casein-kinase-ii and Brain-Neoplasms

Glioblastoma is a highly aggressive brain tumor that is characterized by its unparalleled invasiveness. Invasive glioblastoma cells not only escape surgery and focal therapies but also are more resistant to current radio- and chemo-therapeutic approaches. Thus, any curative therapy for this deadly disease likely should include treatment strategies that interfere with glioblastoma invasiveness. Understanding glioblastoma invasion mechanisms is therefore critical. We discuss the strengths and weaknesses of various glioblastoma invasion models and conclude that robust experimental evidence has been obtained for a pro-invasive role of Ephrin receptors, Rho GTPases, and casein kinase 2 (CK2). Extensive interplay occurs between these proteins, suggesting the existence of a glioblastoma invasion signaling network that comprises several targets for therapy.

Topics: Animals; Antineoplastic Agents; Brain; Brain Neoplasms; Casein Kinase II; Drug Discovery; Extracellular Matrix; Glioblastoma; Humans; Neoplasm Invasiveness; Receptors, Eph Family; rho GTP-Binding Proteins; Signal Transduction

Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.

Topics: Animals; Brain Neoplasms; Carcinogenesis; Casein Kinase II; Cell Cycle Proteins; Cell Line, Tumor; Female; Glioblastoma; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Male; Mice; Mitosis; Neoplasm Proteins; Nuclear Proteins; Protein-Arginine N-Methyltransferases; Signal Transduction; Xenograft Model Antitumor Assays

The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells.. GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence.. TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945.. combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

Topics: Adenosine Triphosphate; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Calreticulin; Casein Kinase II; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Glioblastoma; Humans; Inflammation; Microscopy, Fluorescence; Naphthyridines; Phenazines; Propidium; Signal Transduction; Temozolomide; Treatment Outcome

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Since surviving patients experience severe neurocognitive disabilities, better and more effective treatments are needed to enhance their quality of life. Casein kinase 2 (CK2) is known to regulate cell growth and survival in multiple cancers; however, the role of CK2 in MB is currently being studied. In this study, we verified the importance of CK2 in MB tumorigenesis and discovered that inhibition of CK2 using the small molecule inhibitor, CX-4945, can sensitize MB cells to a well-known and tolerated chemotherapeutic, temozolomide (TMZ). To study the role of CK2 in MB we modulated CK2 expression in multiple MB cells. Exogenous expression of CK2 enhanced cell growth and tumor growth in mice, while depletion or inhibition of CK2 expression decreased MB tumorigenesis. Treatment with CX-4945 reduced MB growth and increased apoptosis. We conducted a high-throughput screen where 4000 small molecule compounds were analyzed to identify compounds that increased the anti-tumorigenic properties of CX-4945. TMZ was found to work synergistically with CX-4945 to decrease cell survival and increase apoptosis in MB cells. O-6-methylguanine-DNA methyltransferase (MGMT) activity is directly correlated to TMZ sensitivity. We found that loss of CK2 activity reduced β-catenin expression, a known MGMT regulator, which in turn led to a decrease in MGMT expression and an increased sensitivity to TMZ. Our findings show that CK2 is important for MB maintenance and that treatment with CX-4945 can sensitize MB cells to TMZ treatment.

Topics: Antineoplastic Agents, Alkylating; Brain Neoplasms; Casein Kinase II; Enzyme Inhibitors; Humans; Medulloblastoma; Prognosis; Temozolomide

