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, 14 (4), 1035-47

Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth

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Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth

Galina Gritsina et al. Mol Cancer Ther.

Abstract

Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy.

Conflict of interest statement

Conflicts of interest: Dennis Huszar is an employee of AstraZeneca. The remaining authors have no conflicts of interest to declare.

Figures

Figure 1
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 AZD1480 (0.05, 0.1, 1, 5, 10 μmol/L) for 24 h 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 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 three 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, 0.5, 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 OC 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 one-way ANOVA test followed by multiple comparison test; proliferation assay data were analyzed with two-way ANOVA followed by multiple comparison test, migration and adhesion data were analyzed with unpaired t test. P values less <0.05 were considered significant (*p<0.05, **p<0.01, ***p <0.001, ****p<0.0001).
Figure 2
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 magnetic resonance imaging (MRI) and drug treatment initiated when tumor volume reached ∼50 mm3. Equal numbers (n=17 mice/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) prior to euthanasia. D) Weekly MRI datasets were subjected 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's exact two-sided test. P values less <0.05 were considered significant (***p <0.001, ****p<0.0001).
Figure 3
Figure 3. AZD1480-mediated tumor growth inhibition is accompanied by reduced STAT3 activation
(A) Gross images of reproductive tracts and light microscopic images of hematoxylin and eosin (H&E) and TAg stained sections of tumors from representative vehicle- (upper panels) or AZD1480-treated (lower panels) 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 electro chemiluminescent ELISA analysis (C) for detection and quantification of activating STAT3 phosphorylation at tyrosine 705 (pSTAT3Y705). ****p<0.0001.
Figure 4
Figure 4. Expression of STAT3 target genes is altered in AZD1480-treated ovarian tumors
TgMISIIR-TAg mice with ∼500 mm3 ovarian tumors were treated with vehicle or 30 mg/kg AZD1480 (n=4 mice/group) and tumors were harvested 6 hours after drug treatment. RNA was isolated from tumors and analyzed by genome wide microarray analysis 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 potenitial normalizing genes Gusb and Hprt1. Quantity was normalized to Ppib. (Error bars indicate ±SEM; data was analyzed using the Mann-Whitney t test, *p<0.05).
Figure 5
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 fluorescent molecular tomography (FMT). Tumor-bearing mice were treated with vehicle or AZD1480 (n=5/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
Figure 6. AZD1480-mediated JAK/STAT3 inhibition reduces T cell populations in the peritoneal tumor microenvironment
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 cytometry 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 cytometry 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 Wilcoxon test, *p<0.05, ** p <0.01.

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