alpha-CaMKII controls the growth of human osteosarcoma by regulating cell cycle progression

Abstract

Osteosarcoma is the most frequent type of primary bone cancer in children and adolescents. These malignant osteoid forming tumors are characterized by their uncontrolled hyperproliferation. Here, we investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the growth of human osteosarcoma. We show that alpha-CaMKII is expressed in human osteosarcoma cell lines and in primary osteosarcoma tissue derived from patients. The pharmacologic inhibition of CaMKII in MG-63 and 143B human osteosarcoma cells by KN-93 resulted in an 80 and 70% decrease in proliferation, respectively, and induced cell cycle arrest in the G(0)/G(1) phase. The in vivo administration of KN-93 to mice xenografted with human osteosarcoma cells significantly decreased intratibial and subcutaneous tumor growth. Mechanistically, KN-93 and alpha-CaMKII siRNA increased p21((CIP/KIP)) gene expression, protein levels, and decreased the phosphorylation of retinoblastoma protein and E2F transactivation. Furthermore, the inhibition of CaMKII decreased membrane-bound Tiam1 and GTP-bound Rac1, which are known to be involved in p21 expression and tumor growth in a variety of solid malignant neoplasms. Our results suggest that CaMKII plays a critical role in the growth of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat conventional high-grade osteosarcoma in children.

Figure 1

Human osteosarcoma expresses high levels of α-CaMKII. MG-63,143B and hMSC cells were used. Representative images from three experiments of (a) RT-PCR using CaMKII isoforms or actin-specific primers. (b) Western blots using antibodies directed against phosphorylated and total α-CaMKII and actin (c) Immunohistochemistry staining using a specific antibody directed against p-α-CaMKII. (d) Primary human osteosarcoma tissue was surgically removed, formalin-fixed and paraffin-embedded. H&E staining (left) shows osteosarcoma. Immunohistochemical staining (right) was performed using an antibody against p-α-CaMKII (brown), counterstained with hematoxylin (blue). NC, without immunoreactivity, is shown in the low left inset. Photomicrographs were obtained at × 400 magnification, and are representative of the four different patients.

Figure 2

The CaMKII antagonist, KN-93, arrests the growth of human osteosarcoma cells. (a) MG-63 cells were treated with either DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for 24 h. Cells were then lysed and protein extracts were separated by SDS-PAGE. Western blots were conducted using antibodies directed against p-α-CaMKII or actin. (b) MG-63 or 143B cells were cultured for 24 h and then treated for 24 and 48 h with DMSO (0.1% v/v) (control), KN-92 (10 μM) or KN-93 (10 μM). At the end of the study, an MTT proliferation assay was performed. Values were obtained from three separate experiments and represent the mean±s.e. *P<0.01. (c) MG-63 Cells were stained with PI and flow cytometric analysis of the DNA content was performed. Analysis of the cell cycle was performed using ModFit LT 3.1 software. Values were obtained from three separate experiments and represent the mean±s.e. *P<0.01. (d) MG-63 cells were seeded in growth factor-reduced Matrigel, supplemented with DMSO, 10 μM KN-92 or 10μM KN-93. Medium and treatment were replaced every day for 7 days. Arrows indicate tumor colonies. Photographs are representative of three different experiments. Colonies were counted from three randomly selected microscopic fields from three different experiments.

Figure 3

The CaMKII antagonist, KN-93, controls the expression and activation of cell cycle regulatory proteins in osteosarcoma. (a) MG-63 cells were treated with 10 μM KN-93 for the indicated time (upper panel), or treated for 24 h with the indicated concentrations of KN-93 (lower panel). A representative immunoblot of p21 and actin from three separate experiments is shown. (b) Real-time PCR was performed using primers specific for p21 or 18S rRNA (upper panel). Values were obtained from three separate experiments and represent the mean±s.e. of p21 messenger RNA expression relative to 18 S rRNA expression. *P<0.01. (Lower panel) MG-63 cells were treated with DMSO (0.1%), KN-93 (10 μM) or KN-93 (10 μM) + actinomycin D (Act D) (0.8 μM) for 12 h. Cells were then lysed and immunoblotting was performed using p21- and actin-specific antibodies. Representative images from three separate experiments are shown. (c) MG-63 cells were treated with DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for the indicated time. Protein was extracted and immunoprecipitation was performed using anti-CDK2 or anti-CDK4 antibodies, followed by Western blotting for p21 or CDK4 (upper panel), and p21 or CDK2 (lower panel). Representative images from three separate experiments are shown. (d) MG-63 cells were treated with either DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for 24 or 48 h. (Upper panel) Western blots using antibodies directed against p-Rb or total Rb were performed. (Lower panel) MG-63 cells were transfected with luciferase reporter plasmids driven by the E2F sequences and a CMV-β-galactosidase reporter constructs using Lipofectamine for 24 h. Cells were then treated with DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for 24 h. Reporter activity was then measured. Data are expressed relative to an internal control (CMV-β-galactosidase) and are the means±s.e. of three separate experiments, each performed in triplicate; *P≤0.01.