Tumours of astroglial origin, both malignant glioblastoma (GBM) and benign subependymal giant cell astrocytoma (SEGA), pose a serious medical problem. Casein kinase 2 (CK2), a member of the serine/threonine kinase family, has antiapoptotic properties and plays a vital role in glial tumour cell survival. It contributes to invasive cell growth and is often upregulated in malignant neoplastic cells; however, its role in benign tumours of astrocytic origin is less understood. In the present study we investigated the effects of small molecule CK2 inhibitors on proliferation and viability of glioma cells in vitro. The experiments were conducted on commercial T98G malignant glioma cell line and the SEGA cell line, derived from a paediatric case of tuberous sclerosis complex (TSC). Cell cultures were incubated with selected CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) at 0.1, 1, 10, 25, 50, 75 and 100 µM concentrations for 24 and 48 hours. Cell proliferation was assessed using a cell counter and cell viability was evaluated by MTT assay. TBB at 75 µM and 100 µM, and TBI starting from 25 µM, both reduced T98G cell proliferation after 24 hours, while DMAT was ineffective. All tested small molecule CK2 inhibitors appear to reduce T98G cell growth and viability after 48 hours, although TBI appeared to be the most effective and reduced cell growth in the 50-100 µM dose range. TBI also showed potential efficacy in reducing the number and viability of SEGA cells after 48 hours. Proliferation and viability of SEGA cells have proven resistant to TBB treatment. DMAT only reduced the viability of SEGA cells at 24 (at 100 µM) and 48 hours (10-100 µM). Importantly, normal human astrocyte cells were found to be moderately resistant to TBB, while their viability was mildly reduced at higher doses of DMAT and TBI. In conclusion, CK2 appears to play a role not only in malignant glioma cells but it can also sustain the viability and proliferation of benign astrocytoma cells. The obtained antitumor effects of CK2 inhibitors significantly exceeded their mild or no effect on normal astrocytes in control, which supports the therapeutic potential of these compounds against gliomas.

Topics: Antineoplastic Agents; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glioblastoma; Humans; Protein Kinase Inhibitors

Tumours of astroglial origin are the most common primary brain malignancy characterized by infiltrative growth and resistance to standard antitumour therapy. Glioma progression is thought to be related to various intracellular signal transduction pathways that involve the activation of protein kinases. Protein kinases play important roles in cell differentiation, proliferation, and survival. Recently, novel, specific inhibitors of constitutively active serine/threonine kinases and structurally similar isothiourea derivatives were suggested to induce apoptosis and inhibit proliferation in several types of human cancer cells.. In this study, we examined the cytotoxic and proapoptotic activities of selected modified pentabromobenzyl isothioureas (ZKKs) in an adult human glioblastoma (T98G) and a subependymal giant cell astrocytoma cell (SEGA) line. We evaluated cell proliferation, viability, and apoptosis.. Two pentabromobenzyl isothiourea bromide derivatives, ZKK-13 and N,N,N'-trimethyl-ZKK1 (TRIM), exhibited the most potent cytotoxic and proapoptotic efficacies against human glioma-derived cells, even at a very low concentration (1 μM). ZKK-13 (25-50 μM) inhibited cell growth by approximately 80-90% in 24 and 48 h of treatment. We showed that selected ZKKs exerted antiproliferative activity against astroglial neoplastic cells of both low- and high-grade tumour malignancy classes. No synergistic effects were detected when ZKKs were combined with serine/threonine kinase inhibitors.. Our findings indicated that modified ZKKs show promise for the treatment of glioma-derived brain tumours.

Topics: Adult; Antineoplastic Agents; Apoptosis; Astrocytes; Astrocytoma; Brain Neoplasms; Casein Kinase II; Cell Division; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Isothiuronium; Molecular Targeted Therapy; Neoplasm Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Thiourea

Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

Topics: Benzoquinones; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Drug Interactions; Drug Resistance, Neoplasm; Glioblastoma; Glucosides; Heat Shock Transcription Factors; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lignans; Protein Kinase Inhibitors; Transcription Factors; Transcription, Genetic; Transcriptome; Up-Regulation

