Non-small Cell Lung Cancer Cell Lines A549 Research Articles

IL‑21/IL‑21R inhibit tumor growth and invasion in non‑small cell lung cancer cells via suppressing Wnt/β‑catenin signaling and PD‑L1 expression.

Lung cancer is considered to be one of the world's deadliest diseases, with non-small cell lung cancer (NSCLC) accounting for 85% of all lung cancer cases. The present study aimed to investigate the role and underlying mechanisms of interleukin-21 (IL-21), and its receptor IL-21R, in NSCLC. Lung tissues and blood samples of NSCLC were used to measure IL-21, IL-21R and programmed death 1 ligand 1 (PD-L1) expression using ELISA, western blot and immunohistochemistry analyses. Following treatment with different doses of IL-21, the proliferation, invasion and migration of human NSCLC cell line A549 was evaluated using a cell counting kit-8, colony formation, Transwell and scratch wound healing assays, respectively. Additionally, IL-21R and PD-L1 expression in A549 cells was detected using western blot analysis and immunofluorescence. IL-21R silencing was subsequently used to investigate its effects in cell proliferation, invasion and migration. PD-L1, IL-1β and tumor necrosis factor α (TNF-α) expression were measured. Finally, Wnt/β-catenin signaling expression was evaluated using western blot analysis following treatment with IL-21. Cells were then treated with lithium chloride (LiCl), which is an agonist of Wnt/β-catenin signaling, and the levels of PD-L1, IL-1β and TNF-α were detected. The results revealed that IL-21 and IL-21R expression in the lung tissues and blood samples of patients with NSCLC were decreased, while PD-L1 expression was increased, compared with normal tissues or healthy controls. Treatment of A549 cells with IL-21 upregulated IL-21R expression, downregulated PD-L1 and inhibited cell growth and metastasis in a dose-dependent manner. Following IL-21R silencing, the effects of IL-21 treatment were reversed, suggesting that IL-21 acted on A549 cells through binding to IL-21R. In addition, the results demonstrated that IL-21 treatment reduced the expression levels of proteins associated with the Wnt/β-catenin signaling, whereas activation of Wnt/β-catenin signaling with the LiCl agonist upregulated PD-L1, IL-1β and TNF-α expression. In conclusion, the IL-21/IL-21R axis reduced the growth and invasion of NSCLC cells via inhibiting Wnt/β-catenin signaling and PD-L1 expression. The present results may provide a novel molecular target for NSCLC diagnosis and therapy.

Non-canonical Raf-1/p70S6K signalling in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer‐related death globally, with non–small‐cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf‐1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf‐1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf‐1 in NSCLC. Raf‐1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf‐1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf‐1 knock‐down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf‐1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki‐67 staining and TUNEL staining, respectively. When Raf‐1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf‐1 knock‐down. Overexpression of p70S6K after Raf‐1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf‐1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf‐1/p70S6K signalling a promising target for NSCLC treatment.

MicroRNA-608 Promotes Apoptosis in Non-Small Cell Lung Cancer Cells Treated With Doxorubicin Through the Inhibition of TFAP4.

Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-associated death worldwide. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules of ∼20–25 nucleotides in length. Single nucleotide polymorphisms are a class of genetic variation in the human genome, which when present in miRNA genes are associated with the risk of developing cancer. This study aimed to identify whether the miRNA (miR)-608 polymorphism rs4919510 influenced the incidence of lung cancer, and to explore the underlying mechanisms of miR-608 in the pathogenesis of the disease. A total of 37 patients with non-small cell lung cancer (NSCLC) were selected to determine the expression levels of miR-608; 96 NSCLC patients and 136 cancer-free healthy controls were recruited to determine the incidence of miR-608 rs4919510 in lung cancer patients. Additionally, the impact of miR-608 on the expression of predicted target genes, cell migration, viability, proliferation, and apoptosis was also assessed. We found that the presence of miR-608 rs4919510 did not affect the susceptibility of patients to NSCLC or the maturation of miR-608. miR-608 expression levels were found to be downregulated in NSCLC tissues. Furthermore, overexpression of miR-608 promoted doxorubicin-induced apoptosis in NSCLC cell lines A549 and HCC4006 by inhibiting the expression of transcription factor activating enhancer-binding protein 4 (TFAP4), and high expression levels of TFAP4 were observed in NSCLC tissues. Therefore, our results may provide valuable insights for the chemotherapeutical treatment of NSCLC.

