The long non-coding RNA, GAS5, enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFR via downregulation of the IGF-1R expression

Journal of Hematology & Oncology

Lin Zhang¹  Mingrui Shao¹  Xitao Chen¹  Shize Yang¹  Peiwen Li¹  Xinwen Zhong¹  Wenya Li¹  Xiaohan Qu¹  Siyuan Dong¹ 

[1] Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang 110001, Liaoning Province, People’s Republic of China

关键词: IGF-1R;    GAS5;    Gefitinib resistance;    NSCLC;   

Others  :  1211899
DOI  :  10.1186/s13045-015-0140-6

 received in 2014-11-16, accepted in 2015-04-20,  发布年份 2015

【 摘 要 】

Background

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are approved for patients with recurrent non-small cell lung cancer (NSCLC). However, the efficacy of EGFR-TKIs in NSCLC therapy is limited by primary and acquired resistance. Recent studies have revealed that long non-coding RNAs (LncRNA) may be involved in EGFR-TKI resistance. Therefore, a better understanding of the interactive mechanisms underlying LncRNA-mediated EGFR-TKIs resistance may help us to improve clinical response rates.

Method

To investigate the expression of growth arrest-specific 5 (GAS5) in lung adenocarcinoma, we performed real-time reverse-transcriptase polymerase chain reaction. The correlation between GAS5 expression levels and the samples’ clinicopathological features was also analyzed. Primary resistance to EGFR-TKIs was identified in the human lung adenocarcinoma cell line A549. Plasmid vectors were used to overexpress GAS5 in A549 cells. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) colony formation assays and EdU (5-ethynyl-2’-deoxyuridine) assays were used to assess cell proliferation, and flow-cytometric analysis was used to evaluate the apoptosis rate. The expression levels of our target proteins, namely, EGFR, p-EGFR, ERK, p-ERK, Akt, p-Akt, IGF-1R (insulin-like growth factor 1 receptor), and p-IGF-1R, were analyzed by western blotting. A549 cells transfected with pcDNA-GAS5 were injected into nude mice. The transplanted mice were treated with gefitinib to study the effect of GAS5 on the resistance to EGFR-TKIs in vivo.

Results

Our results showed that GAS5 was significantly downregulated in lung adenocarcinoma tissues compared with the paired adjacent non-tumorous tissue samples. Furthermore, lower GAS5 expression levels were associated with larger tumor sizes, poor tumor differentiation, and advanced pathological stages. However, GAS5 was almost equally expressed between benign tumors compared with the adjacent normal tissues. GAS5 was also overexpressed in EGFR-TKI sensitive cell lines compared with the resistant cell line. Using MTT, EdU incorporation, and colony formation assays, we showed that GAS5-expressing A549 cells displayed an elevated level of cell death. In addition to its pro-apoptotic effect in the A549 cell line, GAS5 overexpression also suppressed the growth of A549-derived tumors in nude mice treated with gefitinib. GAS5 overexpression was inversely correlated with the expression of the EGFR pathway and IGF-1R proteins.

Conclusions

Collectively, our results indicated that GAS5 LncRNA may represent a potential biomarker for the diagnosis of lung adenocarcinoma and that GAS5 might play a novel role in the development of the resistance to gefitinib, which could be reversed by overexpressing GAS5.

