Cancer Therapy: Preclinical Primary and Acquired Resistance of Colorectal Cancer Cells to Anti-EGFR Antibodies Converge on MEK/ERK Pathway Activation and Can Be Overcome by Combined MEK/EGFR Inhibition Teresa Troiani, Stefania Napolitano, Donata Vitagliano, Floriana Morgillo, Anna Capasso, Vincenzo Sforza, Anna Nappi, Davide Ciardiello, Fortunato Ciardiello, and Erika Martinelli Abstract Purpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPK pathway is one of the resistance mechanisms to cetuximab. Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to cetuximab. Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15, SW620, and SW480) and one with a BRAF mutation (HT29) were resistant to the antiproliferative effects of cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. Combined treatment with cetuximab and BAY 86-9766 induced a synergistic antiproliferative and apoptotic effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2 inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established human HCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and BAY 86-9766 caused significant tumor growth inhibition and increased mice survival. Conclusion: These results suggest that activation of MEK is involved in both primary and acquired resistance to cetuximab and the inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. Ó2014 AACR. Introduction Colorectal cancer is a major cause of morbidity and mortality throughout the world (1). The prognosis of patients diagnosed with metastatic colorectal cancer has improved markedly over the last 15 years, with an increase in median overall survival from 6 months with only best supportive care to more than 2 years with the introduction of active chemotherapy drugs, such as fluropyrimidines, oxaliplatin, and irinotecan, and of molecular targeted drugs, such as bevacizumab, cetuximab, panitumumab, aflibercept, and regorafenib (2, 3). Cetuximab and panitumumab are 2 blocking anti-epi- dermal growth factor receptor (EGFR) monoclonal anti- bodies (mAb) that inhibit the activation of the EGFR and its downstream intracellular signals, the RAS–RAF–MEK– MAPK and the PTEN–PIK3CA–AKT pathways (4–6). In particular, cetuximab is an effective treatment as single agent or in combination with standard chemotherapy regi- mens for a subset of patients with metastatic colorectal cancer (7). Resistance to anti-EGFR therapies is likely because of the constitutive activation in cancer cells of signaling pathways acting downstream and/or indepen- dently of EGFR. In fact, point mutations in codon 12 or 13 within the exon 2 of the KRAS gene have been found as the major negative predictor of efficacy for cetuximab (8, 9). Therefore, cetuximab is currently used in monotherapy or in Authors' Affiliation: Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," Seconda Universit a degli Studi di Napoli, Via S. Pansini 5, Naples, Italy Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). T. Troiani and S. Napolitano contributed equally to this article. Corresponding Author: Teresa Troiani, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," Seconda Universit a degli Studi di Napoli, Via S. Pansini 5, 80131 Naples, Italy. Phone: 39-0815666725; Fax: 39-0815666732; E-mail: tessy75@inwind.it doi: 10.1158/1078-0432.CCR-13-2181 Ó2014 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 3775 on June 15, 2020. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst May 8, 2014; DOI: 10.1158/1078-0432.CCR-13-2181