Abstract 777: Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer
Authors: Haque Inamul , Sharma Mukut , Godwin Andrew K. , Huang Chao H. 
Published: August 2019

Background: Lung cancer is the leading cause of cancer related death in the United States. Epidermal Growth Factor Receptor (EGFR) mutation predicts response to a tyrosine kinase inhibitor (TKI) of EGFR in approximately 25% of patients. Non-small cell lung cancer (NSCLC) patients with EGFR mutation eventually develop resistance to EGFR-TKI. The mechanism of resistance is not fully elucidated but majority of the cases is related to emergence of clones with T790M mutation in EGFR, amplification of cMET and Epidermal Mesenchymal Transition (EMT). Sonic Hedgehog (SHh) signaling activation is involved in EMT through FGF, notch, TGFβ signaling and microRNA (miRs) networks. Several miRs have been shown to correlate with TKI resistance indicating that miRs may serve as novel targets and/or biomarkers for anti-EGFR therapy. MiR-506 is involved in Hedgehog signaling and it is abnormally expressed in several cancers. However, the role of miR-506 in modulating TKI resistance in NSCLC remains unexplored. In this study, we investigated the role of miR-506 in the regulation of SHh in EGFR-TKI-resistant lung cancer cell lines.

Methods: To generate resistant cell lines, two EGFR-TKI sensitive NSCLC cells (HCC4006 and HCC827) were exposed to increasing concentrations of erlotinib up to 20 µM over 6 months. The resultant clones were verified for sensitivity to erlotinib using a crystal violet staining assay. The expression of miR-506 in parental and resistant clones was measured using Taqman microRNA assay. Invasive/migratory ability of parental and resistant clones was measured by Boyden chamber assays. EMT and stemness markers were evaluated by Western blotting.

Results: The studies demonstrated that miR-506 levels were ~90% and 75% lower in most resistant clones of HCC4006 (HCC4006ER4) and HCC827 (HCC827ER3), respectively. These clones showed overexpression of SHh (5-fold in HCC4006ER4 and 2.5-fold in HCC827ER3 as compared to their respective parental cells). Ectopic expression of miR-506 in these resistant cells inhibits the expression of SHh by ~80% as well as its downstream mediator, GLI1 by 55%. Our results showed that the IC50 of erlotinib was 22.4 μM for ER cells transfected with ctrl-miR and 4.7 μM for cells with miR-506 mimics. Moreover, an epithelial marker, E-cadherin is upregulated (~2.7-fold) and mesenchymal/cancer stem cell markers (e.g., N-cadherin, vimentin, Sox-2, Notch1, CD44 and ALDH1A1) were significantly downregulated (75-90%, p-value&lt;0.001) in miR-506 overexpressing cells.

Conclusions: Our data showed that miR-506 downregulation and induction of SHh are associated with EGFR-TKI resistance in EGFR mutated NSCLC cells. MiR-506 interference and inhibition of SHh pathway may be potential therapeutic strategies to reverse resistance to EGFR-TKI in NSCLC with EGFR mutation. (This study was funded by the support from IRG-16-194-07(IH) and Godwin's lab).

Citation Format: Inamul Haque, Mukut Sharma, Andrew K. Godwin, Chao H. Huang. Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 777.