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Afatinib resistance in non-small cell lung cancer involves the PI3K/AKT and MAPK/ERK signalling pathways and epithelial-to-mesenchymal transition

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Abstract

The epidermal growth factor receptor (EGFR) signalling is one of the most deregulated pathways in non-small cell lung cancer (NSCLC). Recently, the development of novel irreversible tyrosine kinase inhibitors (TKI), such as afatinib, has significantly improved the survival of advanced NSCLC patients harbouring activated EGFR mutations. However, treatment with TKI is not always curative due to the development of resistance. In the present study, we investigated the sensitivity to afatinib in two NSCLC EGFR mutated cell lines (NCI-H1650 and NCI-H1975) by expression profile analysis of 92 genes involved in the EGF pathway. Thereafter, the established afatinib resistant clones were evaluated at different biological levels: genomic, by array comparative genomic hybridisation (aCGH) and deep sequencing; transcriptomic, by quantitative polymerase chain reaction (qPCR) and proteomic, by Western blot and immunofluorescence. The baseline gene expression of the two cell lines revealed that NCI-H1650, the less afatinib-responsive cell, showed activation of two main EGFR downstream pathways such as PI3K/AKT and PLCγ/PKC axes. Analysis of the afatinib-resistant cells showed PI3K/AKT and MAPK/ERK pathways activation together with a biological switch from an epithelial-to-mesenchymal phenotype might confer afatinib-resistant properties to this cell line. Our data suggest that the activation of EGFR-dependent downstream pathways might be involved in the occurrence of resistance to afatinib assuming that the EGFR mutational status should not be exclusively considered when selecting TKI treatments. In particular, the epithelial-to-mesenchymal transition might provide a new basis for understanding afatinib resistance.

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Acknowledgments

We kindly thank Dr. Cristina Bruzzo and Dr. Zita Cavalieri for technical support (cell lines maintenance, drug treatments, cytotoxicity tests).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Lung Cancer Unit, IRCCS AOU San Martino—IST, L.go R. Benzi 10, 16132, Genoa, Italy

    Simona Coco, Anna Truini, Angela Alama, Maria Giovanna Dal Bello, Erika Rijavec, Carlo Genova, Giulia Barletta, Claudio Sini & Francesco Grossi

  2. Immunology Unit, IRCCS AOU San Martino—IST, L.go R. Benzi 10, 16132, Genoa, Italy

    Roberta Venè

  3. Clinica di Medicina Interna ad Indirizzo Oncologico, IRCCS AOU San Martino—IST, L.go R. Benzi 10, 16132, Genoa, Italy

    Anna Garuti, Enrico Carminati & Alberto Ballestrero

  4. Dipartimento di Medicina Interna e Specialità Mediche (DIMI), Università di Genova, V.le Benedetto XV 6, 16132, Genoa, Italy

    Anna Truini & Francesco Boccardo

  5. UOC Clinica di Oncologia Medica, IRCCS AOU San Martino—IST, L.go R. Benzi 10, 16132, Genoa, Italy

    Francesco Boccardo

Authors
  1. Simona Coco
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  2. Anna Truini
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  3. Angela Alama
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  9. Carlo Genova
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  11. Claudio Sini
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  14. Francesco Grossi
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Correspondence to Simona Coco.

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Coco, S., Truini, A., Alama, A. et al. Afatinib resistance in non-small cell lung cancer involves the PI3K/AKT and MAPK/ERK signalling pathways and epithelial-to-mesenchymal transition. Targ Oncol 10, 393–404 (2015). https://doi.org/10.1007/s11523-014-0344-7

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