Gefitinib-resistant Lung Adenocarcinoma Cells Research Articles

FTO/m6A mediates miR-138-5p maturation and regulates gefitinib resistance of lung adenocarcinoma cells by miR-138-5p/LCN2 axis

BackgroundLung cancer (LC) occupies an important position in the lethality of cancer patients. Acquired resistance to gefitinib in lung adenocarcinoma (LUAD) seriously affects the therapeutic efficacy of LC. Thus, it is of major scientific and clinical significance to probe the mechanism of gefitinib resistance in LUAD for ameliorating the prognosis of patients.MethodsThe expression of miRNAs in gefitinib-resistant LUAD cells was validated using qRT-PCR. Cell viability was assessed through CCK-8, whereas cell death was examined through PI staining. Changes in the ferroptosis process were evaluated by detecting the intracellular Glutathione (GSH), Malondialdehyde (MDA), and Reactive Oxygen Species (ROS) levels. Downstream targets of miR-138-5p were verified via luciferase reporter and RNA pull-down assays. RIP and qRT-PCR were employed to evaluate pri-miR-138-5p binding to DiGeorge critical region 8 (DGCR8) and the pri-miR-138-5p m6A modification level. Additionally, the impact of fat mass and obesity-associated protein (FTO) on LUAD gefitinib sensitivity was assessed in vivo by constructing a xenograft model.ResultsWe observed that miR-138-5p was notably diminished in gefitinib-resistant cells. Overexpression of miR-138-5p suppressed viability while facilitated cell death and intracellular ferroptosis in gefitinib-resistant cells. Moreover, lipocalin 2 (LCN2) was the downstream target of miR-138-5p. The biological functions of miR-138-5p on gefitinib-resistant cells was reversed by introduction of LCN2. FTO suppressed the binding of DGCR8 to pri-miR-138-5p through m6A modification, thereby restraining the processing of miR-138-5p. Meanwhile, silencing of FTO enhanced the sensitivity of LUAD to gefitinib treatment.ConclusionFTO suppressed the processing of miR-138-5p and then modulated the proliferation, death, and ferroptosis of gefitinib-resistant cells through the miR-138-5p/LCN2 pathway, which may put forward novel insights for clinically ameliorating the therapeutic effect of gefitinib in LUAD.

NEAT1_1 confers gefitinib resistance in lung adenocarcinoma through promoting AKR1C1-mediated ferroptosis defence

Gefitinib is one of the most extensively utilized epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for treating advanced lung adenocarcinoma (LUAD) patients harboring EGFR mutation. However, the emergence of drug resistance significantly compromised the clinical efficacy of EGFR-TKIs. Gaining further insights into the molecular mechanisms underlying gefitinib resistance holds promise for developing novel strategies to overcome the resistance and improve the prognosis in LUAD patients. Here, we identified that the inhibitory efficacy of gefitinib on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection resulted in gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) exhibited significant upregulation in gefitinib-resistant strains of LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) of LUAD patients who received first-generation EGFR-TKI treatment. Knockdown of AKR1C1 partially reversed drug resistance by re-sensitizing the LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the aberrant upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, upregulated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1_1 (NEAT1_1) sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assay and miRNA rescue experiment confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the effects of NEAT1_1/miR-338-3p/AKR1C1 axis-mediated ferroptosis defence in gefitinib resistance in LUAD. Thus, targeting NEAT1_1/miR-338-3p/AKR1C1 axis might be a novel strategy for overcoming gefitinib resistance in LUAD harboring EGFR mutation.

