Cross-suppression of EGFR ligands amphiregulin and epiregulin and de-repression of FGFR3 signalling contribute to cetuximab resistance in wild-type KRAS tumour cells

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. Lentivirus-delivered small hairpin RNAs were employed to...

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Published in:British journal of cancer 2012-04, Vol.106 (8), p.1406-1414
Main Authors: OLIVERAS-FERRAROS, C, CUFI, S, QUERALT, B, VAZQUEZ-MARTIN, A, MARTIN-CASTILLO, B, DE LLORENS, R, BOSCH-BARRERA, J, BRUNET, J, MENENDEZ, J. A
Format: Article
Language:English
Subjects:
Amphiregulin
Antibodies, Monoclonal - pharmacology
Antibodies, Monoclonal, Humanized
Antineoplastic Agents - pharmacology
Apoptosis - drug effects
Biological and medical sciences
Biomarkers
Cancer research
Cell Survival - drug effects
Cetuximab
Drug Resistance, Neoplasm - drug effects
EGF Family of Proteins
Epidermal growth factor
Epidermal Growth Factor - antagonists & inhibitors
Epidermal Growth Factor - biosynthesis
Epidermal Growth Factor - genetics
Epiregulin
Gene expression
Gene Knockdown Techniques
Glycoproteins - antagonists & inhibitors
Glycoproteins - biosynthesis
Glycoproteins - genetics
Humans
Intercellular Signaling Peptides and Proteins - biosynthesis
Intercellular Signaling Peptides and Proteins - genetics
Kinases
Ligands
Medical research
Medical sciences
Metastasis
Oncology
Pyrimidines - pharmacology
Receptor, Epidermal Growth Factor - metabolism
Receptor, Fibroblast Growth Factor, Type 3 - metabolism
Short Communication
Signal Transduction - drug effects
Skin Neoplasms - drug therapy
Skin Neoplasms - pathology
Structure-Activity Relationship
Tumor Cells, Cultured
Tumors
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Summary: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. 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. 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. 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.
ISSN:0007-0920
1532-1827
DOI:10.1038/bjc.2012.103