Abstract 3705: PLK2 phosphorylates TAp73 and prohibits TAp73 tumor suppressor activity in osteosarcoma cells

Abstract

Abstract Background: TAp73, as a member of the p53 tumor suppressor family, is frequently overexpressed in malignant tumors in humans. The abundance of TAp73 expression and phosphorylation modification regulate its transcriptional activity. In previous study, we found that the anti-tumor function of TAp73 was reactivated by protein dephosphorylation. Polo-like kinase 2 (PLK2) was previously found phosphorylated p53 and transcriptionally regulated by p53, and these interactions affected the fate of cells. However whether PLK2 interacts with TAp73, phosphorylates TAp73, and modulate its tumor suppressor function are still unclear. Herein, we investigate how PLK2 phosphorylates TAp73 and affects TAp73 function in human osteosarcoma cells. Materials and Methods: Osteosarcoma cell lines, Saos2 and MG63 were used as models with differential expression levels of TAp73. Phosphorylation predictor software Scansite 3.0 and the predictor GPS-polo 1.0 were used to theoretically analyze the phosphorylation sites on TAp73. Co-immunoprecipitation (Co-IP), phosphor-tag Western Blot (WB), and indirect immunofluorescence assays were used to determine the interaction between PLK2 and TAp73. TAp73 activity was assessed by testing downstream genes, P21 and PUMA using Western blot and RT-PCR. The physiological effects of PLK2 interacting with TAp73 on cell cycle G1 phase, cell proliferation and cell apoptosis were measured by flow cytometry, cell counting kit-8 and terminal-deoxynucleotidyl transferase mediated nick end labeling assays. Results: TAp73 expression was low in Saos2 cells when compared with MG63 cells, at both mRNA and protein levels, but both have the similar level of PLK2 expression. DNA damaging drugs, Cisplatin and Adriamycin, up-regulated TAp73 and PLK2 expression on does-dependent way. We found that PLK2 directly bound to and phosphorylated TAp73 when TAp73 induced by DNA damaging. The phosphorylation predictor software identified the potential phosphorylation sites that PLK2 could phosphorylate TAp73. PLK2 phosphorylated TAp73 at residue Ser48, which prohibited TAp73 translocation to the nucleus. Additionally, combination of PLK2 inhibition by siRNA with DNA-damaging drugs up-regulated both p21 and PUMA mRNA expression to a greater extent than DNA-damaging drug treatment alone. Inhibiting PLK2 in TAp73-induced osteosarcoma cells enhanced the effects of the DNA-damaging drugs on G1 phase arrest. Moreover, inhibiting PLK2 in TAp73-induced cells sensitized the effects of DNA-damaging drugs on cell proliferation and apoptosis. However, TAp73-knockdown decreased the antitumor effects of DNA-damaging drugs. Conclusion: These findings reveal a novel PLK2 function in the phosphorylation of TAp73, which prohibits TAp73 anti-tumor activity in osteosarcoma cells. Further studies could be investigated how to target PLK2 and enhance TAp73 antitumor activity in preclinical and clinical trials. Citation Format: Zhengbo Hu, Hai Lu, Anmin Jin, Xiaohong Liao, Zunying Xu, Chao Dong. PLK2 phosphorylates TAp73 and prohibits TAp73 tumor suppressor activity in osteosarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3705.