【 授权许可】

   
2015 Dong et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Siegel R, Naishadham D, Jemal A. Cancer statistics for Hispanics/Latinos, 2012. CA Cancer J Clin. 2012; 62(5):283-98.
• [2]Witt C. European respiratory society/american thoracic society/international association for the study of lung cancer international multidisciplinary classification of lung adenocarcinoma: state of the art. J Thorac Oncol. 2011; 6(8):1451.
• [3]Kris MG, Natale RB, Herbst RS, Lynch TJ, Prager D, Belani CP et al.. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA. 2003; 290(16):2149-58.
• [4]Perez-Soler R. Phase II, clinical trial data with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (OSI-774) in non-small-cell lung cancer. Clin Lung Cancer. 2004; 6 Suppl 1:S20-3.
• [5]Wheler J, Falchook G, Tsimberidou AM, Hong D, Naing A, Piha-Paul S et al.. Revisiting clinical trials using EGFR inhibitor-based regimens in patients with advanced non-small cell lung cancer: a retrospective analysis of an MD Anderson cancer center phase I population. Oncotarget. 2013; 4(5):772-84.
• [6]Lynch TJ, Bell DW, Sordella R, Gurubhagavatula 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. N Engl J Med. 2004; 350(21):2129-39.
• [7]Perez-Soler R, Chachoua A, Hammond LA, Rowinsky EK, Huberman M, Karp D et al.. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol. 2004; 22(16):3238-47.
• [8]Martin L, Chang HY. Uncovering the role of genomic "dark matter" in human disease. J Clin Invest. 2012; 122(5):1589-95.
• [9]Gibb EA, Vucic EA, Enfield KS, Stewart GL, Lonergan KM, Kennett JY et al.. Human cancer long non-coding RNA transcriptomes. PLoS One. 2011; 6(10):e25915.
• [10]Lin R, Roychowdhury-Saha M, Black C, Watt AT, Marcusson EG, Freier SM et al.. Control of RNA processing by a large non-coding RNA over-expressed in carcinomas. FEBS Lett. 2011; 585(4):671-6.
• [11]Schmidt LH, Spieker T, Koschmieder S, Schaffers S, Humberg J, Jungen D et al.. The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J Thorac Oncol. 2011; 6(12):1984-92.
• [12]Shi X, Sun M, Liu H, Yao Y, Kong R, Chen F, et al. A critical role for the long non-coding RNA GAS5 in proliferation and apoptosis in non-small-cell lung cancer. Mol Carcinog 2013.
• [13]Liu XH, Liu ZL, Sun M, Liu J, Wang ZX, De W. The long non-coding RNA HOTAIR indicates a poor prognosis and promotes metastasis in non-small cell lung cancer. BMC Cancer. 2013; 13:464. BioMed Central Full Text
• [14]Schneider C, King RM, Philipson L. Genes specifically expressed at growth arrest of mammalian cells. Cell. 1988; 54(6):787-93.
• [15]Coccia EM, Cicala C, Charlesworth A, Ciccarelli C, Rossi GB, Philipson L et al.. Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development. Mol Cell Biol. 1992; 12(8):3514-21.
• [16]Mourtada-Maarabouni M, Pickard MR, Hedge VL, Farzaneh F, Williams GT. GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer. Oncogene. 2009; 28(2):195-208.
• [17]Chen MC, Chen CH, Wang JC, Tsai AC, Liou JP, Pan SL et al.. The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells. Cell Death Dis. 2013; 4:e810.
• [18]Selvaggi G, Novello S, Torri V, Leonardo E, De Giuli P, Borasio P et al.. Epidermal growth factor receptor overexpression correlates with a poor prognosis in completely resected non-small-cell lung cancer. Ann Oncol. 2004; 15(1):28-32.
• [19]Goulart BH, Martins RG, Lynch TJ. Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results. J Clin Oncol. 2001; 19(20):4089.
• [20]Herbst RS. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol. 2005; 23(25):5892-9.
• [21]Ponting CP, Belgard TG. Transcribed dark matter: meaning or myth? Hum Mol Genet. 2010; 19(R2):R162-8.
• [22]Mattick JS. The genetic signatures of noncoding RNAs. PLoS Genet. 2009; 5(4):e1000459.
• [23]Liu Z, Wang W, Jiang J, Bao E, Xu D, Zeng Y et al.. Downregulation of GAS5 promotes bladder cancer cell proliferation, partly by regulating CDK6. PLoS One. 2013; 8(9):e73991.
• [24]Sun M, Jin FY, Xia R, Kong R, LI JH, Xu TP et al.. Decreased expression of long noncoding RNA GAS5 indicates a poor prognosis and promotes cell proliferation in gastric cancer. BMC Cancer. 2014; 14(1):319. BioMed Central Full Text
• [25]Noto A, De Vitis C, Roscilli G, Fattore L, Malpicci D, Marra E et al.. Combination therapy with anti-ErbB3 monoclonal antibodies and EGFR TKIs potently inhibits non-small cell lung cancer. Oncotarget. 2013; 4(8):1253-65.
• [26]Smith CM, Steitz JA. Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5'-terminal oligopyrimidine gene family reveals common features of snoRNA host genes. Mol Cell Biol. 1998; 18(12):6897-909.
• [27]Kino T, Hurt DE, Ichijo T, Nader N, Chrousos GP. Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci Signal. 2010; 3(107):ra8.
• [28]García-Claver A, Lorente M, Mur P, Campos-Martín Y, Mollejo M, Velasco G et al.. Gene expression changes associated with erlotinib response in glioma cell lines. Eur J Cancer. 2013; 49(7):1641-53.
• [29]Jeannot V, Busser B, Brambilla E, Wislez M, Robin B, Cadranel J et al.. The PI3K/AKT pathway promotes gefitinib resistance in mutant KRAS lung adenocarcinoma by a deacetylase-dependent mechanism. Int J Cancer. 2014; 134(11):2560-71.
• [30]Ihle NT, Byers LA, Kim ES, Saintigny P, Lee JJ, Blumenschein GR et al.. Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome. J Natl Cancer Inst. 2012; 104(3):228-39.
• [31]Hurbin A, Wislez M, Busser B, Antoine M, Tenaud C, Rabbe N et al.. Insulin-like growth factor-1 receptor inhibition overcomes gefitinib resistance in mucinous lung adenocarcinoma. J Pathol. 2011; 225(1):83-95.
• [32]Cortot AB, Repellin CE, Shimamura T, Capelletti M, Zejnullahu K, Ercan D et al.. Resistance to irreversible EGF receptor tyrosine kinase inhibitors through a multistep mechanism involving the IGF1R pathway. Cancer Res. 2013; 73(2):834-43.
• [33]Guix M, Faber AC, Wang SE, Olivares MG, Song Y, Qu S et al.. Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins. J Clin Invest. 2008; 118(7):2609-19.
• [34]Venkatraman A, He XC, Thorvaldsen JL, Sugimura R, Perry JM, Tao F et al.. Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence. Nature. 2013; 500(7462):345-9.
• [35]Cappuzzo F, Tallini G, Finocchiaro G, Wilson RS, Ligorio C, Giordano L et al.. Insulin-like growth factor receptor 1 (IGF1R) expression and survival in surgically resected non-small-cell lung cancer (NSCLC) patients. Ann Oncol. 2009; 21(3):562-7.
• [36]Zhang Z, Zhu Z, Watabe K, Zhang X, Bai C, Xu M et al.. Negative regulation of lncRNA GAS5 by miR-21. Cell Death Differ. 2013; 20(11):1558-68.
• [37]Kyprianou N, Sheth S, Jajoo S, Kaur T, Mukherjea D, Sheehan K et al.. Resveratrol reduces prostate cancer growth and metastasis by inhibiting the Akt/MicroRNA-21 pathway. PLoS One. 2012; 7(12):e51655.
• [38]Sobin LH, Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours. 7th ed. Wiley, West Sussex; 2009.