Synergistic cytotoxicity of afatinib and cetuximab against EGFR T790M involves Rab11-dependent EGFR recycling

Biochem Biophys Res Commun. 2014 Dec 12;455(3-4):269-76. doi: 10.1016/j.bbrc.2014.11.003. Epub 2014 Nov 11.

Abstract

EGFR is an important therapeutic target for non-small cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, are effective in cases with EGFR-activating mutations. However, most such cases become resistant through a secondary EGFR mutation, T790M. While the second-generation TKI afatinib has a higher affinity for double-mutant EGFRs, better efficacy is needed. Combining afatinib with the anti-EGFR monoclonal antibody cetuximab improves clinical outcomes, but the mechanism is unclear. Here we examined this effect using erythroleukemic K562 cells. The activating EGFR mutation L858R is sensitive to first-generation TKIs, and adding T790M confers resistance to these drugs. This double-mutant EGFR was moderately sensitive to afatinib, but responded weakly to cetuximab. Combined afatinib and cetuximab synergistically increased their cytotoxicity for K562 cells expressing the double-mutant EGFR. Apoptosis in these cells followed induction of the pro-apoptotic protein BIM. Unexpectedly, afatinib caused redistribution of EGFR to the cell surface through Rab11a-dependent recycling. Cetuximab reduced cell-surface EGFR, and total EGFR decreased synergistically when cetuximab was combined with afatinib. Our results suggest that the synergistic effect exerted by afatinib and cetuximab on NSCLCs is associated with BIM induction and alterations in EGFR status.

Keywords: Cytotoxicity; EGFR; EGFR-TKI.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Afatinib
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Antineoplastic Agents / pharmacology*
  • Apoptosis
  • Apoptosis Regulatory Proteins / metabolism
  • Bcl-2-Like Protein 11
  • COS Cells
  • Cell Membrane / metabolism
  • Cetuximab
  • Chlorocebus aethiops
  • Dose-Response Relationship, Drug
  • Drug Resistance, Neoplasm
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • HeLa Cells
  • Humans
  • K562 Cells
  • Membrane Proteins / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Mutation
  • Proto-Oncogene Proteins / metabolism
  • Quinazolines / pharmacology*
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • Apoptosis Regulatory Proteins
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Quinazolines
  • Afatinib
  • ErbB Receptors
  • rab11 protein
  • rab GTP-Binding Proteins
  • Cetuximab