HDAC Inhibitor Vorinostat Enhances the Antitumor Effect of Gefitinib in Squamous Cell Carcinoma of Head and Neck by Modulating ErbB Receptor Expression and Reverting EMT FRANCESCA BRUZZESE, 1 ALESSANDRA LEONE, 1 MONIA ROCCO, 1 CARMINE CARBONE, 1 GENY PIRO, 2 MICHELE CARAGLIA, 1 ELENA DI GENNARO, 1 AND ALFREDO BUDILLON 1 * 1 Experimental Pharmacology Unit, Department of Research, National Cancer Institute ‘‘Fondazione G. Pascale’’, Naples, Italy 2 Centro di Ricerche Oncologiche di Mercogliano (CROM), Mercogliano, Avellino, Italy Potentiation of epidermal growth factor receptor (EGFR) inhibitors is required in squamous cell carcinoma of head and neck (SCCHN) to improve their therapeutic index. We demonstrated that the histone deacetylase inhibitor vorinostat in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in SCCHN cells, including cells resistant to gefitinib. We provided evidence suggesting that differential modulation of ErbB receptors together with reversion of epithelial-to-mesenchymal transition (EMT) by vorinostat represent mechanistic bases for the observed synergism. We demonstrated in epithelial CAL27 cells expressing EGFR, ErbB2, and ErbB3 that vorinostat downregulated the expression and signaling of all three receptors. In gefitinib-resistant KB and Hep-2 cells, both of which had undergone EMT and expressed very low levels of ErbB3, vorinostat reverted the mesenchymal phenotype by inducing both E-cadherin and ErbB3 and downregulating vimentin as well as EGFR and ErbB2. Both transcriptional and post-translational mechanisms were involved in the modulation of ErbB receptors by vorinostat. Attenuation of all ErbB transcripts in CAL27 cells as well as induction of ErbB3 transcript in Hep-2 and KB cells was seen upon vorinostat treatment. We showed that vorinostat induced ubiquitination of EGFR and ErbB2 and targeted them predominantly to lysosome-degradation in all cell lines, while the induction of ErbB3-ubiquitination in CAL27 cells led to proteasomes-degradation. Overall, this study suggests that the vorinostat/gefitinib combination represents a promising therapeutic strategy that warrants further clinical evaluation in SCCHN, including tumors intrinsically resistant to EGFR-inhibitors. J. Cell. Physiol. 226: 2378–2390, 2011. ß 2010 Wiley-Liss, Inc. Despite advances in our understanding of the molecular mechanisms of tumor development, prevention, and treatment, the long-term survival for patients with squamous cell carcinoma of head and neck (SCCHN), a cancer that worldwide accounts for more than 500,000 cases each year (Cohen et al., 2010), remains low. The poor prognosis for SCCHN reflects a limited understanding of the mechanisms of local and regional metastasis present in a significant portion of patients, together with an unsatisfactory responsiveness to conventional systemic therapy in recurrent/advanced disease (Haddad and Shin, 2008; Bernier et al., 2009). Overexpression of epidermal growth factor receptor (EGFR) and of its ligands TGF-a or EGF has been observed in about 90% of SCCHN specimens and correlates with poor disease-free and overall survival, an increased risk of disease recurrence and metastasis, and resistance to chemotherapy and radiotherapy (Kalyankrishna and Grandis, 2006; Sheu et al., 2009). Therefore, EGFR is considered to be an excellent target for this disease, and cetuximab, an anti-EGFR monoclonal antibody although have yielded only modest clinical outcomes in monotherapy, is the only targeted therapy approved for the treatment of SCCHN in patients with locally advanced tumors in association with radiotherapy and in patients with recurrent or metastatic diseases in combination with cisplatin-based chemotherapy (Bonner et al., 2006, 2010; Haddad and Shin, 2008; Vermorken et al., 2008). Small-molecule tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib, which prevent the binding of ATP to the receptor and thereby inactivate EGFR, have also been investigated for SCCHN in preclinical and clinical settings (Kalyankrishna and Grandis, 2006; Haddad and Shin, 2008; Vermorken et al., 2008). Our previous studies have preclinically investigated the molecular mechanisms of the growth inhibition Abbreviations: EGFR, epidermal growth factor receptor; ATP, adenosine triphosphate; MAPK, mitogen-activated protein kinase; PARP, poly ADP ribose polymerase; PI3K, phosphatidylinositol 3-kinase; NH 4 Cl, ammonium chloride; PEG, polyethylene glycol; DMSO, dimethyl sulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: Associazione Italiana per la Ricerca sul Cancro (AIRC), grant number: IG 9332 Contract grant sponsor: Italian Ministry of Health Michele Caraglia’s present address is Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy. *Correspondence to: Alfredo Budillon, Experimental Pharmacology Unit, Department of Research, National Cancer Institute ‘‘Fondazione G. Pascale,’’ Via M. Semmola, 80131 Naples, Italy. E-mail: abudillon@yahoo.com Received 1 September 2010; Accepted 24 November 2010 Published online in Wiley Online Library (wileyonlinelibrary.com), 9 December 2010. DOI: 10.1002/jcp.22574 ORIGINAL RESEARCH ARTICLE 2378 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.