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.
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.


METHODS
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.
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. dsycmu@163.com.
Siyuan
S
Dong
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. han_seal@163.com.
Xiaohan
X
Qu
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. liwenyacmu@163.com.
Wenya
W
Li
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. zhongxinwencmu@163.com.
Xinwen
X
Zhong
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. lipeiwencmu123@163.com.
Peiwen
P
Li
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. yangshizecmu@163.com.
Shize
S
Yang
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. chenxitaocmu@163.com.
Xitao
X
Chen
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. shaomingruicmu@163.com.
Mingrui
M
Shao
Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. zhanglincmu@163.com.
Lin
L
Zhang
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000368:Aged; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D000077156:Gefitinib; D018773:Genes, erbB-1; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D062085:RNA, Long Noncoding / Q000096:biosynthesis*; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; C561567:GAS5 long non-coding RNA, human; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D062085:RNA, Long Noncoding; D017526:Receptor, IGF Type 1; D000077156:Gefitinib