Impact and mechanism of PMs-3 to epithelial mesenchymal transformation of non-small cell lung cancer cell line A549

Objective To explore Impact and mechanism of PMs-3 to epithelial mesenchymal transformation (EMT) of non-small cell lung cancer (NSCLC) cell line A549. Methods Nanocomposite structure synthetized by our team was utilized on human NSCLC cell line A549 and human normal pulmonary epithelial cell line BEAS-2B with concentrations of 0, 20, 40, 60, 80, 100, 120 nmol/L for 48 h. Methyl thiazol tetrazolium (MTT) assay was employed to detect the effect of PMs-3 on A549 cells. Wound assay and Transwell chamber assay were used to test the migration and invasion of PMs-3 on A549 cells. EMT related genes were detected with real time PCR (RT-qPCR) and Western blotting. Results Results of MTT assay detected that PMs-3 could inhibit activity of A549 cells significantly, and inhibition rate was (3.203±1.222)% (0 nmol/L), (11.958±4.000)% (20 nmol/L), (25.348±7.710)% (40 nmol/L), (34.775±9.155)% (60 nmol/L), (41.163±10.988)% (80 nmol/L), (50.430±9.743)% (100 nmol/L), (51.895±6.161)% (120 nmol/L), which showed a dose-dependent manner (F=35.451, P 0.05). Conclusion PMs-3 could inhibit NSCLC cells via regulating EMT. Key words: Non-small cell lung cancer; PMs-3; Epithelial mesenchymal transformation; Drug treatment

MiR-655-3p inhibits cell migration and invasion by targeting pituitary tumor-transforming 1 in non-small cell lung cancer.

miR-655-3p functions as a tumor suppressor in tumor metastases; however, its role and mechanism in regulating cell migration and invasion of non-small cell lung cancer (NSCLC) remain unclear. Here, we found that miR-655-3p expression was markedly decreased in the NSCLC cell lines A549, NCI-H1650, PC14/b, NCI-H1299, and HPAEpiC compared to levels observed in normal human lung fibroblasts. miR-655-3p overexpression significantly inhibited migration and invasion of A549 and PC14/b cells, and pituitary tumor-transforming 1 (PTTG1) expression was up-regulated in the NSCLC cells. Luciferase reporter assays indicated that PTTG1 was a direct target of miR-655-3p. Additionally, PTTG1 overexpression alleviated the inhibitory effect of miR-655-3p on migration and invasion abilities in A549 and PC14/b cells. In conclusion, miR-655-3p inhibits NSCLC migration and invasion by targeting PTTG1, suggesting that miR-655-3p may serve as a therapeutic target to provide a new approach for the clinical treatment of NSCLC.

Circ-ITCH correlates with less advanced tumor features as well as prolonged survival, and it inhibits cells proliferation but promotes apoptosis in non-small cell lung cancer

