii) 0-50 μM S3I-201.1066 (or 50 μM S3I-201.1066, insert) as the analyte to the purified His-tagged Stat3 protein immobilized on HisCap Sensor Chip; and (C) Fluorescence Polarization assay of the binding to the 5-carboxyfluorescein-GpYLPQTV-NH₂ probe of (i) an increasing concentration of purified His-Stat3, or (ii)-(iv) a fixed amount of purified His-Stat3 (150 nM) in the presence of increasing concentrations of (ii) GpYLPQTV-NH₂, (iii) GYLPQTV-NH₂ or (iv) S3I-201.1066. Stat3:DNA complexes in gel are shown, control (-) lane or zero (0) represent 0.05% DMSO. Data are representative of 2-4 independent determinations.

Figure 4. Effect of S3I-201.1066 on the colocalization or association of Stat3 with EGF receptor and on Stat3 nuclear translocation

(A) Immunofluorescence imaging/confocal microscopy of Stat3 colocalization with EGFR and Stat3 nuclear localization in EGF-stimulated (1 μg/ml; 10 min) NIH3T3/hEGFR pre-treated with or without 50 μM S3I-201.1066 for 30 min; or (B) Immunoblotting analysis of (i) EGFR immunecomplex (upper panel) or whole-cell lysates (lower panel) from S3I-201.1066-treated Panc-1 and MDA-MB-231 cells, or (ii) immunecomplexes of EGFR (upper panel) or Stat3 (lower panel) were treated with the indicated concentrations of S3I-201.1066, and subsequent immunecomplexes of EGFR or Stat3 were probed for EGFR, Stat3, Shc, Grb 2, or Erk1/2^MAPK. Data are representative of 3 independent studies.

Figure 5. S3I-201.1066 suppresses viability, survival, malignant transformation and migration of malignant cells that harbor persistently-active Stat3

(A and B) Human breast (MDA-MB-231), pancreatic (Panc-1), and ovarian (A2780S) cancer cells, the v-Src transformed mouse fibroblasts (NIH3T3/v-Src) and their v-Ras transformed counterparts (NIH3T3/v-Ras), the Stat3 null mouse embryonic fibroblasts (Stat3-/-), and the normal human pancreatic duct epithelial cells (HPDEC) were treated once or untreated with 30-100 μM S3I-201.1066 for 24-48 h. Cells were (A) assayed for viability using CyQuant cell proliferation kit; IC₅₀ values (bottom panel) were derived from graphical representation, or (B) allowed to culture until large colonies were visible, which were stained with crystal violet and enumerated; (C) Cells (NIH3T3/v-Src, NIH3T3/v-Ras, A2780S, MDA-MB-231, and Panc-1) growing in soft-agar suspension were treated with or without 30-100 μM S3I-201.1066 every 2-3 days until large colonies were visible, which were stained with crystal violet and enumerated; and (D) Cells (MDA-MB-231, Panc-1, NIH3T3/v-Src and NIH3T3/v-Ras in culture were wounded and treated with or without 50 μM S3I-201.1066 for 12 or 24 h and allowed to migrate into the denuded area in a wound healing assay. Cultures were visualized at 10X magnification by light microscopy and (i) cells that migrated into the denuded area counted and plotted against the concentration of S3I-201.1066 or (ii) cultures were photographed. Values are the mean and S.D. of 3-4 independent determinations, data are representative of 4 independent studies. p values, * - <0.05 , and ** - <0.01.

Figure 6. S3I-201.1066 suppresses c-Myc, Bcl-xL, Survivin, MMP-9 and VEGF expression in vitro and in vivo and inhibits growth of human breast tumor xenografts

(A) SDS-PAGE and Western blotting analysis of whole-cell lysates prepared from the human breast cancer, MDA-MB-231 and pancreatic cancer, Panc-1 cells, and the v-Src-transformed mouse fibroblasts (NIH3T3/v-Src) untreated (DMSO, control) or treated with 50 μM S3I-201.1066 for 24 h and probing with anti-Myc, Bcl-xL, MMP-9, Survivin, VEGF or β-Actin antibodies; (B) Human breast (MDA-MB-231) tumor-bearing mice were given S3I-201.1066 (3 mg kg⁻¹) or vehicle (0.1% DMSO in PBS) i.v. every 2 or 3 days. Tumor sizes, measured every 2 or 3 days, were converted to tumor volumes and plotted against treatment days; (C) Tumor tissue lysates prepared from extracted tumor tissues from one control (C) and three treated (T1-T3) mice were subjected to (i) Stat3 DNA-binding activity and EMSA analysis or (ii) immunoblotting analysis for pY705Stat3, Stat3, c-Myc, Bcl-xL, VEGF, and β-Actin. Positions of proteins in gel are shown. Data are representative of 2-3 independent determinations, values in parenthesis represent the band intensities for the samples from treated cells relative to the respective control (set at 1), data are the mean and S.D. from replicates of 12 tumor-bearing mice in each group. p values, *<0.05.