The kinase Mirk is a potential therapeutic target in osteosarcoma

Abstract

Osteosarcoma is the most common primary malignant bone tumor affecting children and adolescents. The majority of patients are treated by surgery and chemotherapy but have limited alternative therapeutic options. Kinases play an important role in the growth and survival of tumor cells. We aim to identify specific kinases to be vital in the survival of osteosarcoma cells and thus may be a key target in creating novel anticancer therapies. A lentiviral short hairpin RNA kinase library, screened osteosarcoma cells, identified kinase minibrain-related kinase (Mirk) (Dyrk1B) as a potential target. Knockdown Mirk expression could inhibit cell growth and induce apoptosis. Chemically synthetic small interfering RNA knockdown and complementary DNA rescue assay further confirmed the results from the decrease of Mirk gene expression. The relationship between Mirk gene expression and the clinical characteristics of patients with osteosarcoma was investigated using tissue microarray and immunohistochemistry analysis. The data indicate that the overall survival rate of patients with Mirk high staining (high levels of Mirk protein expression) is significantly shorter than those with Mirk low staining and moderate staining. This highlights Mirk's potential to serve as a promising target for molecular therapy in the treatment of osteosarcoma.

Fig. 1.

Mirk is a new regulator of osteosarcoma cell survival and apoptosis. (A) Results of lentiviral shRNA directed against Mirk in KHOS cells from the initial screen. The data are representative of a single well of a 96-well plate of MISSION® LentiExpress™ Human Kinases shRNA library as described in the Materials and Methods. (B) Mirk cDNA rescue assay. Pretransfection of full length of Mirk cDNA constructs that lack the 3′UTR could reverse the cell death phenotype when induced by a specific Mirk lentiviral shRNA, which targets the 3′UTR region. (C) Mirk expression in cDNA rescue assay as determined by western blot.

Fig. 2.

Mirk expression in other tumor cell lines and osteosarcoma tissues. (A) High levels of Mirk expression were visible in tumor cell lines, including osteosarcoma (U-2OS, KHOS), uterine sarcoma (MES-SA), chondrosarcoma (CS-1) synovial sarcoma (SS-1), Ewing sarcoma (TC-71) and ovarian cancer (SKOV-3, 3A, 2008). (B) Mirk expression in osteosarcoma tissues as determined by western blot. OST1–OST6 represent tissue samples from six different patients.

Fig. 3.

Knocked down Mirk expression decreases cell proliferation in multiple osteosarcoma cell lines. (A) Cell proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide on cell lines: U-2OS, Saos, OSA344 and normal osteoblast cell HOB-c, after transduction with Mirk shRNA (NM_004714.x-442s1c1). The absorbance values were normalized by the control cells (non-infected) to 100% survival and the values of Mirk shRNA transduced cells were then quantified to get relative percentage of cell survival. Mirk knockdown decreases cell proliferation in osteosarcoma cells but not in normal osteoblast cells. (B) Phase contrast photomicrographs of cell lines before and after infection of lentiviral Mirk shRNA. Mirk knockdown induces significantly cell death in osteosarcoma cell lines U-2OS, Saos and OSA344 but not in normal osteoblast cell HOB-c.

Fig. 4.

Synthetic siRNA targeting Mirk decreases cell proliferation and induces apoptosis in KHOS cells. (A) KHOS cells were transfected with Mirk siRNA or non-specific siRNA, and cell proliferation posttransfection was determined by CellTiter 96® AQueous One Solution Reagent. (B) Apoptosis was evaluated using the M30-Apoptosense ELISA assay kit. The increase levels of cytokeratin 18 reflect Mirk siRNA induced apoptosis in KHOS transfected cells. (C) Synthetic siRNA Mirk knockdown reduces Mirk protein expression as determined by western blot.

Fig. 5.

Mirk expression is correlated with poor osteosarcoma survival. (A) Kaplan–Meier survival curve of osteosarcoma patients are subgrouped as either Mirk high staining (Mirk staining = 3+), Mirk moderate staining (Mirk staining = 2+) or Mirk low staining (Mirk staining = 1+). The overall survival of patients with Mirk high staining is significantly shorter than those patients with Mirk low staining and moderate staining (P = 0.001). (B) The levels of Mirk staining for samples obtained from patients who terminated in <60 follow-up months were significantly higher than those who survived at 60 follow-up months (P = 0.0012). (C) Representative immunohistochemical staining of Mirk expression levels in osteosarcoma tissues.