title: Synergistic cytotoxicity of afatinib and cetuximab against EGFR T790M involves Rab11-dependent EGFR recycling.
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.
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Zenta
authors.initials: Z
authors.lastname: Watanuki
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Kosai
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Nanae
authors.initials: N
authors.lastname: Osanai
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogama
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan; Department of Cancer Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Tamai
authors.affiliation: Division of Molecular and Cellular Oncology, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Division of Cancer Stem Cell, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Kennichi
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Department of Respiratory Medicine, Miyagi Cancer Center, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Fukuhara
authors.affiliation: Department of Respiratory Medicine, Miyagi Cancer Center, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Maemondo
authors.affiliation: Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ichinose
authors.affiliation: South Miyagi Medical Center, 38-1 Aza-Nishi, Ohgawara 989-1253, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nukiwa
authors.affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan; Department of Cancer Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Electronic address: tanaka@med.tohoku.ac.jp.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Tanaka
mesh_terms: D000077716:Afatinib; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D000068818:Cetuximab; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000077716:Afatinib; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D000068818:Cetuximab; D066246:ErbB Receptors; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011799:Quinazolines; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C000606775:BCL2L11 protein, human
keywords: Cytotoxicity; EGFR; EGFR-TKI