Diffuse and uncontrollable brain invasion is a hallmark of glioblastoma (GBM), but its mechanism is understood poorly. We developed a 3D ex vivo organotypic model to study GBM invasion. We demonstrate that invading GBM cells upregulate a network of extracellular matrix (ECM) components, including multiple collagens, whose expression correlates strongly with grade and clinical outcome. We identify interferon regulatory factor 3 (IRF3) as a transcriptional repressor of ECM factors and show that IRF3 acts as a suppressor of GBM invasion. Therapeutic activation of IRF3 by inhibiting casein kinase 2 (CK2)-a negative regulator of IRF3-downregulated the expression of ECM factors and suppressed GBM invasion in ex vivo and in vivo models across a panel of patient-derived GBM cell lines representative of the main molecular GBM subtypes. Our data provide mechanistic insight into the invasive capacity of GBM tumors and identify a potential therapy to inhibit GBM invasion.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix; Glioblastoma; Humans; Interferon Regulatory Factor-3; Male; Mice; Mice, Nude; Mice, SCID; Neoplasm Invasiveness

With an unsurpassed capacity for invasion into normal brain tissue, glioblastoma multiforme is the most lethal primary brain tumor. New research suggests that altering a subset of extracellular matrix factors, including interferon regulatory factor (IRF)3 and casein kinase (CK)2, may decrease the migratory potential of these aggressive tumors.

Topics: Animals; Brain Neoplasms; Casein Kinase II; Extracellular Matrix; Glioblastoma; Humans; Interferon Regulatory Factor-3; Neoplasm Invasiveness; Neoplasm Proteins

DNA repair gene defects are found in virtually all human glioblastomas, but the genetic evidence for a direct role remains lacking. Here we demonstrate that combined inactivation of the XRCC4 non-homologous end-joining (NHEJ) DNA repair gene and p53 efficiently induces brain tumours with hallmark characteristics of human proneural/classical glioblastoma. The murine tumours exhibit PTEN loss of function instigated by reduced PTEN mRNA, and increased phosphorylated inactivation and stability as a consequence of aberrantly elevated CK2 provoked by p53 ablation and irrevocably deregulated by NHEJ inactivation. This results in DNA damage-resistant cytoplasmic PTEN and CK2 expression, and the attenuation of DNA repair genes. CK2 inhibition restores PTEN nuclear distribution and DNA repair activities and impairs tumour but not normal cell survival. These observations demonstrate that NHEJ contributes to p53-mediated glioblastoma suppression, and reveal a crucial role for PTEN in the early DNA damage signalling cascade, the inhibition of which promotes tumorigenicity and drug-resistant survival.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; DNA Damage; DNA End-Joining Repair; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Glioblastoma; Humans; Male; Mice; PTEN Phosphohydrolase; Signal Transduction; Tumor Suppressor Protein p53

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α') and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133

Topics: AC133 Antigen; Animals; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Embryo, Mammalian; Enzyme Inhibitors; Female; Gefitinib; Glioblastoma; Humans; Mice; Mice, Inbred C57BL; Mice, Nude; Naphthyridines; Neoplastic Stem Cells; Phenazines; Pregnancy; Quinazolines; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is the most prevalent and aggressive human glial tumour with a median survival of 14-15 months. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment. Unfortunately, chemoresistence always ensues with concomitant tumour regrowth. Protein kinase CK2 (CK2) contributes to tumour development, proliferation, and suppression of apoptosis in cancer and it is overexpressed in human GBM. Targeting CK2 in GBM treatment may benefit patients. With this translational perspective in mind, we have studied the CK2 expression level by Western blot analysis in a preclinical model of GBM: GL261 cells growing orthotopically in C57BL/6 mice. The expression level of the CK2 catalytic subunit (CK2α) was higher in tumour (about 4-fold) and in contralateral brain parenchyma (more than 2-fold) than in normal brain parenchyma (p < 0.05). In contrast, no significant changes were found in CK2 regulatory subunit (CK2β) expression, suggesting an increased unbalance of CK2α/CK2β in GL261 tumours with respect to normal brain parenchyma, in agreement with a differential role of these two subunits in tumours.

