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. 2011 Dec;10(12):2384-93.
doi: 10.1158/1535-7163.MCT-11-0480. Epub 2011 Oct 25.

Therapeutic potential of AZD1480 for the treatment of human glioblastoma

Braden C McFarland  1 , Jing-Yuan Ma, Catherine P Langford, G Yancey Gillespie, Hao Yu, Ying Zheng, Susan E Nozell, Dennis Huszar, Etty N Benveniste
Affiliations

Therapeutic potential of AZD1480 for the treatment of human glioblastoma

Braden C McFarland et al. Mol Cancer Ther. 2011 Dec.

Abstract

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in glioblastoma (GBM) progression VSports手机版. To develop a therapeutic strategy to inhibit STAT-3 signaling, we have evaluated the effects of AZD1480, a pharmacologic inhibitor of JAK1 and JAK2. In this study, the in vitro efficacy of AZD1480 was tested in human and murine glioma cell lines. AZD1480 treatment effectively blocks constitutive and stimulus-induced JAK1, JAK2, and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis. Furthermore, we used human xenograft GBM samples as models for the study of JAK/STAT-3 signaling in vivo, because human GBM samples propagated as xenografts in nude mice retain both the hallmark genetic alterations and the invasive phenotype seen in vivo. In these xenograft tumors, JAK2 and STAT-3 are constitutively active, but levels vary among tumors, which is consistent with the heterogeneity of GBMs. AZD1480 inhibits constitutive and stimulus-induced phosphorylation of JAK2 and STAT-3 in these GBM xenograft tumors in vitro, downstream gene expression, and inhibits cell proliferation. Furthermore, AZD1480 suppresses STAT-3 activation in the glioma-initiating cell population in GBM tumors. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial GBM tumors by inhibiting STAT-3 activity, indicating that pharmacologic inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors. .

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Conflict of interest statement

Conflict-of-interest disclosure: The following authors declare no conflicts of interest: B. C. M. , J-Y. M V体育安卓版. , C. P. L. , G. Y. G. , H. Y. , Y. Z. , and S. E. N. E. N. B. is a scientific advisor for The Sontag Foundation and D. H. is a full-time employee of AstraZeneca, and holds company stock.

"VSports手机版" Figures

Figure 1
Figure 1. AZD1480 Inhibits JAK/STAT-3 Signaling in Glioma Cells
A, Cells were treated with AZD1480 for the indicated times, lysed and immunoblotted with the indicated antibodies. B, Cells were treated with AZD1480 for the indicated times, and the WST-1 cell proliferation assay performed. The 48 and 72 h time points were statistically significant. Data represent mean ± SD, replicates of three (**, p<0.001; ANOVA). C, U251-MG cells were treated with AZD1480 for 48 h followed by Annexin V/PI staining and examined by flow cytometry. Data represent mean ± SD, replicates of three (*, p<0.05; **, p<0.001; ANOVA). Representative of two experiments. D, U251-MG cells were treated with AZD1480 for the indicated times, lysed and immunoblotted with the indicated antibodies. E, U251-MG cells were plated in 0.4% soft agar without or with AZD1480. Data represent mean ± SD (**, p<0.001; ANOVA). Representative of three experiments.
Figure 2
Figure 2. AZD1480 Inhibits Stimulus-induced Phosphorylation of JAK1, JAK2 and STAT-3 and Prevents Downstream Gene Transcription
A, Cells were treated with the indicated doses of AZD1480 for 2 h prior to a 15 min stimulation with OSM (1 ng/ml). Cells were lysed and immunoblotted with the indicated antibodies. B, U251-MG cells were treated with 1 µM AZD1480 for 2 h prior to stimulation with OSM (1 ng/ml) for 1 h. Quantitative RT-PCR was performed as described in the Methods. Data represent mean ± SD, replicates of three (**, p<0.001, Student’s t-test). Representative of two experiments.
Figure 3
Figure 3. Human Xenograft GBM Tumors Exhibit Constitutive JAK2/STAT-3 Activation
A&B, Fresh snap frozen xenograft tumor samples were lysed and immunoblotted with the indicated antibodies (A) or analyzed for phosphorylated JAK2 levels using ELISA (B).
Figure 4
Figure 4. AZD1480 Inhibits JAK/STAT-3 Signaling in Human Glioblastoma Xenograft Tumors In Vitro
A, Xenograft X1066 was disaggregated into single cells and treated with AZD1480 for the indicated times, or treated with the indicated doses for 2 h prior to stimulation with OSM (1 ng/ml) for 15 min. Cells were lysed and immunoblotted with the indicated antibodies. B, Xenograft X1016 cells were disaggregated into single cells, treated with 1 µM AZD1480 for 2 h prior to stimulation with OSM (1 ng/ml) for 1 h, and quantitative RT-PCR performed. Data represent mean ± SD, replicates of three (*, p<0.05; **, p<0.01 Student’s t-test). C, Xenograft X1016 was disaggregated into single cells, treated with AZD1480 for the indicated times, and the WST-1 cell proliferation assay was performed. The 48 and 72 h time points were statistically significant. Data represent mean ± SD, replicates of three (*, p< 0.01; **, p<0.001 ANOVA). D, Xenograft X1066 was disaggregated into single cells and separated based on cell surface expression of CD133. Separated cells were then treated with AZD1480 (1 µM) for 2 h followed by treatment with OSM (10 ng/ml) for 15 min, lysed and immunoblotted with the indicated antibodies.
Figure 5
Figure 5. AZD1480 Inhibits In Vivo Growth of Subcutaneous Xenograft GBM Tumors by Inhibiting STAT-3 Activity
A, Xenograft tumor X1046 was removed, disaggregated, roughly minced, and injected subcutaneously into athymic nude mice. Data represent mean ± SEM (*, p<0.05, Student’s t-test at day 29). B&C, Frozen tumor samples were homogenized, and 30 µg was immunoblotted with the indicated antibodies. D, Xenograft tumor X1066 was injected subcutaneously into athymic nude mice as described in (A). Data represent mean ± SEM (**, p<0.01, Student’s t-test at day 21). E, Excised flank tumors were weighed for analysis. Data represent mean ± SD (*, p<0.05, Student’s t-test). F, RNA was isolated from frozen flank tumor samples, followed by generation of cDNA, and quantitative RT-PCR. Data represent mean of group ± SD (*, p<0.01 Mann-Whitney Rank Sum test).
Figure 6
Figure 6. The JAK Inhibitor AZD1480 Increases Survival of Mice Bearing Intracranial Xenograft GBM Tumors
A&B, Xenograft tumor X1046 (A) or X1016 (B) was removed, disaggregated into single cells and injected intracranially into nude mice. Survival was measured and mice were euthanized upon moribund or by 180 days.
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