Translational Cancer Mechanisms and Therapy The IGF2/IR/IGF1R Pathway in Tumor Cells and Myofibroblasts Mediates Resistance to EGFR Inhibition in Cholangiocarcinoma Javier Vaquero 1,2,3 , Cindy Lobe 1 , Sylvana Tahraoui 1 , Audrey Clap eron 1 , Martine Mergey 1 , Fatiha Merabtene 1 , Dominique Wendum 1,4 , C edric Coulouarn 5 , Chantal Housset 1,6 , Christ  ele Desbois-Mouthon 1 , Fran¸ coise Praz 1 , and Laura Fouassier 1 Abstract Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal growth factor receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechan- isms against EGFR inhibition. Experimental Design: Resistant CCA cells to EGFR inhibi- tion were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid growth were determined in vitro, and tumor growth was evaluated in CCA xenograft models. Results: Erlotinib-resistant CCA cells displayed metasta- sis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial–mes- enchymal transition (EMT) and a cancer stem cell (CSC)– like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like growth factor (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expres- sion. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated fibro- blasts (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor growth and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by fibroblasts boosted IR/IGF1R sig- naling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated fibroblast proliferation and activation. Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribu- tion of stromal cells. Clin Cancer Res; 24(17); 4282–96. Ó2018 AACR. Introduction Cholangiocarcinoma (CCA) is a heterogeneous group of malig- nancies that displays a biliary epithelial cell phenotype (1). CCA can emerge at every point of the biliary tree, from the canals of Hering to the main bile duct, and thereby is classified as intra- hepatic, perihilar, and distal CCA, which share some similarities but also present important intertumor and intratumor differences affecting the pathogenesis and outcome (1). The overall prognosis is very poor due to late clinical presentation and the ability of the tumor to develop chemoresistance (1, 2). Late diagnosis com- promises the only effective therapeutic option, surgical resection, that is applicable in 20% of cases. Patients ineligible for surgery undergo a palliative treatment with a combination of gemcitabine and oxaliplatin (GEMOX; refs. 1, 3). In case of tumor progression after this first line of chemotherapy, there is no other treatment approved despite the identification of potential therapeutic targets. Epidermal growth factor receptor (EGFR) plays a critical role in oncogenesis, which places it as one of the promising targets for therapeutic inhibition. Several studies, including ours, have shown the major contribution of EGFR to CCA cell proli- feration, migration, and invasion (4–6). In addition, dysregu- lations of EGFR expression and signaling have been associated with tumor progression and poorer prognosis in CCA patients (7, 8). However, despite EGFR attractive position as a molecular target for therapy, several independent clinical trials have reported very poor responses in patients with CCA treated with different small tyrosine kinase inhibitors (TKI) and mono- clonal antibodies designed to specifically target EGFR (9–14). CCA is characterized by a dense desmoplastic stroma rich in alpha-smooth muscle actin (a-SMA)-positive cells known as cancer-associated fibroblasts (CAF), which have been shown to participate in cancer progression (15). In this context, CAFs are 1 Sorbonne University, INSERM, Saint-Antoine Research Center (CRSA), Paris, France. 2 Fondation ARC, Villejuif, France. 3 LPP, CNRS, Ecole Polytech., Univ. Paris-Sud, Observatoire de Paris, Univ. Paris-Saclay, Sorbonne University, PSL Research University, Paris, France. 4 Assistance Publique-H^ opitaux de Paris, Saint-Antoine Hospital, Department of Pathology, Paris, France. 5 INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMe- Can), Rennes, France. 6 Assistance Publique-H^ opitaux de Paris, Saint-Antoine Hospital, Hepatology Department, Paris, France. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Laura Fouassier, INSERM, 27 rue de Chaligny, 75012 Paris, France. Phone: 33-6-98-77-40-01; E-mail: laura.fouassier@inserm.fr doi: 10.1158/1078-0432.CCR-17-3725 Ó2018 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 24(17) September 1, 2018 4282 on April 25, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst May 1, 2018; DOI: 10.1158/1078-0432.CCR-17-3725