Topics: Animals; Apoptosis; Brain; Brain Neoplasms; Carcinogenesis; Casein Kinase II; Catalytic Domain; Cell Proliferation; Female; Glioblastoma; Glioma; Humans; Mice; Mice, Inbred C57BL

Understanding mechanisms that link aberrant metabolic adaptation and pro-survival responses in glioma cells is crucial towards the development of new anti-glioma therapies. As we have previously reported that CK2 is associated with glioma cell survival, we evaluated its involvement in the regulation of glucose metabolism. Inhibition of CK2 increased the expression of metabolic regulators, PDK4 and AMPK along with the key cellular energy sensor CREB. This increase was concomitant with altered metabolic profile as characterized by decreased glucose uptake in a PDK4 and AMPK dependent manner. Increased PDK4 expression was CREB dependent, as exogenous inhibition of CREB functions abrogated CK2 inhibitor mediated increase in PDK4 expression. Interestingly, PDK4 regulated AMPK phosphorylation which in turn affected cell viability in CK2 inhibitor treated glioma cells. CK2 inhibitor 4,5,6,7-Tetrabromobenzotriazole (TBB) significantly retarded the growth of glioma xenografts in athymic nude mouse model. Coherent with the in vitro findings, elevated senescence, pAMPK and PDK4 levels were also observed in TBB-treated xenograft tissue. Taken together, CK2 inhibition in glioma cells drives the PDK4-AMPK axis to affect metabolic profile that has a strong bearing on their survival.

Topics: Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Cyclic AMP Response Element-Binding Protein; Glioma; Glucose; Humans; Mice, Nude; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; RNA, Messenger; Signal Transduction; Sirtuin 1; Triazoles; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Casein kinase II contributes to the growth and survival of malignant gliomas and attracts increasing attention as a therapeutic target in these tumors. Several reports have suggested that this strategy might be most relevant for specific subgroups of patients, namely Verhaak's classical and TP53 wild-type tumors. Using kinase assays and microarray genetic profiling in a series of 27 proprietary fresh frozen surgical glioma samples, we showed that constitutive CK2 kinase activation is not restricted to tumors that present increased copy numbers or mRNA expression of its catalytic or regulatory subunits, and can result from a functional activation by various cytokines from the glioma microenvironment. Using corresponding primary tumor and human astrocyte cell cultures as well as glioma cell lines, we confirmed that CK2 inhibition is selectively toxic to malignant glial tumors, without any restriction to tumor class or to TP53 status. We finally showed that while the contribution of CK2 to the constitutive NF-κB hyperactivation in malignant gliomas is at best moderate, a delayed activation of NF-κB may associate with the therapeutic resistance of glioma cells to CK2 inhibition.

Topics: Apigenin; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Naphthyridines; Phenazines; Tissue Array Analysis; Tumor Microenvironment; Tumor Suppressor Protein p53

Glioblastoma multiforme (GBM) remains the deadliest brain tumor in adults. GBM tumors are also notorious for drug and radiation resistance. To inhibit GBMs more effectively, polymalic acid-based blood-brain barrier crossing nanobioconjugates were synthesized that are delivered to the cytoplasm of cancer cells and specifically inhibit the master regulator serine/threonine protein kinase CK2 and the wild-type/mutated epidermal growth factor receptor (EGFR/EGFRvIII), which are overexpressed in gliomas according to The Cancer Genome Atlas (TCGA) GBM database. Two xenogeneic mouse models bearing intracranial human GBMs from cell lines LN229 and U87MG that expressed both CK2 and EGFR at different levels were used. Simultaneous knockdown of CK2α and EGFR/EGFRvIII suppressed their downstream prosurvival signaling. Treatment also markedly reduced the expression of programmed death-ligand 1 (PD-L1), a negative regulator of cytotoxic lymphocytes. Downregulation of CK2 and EGFR also caused deactivation of heat shock protein 90 (Hsp90) co-chaperone Cdc37, which may suppress the activity of key cellular kinases. Inhibition of either target was associated with downregulation of the other target as well, which may underlie the increased efficacy of the dual nanobioconjugate that is directed against both CK2 and EGFR. Importantly, the single nanodrugs, and especially the dual nanodrug, markedly suppressed the expression of the cancer stem cell markers c-Myc, CD133, and nestin, which could contribute to the efficacy of the treatments. In both tumor models, the nanobioconjugates significantly increased (up to 2-fold) animal survival compared with the PBS-treated control group. The versatile nanobioconjugates developed in this study, with the abilities of anti-cancer drug delivery across biobarriers and the inhibition of key tumor regulators, offer a promising nanotherapeutic approach to treat GBMs, and to potentially prevent drug resistance and retard the recurrence of brain tumors.