CA916798 predicts poor prognosis and promotes Gefitinib resistance for lung adenocarcinoma

BackgroundOur previous studies have identified CA916798 as a chemotherapy resistance-associated gene in lung cancer. However, the histopathological relevance and biological function of CA916798 in lung adenocarcinoma (LUAD) remains to be delineated. In this study, we further investigated and explored the clinical and biological significance of CA916798 in LUAD.MethodsThe relationship between CA916798 and clinical features of LUAD was analyzed by tissue array and online database. CCK8 and flow cytometry were used to measure cell proliferation and cell cycle of LUAD after knockdown of CA916798 gene. qRT-PCR and western blotting were used to detect the changes of cell cycle-related genes after knockdown or overexpression of CA916798. The tumorigenesis of LUAD cells was evaluated with or without engineering manipulation of CA916798 gene expression. Response to Gefitinib was evaluated using LUAD cells with forced expression or knockdown of CA916798.ResultsThe analysis on LUAD samples showed that high expression of CA916798 was tightly correlated with pathological progression and poor prognosis of LUAD patients. A critical methylation site in promoter region of CA916798 gene was identified to be related with CA916798 gene expression. Forced expression of CA916798 relieved the inhibitory effects of WEE1 on CDK1 and facilitated cell cycle progression from G2 phase to M phase. However, knockdown of CA916798 enhanced WEE1 function and resulted in G2/M phase arrest. Consistently, chemical suppression of CDK1 dramatically inhibited G2/M phase transition in LUAD cells with high expression of CA916798. Finally, we found that CA916798 was highly expressed in Gefitinib-resistant LUAD cells. Exogenous expression of CA916798 was sufficient to endow Gefitinib resistance with tumor cells, but interference of CA916798 expression largely rescued response of tumor cells to Gefitinib.ConclusionsCA916798 played oncogenic roles and was correlated with the development of Gefitinib resistance in LUAD cells. Therefore, CA916798 could be considered as a promising prognostic marker and a therapeutic target for LUAD.

LncRNA AFAP1-AS1 Induces Gefitinib Resistance of Lung Adenocarcinoma Through the miR-653-5p/AGR2 Axis.

Gefitinib resistance limits the therapeutic efficacy of gefitinib to lung adenocarcinoma (LUAD). The goal of this research is to learn more about the lncRNA AFAP1-AS1 and how it functions in gefitinib-resistant LUAD cells. RT-qPCR was performed to test the expression of AFAP1-AS1, miR-653-5p and AGR2 in LUAD tissues with acquired resistance to gefitinib or not as well as in gefitinib-resistant LUAD cells. Cell proliferation, invasion and apoptosis were measured by CCK8 assays, transwell invasion assays and flow cytometry, respectively. Luciferase reporter assay showed that miR-653-5p and AFAP1-AS1 or AGR2 interactions. In gefitinib-resistant LUAD cells and tissues, AFAP1-AS1 was overexpressed. Meanwhile, silencing AFAP1-AS1 reduced proliferation and migration while increasing apoptosis and gefitinib sensitivity. Mechanically, AFAP1-AS1 sequestered the miR-653-5p and blocked the inhibition of miR-653-5p to AGR2 and stepwise upregulated AGR2 overexpression in LUAD gefitinib resistant cells, resulting gefitinib resistance in LUAD. AFAP1-AS1 promotes gefitinib-resistance LUAD cells through a previously unrecognized miR-653-5p/AGR2 axis, suggesting targeting AFAP1-AS1/miR-653-5p/AGR2 axis might be a promising way for LUAD intervention.

Y-box binding protein 1 (YB-1) promotes gefitinib resistance in lung adenocarcinoma cells by activating AKT signaling and epithelial-mesenchymal transition through targeting major vault protein (MVP).

