Article Figures & Data
Figures
- Figure 1.
AZD1480 treatment reduces phosphorylated STAT3 levels and inhibits ovarian carcinoma cell migration and adhesion. A, human (A1847, OVCAR-5, and OVCAR-8) and murine (MOVCAR-5447 and MOVCAR-5009) ovarian carcinoma cells were treated with DMSO (vehicle) or increasing concentrations of AZD1480 (0.05, 0.1, 1, 5, 10 μmol/L) for 24 hours and protein lysates subjected to immunoblot analysis with antibodies recognizing pSTAT3Y705 and total STAT3. B, A1847, OVCAR-5, and OVCAR-8 cells were grown in the presence of DMSO (vehicle) or increasing concentrations of AZD1480 (0.05, 0.1, 0.5, 1, 5, 10 μmol/L) for 72 hours and cell viability was determined by CellTiter Blue Viability Assay. Data indicate the mean percentage viability calculated from triplicate samples from 3 independent experiments (±SEM). C, OVCAR-5 and OVCAR-8 cells were treated with 0, 0.5, 1, or 5 μmol/L AZD1480 for 48 hours and analyzed for the presence of Annexin V-PE+ cells and PARP cleavage. Data shown are the mean values (±SEM) from 3 independent experiments. Cleaved PARP levels were detected by immunoblot analysis. D, the effect of increasing concentrations of AZD1480 on cell proliferation was determined by exposure of A1847, OVCAR-5, and OVCAR-8 cells to 0, 0.1, 1, or 5 μmol/L AZD1480 for 6, 24, 48, and 72 hours. Cells were fixed and stained with crystal violet, and plates read on a spectrophotometer to determine the optical density (OD590 nm). Data are presented as the mean OD590 nm ± SEM (n = 3). E, chemotactic migration assays were performed to determine the effects of 1.0 μmol/L AZD1480 treatment on migration of A1847, OVCAR-5, and OVCAR-8 cells. F, the effects of 1.0 μmol/L AZD1480 treatment on ovarian carcinoma cell adhesion to fibronectin and type I collagen was determined. The bars depict the mean number of migrated or adherent cells ± SEM (n = 3). Statistical analysis for data collected from viability and apoptosis assays was performed using a one-way ANOVA test followed by a multiple comparison test; proliferation assay data were analyzed with 2-way ANOVA followed by the multiple comparison test; migration and adhesion data were analyzed with an unpaired t test. P < 0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
- Figure 2.
AZD1480 treatment inhibits ovarian tumor growth and ascites production in MISIIR-TAg mice. Spontaneous tumor development and growth in MISIIR-TAg mice was monitored by weekly MRI and drug treatment initiated when tumor volume reached about 50 mm3. Equal numbers (n = 17 mice per group) of MISIIR-TAg mice were treated with vehicle or 30 mg/kg AZD1480. A, representative images of MRI scans at baseline and after 7 weeks of treatment. Ovarian tumors are outlined in yellow dashed lines. Tumor volume calculated from MRI data for all mice at baseline (B) and final scans (C) before euthanasia. D, weekly MRI datasets were subjected to volumetrics analysis and slopes of log-transformed tumor growth rate calculated for each mouse. At necropsy, final tumor volume was determined by caliper measurements (E) and the presence or absence of malignant ascites was determined (F). The MRI data for tumor growth rate were analyzed by the Wilcoxon signed-rank test, final tumor volumes by the Mann–Whitney t test and ascites fraction by the Fisher exact 2-sided test. P < 0.05 was considered significant (***, P < 0.001; ****, P < 0.0001).
- Figure 3.
AZD1480-mediated tumor growth inhibition is accompanied by reduced STAT3 activation. A, gross images (a, a') of reproductive tracts and light microscopic images of H&E (b-d, b'-d') and TAg (e, e') stained sections of tumors from representative vehicle- (top) or AZD1480-treated (bottom) mice. Tumor tissue specimens from vehicle- and AZD1480-treated mice were harvested 6 hours after the last drug dose and analyzed by immunoblot (B) and electrochemiluminescent ELISA analysis (C) for detection and quantification of activating STAT3 phosphorylation at tyrosine 705 (pSTAT3Y705). ****, P < 0.0001.
- Figure 4.
Expression of STAT3 target genes is altered in AZD1480-treated ovarian tumors. TgMISIIR-TAg mice with about 500 mm3 ovarian tumors were treated with vehicle or 30 mg/kg AZD1480 (n = 4 mice per group), and tumors were harvested 6 hours after drug treatment. RNA was isolated from tumors and analyzed by genome-wide microarray analysis to determine the effects of drug treatment on gene transcription. A, heat map showing 10 upregulated and 87 downregulated genes (2-fold change and P < 0.01 cutoff) in AZD1480-treated tumors. B, qRT-PCR validation differential expression of selected genes. C, qRT-PCR amplification of potential normalizing genes Gusb and Hprt1. Quantity was normalized to Ppib. Error bars indicate ±SEM; data were analyzed using the Mann–Whitney t test. *, P < 0.05.
- Figure 5.
AZD1480 inhibits tumor-associated integrin αvβ3 expression and MMP activity. The tumor-associated integrin αvβ3 expression and MMP activity was detected by combined anatomic imaging (MRI) and FMT. Tumor-bearing mice were treated with vehicle or AZD1480 (n = 5 per group) for 4 weeks and imaged weekly by MRI to monitor tumor growth and FMT to monitor integrin αvβ3 probe binding and MMP probe activation. A, representative MRI images, IntegriSense probe binding, and MMPSense probe activation in ovarian tumors in vehicle- and AZD1480-treated mice. Quantification of tumor volume by MRI (B) and IntegriSense probe retention (C) and MMP probe activation (D) by FMT. (*, P < 0.05; **, P < 0.01).
- Figure 6.
AZD1480-mediated JAK/STAT3 inhibition reduces T-cell populations in the peritoneal TME. Cell suspensions were prepared from peritoneal washes and spleens harvested from vehicle (n = 12)- and AZD1480-treated (n = 19) mice and analyzed by flow cytometry. A, representative flow cytometric dot plots and pooled data showing the number and percent (normalized to CD45+ leukocytes) of CD45+CD4+ T cells in peritoneal cavity and spleen. B, representative flow cytometric dot plots and pooled data showing the absolute number and percent (normalized to CD45+ leukocytes) of CD45+CD4+FoxP3+ T cells in peritoneal cavity and spleen. Bars show mean ± SEM (vehicle, n = 10; AZD1480, n = 19). Statistical analysis is based on the Wilcoxon test. *, P < 0.05; **, P < 0.01.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures S1-S3 - Supplementary Figures S1-S3: Supplementary Figure S1. AZD1480 abrogates STAT3 DNA binding while targeting JAK2 or STAT3 has little effect on viability or proliferation in cultured cells. Supplementary Figure S2. Pharmacodynamics of AZD1480 treatment. Supplementary Figure S3. Flow cytometry analysis of immune cell subpopulations in tumors and peritoneal washes from vehicle- and AZD1480-treated mice.
- Supplemental Table 1 - Supplemental Table 1. Differentially expressed genes in AZD1480 treated tumors vs. vehicle treated controls (2-fold change and p<0.01 cutoff).
- Supplemental Table 2 - Supplemental Table 2. Gene ontology (GO) enrichment categories.
Volume 14, Issue 4