Topics: Adult; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Blood-Brain Barrier; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; ErbB Receptors; Female; Glioblastoma; Humans; Malates; Mice; Mice, Nude; Nanoconjugates; Neoplastic Stem Cells; Oligonucleotides, Antisense; Polyethylene Glycols; Polymers; Signal Transduction; Surface Properties

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.

Topics: Animals; Benzimidazoles; beta Catenin; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Mice; Naphthyridines; Neoplasm Transplantation; Neoplastic Stem Cells; Phenazines; Prognosis; Signal Transduction; Survival Analysis

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α' or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2-PP2A pathway.

Topics: Animals; bcl-X Protein; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Glioma; HEK293 Cells; Humans; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; Serine; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transplantation, Heterologous

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

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Casein Kinase II; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Enzyme Activation; Female; Gene Amplification; Gene Dosage; Glioblastoma; Humans; Mice; Mice, Nude; Naphthyridines; Phenazines; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Glioblastoma is the most prevalent and malignant primary brain tumor in adults, and its response to current therapies is limited. Protein kinase CK2 is overexpressed in glioblastoma and regulates glioblastoma cell survival, proliferation, and migration and brain tumorigenesis. Targeting CK2 for glioblastoma treatment may benefit patients with glioblastoma.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Casein Kinase II; Female; Glioblastoma; Humans; Naphthyridines; Phenazines; Signal Transduction

Glioblastoma multiforme (GBM) are resistant to TNFα-induced apoptosis and blockade of TNFα-induced NF-κB activation sensitizes glioma cells to apoptosis. As Casein kinase-2 (CK2) induces aberrant NF-κB activation and as we observed elevated CK2 levels in GBM tumors, we investigated the potential of CK2 inhibitors (CK2-Is) - DRB and Apigenin in sensitizing glioma cells to TNFα-induced apoptosis. CK2-Is and CK2 small interfering RNA (siRNA) reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation, and sensitized cell to TNFα-induced apoptosis. Importantly, CK2-Is activated p53 function in wild-type but not in p53 mutant cells. Activation of p53 function involved its increased transcriptional activation, DNA-binding ability, increased expression of p53 target genes associated with cell cycle progression and apoptosis. Moreover, CK2-Is decreased telomerase activity and increased senescence in a p53-dependent manner. Apoptotic gene profiling indicated that CK2-Is differentially affect p53 and TNFα targets in p53 wild-type and mutant glioma cells. CK2-I decreased MDM2-p53 association and p53 ubiquitination to enhance p53 levels. Interestingly, CK2-Is downregulated SIRT1 activity and over-expression of SIRT1 decreased p53 transcriptional activity and rescued cells from CK2-I-induced apoptosis. This ability of CK2-Is to sensitize glioma to TNFα-induced death via multiple mechanisms involving abrogation of NF-κB activation, reactivation of wild-type p53 function and SIRT1 inhibition warrants investigation.

Topics: Apigenin; Apoptosis; Brain; Brain Neoplasms; Casein Kinase II; Cell Cycle Checkpoints; Cell Line, Tumor; Dichlororibofuranosylbenzimidazole; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; NF-kappa B; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Signal Transduction; Sirtuin 1; Telomerase; Transfection; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis.

