- Letter to the Editor
- Open Access
Proteomic analysis of HEK293 cells expressing non small cell lung carcinoma associated epidermal growth factor receptor variants reveals induction of heat shock response
Experimental Hematology & Oncology volume 4, Article number: 16 (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.
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.
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 . Hsp70 expression was already reported in primary NSCLC tumors  and serum samples collected from NSCLC patients  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 . 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.
Bell DW, Brannigan BW, Matsuo K, Finkelstein DM, Sordella R, Settleman J, et al. Increased prevalence of EGFR –mutant in lung cancer in women and in East Asian populations: analysis of estrogen related polymorphism. Clin Cancer Res. 2008;14(13):4079–84.
Mehta J. Molecular epidemiology of epidermal growth factor receptor mutations in lung cancers in Indian population. Ind J Cancer. 2013;50(2):102–6.
Shin DY, Na II, Kim CH, Park S, Baek H, Yang SH. EGFR mutation and brain metastasis in pulmonary adenocarcinomas. J Thorac Oncol. 2014;9(2):195–9.
Bos PD, Zhang XH, Nadal C, et al. Genes that mediate breast cancer metastasis to the brain. Nature. 2009;459:1005–9.
Pilot K, Zakowski MF, Fan PD, Sconfeld EA, Pao W, Varmus HE. Lung adenocarcinomas induced in mice by mutant EGF receptors founding human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev. 2006;20(11):1496–510.
Lynch TJ, Bell DW, Sordell R, Gurubhagavatulu S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small cell lung cancer to Gefitinib. New Eng J Med. 2004;350(21):2129–39.
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response Gefitinib therapy. Science. 2004;304:1497–500.
Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. New Engl J Med. 2005;352(8):786–92.
Bloomston M, Zhou JX, Rosemurgy AS, Frankel W, Muro-Cacho CA, Yeatman TJ. Fibrinogen gamma over expression in pancreatic cancer identified by large-scale proteomic analysis of serum samples. Cancer Res. 2006;66(5):2592–9.
Lai KC, Liu CJ, Chang KW, Lee TC. Depleting IFIT2 mediates atypical PKC signaling to enhance the migration and metastatic activity of oral squamous cell carcinoma cells. Oncogene. 2013;32(32):3686–97.
Wang HX, Liu YF, Yang SJ, Duan CG, Wang YX, Zhao J, et al. Expression of HSP70 Grp94 and IgG in human lung carcinoma. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008;24(5):447–9.
Małusecka E, Zborek A, Krzyzowska-Gruca S, Krawczyk Z. Expression of heat shock proteins HSP70 and HSP27 in primary non-small cell lung carcinomas.An immunohistochemical study. Anticancer Res. 2001;21(2A):1015–21.
Zimmermann M, Nickl S, Lambers C, Hacker S, Mitterbauer A, Hoetzenecker K, et al. Discrimination of clinical stages in non-small cell lung cancer patients by serum HSP27 and HSP70: A multi-institutional case–control study. Clin Chim Acta. 2012;413:1115–20.
Li S, Wang R, Zhang M, Wang L, Cheng S. Proteomic analysis of non-small cell lung cancer tissue interstitial fluids. World J Surgical Oncol. 2013;11:173.
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.
The authors declare that they have no competing interests.
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.
Additional file 1:
Additional file 2:
EGFR mutation analysis on lung tumor tissues.
Additional file 3:
Ligand dependent phosphorylation of EGFR mutants.
Additional file 4:
Sensitivity of mutant receptors to Gefitinib.
Additional file 5:
Sensitivity of mutant receptors to Erlotinib.
Additional file 6:
2D gel with resolved proteins of EGFR expressing cells.
Additional file 7:
Different densities of protein spots in cells expressing mutants vs. wild type receptor.
Additional file 8:
List of proteins differentially regulated in response to mutant L861Q vs. wild type receptor identified by MS analysis.
Additional file 9:
List of real time PCR primers.
Additional file 10:
Quantitative real time PCR of regulated gene transcripts in cells expressing EGFR mutants.
Rights and permissions
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.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Kamath, A., Joseph, A.M., Gupta, K. et al. Proteomic analysis of HEK293 cells expressing non small cell lung carcinoma associated epidermal growth factor receptor variants reveals induction of heat shock response. Exp Hematol Oncol 4, 16 (2015). https://doi.org/10.1186/s40164-015-0010-5
- Heat shock proteins
- Tumor progression
- Drug sensitivity