Abstract LB-266: Expression of AGR2 in lung cancer cells needs an active Hippo pathway co-activator, YAP1

Abstract

Abstract Background/Objective: We have previously established that Anterior Gradient-2 (AGR2) affects cancer cell growth by activating YAP1 of the Hippo pathway and inducing AREG of the EGF pathway. Although significant advances have been made concerning the growth promoting effects of AGR2 on cancer cells, including those derived from the pancreas, lung and esophagus, little is known about how AGR2 expression is regulated. Because of AGR2's potential role in human cancer and its established role in organ development in lower vertebrates, we explored the mechanisms responsible for its expression. Methods: ShRNA mediated specific knockdown of YAP1 was conducted in H460 human lung cancer cells. Quantitative real-time PCR was performed to confirm the knockdown efficiency. The expression of AGR2, AREG, and the YAP1-induced genes CTGF, CYR61, COL8A1 was assayed by quantitative real time PCR after YAP1knockdown. The effect of YAP1 knockdown on AGR2 protein was evaluated by immunoblotting. Finally, an AGR2 expression reporter based on the firefly luciferase gene was employed to evaluate whether YAP1 acts on AGR2 promoter activity. Results: Reduced YAP1 expression by lentivirus mediated shRNAmir transduction was further confirmed by western blotting and was also supported by the decrease of YAP1 readouts, such as CTGF. A 95% knockdown of YAP1 expression in H460 cells resulted in a significant morphological change and a 275-fold decrease in AGR2 mRNA. As demonstrated in previously published work, AREG expression was also decreased by 40-fold. Western blots showed that in YAP1 knockdown cells (YAP1KD), there was only negligible levels of AGR2 protein. The data suggested that active YAP1 protein is required for AGR2 expression. The decrease of AGR2 mRNA expression by YAP1KD could be transcriptional or posttranscriptional. To address this question, an AGR2 promoter assay to answer whether YAP1 affects AGR2 transcription. Our AGR2 reporter assay showed that there was more than 5 times more luciferase activity in YAP1KD cells compared to cells infected with the control lentivirus not targeting to any known gene, suggesting that AGR2 expression is regulated in a post-transcriptional manner. Conclusion: YAP1 decreases AGR2 mRNA through a post-transcriptional mechanism, which may include microRNAs. There are four potential miRNA binding sites in the 3-UTR region of AGR2 transcripts. Future experiments will explore if any of these miRNAs are induced upon YAP1 knockdown. This work provides insight into the crosstalk between YAP1 and AGR2, which may have significant implications in lung cancer carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-266. doi:1538-7445.AM2012-LB-266