Abstract 4355: A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: We endeavored 1) to understand the contribution of TGFβ signaling on expression of a 13-gene microRNA (miRNA) signature and 2) to assess the activity of the signature miRNA genes on mRNA targets that impact the mechanism of activity of erlotinib, the mechanisms by which resistance to erlotinib arises, and the influence of epithelial-to-mesenchymal transition (EMT) on sensitivity to erlotinib. Background: Previous findings from this lab identified a 13-gene miRNA signature that can predict response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in non-small cell lung cancer (NSCLC) cell lines. Bioinformatic analysis of the miRNA signature showed a convergence of the 13 miRNA genes on the TGFβ signaling pathway. We hypothesize that TGFβ signaling controls expression of the miRNAs comprising a signature of response to erlotinib in NSCLC. Methods: Two signature miRNA genes (miR-140-3p and miR-141) were chosen for study because they demonstrate opposing expression levels in erlotinib-resistant and -sensitive NSCLC cell lines. Promoters of both miRNA genes harbor potential Smad Binding Elements (SBEs), binding sites of the TGFβ-responsive Smad transcription factors. TGFβ activity was assessed in erlotinib-resistant (A549) and -sensitive (PC9) NSCLC cell lines using western blot analysis of proteins of interest in the TGFβ signaling cascade and quantitative real-time PCR for expression of miR-140 and -141. The activity of the potential SBEs controlling each of the two miRNA genes was measured by sub-cloning relevant promoter regions into luciferase-reporter constructs. The resulting miR-140 and -141 promoter constructs were transfected into A549 and PC9 cells, and luciferase activity was measured in the presence of TGFβ, the TGFβ inhibitor, SB-431542, and pharmacological inhibitors of EGFR and MAPK. Results: TGFβ acts through the Smad-responsive, canonical signaling pathway in A549 cells. In PC9 cells, TGFβ utilizes the Ras/MAPK pathway for similar signaling endpoints. Analysis of endogenous miRNA expression supports the hypothesis that TGFβ, in the presence and absence of SB-431542 treatment, alters the expression of both microRNAs in both cell lines. Analysis of the influence of the possible SBE sites in each miRNA gene promoter suggests a cell-line specific response. Conclusion: TGFβ can control the expression of signature microRNA genes using different downstream effectors in erlotinib-sensitive and in erlotinib-resistant cell lines. We hypothesize that the erlotinib sensitivity signature miRNA genes can control sensitivity to erlotinib and EMT. Citation Format: Madeline J. Krentz, Rebecca Astley, Andrew Stacy, Esther P. Black. A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2014-4355