Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma

Abstract

Purpose: Gliomas are the most frequently occurring primary malignancies in the brain, and glioblastoma is the most aggressive of these tumors. Protein kinase CK2 is composed of two catalytic subunits (α and/or α') and two β regulatory subunits. CK2 suppresses apoptosis, promotes neoangiogenesis, and enhances activation of the JAK/STAT, NF-κB, PI3K/AKT, Hsp90, Wnt, and Hedgehog pathways. Aberrant activation of the NF-κB, PI3K/AKT, and JAK/STAT-3 pathways is implicated in glioblastoma progression. As CK2 is involved in their activation, the expression and function of CK2 in glioblastoma was evaluated.

Experimental design and results: Analysis of 537 glioblastomas from The Cancer Genome Atlas Project demonstrates the CSNK2A1 gene, encoding CK2α, has gene dosage gains in glioblastoma (33.7%), and is significantly associated with the classical glioblastoma subtype. Inhibition of CK2 activity by CX-4945, a selective CK2 inhibitor, or CK2 knockdown by siRNA suppresses activation of the JAK/STAT, NF-κB, and AKT pathways and downstream gene expression in human glioblastoma xenografts. On a functional level, CX-4945 treatment decreases the adhesion and migration of glioblastoma cells, in part through inhibition of integrin β1 and α4 expression. In vivo, CX-4945 inhibits activation of STAT-3, NF-κB p65, and AKT, and promotes survival of mice with intracranial human glioblastoma xenografts.

Conclusions: CK2 inhibitors may be considered for treatment of patients with glioblastoma.

Figure 1. Gene dosage gain of CSNK2A1 in GBM

(A) Gene-level CNV analysis across chromosome 20 in 537 GBMs. Gene dosages are mapped according to gene order on chromosome 20. CSNK2A1 shows gene dosage gains in 33.7% of tumors (green line, CSNK2A1 locus on 20p13∥C). The RefSeq genes track shows coding gene locations. The proportions diagram shows the frequency of gene dosage gains along chromosome 20. Color intensity is proportional to the deviation from zero. Gene-dosage values indicate the log2 ratio of red (R, Cy5) to green (G, Cy3) intensity of the fluorescence dye (or log2R/G). (B) Gene dosage gain of CSNK2A1 is associated with significant gain of CSNK2A1 mRNA expression in 482 and 428 patients whose tumors were profiled on two different platforms at the Broad Institute (BI) and University of North Carolina at Chapel Hill (UNC), respectively. Values for gene dosage and gene expression are presented as log2R/G ratios. Locally weighted least squares (LOWESS) smooth fits (in red) confirmed the appropriateness of the linear regression models; p value indicates statistical significance according to estimated slope of the regression line. The corresponding box plots show the distribution of CSNK2A1 expression in tumors containing and lacking CSNK2A1 gains; p value calculated via Wilcoxon rank-sum test. (C) Gene-dosage profiles for CSNK2A1 across 490 GBMs along with its relationship to four molecular subtypes of GBM. A corresponding two-way contingency-table analysis reveals a significant association of CSNK2A1 gain with the classical subtype. CI denotes confidence interval.

Figure 2. Inhibition of CK2 Suppresses STAT Activation

(A) 20 μg of Xenografts X1016, X1046 and X1066, GBM lines U251-MG and U87-MG, and normal brain (NB) lysate were immunoblotted with indicated antibodies. (B) U251-MG cells were transfected with 100 nM of non-target, CK2α, CK2β or CK2α’ siRNAs for 48 h, then stimulated with 10 ng/ml of IL-6 and 25 ng/ml of sIL-6R for 10 min. (C-D) Xenograft X1066 was treated with CX-4945 in serum-free medium for 4 h, and then stimulated as above. Lysates were immunoblotted with indicated antibodies. (E) U251-MG cells were pretreated with CX-4945 in serum-free medium for 4 h, and then stimulated with OSM (5 ng/ml) for 1 h. mRNA was analyzed by qRT-PCR. *, p<0.05. (F) U251-MG cells were pretreated with CX-4945 in serum-free medium for 4 h, and then stimulated with EGF (50 ng/ml) for 10 min. (G) U251-MG cells were serum-starved overnight, treated with CX-4945 for 4 h, and stimulated with EGF for 1 h. mRNA was analyzed by qRT-PCR. *, p<0.05.

Figure 3. Inhibition of CK2 Suppresses NF-κB Activation

(A) U251-MG cells were transfected as described in Figure 2B, then stimulated with TNF-α (1 ng/ml) for 10 min. (B) X1066 was treated with CX-4945 for 4 h in serum-free medium, and then stimulated as above. (C) X1066 was treated with CX-4945 (10 μM) for 4 h in serum-free medium, and then stimulated with TNF-α for 1 h. mRNA was analyzed by qRT-PCR. *, p<0.05. (D) X1046 was pretreated with CX-4945 (10 μM) for 4 h in serum-free medium, and then stimulated with IL-1β for 10 min.

