Proteomic analysis of HEK293 cells expressing non small cell lung carcinoma associated epidermal growth factor receptor variants reveals induction of heat shock response
© Kamath et al. 2015
Received: 29 May 2015
Accepted: 3 June 2015
Published: 12 June 2015
In view of the fact that certain non small cell lung carcinoma associated epidermal growth factor receptor mutations keep the receptor constitutively active, the downstream effectors of altered activity of mutant receptors are largely unknown. By 2D gel electrophoresis and MALDI-TOF/MS analysis, we showed that increased activity of EGFR mutants, L858R, L861Q and A871G induce heat shock proteins such as Hsp70, Hsp60, Hsp90B1, Hsp5a, Hsp71 and few transcriptional factors. Of which, Hsp70 was observed to be regulated more selectively to L861Q mutant. Our results suggest the possible role of heat shock proteins in lung tumor progression considering EGFR mutations.
KeywordsEGFR NSCLC TKI Heat shock proteins Tumor progression Drug sensitivity
To the editor
Large number of studies reported epidermal growth factor receptor mutations (EGFR) in non small cell lung carcinoma (NSCLC) patients worldwide, most commonly in Asian countries including India in the last decade [1, 2]. In vitro studies have demonstrated the contribution of EGFR mutations to uncontrolled tumor proliferation and evasion of programmed cell death in various cancers [3, 4] including lung tumorigenesis in transgenic mice models . It is known that a set of NSCLC associated EGFR mutations especially in tyrosine kinase (TK) domain have accounted for to have prognostic significance as they sensitize the receptor to TKI [6, 7]. Therefore, the question remains unanswered why NSCLC tumors with certain mutations respond to targeted drugs while others make the tumor resistant to the same drug . This implies the complexity of drug sensitivity in patients harboring EGFR mutations and the complexity may be due to altered activity of mutant receptors affecting various downstream molecules for tumor survival which are largely unknown. Till date, detection of EGFR mutations remains an important prognostic test, as FDA approved drugs including the drugs which are currently under development for NSCLC treatment target EGFR. Unfortunately, in spite of considering mutations for treatment prediction, prognosis of advanced stage tumors remains poor. At this juncture, identification of downstream effectors of altered mutant receptor activity with prognostic importance is essential. Uncovering of such molecules may also allow us to understand the lung tumor progression and complexity of drug sensitivity driven by receptor variants.
We initiated our study by screening FFPE lung tumor tissues derived from NSCLC patients from north Indian population for EGFR mutations by RT-PCR followed by sequencing after obtaining ethical approval and informed consent from patients. Detailed methodology was given in Additional file 1. Receptor activity and drug sensitivity was determined by measuring phosphorylation on tyr1068 residue of each mutant generated by site directed mutagenesis. Two amino acid substitutions, L861Q (10.5 %) and A871G (2.1 %) in exon21 and K879R (24.2 %) in exon22 of TK domain were detected (Additional file 2). Former two demonstrated increased receptor activity (Additional file 3), and sensitivity to TKI, Gefitinib more selectively (Additional files 4 and 5). While the latter one was found to be indistinguishable from wild type receptor with respect to its activity and drug sensitivity.
Differentially expressed proteins in cells expressing mutants vs. wild type EGFR identified by MALDI-TOF/MS analysis
L861Q vs. WT
A871G vs. WT
Heat shock cognate 71 kDa protein
Heat shock 70 kDa protein 1
Endoplasmin (GRP94) HSP90B1
60 kDa heat shock protein, mitochondrial
78 kDa GRP (HSP5A)
T-complex protein 1 subunit epsilon
Tubulin alpha-1B chain
Keratin, type I cytoskeletal 18, 19
Keratin, type II cytoskeletal 8
Actin, cytoplasmic 2
This study was funded by Indian Council of Medical Research (No. 5/13/94/2008- NCD III), New Delhi. India. We are grateful to Dr. Raju Rajala, OUHSC, USA for providing us with EGFR full length plasmid from which all receptor mutants were generated.
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