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.
The online version of this article (doi:10.1186/s40164-015-0010-5) contains supplementary material, which is available to authorized users.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived, designed the study and manuscript preparation: MSR; Screening of all clinical samples for mutations, performed 2D GE and MS analysis: AK, AMJ; Pathological evaluation: KG; Lung tumor surgeon by whom tumor tissues were collected: RD; Enrollment of NSCLC patients and collection of biopsies from patients: DB, AJ; All authors read and approved the final manuscript.
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 [5]. 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 [8]. 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.
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Further, we investigated proteomic profile of HEK293 cells in response to altered activity of mutants L861Q, A871G and the most widely reported mutation, L858R by 2D GE followed by MALDI-TOF/Mass spectrometry analysis. Representative 2D gel with resolved protein spots of wild type EGFR expressing cells and different densities of analyzed spots of wild type vs. mutant receptors were shown in Additional files 6 and 7. Protein profile of cells expressing each mutant upon EGF stimulation was compared with protein profile of wild type receptor. Heat shock proteins such as Hsp70, Hsp71, Hsp90B1, Hsp60, and Hsp5a were identified to be differentially regulated largely in response to EGFR mutants (Table 1). Other effectors were FGG, IFIT2, cytoskeletal proteins and transcriptional factors such as HOXD11, HOX B4 (Additional file 8). Most of the proteins identified herein including IFIT2 and FGG were reported to be associated with various cancers with the potential to metastasize the tumor [9, 10]. Few of the identified proteins were validated at transcript level by quantitative real time PCR (Fig. 1a, b, Additional files 9 and 10). Up regulation of Hsp70 more selectively to L861Q mutant activity was consistent in our experiments. Reduction of its expression with Gefitinib treatment (Fig. 1c and d) suggests the possible role of this mutant in Hsp70 regulation. Published literature strongly argues that certain molecular chaperones, more importantly Hsp70 play a significant role in tumor survival [11]. Hsp70 expression was already reported in primary NSCLC tumors [12] and serum samples collected from NSCLC patients [13] as well. However, we demonstrated first time that heat shock proteins are the major downstream effectors of NSCLC associated EGFR variants. Supporting our data, some proteins detected in our study were recently reported to be differentially expressed in a proteomic study carried out on interstitial fluids collected from NSCLC patients [14]. Considering the importance of molecular chaperones in tumor survival and with their change of expression in response to altered EGFR activity, we hypothesize that they may also regulate progression of NSCLC tumors harboring EGFR mutations. So, the molecular mechanism involved in tumor progression, drug complexity and the prognostic implications of these heat shock proteins in NSCLC patient management are worth exploring.
Table 1
Differentially expressed proteins in cells expressing mutants vs. wild type EGFR identified by MALDI-TOF/MS analysis
Spot No.
Protein name
Acc. No
Mol. Wt
PI
Mascot Score
L858Rvs. WT
L861Q vs. WT
A871G vs. WT
6601
Heat shock cognate 71 kDa protein
P11142
71082
5.37
24
1.8
1.2
1.13
7501
Heat shock 70 kDa protein 1
P08107
70294
5.48
147
1.7
2.1
0.93
3702
Endoplasmin (GRP94) HSP90B1
P14625
92696
4.76
88
0.02
0.28
0.02
5503
60 kDa heat shock protein, mitochondrial
P10809
61187
5.7
75
1.4
1.2
1.03
4701
78 kDa GRP (HSP5A)
P11021
72402
5.07
158
1.1
1.31
0.34
7502
T-complex protein 1 subunit epsilon
P48643
60089
5.45
63
0.29
0.36
0.56
7403
Protein disulfide-isomerase
P30101
57146
5.98
84
0.73
0.96
0.63
4501
Tubulin alpha-1B chain
P68363
50804
4.94
128
0.58
0.69
0.33
3401
Tubulinbeta-2C chain
P68371
50255
4.79
239
0.63
1.2
0.72
4501
Vimentin
P08670
53676
5.06
24
0.58
0.69
0.33
6302
Keratin, type I cytoskeletal 18, 19
P05783
48029
5.34
319
0
1.5
0.86
7402
Keratin, type II cytoskeletal 8
P05787
53671
5.52
59
0.73
0.96
0.63
5203
Actin, cytoplasmic 2
P63261
42108
5.31
107
1.2
1.31
0.79
×
Acknowledgments
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.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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The authors declare that they have no competing interests.
Authors’ contributions
Conceived, designed the study and manuscript preparation: MSR; Screening of all clinical samples for mutations, performed 2D GE and MS analysis: AK, AMJ; Pathological evaluation: KG; Lung tumor surgeon by whom tumor tissues were collected: RD; Enrollment of NSCLC patients and collection of biopsies from patients: DB, AJ; All authors read and approved the final manuscript.
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