BackgroundThis study was to investigate the correlation of circ-ITCH expression with tumor features, disease free survival (DFS) and overall survival (OS) in non-small cell lung cancer (NSCLC) patients, and the effect of its dysregulation on cells proliferation and apoptosis of NSCLC cells.MethodsTumor tissue and paired adjacent normal tissue samples from 190 surgical NSCLC patients were collected and used for detecting circ-ITCH expression by qPCR. Circ-ITCH expression was also measured in normal pulmonary epithelial cell line and 4 NSCLC cell lines by qPCR. Patients’ baseline characteristics were recorded and DFS as well as OS were calculated. Blank, circ-ITCH overexpression, blank shRNA, circ-ITCH shRNA plasmids were transferred into NSCLC cell line (A549 cells) as NC(+), Circ-ITCH(+), NC(−) and Circ-ITCH(−) groups. Cells proliferation was measured using CCK-8 and cells apoptosis rate was measured using annexin V (AV)/propidium iodide (PI).ResultsCirc-ITCH expression was decreased in tumor tissue compared with paired adjacent tissue, and was negatively correlated with tumor size, lymph node metastasis and TNM stage. And circ-ITCH high expression was associated with favorable DFS and OS. In vitro experiments revealed that compared with normal pulmonary epithelial cell line BEAS-2B, circ-ITCH expression was reduced in NSCLC cell lines HCC827, A549, NCI-H1299 and NCI-H460. And cells proliferation was decreased in Circ-ITCH(+) group and elevated in Circ-ITCH(−) group at 72 h. Cells apoptosis rate was increased in Circ-ITCH(+) group and decreased in Circ-ITCH(−) group at 72 h.ConclusionsCirc-ITCH associates with less advanced tumor features and prolonged survival, and it inhibits cells proliferation while promotes cells apoptosis in NSCLC.

MiR‑497 inhibits the proliferation and migration of A549 non‑small‑cell lung cancer cells by targeting FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) signaling has been reported to contribute to the carcinogenic progression of various cancer types. Previous studies have demonstrated that FGFR1 expression is increased in non‑small cell lung cancer (NSCLC) and promotes cancer cell metastasis. However, the molecular mechanisms underlying increased FGFR1 expression in NSCLC remains largely unknown. In the current study, microRNA (miR)‑497 levels were observed to be inversely correlated with FGFR1 expression in tumor samples from patients with NSCLC. In the NSCLC cell line A549, miR‑497 overexpression inhibited cell proliferation and migration. Increased expression of miR‑497 led to a reduction in FGFR1 expression, at the mRNA and protein levels. In addition, transfection of miR‑497 mimics inactivated the protein kinase B (AKT) and c‑Jun N‑terminal kinase (JNK) signaling pathways, as reduced matrix metallopeptidase 26 expression; all of which are regulated by FGFR1. Using TargetScan software, FGFR1 was also identified as a predicted target gene of miR‑497, and a dual luciferase reporter assay confirmed that miR‑497 directly regulated FGFR1. Transfection of a recombinant FGFR1 overexpression vector reversed miR‑497 mimic‑induced arrest of cell growth and migration in A549 cells. In conclusion, the results of the present study identified miR‑497 as a potential tumor suppressor gene in NSCLC that may function via repressing FGFR1 expression, and AKT and JNK signaling.

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: Involvement of ROS and mitophagy.

Despite significant strides in understanding the pathophysiology of non-small cell lung cancer (NSCLC), these neoplasms typically present with intrinsic chemo- and radiotherapeutic resistance. Transcriptomic analyses of patient NSCLC tumors stratified by survival times have identified the PTEN-induced putative kinase 1 (PINK1) as a molecular governor of tumor aggressiveness and patient survival time. PINK1 has been shown to confer neuroprotection in models of Parkinson Disease by ensuring proper mitochondrial turnover (mitophagy), the upkeep of ATP production and sequestering of reactive oxygen species (ROS). We utilized an shRNA against PINK1 and the glycolytic inhibitor 3-BP to assess effects on NSCLC viability via MTS cell viability assay. ATP levels, caspase-9 activation, mitophagy and ROS production were determined with standardly available kits. Cytochrome c cellular localization and phosphorylated parkin levels were determined using an ELISA. Our results demonstrate that PINK1 depletion in the NSCLC cell line A549 via shRNA, reduced cancer cell proliferation, increased cell death, reduced ATP production, inhibited mitophagy and increased ROS and caspase-9-dependent apoptosis. PINK1 depleted cells were more susceptible to the glycolytic inhibitor 3-bromopyruvate (3-BP), which further perturbed ATP production. PINK1 depletion and 3-BP synergistically increased ROS production, caspase-9-dependent apoptosis and additively repressed mitophagy. These results suggest that PINK1 depletion alters energetic metabolism and confers sensitivity to agents that inhibit glycolysis. Targeting accelerated tumor cell metabolism may prove useful in the clinical setting while sparing non-malignant tissue.