Figure 4

The α-isoform of CaMKII regulates the proliferation of MG-63 osteosarcoma cells and p21 protein levels. MG-63 cells were transfected with control or α-CaMKII siRNA, WT or dominant-negative α-CaMKII (K42M) plasmids 24 h after plating, and were harvested 72 h after transfection. Cells were lysed for whole-cell protein extraction. Immunoblots were developed using antibodies directed against (a) p-α-CaMKII, total α-CaMKII and actin or (b) p21 and actin. The blots are representative of three separate experiments. (c) MG-63 cells were either transfected with WT or dominant-negative α-CaMKII (K42M) plasmids 24 h after plating, or cultured without transfection (NT). MTT proliferation assay was performed 48 h after transfection. Values were obtained from three separate experiments and represent the mean±s.e; *P<0.01. (d) MG-63 cells were transfected for 24 h with control or p21 siRNA. Cells were then treated with DMSO or 10 μM KN-93 for 24 h, followed by an MTT proliferation assay. Values were obtained from three separate experiments and represent the mean±s.e. *P<0.01. (e) MG-63 cells were transfected with control or α-CaMKII siRNA. Immunoblots were developed using antibodies directed against CaMKK, p-CaMKI, CaMKI and actin. The blots are representative of three separate experiments.

Figure 5

The CaMKII antagonist, KN-93, decreases the in vivo growth of human osteosarcoma. MG-63 cells were subcutaneously or intratibially injected into 5-week-old male athymic nu/nu mice that were divided into untreated and KN-93-treated groups. Mice were treated every other day for 6 weeks. (a) After mice were killed, tibiae were removed, formalin fixed and MicoCT scanning was performed. The upper panel shows a two-dimensional section in the proximal tibia, where osteosarcoma has grown (indicated by broken white circle), whereas the lower panel shows a 3D image of the cross-sectional proximal tibia also showing the tumor. (b) Subcutaneous tumors were removed, formalin fixed and paraffin embedded. H&E and IHC staining were performed for p21, p-Rb and p-α-CaMKII. All images were obtained at × 200 (lower left insets) and × 400. The results showed no change in the cytomorphology comparing the control to the KN-93-treated tumors, but upregulation of p21 and downregulation of p-α-CaMKII and p-pRb, as assessed by IHC.

Figure 6

The CaMKII antagonist, KN-93, decreases the activation of Rac1 and Tiam1 in osteosarcoma cells. MG-63 cells were treated with vehicle only (control) or 10 μM KN-93 for 1, 2, 4, 8 and 24 h. (a) Rac1 activation assay was performed by a pulldown assay, whereby the activated (GTP bound) Rac1 protein is pulled down from whole-cell protein lysates with PAK-1 PBD-conjugated agarose beads. (b) Cytoplasmic membrane proteins were extracted and Western blots analyses were performed using antibodies directed against Tiam1 and integrin β1. Integrin β1 was examined as a loading control. Representative images from three experiments are shown. The band intensities of both GTP-Rac1 relative to Rac1 (a, lower panel) and Tiam 1 relative to integrin β1 (b, lower panel) are plotted as a graph. Values were obtained from three separate experiments and represent the mean±s.e; *P<0.05. (c) A proposed mechanism depicting the role of CaMKII in cell cycle progression in osteosarcoma. The increase in the expression/activation of α-CaMKII in osteosarcoma cells results in an increase in the phosphorylation of Tiam1 and its membrane localization. This increases the activation of Rac1 and ultimately inhibits the expression of p21 protein. This leads to a disruption of cell cycle regulation and results in an unregulated growth of these cells, and therefore development of osteosarcoma.