Abstract 3431: Regulation of integrins AlphaV Beta3 and focal adhesion kinase signaling by PTTG in induction of EMT

Abstract

Abstract Pituitary tumor transforming gene (PTTG) is a well-studied oncogene for its role in tumorigenesis. PTTG expression levels correlate with the tumor development, growth and metastasis and serve as a marker of malignancy in several cancer types including lung. Epithelial to mesenchymal transition (EMT) is a crucial step in tumor progression and metastasis, during which epithelial cells lose their polarity and cell-cell adhesion. Integrins are transmembrane proteins which bind ligands found in the extracellular matrix. Interactions between the extracellular matrix and the actin cytoskeleton commonly take place at focal adhesions on the cell surface that contain localized concentrations of integrins, signaling molecules, and cytoskeletal elements. Talin forms a direct interaction with the integrin cytoplasmic domain, and interacts with cytoskeletal elements (actin) and signaling factors. The present study was focused to define the regulation of integrins and FAK pathway by PTTG in induction of EMT. For our studies, we used lung cancer cell lines A549 and H1299 and infected with adenovirus expressing PTTG cDNA or adenovirus expressing control GFP. Using Real Time PCR and immunohistochemistry and FACS analysis, we observed that overexpression of PTTG in cells resulted in a significant increase in the expression of integrins αv and β3 in both cell lines compared to uninfected cells or cells infected with adenovirus expressing GFP. These results were confirmed by the down-regulation of PTTG expression using adenovirus expressing PTTG-specific siRNA that showed a significant decrease in the expression of integrins αv and β3. Western blot analysis revealed increased phosphorylation of FAK on overexpression of PTTG. Analysis of downstream signaling genes such as Paxillin, Metavinculin and Rac 1 by quantitative real time PCR analysis showed positive correlation between levels of PTTG and these genes. Treatment of A549 and H1299 cells with the αvβ3 antagonist echistatin (RGD peptide) and siRNA specific for integrin αv resulted in a decreased in phosphorylation of FAK as well as expression of downstream signaling genes Paxillin, Metavinculin and Rac 1. The Vinculin and Phallodoin are responsible for the functional link of FAK to the actin cytoskeleton. Disruption of actin cytoskeleton was confirmed with immunostaining. Taken together our results suggest for the first time an important role of PTTG in regulation of integrins αv and β3 leading to change in Focal Adhesion Kinase (FAK) pathway resulting in induction of EMT. 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 3431. doi:10.1158/1538-7445.AM2011-3431