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      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

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          Abstract

          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.

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          Most cited references 29

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          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.

          More persons in the United States die from non-small cell lung cancer (NSCLC) than from breast, colorectal, and prostate cancer combined. In preclinical testing, oral gefitinib inhibited the growth of NSCLC tumors that express the epidermal growth factor receptor (EGFR), a mediator of cell signaling, and phase 1 trials have demonstrated that a fraction of patients with NSCLC progressing after chemotherapy experience both a decrease in lung cancer symptoms and radiographic tumor shrinkages with gefitinib. To assess differences in symptomatic and radiographic response among patients with NSCLC receiving 250-mg and 500-mg daily doses of gefitinib. Double-blind, randomized phase 2 trial conducted from November 2000 to April 2001 in 30 US academic and community oncology centers. Patients (N = 221) had either stage IIIB or IV NSCLC for which they had received at least 2 chemotherapy regimens. Daily oral gefitinib, either 500 mg (administered as two 250-mg gefitinib tablets) or 250 mg (administered as one 250-mg gefitinib tablet and 1 matching placebo). Improvement of NSCLC symptoms (2-point or greater increase in score on the summed lung cancer subscale of the Functional Assessment of Cancer Therapy-Lung [FACT-L] instrument) and tumor regression (>50% decrease in lesion size on imaging studies). Of 221 patients enrolled, 216 received gefitinib as randomized. Symptoms of NSCLC improved in 43% (95% confidence interval [CI], 33%-53%) of patients receiving 250 mg of gefitinib and in 35% (95% CI, 26%-45%) of patients receiving 500 mg. These benefits were observed within 3 weeks in 75% of patients. Partial radiographic responses occurred in 12% (95% CI, 6%-20%) of individuals receiving 250 mg of gefitinib and in 9% (95% CI, 4%-16%) of those receiving 500 mg. Symptoms improved in 96% of patients with partial radiographic responses. The overall survival at 1 year was 25%. There were no significant differences between the 250-mg and 500-mg doses in rates of symptom improvement (P =.26), radiographic tumor regression (P =.51), and projected 1-year survival (P =.54). The 500-mg dose was associated more frequently with transient acne-like rash (P =.04) and diarrhea (P =.006). Gefitinib, a well-tolerated oral EGFR-tyrosine kinase inhibitor, improved disease-related symptoms and induced radiographic tumor regressions in patients with NSCLC persisting after chemotherapy.
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            GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer.

            Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Using functional expression cloning, we have identified GAS5 (growth arrest-specific transcript 5) as critical to the control of mammalian apoptosis and cell population growth. GAS5 transcripts are subject to complex post-transcriptional processing and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers. We have found that, in some cell lines, GAS5 expression induces growth arrest and apoptosis independently of other stimuli. GAS5 transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues. The GAS5 gene has no significant protein-coding potential but expression encodes small nucleolar RNAs (snoRNAs) in its introns. Taken together with the recent demonstration of tumor suppressor characteristics in the related snoRNA U50, our observations suggest that such snoRNAs form a novel family of genes controlling oncogenesis and sensitivity to therapy in cancer.
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              The Genetic Signatures of Noncoding RNAs

              The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses.
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                Author and article information

                Contributors
                dsycmu@163.com
                han_seal@163.com
                liwenyacmu@163.com
                zhongxinwencmu@163.com
                lipeiwencmu123@163.com
                yangshizecmu@163.com
                chenxitaocmu@163.com
                shaomingruicmu@163.com
                zhanglincmu@163.com
                Journal
                J Hematol Oncol
                J Hematol Oncol
                Journal of Hematology & Oncology
                BioMed Central (London )
                1756-8722
                29 April 2015
                29 April 2015
                2015
                : 8
                Affiliations
                Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province 110001 People’s Republic of China
                Article
                140
                10.1186/s13045-015-0140-6
                4419450
                25925741
                © Dong et al.; licensee BioMed Central. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Categories
                Research
                Custom metadata
                © The Author(s) 2015

                Oncology & Radiotherapy

                igf-1r, gas5, gefitinib resistance, nsclc

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