Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription

Abstract

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment beta-catenin-T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of beta-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP-CK2alpha in HEK-293T cells resulted in beta-catenin-Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented beta-catenin-Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP-CK2alpha expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP-survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2alpha in HEK-293T cells coincided with reduced beta-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via beta-catenin-Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies.

Fig. 1.

TBB reduces viability, augments cell death, and alters the cell cycle in human cancer cell lines. (A) Viability was determined by the MTS assay in cells either untreated (white bars) or treated with 100 μM TBB for 24 h (black bars). Reductions in viability were significant in all cases (∗, P ≤ 0.05). (B) Cell cycle distribution of HT29(US) cells in the absence (NT) or presence of 40 or 80 μM TBB was determined by flow cytometry. TBB indicates the presence of this inhibitor (80 μM) during the 24-h treatment period. Results averaged from three independent experiments are shown (mean ± SE). Significant changes with respect to controls are indicated (∗, P ≤ 0.05).

Fig. 2.

TBB reduces survivin mRNA and protein levels in human cancer cell lines. (A) Western blot analysis of survivin and β-actin levels in lysates (25 μg) from human colon [HT29(US), DLD-1, and SW-480] cancer cells and breast cancer cells (ZR-75) grown for 24 h in the absence (−) or presence (+) of 100 μM TBB. One experiment representative of three with similar outcomes is shown. (B) RT-PCR analysis of survivin and actin mRNA levels in HT29(US) cells treated for 24 h with increasing concentrations of TBB. (C) Western blot showing survivin and β-actin protein levels in HT29(US) cells treated as in B. Values shown for survivin mRNA and protein levels in the respective lanes were averaged from three independent experiments after standardization to actin (mean ± SE). Significant changes with respect to controls are indicated (∗, P ≤ 0.05; #, P ≤ 0.01).

Fig. 3.

TBB reduces survivin levels and blocks CK2α-induced β-catenin–Tcf/Lef transcriptional activity in HEK-293T cells. (A) RT-PCR analysis of survivin and actin mRNAs in HEK-293T cells treated for 24 h with increasing concentrations of TBB. (B) Western blot showing survivin and β-actin protein levels in HEK-293T cells treated as mentioned in A. Values shown for survivin mRNA and protein levels were standardized to actin and averaged from three independent experiments (mean ± SE). (C) β-Catenin–Tcf/Lef-dependent luciferase reporter activity expressed in relative units (RU) in HEK-293T cells cotransfected with increasing amounts of cDNA encoding HA–CK2α (pCEFLHA–CK2α) and wild-type Tcf/Lef (TOP-FLASH, black bars) or mutated Tcf/Lef (FOP-FLASH, white bars) luciferase reporter plasmids. TBB indicates the presence of 100 μM TBB for 24 h after transfection. A β-galactosidase-encoding plasmid was cotransfected in all experiments to standardize results. Values in A–C were averaged from three independent experiments, each in triplicate (mean ± SE) (∗, P ≤ 0.05; #, P ≤ 0.01).

Fig. 4.

Ectopic GFP–survivin, but not GFP–CK2α, precludes TBB-induced apoptosis in HEK-293T cells. HEK-293T cells transfected with 2 μg of the plasmids pEGFP–C1, pEGFP–CK2α, or pEGFP–survivin were subsequently treated with either 100 μM TBB (+) or DMSO control (−) for 24 h. Survivin and actin mRNA (A) and protein (B) levels were determined by RT-PCR or Western blotting, respectively. For survivin mRNA, endogenous and pEGFP–survivin-derived (endo + ecto) levels combined are shown. At the protein level, GFP–survivin (ecto) and endogenous survivin (endo) are shown separately. Quantitative data for survivin standardized to actin averaged from three independent experiments are shown in the respective lanes (mean ± SE) (∗, P ≤ 0.05; #, P ≤ 0.01). (C) Apoptosis was analyzed by FACS in HEK-293T cells for the conditions indicated. Results averaged from three experiments are shown (mean ± SE). Values obtained in mock-transfected cells were compared with those of the other experimental conditions by using ANOVA (∗, P ≤ 0.05). Note that increments in TBB-induced apoptosis were significant only in the two cases indicated.

Fig. 5.

DMAT or CK2α silencing using siRNAs has effects similar to those observed with TBB in HT29(US) and HEK-293T cells. (A) Western blot showing survivin and β-catenin protein levels in HT29(US) cells treated with increasing concentrations of DMAT. An experiment representative of two is shown. (B) Luciferase reporter assay of β-catenin–Tcf/Lef activity in HEK-293T cells cotransfected with increasing amounts of cDNA encoding HA–CK2α (pCEFLHA–CK2α) and the TOP-FLASH (black bars) or FOP-FLASH (white bars) vectors. DMAT indicates the presence of this inhibitor (40 μM) during the 24-h period after transfection. As an additional control, transfection with a plasmid (2 μg) encoding a kinase-inactive mutant HA–CK2α^D156A (KI) was used under the same conditions. Data shown were averaged from two experiments in triplicate (mean ± SE). (C) Western blot showing CK2α, β-catenin, and survivin protein levels in HEK-293T cells transfected with either control siRNA (100 nM) or an equimolar mixture (mix) of two CK2α-specific siRNAs (α-siRNA and α10-siRNA) at the indicated final concentrations. An experiment representative of two is shown.