Abstract 3987: Src regulates inhibitor of differentiation 1 (Id1) via c-Myc induced repression of microRNA-29b: Implications for invasion of lung cancer cells and their resistance against Src inhibitors
Authors: Rothschild Sacha I. , Tschan Mario P. , Jaggi Rolf , Fey Martin F. , Gugger Mathias , Gautschi Oliver 
Published: June 2012

Abstract
Background: Src and Id1 mediate cancer cell invasion and are frequently co-expressed in non-small cell lung cancer (NSCLC). We previously demonstrated that Src regulates Id1 in human lung cancer cells. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by inducing translational inhibition or cleavage of their target mRNAs. The current study aimed at identifying miRNAs controlled by Src that target Id1, and to test if modulation of these miRNAs and Id1 in NSCLC cells leads to resistance against Src inhibitors.
Methods: The study included the following human lung cancer cell lines: A549, H460, H1299, H820, HCC827. Cells were incubated with the Src inhibitors Saracatinib and Dasatinib and miRNA was extracted. To assess miRNA expression profiles, we used TaqMan® Array MicroRNA Cards (Applied Biosystems). Western Blotting was performed using antibodies for Src, phospho-Src, Id1, c-Myc, active beta-Catenin and Actin. Id1 and miR-29b transcript levels were measured using real-time quantitative RT-PCR. Lentiviral vectors expressing pre-miR-29b, anti-sense miR-29b, Id1 and c-Myc were used. Migration was tested with wound healing assays. For invasion matrigel coated membranes for transwell assays were used. Formalin-fixed and paraffin-embedded adenocarcinoma samples and matched lung tissue from 23 patients were used for miRNA expression analysis.
Results: Upon incubation of A549 cells with Saracatinib, miR-29b was the most highly upregulated miRNA with a predicted binding site in the Id1 3’UTR. Indeed, Id1 3’UTR luciferase reporter assays revealed direct binding of miR-29b to the Id1 3’UTR, but not to a mutated Id1 3’UTR. Anti-miR-29b enhanced Id1 mRNA and protein levels and significantly increased cell migration and invasion (p=0.0006). Anti-miR-29b or Id1 overexpression abolished the effects of Src inhibitors on invasion and migration. In contrast, transfection with pre-miR-29b suppressed Id1 and significantly reduced cell migration and invasion (p&lt;0.00001). Overexpression of c-Myc repressed the level of miR-29b and induced the expression of Id1. Saracatinib and Dasatinib decreased active beta-Catenin, c-Myc and Id1 levels, and increased miR-29b levels in a dose-dependent manner. In 19 of 23 human lung adenocarcinoma biopsies, miR-29b was significantly downregulated compared with matched normal lung tissue (p=0.031). miR-29b was a strong prognostic factor for event-free (p=0.003) and overall survival (p=0.04)
Conclusions: These results demonstrate that Id1 is a direct target of miR-29b, which in turn is regulated by Src, active beta-Catenin and c-Myc. Therefore, miR-29b is involved in lung cancer cell migration and invasion. Furthermore, miR-29b is downregulated in human lung adenocarcinomas, and may be involved in the resistance of lung cancer cells against Src kinase inhibitors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2011-3987