Prognostic Impact of Melatonin Receptors MT1 and MT2 in Non-Small Cell Lung Cancer (NSCLC).

Background: Several studies have investigated the inhibitory effect of melatonin on lung cancer cells. There are no data available on the prognostic impact of melatonin receptors MT1 and MT2 in non-small cell lung cancer (NSCLC). Materials and Methods: Immunohistochemical studies of MT1 and MT2 were conducted on NSCLC (N = 786) and non-malignant lung tissue (NMLT) (N = 120) using tissue microarrays. Molecular studies were performed on frozen fragments of NSCLC (N = 62; real time PCR), NMLT (N = 24) and lung cancer cell lines NCI-H1703, A549 and IMR-90 (real time PCR, western blot). Results: The expression of both receptors was higher in NSCLC than in NMLT. Higher MT1 and MT2 expression levels (at protein and mRNA) were noted in squamous cell carcinomas (SCC) compared to adenocarcinomas (AC). MT1 immunoexpression decreased as both the tumour size and the cancer stage increased in the whole cohort, while MT2 decreased as the cancer stage increased, with lymph node involvement (in the whole study group) and increasing malignancy grade (in SCC). Higher expression of MT2 was associated with a favorable prognosis. MT2 was an independent prognostic factor for overall survival (OS) in all analyzed NSCLC and in smoking patients. Conclusions: Our observations may point to the potential prognostic significance of MT2 in NSCLC.

Dieckol inhibits non-small-cell lung cancer cell proliferation and migration by regulating the PI3K/AKT signaling pathway.

Non‐small–cell lung cancer (NSCLC) is one of the most prevalent type of lung cancers with an increased mortality rate in both developed and developing countries worldwide. Dieckol is one such polyphenolic drug extracted from brown algae which has proven antioxidant and anti‐inflammatory properties. In the present study, we evaluated the anticancer property of dieckol against NSCLC cell line A549. The LC50 value of dieckol was found to be 25 µg/mL by performing 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and the antiapoptotic property of dieckol was analyzed by dual staining technique with acridine orange/propidium iodide (AO/PI) stains. It was further confirmed with flow cytometry analysis with Annexin FITC and JC‐1 staining and the anti‐invasive property was assessed by Transwell assay. The molecular mechanism of dieckol anticancer activity was confirmed by estimating the levels of caspases and by estimating the signaling proteins of Pi3K/AKT/mTOR signaling pathway using the immunoblotting technique. Our data suggest that dieckol is potent anticancer agent, it effectively inhibits the invasive and migratory property A549 cells and it also induces apoptosis via inhibiting Pi3K/AKT/mTOR signaling, activating the tumor suppressor protein E‐cadherin signifying that dieckol is potent natural anticancer drug to treat NSCLC.

