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Casein Kinase 2&Alpha Oncogene (2015) 34, 3688–3699 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Casein kinase 2α regulates glioblastoma brain tumor-initiating cell growth through the β-catenin pathway RT Nitta1, S Gholamin1,2, AH Feroze2, M Agarwal1, SH Cheshier1,2, SS Mitra1,2 and G Li1 Glioblastoma (GBM) is the most common and fatal primary brain tumor in humans, and it is essential that new and better therapies are developed to treat this disease. Previous research suggests that casein kinase 2 (CK2) may be a promising therapeutic target for GBMs. CK2 has enhanced expression or activity in numerous cancers, including GBM, and it has been demonstrated that inhibitors of CK2 regressed tumor growth in GBM xenograft mouse models. Our studies demonstrate that the CK2 subunit, CK2α,is overexpressed in and has an important role in regulating brain tumor-initiating cells (BTIC) in GBM. Initial studies showed that two GBM cell lines (U87-MG and U138) transduced with CK2α had enhanced proliferation and anchorage-independent growth. Inhibition of CKα using siRNA or small-molecule inhibitors (TBBz, CX-4945) reduced cell growth, decreased tumor size, and increased survival rates in GBM xenograft mouse models. We also verified that inhibition of CK2α decreased the activity of a well- known GBM-initiating cell regulator, β-catenin. Loss of CK2α decreased two β-catenin-regulated genes that are involved in GBM- initiating cell growth, OCT4 and NANOG. To determine the importance of CK2α in GBM stem cell maintenance, we reduced CK2α activity in primary GBM samples and tumor spheres derived from GBM patients. We discovered that loss of CK2α activity reduced the sphere-forming capacity of BTIC and decreased numerous GBM stem cell markers, including CD133, CD90, CD49f and A2B5. Our study suggests that CK2α is involved in GBM tumorigenesis by maintaining BTIC through the regulation of β-catenin. Oncogene (2015) 34, 3688–3699; doi:10.1038/onc.2014.299; published online 22 September 2014 INTRODUCTION A genome-wide analysis of GBMs revealed an increase in the CK2α Glioblastoma (GBM) is the most common malignant primary brain gene (CSNK2a1),10 and enhanced CK2α expression has been tumor in adults. Four out of every 100 000 Americans are observed compared with pair-matched normal brain samples.11 diagnosed with GBM, which represents ~ 15% of all primary brain In addition to its possible role in GBM tumorigenesis, there are tumors.1 Once the diagnosis of GBM has been made, the overall recent reports indicating that CK2α may have a role of BTIC median survival time for patients treated with surgery, radiation maintenance and growth in GBM. For example, inhibition of CK2α and chemotherapy is only 14–15 months.2 The identification of was shown to impact the self-renewal capabilities of leukemia GBM-associated cancer stem cells or GBM-associated brain tumor- stem cells12 and non-small cell lung carcinoma stem cells.13 initiating cells (BTIC) has fueled research into the contributing role CK2α has also been shown to have an important role in neural of this cell type in GBM pathogenesis and chemotherapy stem cell proliferation and differentiation, indicating a potential 3 resistance. The BTIC were shown to be capable of self-renewal role in BTIC in GBM.14 Additionally, in vitro studies indicate and multi-lineage differentiation, they initiate tumor formation that CK2α may also be involved in BTIC growth by controlling upon intracranial implantation within mice and exhibit elevated β 4 well-known mediators of GBM, including the Wnt/ -catenin therapeutic resistance relative to bulk glioma cells. Therefore, pathway.15–17 elucidation of the molecular mechanisms underlying BTIC main- To determine whether CK2α does have an integral role in GBM tenance and proliferation offers new molecular targets for tumorigenesis and in BTIC growth, we first generated immorta- developing more effective GBM treatments. lized GBM cell lines that had modulated CK2α expression. We An intriguing molecular target for GBM therapy is casein kinase verified that inhibition of CK2α using short interfering RNA (siRNA), 2 (CK2), which is a highly ubiquitous and conserved protein serine/ short hairpin (shRNA) or small-molecule inhibitors decreased threonine kinase composed of two catalytic subunits (CK2α or 5 growth, colony formation and tumor size in mice. Moreover, we CK2α’) and two regulatory subunits (CK2β). CK2 had increased protein expression or activity in the majority of cancers examined also discovered that an important regulator of BTIC in GBM, β α and its growth-related functions have been supported by the -catenin, was decreased when CK2 activity was inhibited. We fi identification of a large number of growth-related protein extended our ndings to tumor spheres generated from