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// Valerio Gelfo 1, 2, * , Maria Teresa Rodia 1, * , Michela Pucci 1 , Massimiliano Dall’Ora 1 , Spartaco Santi 3, 4 , Rossella Solmi 1 , Lee Roth 5 , Moshit Lindzen 5 , Massimiliano Bonafe 1, 2 , Andrea Bertotti 6 , Elisabetta Caramelli 1 , Pier-Luigi Lollini 1 , Livio Trusolino 6 , Yosef Yarden 5 , Gabriele D’Uva 7, ** , Mattia Lauriola 1, 2, ** 1 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 2 Center for Applied Biomedical Research (CRBA) S. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy 3 Institute of Molecular Genetics, National Research Council of Italy, Bologna, Italy 4 Laboratory of Musculoskeletal Cell Biology, IOR-IRCCS, Bologna, Italy 5 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel 6 Candiolo Cancer Institute-Fondazione del Piemonte per l’Oncologia (FPO), IRCCS, Department of Oncology, University of Torino, Candiolo, Italy 7 Scientific and Technology Pole, IRCCS MultiMedica, Milan, Italy * These authors have contributed equally to this work ** Co-last authors Correspondence to: Mattia Lauriola, email: mattia.lauriola@gmail.com Keywords: EGFR, transcriptional response, colon cancer, resistance, cetuximab Received: June 13, 2016 Accepted: September 21, 2016 Published: September 30, 2016 ABSTRACT Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A , IL1B and IL8 , which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro . This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression.