Abstract 4610: Characterizing TGFβ1-inducible EMT models in NSCLC helps define critical EMT-associated genes for therapeutic targeting
Authors: Schmitt Anthony D. , Fuchs Bryan C. , Wei Lan , Tanabe Kenneth K. , Lanuti Michael 
Published: August 2012

Abstract
Background: Mesenchymal phenotype in solid tumors has been associated with poor prognosis and resistance to treatment. Understanding epithelial to mesenchymal transition in lung cancer has implications for treatment and outcomes. We hypothesized that examining EMT expression profiles in multiple established non small cell lung cancer cell lines (NSCLC) and comparing to TGFβ-1-induced NSCLC cell lines would provide insight for new targeted strategies to manipulate EMT status. Methods: Human NSCLC cell lines were obtained from ATCC and other sources. Cell lines phenotypically characterized as epithelial or mesenchymal were treated with recombinant TGFβ-1 or SB-431542 (TGFβRI inhibitor), respectively, and evaluated for expression of E-cadherin, Vimentin, and pSmad2 via Western blot. Comprehensive EMT expression profiles were generated by RT-PCR using a modified commercial 96-well plate containing 87 genes associated with EMT. Cell motility was evaluated by transwell migration assay. Cytotoxicity evaluations with erlotinib and an oncolytic HSV-1 virus (hrR3) were quantified via MTT assay. Results: Western Blots on cell lysates from NSCLC cell lines confirmed expression of well-established EMT surface markers. Western blots on all TGFβ-1-treated epithelial cells indicated total loss of E-cadherin after 7 days of treatment. Inhibition with TGFβRI in mesenchymal cells resulted in only modest increases in E-cadherin, but robust reduction of Vimentin. Cell migration assays indicated that mesenchymal cells and TGFβ-1-treated cells were more invasive than epithelial cells. Comparative expression analyses revealed ErbB3 as 1 of 2 common epithelial genes down-regulated in established mesenchymal cell lines and in TGFβ1-inducible models. This finding was validated with functional assays: small molecule inhibition of TGFβRI provided only a modest sensitizing effect to erlotinib in mesenchymal cell lines. Comparative gene expression examining downstream TGFβ-1 signaling revealed similar up-regulation of well-characterized EMT-associated genes (ZEB-1 and Vimentin), but also genes currently lacking a defined role with respect to EMT in cancer biology. Mesenchymal and TGFβ1-treated cells were also observed to be more resistant to hrR3 viral oncolysis via down-regulation of Nectin-1. Conclusion: There are critical signaling commonalities between established mesenchymal cells and TGFβ1-inducible cell lines. Our approach provides one method to assess similarities in expression of EMT-associated genes in established NSCLC cell lines, and TGFβ1-induced EMT. Future studies will focus on manipulating downstream components of the TGFβ1 pathway to sensitize cells to treatment modalities and explore the potential of altering the mesenchymal phenotype for other therapeutic interventions.
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 4610. doi:1538-7445.AM2012-4610