Abstract 1731: Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness
Authors: Liu Wei-Lin , Hou Tommy Wei-Hsien , Cheng Jason Chia-Hsien 
Published: May 2015

Abstract
Background: Radiotherapy (RT) is an essential component in multi-modality cancer management. While RT yields effective local disease control, distant failures remain great challenges that hinder cancer cure. Tumor cells that survive sublethal irradiation become invasive and metastasize, through up-regulation of matrix metalloproteinase-9 (MMP-9) and remodeling of extracellular matrix. Studies have shown that RT induces MMP-9 production in tumor cells by activating NF-κB through PI3K/Akt and MAPK/ERK pathways. However, clinical applications of inhibitors targeting these pathways are lacking due to toxicity and low efficacy. EGFR and HER2 are canonical upstream signaling molecules that activate these pathways. Additionally, EGFR and HER2, when stimulated with ligands, have been found to induce MMP-9 expression. In this study, we aim to determine the roles of EGFR and HER2 in RT-induced MMP-9 expression, tumor cell invasiveness and metastasis using EGFR inhibitor, erlotinib and EGFR/HER2 inhibitor, afatinib.
Methods and Materials: We used western blot analysis to determine EGFR, HER2, and Akt phosphorylations in irradiated Lewis lung carcinoma (LLC) cells (7.5 Gy) pretreated with erlotinib or afatinib. We then used RT-PCR, western blot assay, ELISA, and gelatin zymography to evaluate MMP-9 expression in irradiated LLC cells pretreated with erlotinib or afatinib. Clonogenic assays estimated irradiated LLC cell survival treated with erlotinib or afatinib. Matrigel-coated Boyden chamber assay was used to evaluate invasion capability of irradiated LLC cells. Lastly, tissue western blot analyses of EGFR/HER2/Akt phosphorylations and MMP-9 were performed in LLC tumor grown on the thigh of C57BL/6 mice treated with combined RT and either drug.
Results: RT induced EGFR, HER2, and Akt phosphorylations as well as increased MMP-9 expression in LLC cells. Pretreatment with afatinib suppressed RT-induced MMP-9 mRNA and protein expression more significantly than erlotinib. Afatinib inhibited RT-induced invasiveness of LLC cells more than erlotinib did. Tissue western blot analysis of irradiated LLC ectopic xenografts showed more inhibition of RT-induced in vivo MMP-9 expression by afatinib than erlotinib. Afatinib was a stronger inhibitor than erlotinib for RT-induced MMP-9 up-regulation and cell invasiveness in LLC cells and ectopic xenografts. However, clonogenic assay of LLC cells showed that neither erlotinib nor afatanib sensitized LLC cells to RT in survival.
Conclusion: Our results suggest that sublethal irradiation induces MMP-9 expression through EGFR and HER2 activation. Dual inhibition of EGFR and HER2 kinase better suppress RT-induced MMP-9 expression and abolish tumor cell invasiveness. Afatinib may serve as an orally bioavailable agent that ameliorates distant metastasis from inadequately irradiated primary lung tumor.
Citation Format: Wei-Lin Liu, Tommy Wei-Hsien Hou, Jason Chia-Hsien Cheng. Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness. [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 1731. doi:10.1158/1538-7445.AM2014-1731