Biochemical and Biophysical Research Communications
Role of tyrosine kinase-independent phosphorylation of EGFR with activating mutation in cisplatin-treated lung cancer cells
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.
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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.