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. 2012 Dec;41(6):1967-76.
doi: 10.3892/ijo.2012.1635. Epub 2012 Sep 21.

Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells

Birgitte B Olsen  1 , Tina H Svenstrup, Barbara Guerra
Affiliations

Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells

Birgitte B Olsen et al. Int J Oncol. 2012 Dec.

Abstract

Glioblastoma multiforme is the most common primary brain tumor and one of the most aggressive types of cancer in adults. Survival signaling and apoptosis resistance are hallmarks of malignant glioma cells. However, recent studies have shown that other types of cell death such as autophagy can be induced in malignant glioma cells. This suggests that stimulation of this process may be explored in new therapeutic strategies against glioblastoma multiforme. Protein kinase CK2 is a highly conserved and constitutively active enzyme that promotes numerous cellular processes such as survival, proliferation and differentiation. CK2 has been found elevated in several malignancies including brain tumors, and to confer resistance against chemotherapeutic agents and apoptotic stimuli. Recently, we have shown that the siRNA-mediated downregulation of CK2 leads to cell death in DNA-PK-proficient human glioblastoma cells. We show, here, that lack of CK2 results in significant induction of autophagic cell death in two human glioblastoma cell lines, M059K and T98G, as indicated by the positive staining of cells with the acidotropic dye acridine orange, and the specific recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosome membranes. Induction of autophagy is accompanied by CK2-dependent decreased phosphorylation of p70 ribosomal S6 and AKT kinases and significantly reduced expression levels of Raptor. In contrast, phosphorylation and activity levels of ERK1/2 are enhanced suggesting an inhibition of the PI3K/AKT/mTORC1 and activation of the ERK1/2 pathways. Furthermore, siRNA-mediated silencing of CK2 results in increased mitochondrial superoxide production in both glioblastoma cell lines. However, mitochondrial reactive oxygen species release correlates with induction of autophagy only in T98G cells. Taken together, our findings identify CK2 as a novel component of the autophagic machinery and underline the potential of its downregulation to kill glioblastoma cells by overcoming the resistance to multiple anticancer agents.

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Figures

Figure 1
Figure 1
NCS induces autophagy in human glioblastoma cells. (A) Phase-contrast microscopy pictures of M059K and T98G cells left untreated (1 and 6) and incubated with 0.5 μg/ml (2 and 7), 1 μg/ml (3 and 8), 2 μg/ml (4 and 9), 5 μg/ml (5 and 10), 10 μg/ml (11) NCS for 24 h, respectively. Pictures showing morphological changes indicating various degrees of toxicity induced by the aforementioned treatments were taken at ×400 magnification. (B) Cells were treated as described above. Induction of autophagy was determined by flow cytometry analysis of acridine orange-positive cells (FL3-H indicates red-fluorescence intensity). Experiments were repeated three times and data from one representative experiment are shown.
Figure 2
Figure 2
Downregulation of CK2 leads to induction of autophagy. (A) Cells were transfected with scramble siRNA (Scr-siRNA) or with siRNAs against the individual CK2 catalytic subunits for 72 h. Where indicated, NCS was added at the indicated concentrations in the last 24 h of incubation with siRNA. Induction of autophagy was analyzed by flow cytometry as described in Fig. 1. A control experiment is also shown where cells were incubated with rapamycin for 24 h. (B) Western blot analysis of total cell lysates from cells treated as indicated. Proteins were visualized by probing the membranes with antibodies against the indicated proteins. (C) Photomicrographs of cells transfected with Scr-siRNA or CK2-siRNAs for 48 h. In the last 24 h of incubation time, cells were transduced with Premo Autophagy Sensor LC3B-GFP and added 0.5 μg/ml (M059K) and 5 μg/ml (T98G) NCS, respectively. (D) Determination of the average number of puncta, as distinct fluorescence green spots, per cell was performed by analyzing 200 cells per sample. The mean values ± SD are shown. Two independent data set were obtained showing similar results. *P<0.001 indicates statistical significance.
Figure 3
Figure 3
Regulation of autophagic flux by cellular depletion of CK2. (A) Representative fluorescence-based images of cells expressing or lacking CK2 exposed to 50 nM bafilomycin A (Baf) for 6 h before the analysis and NCS, as indicated in the figure, for 24 h before cell analysis. (B) Bar graph indicates the average number ± SD of puncta per cell determined by analyzing 200 cells per sample. Three independent experiments were performed obtaining similar results. *P<0.001 indicates statistical significance.
Figure 4
Figure 4
Downregulation of protein kinase CK2 affects the mTOR and ERK1/2 signaling pathways. Cell lysates (30 μg) from cells treated as indicated in the figure were analyzed by western blot analysis using antibodies against the indicated proteins or their phosphorylated form. Anti-β-actin was applied to confirm equal protein loading. At least 4 separate experiments were performed obtaining similar results. Data from one representative experiment are shown.
Figure 5
Figure 5
CK2-knockout leads to enhanced ERK1/2 kinase activity in the presence of NCS. Cells were transfected with Scr-siRNA or CK2-siRNAs for 72 h. Where indicated, NCS was added 24 h before harvesting. Whole cell lysates (500 μg) were subjected to a non-radioactive kinase activity assay in the presence of a GST-Elk-1 fusion protein after immunoprecipitation of phosphorylated ERK1/2. The phosphorylation levels of Elk-1 were detected by western blot analysis with an antibody directed against phospho-Elk1-1 (S383). Representative results from two independent experiments are shown.
Figure 6
Figure 6
ROS involvement in autophagy induction mediated by CK2-siRNA silencing. (A) Cells were treated as indicated in the figure. Mitochondrial superoxide production was measured in two independent experiments by flow cytometry after labeling cells with MitoSOX™ Red as described in the Materials and methods. FL2-H intensity indicates red-positive cells ± SD expressed in percentage. *P<0.001 indicates statistical significance. NS, not significant. (B) Cells were treated as described above. Where indicated, cells were added 100 μM BHA 24 h before harvesting. Induction of autophagy was determined by flow cytometry of acridine orange-positive cells. At least three independent experiments were performed obtaining similar results. Data from one representative experiment are shown.
Figure 7
Figure 7
Model of the proposed influence of CK2 on autophagy induction. The model suggests the placement and function of CK2 with respect to the major signaling cascades regulating autophagy. CK2 has been reported to be a master regulator of cellular functions in virtue of its ability to play a ‘lateral means’ of pathways intervention (12). Here, we show that effective CK2-mediated autophagy induction is achieved by the simultaneous targeting of the ERK1/2-and mTOR signaling pathways. Additional details are reported in the text.
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