Topics: Brain Neoplasms; Casein Kinase II; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, cdc; Glioblastoma; Humans; Immunohistochemistry; Protein Interaction Mapping; Protein Phosphatase 1; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the nonhomologous end-joining pathway of DNA double-strand breaks repair. Although DNA-PK has been biochemically characterized in vitro, relatively little is known about its functions in the context of DNA repair and how its kinase activity is precisely regulated in vivo. Here, we report that cellular depletion of the individual catalytic subunits of protein kinase CK2 by RNA interference leads to significant cell death in M059K human glioblastoma cells expressing DNA-PKcs, but not in their isogenic counterpart, that is M059J cells, devoid of DNA-PKcs. The lack of CK2 results in enhanced DNA-PKcs activity and strongly inhibits DNA damage-induced autophosphorylation of DNA-PKcs at S2056 as well as repair of DNA double-strand breaks. By the application of the in situ proximity ligation assay, we show that CK2 interacts with DNA-PKcs in normal growing cells and that the association increases upon DNA damage. These results indicate that CK2 has an important role in the modulation of DNA-PKcs activity and its phosphorylation status providing important insights into the mechanisms by which DNA-PKcs is regulated in vivo.

Topics: Brain Neoplasms; Calcium-Binding Proteins; Casein Kinase II; Catalytic Domain; Cell Death; Cell Line, Tumor; DNA Damage; DNA-Activated Protein Kinase; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Humans; Phosphorylation

The tumor suppressor p53 is an important cellular protein, which controls cell cycle progression. Phosphorylation is one of the mechanisms by which p53 is regulated. Here we report the interaction of p53 with another key regulator, cdk9, which together with cyclin T1 forms the positive transcription elongation complex, p-TEFb. This complex cooperates with the HIV-1 Tat protein to cause the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II and this facilitates the elongation of HIV-1 transcription. We demonstrate that cdk9 phosphorylates p53 on serine 392 through their direct physical interaction. Results from protein-protein interaction assays revealed that cdk9 interacts with the C-terminal domain (aa 361-393) of p53, while p53 interacts with the N-terminal domain of cdk9. Transfection and protein binding assays (EMSA and ChIP) demonstrated the ability of p53 to bind and activate the cdk9 promoter. Interestingly, cdk9 phosphorylates serine 392 of p53, which could be also phosphorylated by casein kinase II. Kinase assays demonstrated that cdk9 phosphorylates p53 independently of CKII. These studies demonstrate the existence of a feedback-loop between p53 and cdk9, pinpointing a novel mechanism by which p53 regulates the basal transcriptional machinery.

Topics: Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Glioblastoma; HIV-1; Humans; Kinetics; Lung Neoplasms; Phosphorylation; Recombinant Proteins; Serine; Transcription, Genetic; Tumor Suppressor Protein p53

The biochemical regulation of human O6-alkylguanine-DNA alkyltransferase (AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poorly understood. We investigated the regulation of AGT by protein phosphorylation in a human medulloblastoma cell line. Incubation of cell extracts with [gamma-32P]ATP resulted in Mg(2+)-dependent phosphorylation of the endogenous AGT. Immunoprecipitation after exposure of the cells to 32P-labeled inorganic phosphate showed that AGT exists as a phosphoprotein under physiological conditions. Western analysis and chemical stability studies showed the AGT protein to be phosphorylated at tyrosine, threonine, and serine residues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiometry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), respectively. Residual phosphorylation of the endogenous AGT by the PKs present in cell homogenates and phosphorylation of the recombinant AGT by purified serine/threonine kinases, PKA, PKC, and CK II reduced AGT activity by 30-65%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation motifs for many cellular kinases, including PKA and CK II in the AGT protein. These data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of this therapeutically important DNA repair protein in human normal and cancer cells.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Brain Neoplasms; Casein Kinase II; Cyclic AMP-Dependent Protein Kinases; Homeostasis; Humans; Kinetics; Magnesium; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Phosphates; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Recombinant Proteins; Tumor Cells, Cultured

We showed recently that human O(6)-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg(2+) more efficiently than Mn(2+) for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-(32)P]ATP and [gamma-(32)P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Brain Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Casein Kinase II; Cations; Chromatography, Agarose; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Genistein; Guanosine Triphosphate; Humans; Magnesium; Manganese; Medulloblastoma; Mice; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Phosphates; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Threonine; Tumor Cells, Cultured; Tyrosine; Tyrphostins