Figure 4. Inhibition of CK2 Suppresses Adhesion and Migration of GBM Cells

(A) X1016 was treated with CX-4945 (10 μM) for 16 h (upper panel), and U87-MG (middle panel) and U251-MG cells (lower panel) were treated for 12 h. Scale bar is 2 μm. (B) X1016 was treated with CX-4945 (10 μM) for 16 h, and stained with Alexa Fluor 488 phalloidin and DAPI. (C) U251-MG cells were transfected with 100 nM of non-target, CK2α or CK2α’ siRNAs, or CK2α (50 nM) plus CK2α’ (50 nM) siRNAs for 48 h, and stained with Alexa Fluor 488 phalloidin and DAPI. The scale bar is 14 μm. (D) X1016 cells were treated with CX-4945 (10 μM) for 16 h. mRNA was analyzed by qRT-PCR. *, p<0.05. (E) X1046 was treated with CX-4945, and lysates immunoblotted with indicated antibodies. (F) U251-MG cells were transfected with 100 nM of non-target, CK2α or CK2α’ siRNAs for 48 h, and lysates immunoblotted with indicated antibodies. (G) X1046 was treated with CX-4945 for 16 h, and seeded onto fibronectin-coated plates for 1 h. The cells were then fixed, stained with crystal violet, and absorbance measured at 540 nm. The normalized value of untreated cells was set to 1. *, p<0.001. (H) X1066 cells were incubated in the absence or presence of CX-4945 for 4 h, scratched with a p200 pipette tip, and images taken at 0 and 16 h later. The scale bar is 1 μm. (I) Images from the assay were quantified. Representative of three experiments (*, p<0.01; **, p<0.001).

Figure 5. Inhibition of CK2 Suppresses GBM Cell Survival

X1046 (A) and X1066 (B) cells were treated with CX-4945, and cell survival measured by the WST-1 assay. (C) U251-MG cells were transfected with 100 nM of non-target, CK2α or CK2α’ siRNAs, or CK2α (50 nM) plus CK2α’ (50 nM) siRNAs for 48 h, and survival determined by the WST-1 assay. Triplicate experiments, and error bars show ± S.D.. *, p<0.05. (D) U251-MG cells were treated with CX-4945 for 24 or 48 h. Cells were stained with Annexin V and propidium iodide and examined by flow cytometry. Triplicate experiments, and error bars show ± S.D.. *, p<0.05. (E) U251-MG cells were treated with CX-4945 for 24 h, fixed overnight, stained with propidium iodide and digested with RNase. The percentage of cells in the sub-G1, G1, S and G2/M phase was examined by flow cytometry.

Figure 6. CX-4945 Inhibits In Vivo Growth of Xenograft GBM Tumors

(A-D) Xenograft X1046 was injected subcutaneously into nude mice. Cages were randomized, and vehicle (n=4) or 75 mg/kg of CX-4945 (n=4) was administered twice a day by i.p. from days 3-5, and by oral gavage twice a day from day 6. Tumor size (A) and body weight (C) were measured on the indicated days. On day 40, all mice were euthanized. Data represent mean ± SEM. *, p<0.05; **, p<0.005; ***, p<0.001. (B) Tumors were homogenized, and lysates immunoblotted with indicated antibodies. (D) Blood from vehicle and CX-4945-treated mice was obtained by cheek bleeding before euthanasia on day 40, and analyzed with HEMAVET®950. The numbers of white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells and hemoglobin are shown in arbitrary units. n.s., not significant. (E) Five × 10⁵ cells/5 μl of Xenograft X1046 were injected intracranially. Cages were randomized, and vehicle (n=14) or 75 mg/kg of CX-4945 (n=15) was administered orally twice a day for 28 days starting at day 5. Survival was monitored and mice were euthanized upon moribund. Kaplan-Meier survival with LogRank analysis was performed. (F) Five × 10⁵ cells/5 μl of Xenograft X1016 were injected intracranially. Cages were randomized, and vehicle (n=3) or 75 mg/kg of CX-4945 (n=3) was administered orally twice a day starting at day 3, and mice euthanized between days 13-15. Tumors were homogenized, and lysates immunoblotted with indicated antibodies. (G) Five × 10⁵ cells/5 μl of X1016 were injected intracranially. Cages were randomized, and vehicle (n=2) or 75 mg/kg of CX-4945 (n=2) was administered orally twice a day starting at day 3, and mice were euthanized at day 18. Brain tissue from vehicle and CX-4945-treated mice was fixed in formalin, embedded in paraffin and sections stained with anti-Ki-67 antibody and counter stained with Hematoxylin. Representative images and Ki-67+ percentages (in bracket) are shown. The scale bar is 50 μm.