Role of tyrosine kinase-independent phosphorylation of EGFR with activating mutation in cisplatin-treated lung cancer cells

https://doi.org/10.1016/j.bbrc.2015.02.044Get rights and content

Highlights

  • Cisplatin induces Ser/Thr phosphorylation of wild type EGFR via MAPKs.

  • EGFR with active mutation is also phosphorylated by cisplatin in lung cancer cells.

  • Cisplatin causes downregulation of EGFR tyrosine phosphorylation via ERK pathway.

  • Internalization is switched to a p38-dependent mechanism by cisplatin treatment.

  • p38 inhibitor enhances cisplatin-induced apoptotic cell death.

Abstract

Epidermal growth factor receptor (EGFR) mutation is one of the hallmarks of cancer progression and resistance to anticancer therapies, particularly non-small cell lung carcinomas (NSCLCs). In contrast to the canonical EGFR activation in which tyrosine residues are engaged, we have demonstrated that the non-canonical pathway is triggered by phosphorylation of serine and threonine residues through p38 and ERK MAPKs, respectively. The purpose of this study is to investigate the role of non-canonical EGFR pathway in resistance mechanism against cisplatin treatment. Wild type and mutated (exon 19 deletion) EGFR-expressing cells responded similarly to cisplatin by showing MAPK-mediated EGFR phosphorylation. It is interesting that internalization mechanism of EGFR was switched from tyrosine kinase-dependent to p38-dependent fashions, which is involved in a survival pathway that counteracts cisplatin treatment. We therefore introduce a potential combinatorial therapy composed of p38 inhibition and cisplatin to block the activation of EGFR, therefore inducing cancer cell death and apoptosis.

Introduction

Non-small cell lung carcinomas (NSCLCs), the most common type of lung cancer, are relatively insensitive to chemotherapeutics. Combinatorial chemotherapeutics based on cisplatin (CDDP) has been a widely successful treatment for many types of cancer, including lung cancers [1], [2], [3], [4]. Combinatorial therapies based on cisplatin were significantly used for NSCLCs to tackle its resistance. Nevertheless, novel approaches and strategies are required to face newly recognized mechanisms of resistance [5], [6], [7], [8].

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTKs) [9]. Normally, EGF binds to EGFR to induce preferentially tyrosine autophosphorylation, which causes activation of MAPK and Akt pathways. Mutations in the tyrosine kinase (TK) domain of the EGFR found in high percentage of patients, especially in Asian countries, are associated with the development of cancer due to its constitutive activation [10], [11], [12]. Exon 19 deletions or L858R point mutation are the most prevalent of the EGFR mutations in NSCLCs, and cause the enhancement of sensitivity to EGFR TK inhibitors (EGFR-TKIs) [13]. We have demonstrated that, in the presence of cellular stresses, including tumor necrosis factor-α (TNF-α), the activation of p38/ERK induces TK-independent phosphorylation of EGFR at serine/threonine residues [15], [16]. This non-canonical EGFR pathway can eventually cause a p38-dependent internalization of EGFR [15], [16], [17]. The role of both EGFR and MAPKs on the development of resistance, especially against cisplatin, has been reported [18], [19], [20], [21], [22], [23], [24]. Several reports have shown the role of cisplatin in the activation of p38 MAPK, and subsequent non-canonical EGFR pathway [17], [25]; however, a clear identification of the phosphorylated residue and its implication was not shown.

In this study, we are investigating the role of cisplatin in activating the non-canonical pathway in human lung adenocarcinoma cells harboring EGFR mutation. We demonstrated the effect of blocking the p38-mediated EGFR internalization on enhancing the apoptotic potential of cisplatin.

Section snippets

Antibodies and reagents

Phospho-specific antibodies against p38 (Thr-180, Tyr-182), ERK (Thr-202, Tyr-204), and EGFR (Tyr-845, 998, 1045 and 1068, Thr-669, and Ser-1046/1047), in addition to PARP-1 and caspase-3 were purchased from Cell Signaling Technology. Antibodies against EGFR (1005), α-Tubulin (B-7) and β-actin (C-11) were obtained from Santa Cruz Biotechnologies. Recombinant human TNF-α and EGF were obtained from R&D System, and HGF was obtained from PeproTech. LY294002, SB203580, U0126 and PD153035 were from

CDDP induces a non-canonical pathway of wild type EGFR through p38/ERK MAPKs

We and others have reported the role of p38 and ERK in phosphorylation of EGFR at Ser-1046/1047 and Thr-669, respectively, after the stimulation with TNF-α [15], [16], [17]. To examine the effect of CDDP on EGFR phosphorylation, we used HeLa cells as well as other EGFR-wild type cell lines, including A549 (lung adenocarcinoma) and MDA-MB-231 (breast cancer) cells. CDDP caused phosphorylation of ERK, p38, and EGFR at non-canonical serine/threonine sites starting from 60 μM, and gradually

Discussion

We have shown that various cellular stimuli, including TNF-α and Helicobacter pylori infection, trigger the non-canonical EGFR phosphorylation [15], [30], [31]. In contrast to the tyrosine autophosphorylation-dependent canonical EGFR pathway, the non-canonical pathway is regulated via an intracellular feedback mechanism which is independent of direct ligand/receptor interaction. Apparently, these stimuli employed p38/ERK MAPK pathways to induce feedback phosphorylation of EGFR at

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This study has been supported by Grants-in-Aid for Scientific Research on Innovative Areas (No. 23117516) and Scientific Research (C) (No. 23590071) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and a short-term fellowship grant (No. 6093) from the Science and Technology Development Fund (STDF), Egypt.

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    These authors contributed equally to this work.

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