Gefitinib is a first-line treatment option for epidermal growth factor receptor (EGFR)-mutatedlung adenocarcinoma. However, most patients inevitably develop gefitinib resistance. The mechanism underlying this resistance is not fully understood. Y-box binding protein 1 (YB-1) has been reported to play a role in modulating drug sensitivity, but its role in gefitinib resistance is currently unknown. Here, we investigated the role of YB-1 in gefitinib resistance of lung adenocarcinoma. We determined the expression of YB-1, epithelial-mesenchymal transition (EMT) and AKT signaling markers, as well as the viability of lung adenocarcinoma cell lines bearing mutant (HCC827, PC-9) or wild-type (H1299) EGFR. We also evaluated PC-9 cell migration and invasion using transwell assays. The clinical importance of YB-1 and major vault protein (MVP) was evaluated using primary lung adenocarcinoma patient samples. We found that YB-1 was significantly upregulated in gefitinib-resistant lung adenocarcinoma cells compared to gefitinib-sensitive cells. YB-1 augmented gefitinib resistance by activating the AKT pathway and promoting EMT. Decreased migration and invasion was observed upon MVP silencing in YB-1-overexpressing PC-9 cells, as well as restored gefitinib sensitivity. A retrospective analysis of 85 patients with lung adenocarcinoma revealed that YB-1 levels were significantly increased in tyrosine kinase inhibitor (TKI)-resistant patients compared to those in TKI-sensitive patients, indicating that YB-1 may serve as a biomarker to clinically predict acquired gefitinib resistance. YB-1 activates AKT signaling and promotes EMT at least in part by directly activating MVP. Hence, targeting the YB-1/MVP axis may help to overcome gefitinib resistance in lung adenocarcinoma patients.

LncRNA SNHG15 regulates EGFR-TKI acquired resistance in lung adenocarcinoma through sponging miR-451 to upregulate MDR-1

Lung adenocarcinoma (LUAD) is the main component of non-small-cell lung cancer (NSCLC) and causes a great health concern globally. The top priority of LUAD treatment is to deal with gefitinib resistance. Long non-coding RNAs are certified to modify gefitinib resistance in the course of tumor aggravation. The study focuses on addressing the function of small nucleolar RNA host gene 15 (SNHG15) on modifying gefitinib resistance in LUAD. Previously, NOTCH pathway is implicated in LUAD chemo-resistance. SNHG15 level was boosted following the depletion of NOTCH-1 in A549/GR and H1975/GR cells. Functional studies indicated that SNHG15 and multidrug resistance protein 1 (MDR-1) were overexpressed and possess tumor-promoting functions in gefitinib-resistant LUAD cells while miR-451 was downregulated and possess tumor-suppressive behaviors in gefitinib-resistant LUAD cells. Mechanically, the SNHG15 was cytoplasmically distributed in GR LUAD cells. In addition, SNHG15 released MDR-1 from the suppression of miR-451, leading to MDR-1 promotion. In addition, the elevation of SNHG15 could be attributed to ZEB1. Rescue assays highlighted that downstream molecules MDR-1 and miR-451 could reverse the effects of SNHG15 downregulation on gefitinib-resistant LUAD cells. SNHG15 could alter chemo-resistance of LUAD cells to Gefitinib via regulating miR-451/MDR-1, which could be inspiring findings for the advancement of chemo-therapies for LUAD.

Paris Saponins enhance radiosensitivity in a gefitinib-resistant lung adenocarcinoma cell line by inducing apoptosis and G2/M cell cycle phase arrest.

Acquired resistance to epidermal growth factor inhibitors has been reported to be associated with cross‑resistance to radiation. Paris Saponins (PSs) exert a wide range of pharmacological activities, including cell apoptosis induction, multidrug resistance inhibition, angiogenesis inhibition and tumor cell migration by modulating various signaling pathways. The present study aimed to investigate the radiosensitization effects of PSII, PSVI and PSVII in a gefitinib‑resistant PC‑9‑ZD lung adenocarcinoma cell line, and the possible mechanism underlying their function. A clonogenic assay was performed to determine the effects of PS radiosensitization on the PC‑9‑ZD cell line. The cell cycle was analyzed by flow cytometry, and cell apoptosis was analyzed with Annexin V/propidium iodide and Hoechst staining. Protein expression levels were detected by western blotting. The results of the present study revealed a significant increase in PC‑9‑ZD cell line radiosensitivity following treatment with PSs. PSs induced G2/M cell cycle phase arrest and apoptosis of the irradiated PC‑9‑ZD cells. Notably, the expression levels of B cell lymphoma 2 (Bcl‑2) were downregulated, and those of caspase‑3, Bcl‑2‑associated X protein (Bax) and p21/Waf1/Cip1 were upregulated following treatment with PSs. The present results demonstrated that PSs induced radiosensitivity in gefitinib‑resistant cells by inducing G2/M phase arrest and by enhancing the apoptotic response via the modulation of caspase‑3, Bax, Bcl‑2 and p21/Waf1/Cip1 expression.

