Regulation of TGF-β signaling by PLK1 in non-small cell lung cancer

Abstract

Lung cancer is a cancer with a high mortality in the world. Five-year survival rate of non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers, is approximately 10-20%, and the median survival rate is 8-10 months, showing a low survival rate. The reason why lung cancer patients have low survival rate, is that about 40% of patients have metastasized at the time of diagnosis. Metastasis is known to be the leading cause of death in cancer patients than primary tumors. For metastasis motility and invasiveness is acquired through epithelial-mesenchymal transition (EMT). EMT is induced by upregulation of transcription factors that express the genes for the characteristics of mesenchymal cells by activation of TGF-β signaling. Previously our group found that active PLK1 amplified TGF-β signaling and induces metastasis in NSCLC. In addition, phosphorylation of vimentin by PLK1 induced immune evasion through TGF-β signaling. Although it is known that PLK1 activates TGF-β signaling, the mechanism how it activates is unknown. Based on the research background, the aim of this study is to elucidate the mechanism how PLK1 regulates TGF-β signaling. KEGG pathway in microarray analysis using cells expressing PLK1-TD, showed that TGF-β signaling is involved within top 5 pathways. It was observed that the expression of Smad2 was increased in cells expressing the constitutively active PLK1 having invasiveness. Based on the preliminary data, the relationship between PLK1 and Smad2 were observed, when the constitutively active mutant of PLK1 was expressed in A549 cells. In this condition, the levels of p-Smad2 at S465/S467 were upregulated. Conversely, it was observed that the level of pSmad2 decreased when PLK1 was inhibited with shRNA or inhibitor. In the NSCLC A549 and NCI-H460 cells, the interaction of PLK1 and Smad2 appeared when TGF-β was treated. To observe whether PLK1 phosphorylates Smad2, in vitro PLK1 kinase assay was performed observe. PLK1 phosphorylated Smad2 in an in vitro kinase assay. The phosphorylation site of Smad2 were identified using LC/MS-MS spectrometry analysis and site-directed mutagenesis. Expression of phosphomimetics of Smad2 increased the expression of N-cadherin and decreased the expression of E-cadherin. In addition, the invasiveness and migration ability increased by the expression of phosphomimetics of Smad2. Therefore, phosphorylation of Smad2 by PLK1 enhanced cell motility and promoted invasiveness. Collectively, I suggest that PLK1 directly regulates TGF-β signaling for cancer metastasis.