Experimental Studies Of Targeting Protein Kinase Polo-like Kinase 1 In The Treatment Of Osteosarcoma

Osteosarcoma accounts for 60%of primary malignant bone tumors diagnosed in the first two decades of life. Standard treatment for osteosarcoma is surgery and neo-adjuvant chemotherapy. Chemotherapy has significantly improved the survival rate from 11%with surgery alone to 60-70%when surgery is combined with chemotherapy. Patients with advanced osteosarcoma after front-line chemotherapy usually receive further treatment with additional chemotherapy, which may be considered toxic. Many patients eventually relapse, resulting in an unsatisfactory outcome. Unfortunately, not much progress has been made on improving survival over the past 20 years with regard to the treatment of osteosarcoma. Thus, identification of new targeted therapies is a crucial step forward in the drive towards personalized medicine.In this study we describe an optimized systematic screen of known kinases using osteosarcoma cell lines (KHOS and U-2OS) and a lentiviral-based short hairpin RNA (shRNA) human kinase library. We identified several kinases, including human polo-like kinase (PLKl), which inhibits cell growth and induces apoptosis in osteosarcoma cells when knocked down. cDNA rescue and synthetic siRNA assays confirm that the observed phenotypic changes result from the loss of PLKl gene expression. Western blotting analysis confirmed that PLKl is highly expressed and activated in several osteosarcoma cell lines as well as in resected tumor samples.The PLKl inhibitor BI 2536 was confirmed to inhibit proliferation and induce apoptosis in 2D and 3D cultures of osteosarcoma cell lines KHOS and U-2OS. Proliferation assay was performed both in 2D and 3D culture demonstrated that the growth of both cell lines could be inhibited by BI 2536. Cell cycle analysis showed that cells treated with BI 2536 were mainly arrested in G2/M phase. Immunofluorescence and Western blotting analysis confirmed that administration of BI 2536 led to significant decrease of PLKl and Mcl-1 expression in dose-and time-dependent manners. In addition, BI 2536 induced apoptosis was demonstrated by PARP cleavage and caspase assay. Finally, in mouse osteosarcoma xenografts, BI 2536 treated mice had significantly smaller tumors compared with the control mice. Immunohistochemistry showed the overall survival of patients with high expression levels of PLKl was significantly shorter than those patients with lower levels.These results demonstrate the capability and feasibility of a high-throughput screen with a large collection of lentiviral kinases and its effectiveness in identifying potential drug targets. These findings also offer evidence for the potential role for targeting PLKl in osteosarcoma therapy.