Abstract 742: Effect of Hsp90 inhibitor NVP-AUY922 with radiation on lung adenocarcinoma cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors

Abstract Recent progress in understanding of molecular pathology in lung adenocarcinoma has been changing the therapeutic strategy. Especially, tyrosine kinase inhibitors (TKIs) for epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma have improved the outcomes. However acquired resistances to TKIs are observed in most of cases and how to overcome the resistances must be the key factor to make them curable. 90-kDa heat shock protein (Hsp90) is a chaperone protein that assists maturation of client proteins and EGFR is one of them. Reportedly, Hsp90 inhibitors have anti-tumor effect against not only EGFR-TKI sensitive lung adenocarcinomas but also those with EGFR-TKI resistantce. Furthermore Hsp90 inhibitor can increase radiosensitivity through inhibition of hypoxia-inducible factor-1α (HIF-1α), checkpoint kinase 1 (CHK1) and WEE1 G2 checkpoint kinase (WEE1). These facts prompted us to investigate the combined effect of Hsp90 inhibitors and radiation to EGFR-TKI resistant cells. We exposed Hsp90 inhibitor NVP-AUY922 (AUY) to Gefitinib-resistant lung adenocarcinoma cell lines, PC-9 with T790M (PC-9 GRt790m) which is well known secondary mutation with EGFR-TKI resistant cells and HCC827 with MET amplification (HCC827-GRmet) which is also well known mechanism of EGFR-TKI resistant. Our earlier work showed another EGFR-TKI resistant mechanism, acquiring cancer stem cell properties. We also used HCC827 with stem-cell like properties (HCC827-GRstem). To examine the anti-tumor effect, we conducted colonogenic assay with radiation. Then we conducted immunofluorescence staining for γH2AX and counted the foci to observe DNA double strand breaks. We also conducted cell cycle assay with propidium iodide-staining method using flow cytometry. The colonogenic assay showed anti-tumor effect in PC-9 GRt790m and HCC827-GRmet but HCC827-GRstem showed limited effect to AUY and radiation. In γH2AX staining, HCC827-GRmet showed prolonged DNA double strand breaks suggesting leading to apoptosis. In contrast, HCC827-GRstem showed rapid decrease of DNA double strand breaks. In cell cycle analysis, HCC827-GRstem showed smaller population of G2/M compared to HCC827-GRmet and parental HCC827. Taken together, AUY with radiation can be a effective therapy to EGFR-TKI resistant lung adenocarcinoma but stem-cell like properties cause resistant even these therapies. Citation Format: Yuho Maki, Shinsuke Hashida, Hiromasa Yamamoto, Kazuhiko Shien, Tomoaki Ohtsuka, Ken Suzawa, Masashi Furukawa, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Susumu Kanazawa, Shinichi Toyooka. Effect of Hsp90 inhibitor NVP-AUY922 with radiation on lung adenocarcinoma cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 742. doi:10.1158/1538-7445.AM2015-742

Rhodomycin A, a novel Src-targeted compound, can suppress lung cancer cell progression via modulating Src-related pathways.

