Abstract Introduction: Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting EGFR, is widely used to treat patients with HNSCC. However, the majority of patients do not respond or develop resistance to cetuximab. Recent studies have shown that epithelial-to-mesenchymal transition (EMT) is associated with EGFR inhibitor resistance in diverse tumors including HNSCC. To further understand the role of EMT in cetuximab resistance, we examined the HNSCC cell lines for EMT- related genes and proteins and correlated their expression with cetuximab response in vitro. Methods: Responses to cetuximab in the panel of 25 HNSCC cell lines and a spontaneously immortalized keratinocyte (HaCaT) were determined by matrigel colony formation assay. Global gene and microRNA expression of the cell lines were determined using Affymetrix and Taqman microRNA arrays. Differential expression of EMT-related genes was confirmed by qRT-PCR and western blots. Collagen migration assay was performed to assess migration and stable knockdowns of Smad4 and EGFR were achieved using lentiviral shRNAs. The ligand levels in cell cultures were quantified by enzyme-linked immunosorbent assays (ELISA). Results: Both the array and qRT-PCR data confirmed that the EMT-related genes were generally correlated with cetuximab resistance. In two pairs of isogenic cell lines with acquired resistance to cetuximab, SCC1/1Cc8 and SCC25/CTX-10 and CTX-11, the resistant clones show lower E-cadherin, higher vimentin expression and increased migration rate compared to parental cells. We further examined the TGF-β pathway known to induce EMT in HNSCC. Smad4 levels were lower in the resistant clones. Knockdown of Smad4 rendered resistance in the cetuximab sensitive parent cells by activating TGF-β and c-Met pathways. In addition, knockdown of Smad4 resulted in increased p-AKT levels and EMT cellular phenotypes in the SCC1. To further examine the regulation of Smad4 by microRNAs, we determined differentially expressed microRNAs between cetuximab sensitive and resistant isogenic cell lines. We found that miRNA-146a was upregulated in the resistant clones, and Smad4 and EGFR expression were regulated by miRNA-146a mimic and inhibitor. TGFβR1 inhibitor reversed acquired and intrinsic resistance to cetuximab in vitro. Conclusion: We confirmed that EMT is associated with cetuximab resistance and TGF-β and c-MET signaling pathways may play an important role in induction of EMT in a subset of HNSCC. Our results suggest a combination strategy to improve the therapeutic index of cetuximab therapy in HNSCC. 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 1889. doi:1538-7445.AM2012-1889