GBM substrates.6 CK2 is believed to be involved in GBM tumorigenesis, patients and determined that inhibition of CK2α decreased tumor as inhibition of CK2 activity through small-molecule inhibitors or sphere self-renewal, size and in vivo tumorigenic potential of these siRNA induced apoptosis and reduced growth in mouse xenograft cell lines. Through our work, we demonstrate for the first time that models of human GBMs.7–11 In addition, there is evidence CK2α may have an important role in BTIC maintenance through suggesting that the CK2α subunit has an important role in GBM. the regulation of β-catenin in GBM. 1Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA and 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. Correspondence: Dr G Li, Department of Neurosurgery, Stanford University School of Medicine, 1201 Welch Road P309, Stanford, CA 94305, USA. E-mail: [email protected] Received 17 March 2014; revised 28 July 2014; accepted 31 July 2014; published online 22 September 2014 CK2α regulates GBM BTICs through β-catenin RT Nitta et al 3689 NB GBM 1* GBM 2 GBM 3* GBM 4 GBM 5 GBM 6* GBM 7* -CK2α 1.0 2.1 0.2 4.9 1.1 1.5 2.8 2.9 -GAPDH 8 CK2α CK2β 7 6 5 4 3 2 1 primary GBM samples Fold change in mRNA 0 Normal GBM1 GBM2 GBM3 GBM4 GBM5 GBM6 GBM7 100 CK2α High Expression 90 CK2α Low Expression 100 Casein Kinase 2 α High Expression Casein Kinase 2 α Low Expression 80 p = 0.034 80 70 60 60 50 40 40 Suvival (%) Survival (%) 30 20 20 0 10 0 50 100 150 200 0 Weeks 0 12243648 All GBM patients Months Mesenchymal GBM Figure 1. Increased CK2α expression may lead to a worse prognosis for GBM patients. (a) Western blotting measuring the fold change in CK2α in seven primary GBM samples compared with normal brain (NB). GAPDH was used to control for protein load. (b) QPCR analysis of primary GBM samples. *Represents samples with higher expression of CK2α.(c) Survival curve for all GBM patients with high or low expression of CK2α using the REMBRANDT database. (d) Survival curve for the mesenchymal subtype of GBM patients with high or low expression of CK2α using the TCGA. RESULTS that the difference was statistically significant (P = 1.2 × 10 − 10) GBM patients with increased expression of CK2α may lead to a using one-way analysis of variance. We also analyzed a data set worse prognosis that contained 101 tumor stem cells that were derived from GBM 19 α patients (Supplementary Figure S1A). Consistently, we saw a Enhanced CK2 expression or activity has been observed in a α variety of solid tumors, including GBM. To verify that CK2α is reduction in CK2 expression in the normal brain data set when compared with the GBM data set that was statistically significant overexpressed in GBM, we analyzed primary samples from GBM − (P = 1.3 × 10 8). We also conducted a preliminary prognosis patients. Consistent with previous reports, we discovered that 57% analysis of CK2α expression in GBM patients using the Repository (4/7) of the GBM samples had a 2–5-fold increase in CK2α protein 10,11 of Molecular Brain Neoplasia Data (Rembrandt). By sorting the expression compared with normal brain samples. We also GBM patients into high or low expression of CK2α, our findings fi α conducted quantitative PCR (QPCR) and veri ed that CK2 mRNA suggest that GBM patients with high CK2α expression has a trend expression was enhanced in the same GBM patient samples towards a worse prognosis compared with their low-expressing (Figures 1a and b). To expand on our initial findings, we also counterparts (Figure 1c) Although our findings were not analyzed CK2α expression using the R2 microarray analysis and statistically significant (P = 0.08), we expanded our initial findings visualization platform (R2: microarray analysis and visualization to The Cancer Genome Atlas (TCGA). We discovered that when the platform (http://r2.amc.nl)). We discovered that compared with an GBM patients were separated by subtype (classical, mesenchymal, expression data set containing 172 normal brain sections, CK2α neural and proneural) only the mesenchymal subgroup had a expression was significantly increased in a data set derived from statistically significant change (P = 0.034) in patient prognosis 84 GBM samples18 (Supplementary Figure S1A). We determined when comparing patients with high versus low CK2α expression © 2015 Macmillan Publishers Limited Oncogene (2015) 3688 – 3699 CK2α regulates GBM BTICs through β-catenin RT Nitta et al 3690 40 35 YFP α 30 YFP-CK2 * β 25 YFP-CK2 U87-MG U138 20 α β α β 15 * cell number 10 5 Fold change in U87-MG YFP-CK2 YFP-CK2 YFP YFP YFP-CK2 YFP-CK2 0 ∗ 1 235 ∗∗ YFP Days ∗ 18 16 CK2α YFP ∗ 14 YFP CK2α 12 ∗∗ YFP CK2β ∗ CK2β 10 8 6 cell number GAPDH 4 Fold change in U138 2 0 123456 Days 140 120 * 100 80 60 40 Number of Colonies 20 0 YFP YFP-CK2α YFP-CK2β Figure 2.
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