Src activation is involved in cancer progression and the interplay with EGFR. Inhibition of Src activity also represses the signalling pathways regulated by EGFR. Therefore, Src has been considered a target molecule for drug development. This study aimed to identify the compounds that target Src to suppress lung cancer tumourigenesis and metastasis and investigate their underlying molecular mechanisms. Using a molecular docking approach and the National Cancer Institute (NCI) compound dataset, eight candidate compounds were selected, and we evaluated their efficacy. Among them, rhodomycin A was the most efficient at reducing the activity and expression of Src in a dose-dependent manner, which was also the case for Src-associated proteins, including EGFR, STAT3, and FAK. Furthermore, rhodomycin A significantly suppressed cancer cell proliferation, migration, invasion, and clonogenicity in vitro and tumour growth in vivo. In addition, rhodomycin A rendered gefitinib-resistant lung adenocarcinoma cells more sensitive to gefitinib treatment, implying a synergistic effect of the combination therapy. Our data also reveal that the inhibitory effect of rhodomycin A on lung cancer progression may act through suppressing the Src-related multiple signalling pathways, including PI3K, JNK, Paxillin, and p130cas. These findings will assist the development of anti-tumour drugs to treat lung cancer.

Novel curcumin analogs to overcome EGFR–TKI lung adenocarcinoma drug resistance and reduce EGFR–TKI-induced GI adverse effects

Curcumin (1) down-regulates the expression as well as phosphorylation of epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells expressing gefitinib-resistant EGFR. Thirty-seven newly synthesized curcumin analogues including dimethoxycurcumin (2, DMC) were evaluated for their effects on EGFR expression as well as phosphorylation in two gefitinib-resistant lung adenocarcinoma cell lines, CL1-5 (EGFRwt) and H1975 (EGFRL858R+T790M). Based on the identified structure–activity relationships, methoxy substitution at C-3′, C-4′, or both positions favored inhibitory activity (compounds 1, 2, 5, 8–15, 17, 36), while compounds with more polar substituents were generally less active in both cell lines. Compound 36 with a fluorine substituent at C-6′ and its protonated counterpart 2 did not lose activity, suggesting halogen tolerance. In addition, a conjugated linker was essential for activity. Among all evaluated curcumin derivatives, compound 2 showed the best inhibitory effects on both wild-type and mutant EGFR by efficiently inducing gefitinib-insensitive EGFR degradation. Compound 23 also reduced gefitinib-induced gastrointestinal damage in the non-transformed intestinal epithelial cell line IEC-18.

Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells.

Despite good initial responses, drug resistance and disease recurrence remain major issues for lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations taking EGFR-tyrosine kinase inhibitors (TKI). To discover new strategies to overcome this issue, we investigated 40 essential oils from plants indigenous to Taiwan as alternative treatments for a wide range of illnesses. Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects. In this study, we demonstrated that hinokitiol inhibited the proliferation and colony formation ability of lung adenocarcinoma cells as well as the EGFR-TKI-resistant lines PC9-IR and H1975. Transcriptomic analysis and pathway prediction algorithms indicated that the main implicated pathways included DNA damage, autophagy, and cell cycle. Further investigations confirmed that in lung cancer cells, hinokitiol inhibited cell proliferation by inducing the p53-independent DNA damage response, autophagy (not apoptosis), S-phase cell cycle arrest, and senescence. Furthermore, hinokitiol inhibited the growth of xenograft tumors in association with DNA damage and autophagy but exhibited fewer effects on lung stromal fibroblasts. In summary, we demonstrated novel mechanisms by which hinokitiol, an essential oil extract, acted as a promising anticancer agent to overcome EGFR-TKI resistance in lung cancer cells via inducing DNA damage, autophagy, cell cycle arrest, and senescence in vitro and in vivo.

Effect of Paris saponin I on radiosensitivity in a gefitinib-resistant lung adenocarcinoma cell line.

