title: Cross-suppression of EGFR ligands amphiregulin and epiregulin and de-repression of FGFR3 signalling contribute to cetuximab resistance in wild-type KRAS tumour cells.
abstract: BACKGROUND
  In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.
  
  
  METHODS
  Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.
  
  
  RESULTS
  Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.
  
  
  CONCLUSIONS
  Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.
authors.affiliation: Unit of Translational Research, Catalan Institute of Oncology, Girona, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Oliveras-Ferraros
authors.forename: S
authors.initials: S
authors.lastname: Cufí
authors.forename: B
authors.initials: B
authors.lastname: Queralt
authors.forename: A
authors.initials: A
authors.lastname: Vazquez-Martin
authors.forename: B
authors.initials: B
authors.lastname: Martin-Castillo
authors.forename: R
authors.initials: R
authors.lastname: de Llorens
authors.forename: J
authors.initials: J
authors.lastname: Bosch-Barrera
authors.forename: J
authors.initials: J
authors.lastname: Brunet
authors.forename: J A
authors.initials: JA
authors.lastname: Menendez
mesh_terms: D066258:Amphiregulin; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000068818:Cetuximab; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066255:EGF Family of Proteins; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D066260:Epiregulin; D066246:ErbB Receptors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D008024:Ligands; D011743:Pyrimidines / Q000494:pharmacology; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D012878:Skin Neoplasms / Q000188:drug therapy / Q000473:pathology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D066255:EGF Family of Proteins; C587880:EREG protein, human; D066260:Epiregulin; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; C115711:PD 173074; D011743:Pyrimidines; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D051498:Receptor, Fibroblast Growth Factor, Type 3; D000068818:Cetuximab