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

https://doi.org/10.1016/j.bbrc.2014.11.003Get rights and content

Highlights

  • Afatinib and cetuximab were synergistically toxic for K562 cells carrying T790M EGFR.

  • Afatinib induced BIM, and cetuximab augmented this expression.

  • Afatinib increased cell-surface T790M EGFR through Rab11-dependent 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.

Introduction

NSCLCs account for more than 80% of all lung cancer cases and are the leading cause of cancer-related deaths worldwide. Aberrant EGFR signaling, caused by activating somatic EGFR mutations, is implicated in the oncogenesis of a subset of NSCLCs [1], [2]. L858R and exon 19 deletion (Ex19del) represent 90% of these mutations. These NSCLCs are responsive to two EGFR-TKIs, gefitinib and erlotinib, which have higher affinity for the mutants’ ATP-binding pocket than for wild-type EGFR’s [3], [4], [5], [6]. The EGFR-TKIs inhibit EGFR’s kinase activity, autophosphorylation, and downstream signaling, including the PI3K, Akt, and MAPK pathways; apoptosis via the induction of pro-apoptotic Bcl-2 family protein BIM follows [7]. BIM is implicated in the activity of various anti-cancer drugs, and its induction is tightly linked to TKI-sensitivity [8].

Acquisition of resistance is a serious setback for NSCLC patients receiving EGFR-TKIs [9]. Although 60–70% of patients initially respond to first-generation TKIs, many eventually acquire resistance, conferred by a new activating mutation. This EGFR gatekeeper mutation, T790M, occurs in approximately 60% of resistant cases [10]. To overcome this clinical resistance, second-generation TKIs with a higher affinity for EGFRs harboring T790M have been developed. Afatinib (BIBW2992) is a pan-HER inhibitor that blocks not only EGFR, but also HER2 and HER4 [11]. It binds covalently to EGFR’s activation site, inhibiting ATP binding to EGFRs bearing a single Ex19del or L858R mutation or to double mutants carrying T790M. Afatinib is a promising treatment for patients who become resistant to erlotinib or gefitinib [12], [13]; however, better efficacy is needed to overcome TKI resistance.

Monoclonal antibodies such as cetuximab (Erbitux) are also used to target EGFR [14]. Clinically, cetuximab is an effective treatment for colorectal or head and neck cancers. Cetuximab binds EGFR’s extracellular domain III, preventing its ligand binding, conformational activation, and receptor dimerization, which leads to EGFR’s endocytosis and downregulation. Under physiological conditions, internalized EGFR is translocated to early endosomes, then either returned to the surface by recycling endosomes, assisted by Rab-family proteins, or ubiquitinated and captured by the endosomal sorting complex required for transport (ESCRT) [15], [16]. Antibody-mediated EGFR downregulation uses the latter pathway, and lysosome-dependent EGFR degradation supports cetuximab’s therapeutic effect. However, cetuximab alone is not effective for most NSCLC cases, indicating that other EGFR-targeting drugs are needed.

Combining EGFR-TKIs with other agents could potentially overcome the drug resistance, and afatinib–cetuximab combination treatment is a promising new strategy for TKI-resistant tumors [17]. The combination induces tumor shrinkage in a transgenic mouse model of EGFR T790M-positive lung cancer [18], but it is unclear how these drugs induce apoptosis. Here, we investigated the physiological basis of this combination therapy using human K562 cells expressing mutant EGFRs. Our results reveal a cell-based rationale for this therapy and shed light on the drugs’ synergistic effect.

Section snippets

Cells

K562, HeLa, COS-7 (RIKEN BioResource), NCI-H1975 (EGFRL858R+T790M, American-Type Culture Collection) and PLAT-A were cultured in RPMI1640 or DMEM containing 10% fetal calf serum (FCS) and antibiotics at 37 °C in 5% CO2. Plasmids were transferred into cells using FuGENE HD (Roche Diagnostics). Retroviruses were transferred into K562 cells by spinfection.

Plasmids

Human EGFR cDNA was subcloned into a retrovirus vector, pMXs-Puro-3xHA-hEGFRWT. Mutated EGFR-expression vectors for L858R (pMXs-Puro-3xHA-hEGFR

K562 cells expressing exogenous EGFRs acquire EGFR-dependence in the presence of imatinib

To study EGFR-TKI’s effects, we needed an EGFR-negative cancer cell line to use as an evaluation system. Our screen for EGFR-negative human cell lines showed that most, including HeLa and COS-7, expressed considerable EGFR, but the erythroleukemic line K562 lacked endogenous EGFR (Fig. 1A), HER2, or HER3, expression (data not shown). We then introduced wild-type EGFR (EGFRWT), a point-mutated L858R EGFR (EGFRL858R), or T790M-mutated EGFRs (EGFRT790M and EGFRL858R+T790M) (Fig. 1B) into K562

Discussion

Here we demonstrated that cetuximab complements afatinib therapy by enhancing afatinib’s ability to induce apoptosis. We found that afatinib and cetuximab together were strongly cytotoxic for gefitinib-resistant EGFRL858R+T790M-expressing K562 cells, compared with either drug alone. This combination also induced BIMEL in these cells. A likely mechanism underlying this synergistic effect is that the afatinib-induced redistribution of EGFR to the cell surface, through Rab11a-dependent recycling,

Acknowledgments

We thank Dr. T. Kitamura for PLAT-A, and Ms. Y. Tada for technical assistance. This work was supported by JSPS-KAKENHI grants 24790829, 24300326, 24591184 and 24592613.

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