Previous studies have observed that Paris saponin I (PSI) exerts a wide range of pharmacological activities, including cytotoxic activity against a number of malignancies, such as non-small cell lung cancers. The present study aimed to investigate the radiosensitization of PSI treatment on a gefitinib-resistant lung adenocarcinoma cell line, PC-9-ZD, and its possible mechanism. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to determine the growth inhibition effect of PSI. A clonogenic assay was performed to determine the radiosensitizing effect of PSI treatment on the PC-9-ZD cell line. A single-hit multi-target model was used to plot survival curves and calculate sensitizing enhancement ratios. The cell cycle was analyzed by flow cytometry and cell apoptosis was analyzed with fluorescein-isothiocyanate-Annexin V/propidium iodide and Hoechst staining. The expression levels of the proteins were detected by western blotting. There was a significant reduction observed in the proliferation of the PC-9-ZD cell lines that were treated with PSI. PSI enhanced the radiosensitivity of the PC-9-ZD cells with a sensitization enhancement ratio of 1.77. Furthermore, PSI induced G2/M arrest and apoptosis of the irradiated PC-9-ZD cells. Notably, B-cell lymphoma 2 (Bcl-2) was downregulated, and caspase-3, Bcl-2-like protein 4 (Bax) and cyclin-dependent kinase inhibitor 1 (P21waf1/cip1) were upregulated by the PSI treatment. The present study showed that PSI treatment exhibited potent radiosensitivity against gefitinib-resistant PC-9-ZD cells in vitro. This radiosensitivity was associated with cell cycle arrest at the G2/M phase, and apoptosis via an increase in caspase-3, Bax and P21waf1/cip1 as well as a decrease in Bcl-2 production.

Abstract 4041: Lipofection of the designed plasmid expressing microRNA-7 has antitumor effect in gefitinib-resistant lung adenocarcinoma in vivo

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is strikingly effective in tumors harboring activating EGFR mutations since the tumor cells depend on “oncogene addiction” to EGFR signaling that is inhibited effectively by a change in the affinity of ATP and EGFR-TKI to ATP binding site of mutated EGFR. Unfortunately, approximately 50% of patients who responded to EGFR-TKI acquired the resistance to EGFR-TKI due to additional mutation T790M in EGFR. Since T790M mutation restores the affinity of ATP to the ATP binding site of mutated EGFR, the oncogene addiction by EGFR pathway is presumed to sustain in the gefitinib-resistant cells harboring T790M mutation. MicroRNA-7 (miR-7) is predicted to suppress EGFR expression by targeting three sites in the 3′-untranslated region (UTR) of EGFR mRNA in silico. It was proved to be a potential tumor suppressor in EGFR-TKI-resistant lung adenocarcinoma cells by targeting the 3′-UTR of EGFR mRNA (#1396: Annual AACR meeting, 2009). The object of this experiment is to elucidate whether the miR-7 is effective or not in vivo. We established gefitinib-resistant lung adenocarcinoma cell line (RPC-9) carrying a secondary mutation T790M (Cancer Res 67: 7807-14, 2007) from gefitinib-sensitive EGFR mutated (delE746-A750 in exon 19) PC-9 cell line. RPC-9 cells (2 × 106) were injected s.c. into the backs of the female athymic mice at 7 week of age. At 1 week after injection, the mice were randomly assigned into one of three groups (five mice per group) that received either 3 μg/body of miR-7 expressing plasmid, control plasmid or mock using cationic liposome by direct injection. Marked tumor regression was observed within 2 weeks, and 60% of xenograft tumors disappeared. The immunostaining of residual tumor showed the suppression of EGFR expression on the tumor cells. To our knowledge, this is the first report to show the dramatic effect of miR-7 against EGFR-TKI resistant lung cancer model in vivo.Tumor size after treatment with miR-7 Day 1Day 5Day 11Day 18miR-710094.9445.7914.62*Control100144.0103.3242.6*tumor size(%), *p=0.00253, tumor diminished macroscopically in miR-7 group(3/5) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4041.