Mechanism Study of EGFR Expression Regulated by Nek2 via ATDC-Mediated β-catenin Pathway in Liver Cancers

• Main Authors: Chih-Hsin Ou-Yang, 歐陽志欣
• Other Authors: Yann-Jang Chen
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/26at39

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 105 === In our lab’s studies, several chromosome aberrations were found in hepatocellular carcinoma (HCC) by comparative genomic hybridization (CGH). The most frequent aberration was gain change of chromosome 1q. Through array CGH analysis, a candidate gene, NIMA-related kinase 2 (Nek2) located on 1q32.2 was selected. The levels of mRNA and protein of Nek2 were exactly elevated in all HCC cell lines and clinic samples. The abilities of cell proliferation and colony formation were significantly decreased in two HCC cell lines, HA22T and Mahlavu cells with Nek2 knockdown, and even tumor size was reduced as injecting Nek2-knockdown Mahlavu cells into nude mice. We further found activity of epidermal growth factor receptor (EGFR) pathway and expression of EGFR were reduced in Nek2-knockdown Mahlavu cells. We are interesting how Nek2 regulates EGFR expression in tumor cells. 　　Through literature searching, mRNA microarray data, and real-time polymerase chain reaction (RT-PCR) results, we hypothesize that Ataxia-Telangiectasia group D Complementing gene (ATDC)-mediated β-catenin pathway may involve in Nek2-regulated EGFR expression in tumor cells. In order to investigate the detail mechanism, two approaches, loss and gain-of-function for Nek2 were performed. For loss-of-function, Nek2 expression was repressed by Nek2 shRNA in two HCC cell lines, HA22T and Mahlavu. For gain-of-function, overexpression vector, pLPS-Nek2-EGFP was used and ectopically expressed Nek2 in HEK293 and U2OS cell lines, with lower expression of Nek2 and EGFR. The results demonstrated that the protein levels of EGFR, β-catenin, Dvl-2, and ATDC were all decreased in Nek2-knockdown HA22T and Mahlavu cells and that the protein levels of EGFR, β-catenin, and ATDC were all increased only in Nek2-overexpressed U2OS cells. 　　Next, to investigate whether β-catenin is required for Nek2-regulated EGFR expression, we utilized β-catenin inhibitor, BC21, to treat with Nek2-overexpressed U2OS cells. The results showed both levels of EGFR mRNA and protein were reduced in Nek2-overexpressed U2OS cells after BC21 treatment. 　　To further study for whether this mechanism can be applied to other cancers, we used MDA-MB-231, A549, and H1299 cells to silence Nek2 expression. The protein levels of EGFR, β-catenin, and ATDC were all decreased in Nek2-knockdown MDA-MB-231 and H1299 cells. 　　On the other hand, we examined whether EGFR-signal transducers and activators of transcription 3 (STAT3) signaling is down-regulated in Nek2-knockdown cells. The results exhibited the activation of STAT3 and the expression of downstream target genes were inhibited in Nek2-knockdown HA22T and Mahlavu cells. In addition, Nek2-knockdown Mahlavu cells also showed the increased E-cadherin and decreased N-cadherin and suppression of drug resistance against cisplatin treatment. 　　Taken together, we found that Nek2 regulates EGFR expression via ATDC-mediated β-catenin pathway, modulates EGFR-STAT3 signaling, alters the epithelial-mesenchymal transition (EMT) markers, and promotes drug resistance. Thus, we evaluate Nek2 is a potential therapeutic target for HCC.