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 mechanisms against EGFR inhibition.Experimental Design: Resistant CCA cells to EGFR inhibition 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 metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial-mesenchymal 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 expression. 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 fibroblasts (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 signaling 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 contribution of stromal cells.
Model depicting the adaptive mechanisms of CCA to EGFR inhibition. Chronic treatment with an EGFR inhibitor, erlotinib, promotes the overexpression of IGF2, IR and IGF1R in CCA cells. IGF2/IR/IGF1R overexpression leads to the induction of a mesenchymal state in erlotinib-resistant CCA cells characterized by the adoption of EMT/CSC traits and chemoresistance. Resistant CCA cells to EGFR inhibition promote CAFs proliferation by unknown mechanisms. In tumor microenvironment, CAFs produce IGF2 that contributes to chemoresistance of CCA cells by a paracrine loop. Thus, addition of IR/IGF1R inhibitor linsitinib reverses chemoresistance by acting on tumor cells and on CAFs proliferation/activation. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma; TKI, tyrosine kinase inhibitor.