Abstract 2944: Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer
Authors: Chen Chung-Yu , Chen Ying-Yin , Chen Kuan-Yu , Shih Jin-Yuan , Change Yih-Leong , Yu Chong-Jen , Yang Pan-Chyr 
Published: July 2016

Purpose: Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, is elevated in multiple cancers. Thymidylate synthase (TS) is an important enzyme in purine synthesis and DNA replication. In this study, we investigated the AEG-1 expression characteristics in non-small cell lung cancer (NSCLC) to determine whether its expression is correlated with TS regulation of NSCLC.

Materials and Methods: A549, H157, CL1-0, CL1-5, PC9, H1975, H520 and H292 human lung cancer cell-lines were cultured in medium with RPMI1640 + 10% FBS. Immunohistochemical stain and Western blot of AEG-1 and TS were studied in all cell-lines. The primary antibodies used were anti-AEG-1 (1:1,000; chicken polyclonal), anti-actin (1:1,000; rabbit polyclonal; Santa Cruz), and anti-TS (1:1,000; mouse monoclonal; Abcam). Total RNA was extracted using a Qiagen mRNA easy mini kit (Qiagen). Real-time PCR was performed using ABI 7900 fast real-time PCR system to detect expression of AEG-1 and TS. Cell viability was determined by standard MTS assays to recognize IC50 of Pemetrexed in lung cancer cell-lines. Transfection of siRNA for AEG-1 was carried out to knock down AEG-1. AEG-1 pcDNA (Life technologies, CA, USA) were used to quantitatively examine AEG-1 activity. Expressive change of AEG-1associated with TS and signaling pathways were analyzed by Western blot and PCR.

Results: TS and AEG-1 in H157, Cl1-0, CL1-5, H520 and H292 were higher detected by IHC stain, Western blot and RT-PCR mRNA level. The expression of TS was positive correlated with AEG-1 in lung cancer cell-lines. The IC50 values of Pemetrexed for cells were lower in the lung cancer cell-lines with lower TS gene expression. After knockdown of AEG-1 by siRNA in Pemetrexed-resistance PC-9 and A549 cell-lines, the expression of TS decreased associated with decreasing change IC50 of Pemetrexed. In contrast, transfection with AEG-1 pcDNA in PC-9 and A549 cell-lines overexpressed AEG-1 gene, TS level also enhanced associated with increasing change IC50 of Pemetrexed. AEG-1 expression can also activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that augments binding of c-Myc thereby regulating AEG-1 transcription.

Conclusions: Our data indicated a significant correlation between TS and AEG-1 gene expression in NSCLC cell-lines. TS expression might be regulated by AEG-1 through activation of PI3K/AKT and c-Myc signaling pathways associated with development of chemoresistance to pemetrexed in NSCLC treatment

Citation Format: Chung-Yu Chen, Ying-Yin Chen, Kuan-Yu Chen, Jin-Yuan Shih, Yih-Leong Change, Chong-Jen Yu, Pan-Chyr Yang. Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer. [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 2944.