Targeting protein kinase CK2 suppresses bladder cancer cell survival via the glucose metabolic pathway

Abstract

Casein kinase 2 (CK2) is a constitutively active serine/threonine kinase that promotes cell proliferation and resists apoptosis. Elevated CK2 expression has been demonstrated in several solid tumors. The expression of CK2α in bladder cancer was elevated in tumor tissues compared with that in adjacent normal tissues. Amplified expression of CK2α was highly correlated with histological grade in bladder cancer(P = 0.024). Knockdown of CK2α in bladder cancer cell lines resulted in a reduction in tumor aerobic glycolysis, accompanied with lower phosphorylated AKT. Moreover, low CK2α levels suppressed cell growth, and similar results could be reproduced after treatment with CX-4945 with a dose-dependent response. CX-4945 inhibited migration and induced apoptosis. Furthermore, knockdown of CK2α decreased the tumorigenicity of bladder cancer cells in vivo. This study is the first to report that CK2 increases glucose metabolism in human bladder cancer. Blocking CK2 function may provide novel diagnostic and potential therapeutic.

Keywords: CK2α; bladder cancer; glycolysis; oncogene; prognosis.

Figure 1. Expression level of CK2α mRNA and protein in human primary bladder cancer cell lines and surgical specimens as evaluated

A, B. RT-qPCR showed that relative CK2α mRNA expression in 22 BCa and adjacent tissues (P < 0.05). C. CK2α protein in seven BCa and adjacent tissues (N, non-tumor, T, tumor). D. CK2α mRNA levels was up-regulated in seven bladder cancer cell lines compared with those in the normal urinary epithelial cell line sv-Huc.

Figure 2. IHC analyses of CK2α protein expression in primary BCa surgical specimens and Kaplan–Meier survival analyses of the primary BCa patients (n = 137)

A. CK2α staining in BCa, scored as CK2α (-) (+) (++) (+++). All images are shown at × 100 magnification and × 200 magnification. B. BCa patients were divided into low-CK2α expression (n = 54, CK2α- or CK2α+) and high-CK2α expression (n = 83, CK2α++ or CK2α+++) groups. Survival analyses did not reveal a significant association between high CK2α expression and poor prognosis (P = 0.694, log-rank test).

Figure 3. CK2α was essential for bladder cancer cell proliferation

A, B, C. Knockdown efficiency of selected CK2α-targeting siRNAs in transfected cells was evaluated by Western blot and qRT-PCR. D, E. CCK8 assay showed that silencing of CK2α suppressed proliferation of T24 (D) and EJ (E) cell lines.

Figure 4. CK2α inhibition induced a metabolic shift in bladder cancer cells

A. Glucose uptake assays. CK2α inhibition decreased glucose uptake. B. Lactate production assays. CK2α inhibition suppressed lactate secretion. C. CK2α inhibition decreased protein expression in cancer aerobic glycolysis.

Figure 5. Effects of inhibition of CK2 on bladder cancer cell function

A, B. CX-4945 suppressed proliferation of T24 (A) and EJ (B) cell. C, E. Western blot analysis showed that protein expression in cancer aerobic glycolysis was downregulated in T24 and EJ cells after treatment with CX4945. AKT and p-AKT(ser473) (C). GLUT1, G6PD, HK1, HK2, LDHA, LDHB and PKM (E). D. CX-4945 inhibited the migration ability in T24 and EJ cell lines. F. Glucose uptake assays. CX-4945 decreased glucose uptake. G. Lactate production assays. CX-4945 suppressed lactate secretion.

Figure 6. Effect of inhibition of CK2 on apoptosis in bladder cancer cells

CX-4945 induced apoptosis in T24(A) and EJ(B) cell lines.

Figure 7. CK2α inhibition decreased tumorigenesis and reduced cancer cell growth in vivo

A, B. CK2αknockdown EJ cells formed smaller tumour volume compared to the control cells (NC). C. The tumor growth curves in the two groups (P < 0.01). D. CK2αknockdown reduced tumor weight compared to the control cells (NC) (P < 0.01). E. CK2α immunohistochemistry staining of xenografted tumors retrieved from treated nude mice. Representative images are shown. (magnification, ×100)