Abstract 1027: Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer

Abstract Mounting preclinical and clinical evidence suggest an involvement of estrogen signaling in promoting non-small cell lung cancer (NSCLC) progression. In estrogen receptor β-positive (ERβ) cases, we previously found seven genes within the prediction analysis of microarrays 50 gene (PAM50) panel that showed strong association in predicting prognosis of NSCLC patients, with Myc, MIA, FGFR4, CXXC5, Grb-7 and FOXC1 being upregulated while PR was downregulated in highly aggressive lung tumors. These genes described one network containing ERβ and human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, which suggests that these two interacting pathways define lung tumors with more aggressive biology. Here, we sought to evaluate the therapeutic potential of combining the pan-HER tyrosine kinase inhibitor, Dacomitinib, and the ER antagonist, Fulvestrant in NSCLC. Cell viability assay in three different human NSCLC cell lines 201T (EGFR wild-type), A549 (K-ras mutant) and HCC827 (EGFR exon 19 deletion) showed strong synergy between Dacomitinib and Fulvestrant, as determined by a combination index < 1. The combination strongly inhibited p-EGFR, p-HER2, p-HER3, p-HER4, and the downstream signaling p-AKT and p-MAPK compared to single treatments. Mechanistically, the transcription activity of activator protein-1 (AP-1) family members Fos and Jun were significantly reduced, as detected by the ability of AP-1 to bind to DNA. Expression of c-Myc and CyclinD1 were also significantly inhibited by the combination. In NSCLC cell lines, the combination reversed the gene signature associated with poor prognosis with c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 being downregulated while PR was upregulated. Similarly, the AP-1 inhibitor t-5224 mimicked the effects of the combination in reversing the PAM50 gene signature, suggesting that the combination’s effect is largely mediated by AP-1 downregulation. In vivo, the combination also revealed a synergistic effect, measured by the combination ratio method (> 1), and induced tumor regression in 201T and A549 xenografts, with average tumor volume significantly lower than the single treatment groups. The immunohistochemical analysis of the xenografts revealed significant downregulation in Ki67. Western blot analysis of the tumor lysates from the combination treatment groups showed an increase in cleaved caspase-3 and a decrease in both ER and HER signaling. The PAM50 gene signature was also reversed in both xenograft studies detected by real-time-qPCR. Similar effects were seen for c-Myc and PR at the protein levels detected by immunohistochemistry staining. These observations encouragingly support the use of this combination clinically in NSCLC, considering its ability to suppress tumor growth and produce a gene signature that predicts better clinical outcomes, as assessed by the PAM50 genes. Citation Format: Abdulaziz A. Almotlak, Mariya Farooqui, Jill M. Siegfried. Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1027.

Abstract 1267: Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117)

Abstract RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Btissame El Hassouni, Richard J. Honeywell, Joris R. Julsing, Beatrice Balboni, Daniel J. De Klerk, Safet Zekanovic, Kees Smid, Elisa Giovannetti, Young B. Lee, Deog J. Kim. Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1267.

Abstract 1773: The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer

Abstract Immune checkpoint inhibitors (ICIs), specifically those which target the T-cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated substantial clinical benefit in patients with non-small cell lung cancer (NSCLC). Tumoral PD-L1 expression may be important for response to therapy, but the precise mechanism of regulation has yet to be fully elucidated. It is known that PD-L1 expression is elevated in cancers harboring mutations in the RAS family of genes, specifically KRAS. Mutations in the KRAS gene are found in up to 30% of NSCLC tumors, and there are no targeted treatment options available for this subset of patients. The tyrosine phosphatase, SHP-2, has been shown to be a critical regulator of the KRAS signaling cascade. Therefore, genes regulated by the RAS/MAPK signaling cascade, including PD-L1, may be modulated by SHP-2 inhibition. Thus, this study aims to investigate the impact of SHP-2 activity on PD-L1 expression in NSCLC. The allosteric inhibitor of SHP-2, SHP099, was used to ablate SHP-2 activity in KRAS-mutant NSCLC cell lines, H460, A549, H2122, and EGFR-mutant NSCLC cell line PC9. PD-L1 levels were quantified by western blot and flow cytometry analyses. PD-L1 mRNA was measured by quantitative real time PCR (qRT-PCR) analysis. SHP099 activity was confirmed by SHP-2, siRNA transfection in KRAS active cell lines. Inhibition of SHP-2 led to increased levels of PD-L1 expression in the KRAS-mutant cell lines, but not in the EGFR-mutant cells. Analysis of PD-L1 surface expression by flow cytometry confirmed that surface PD-L1 levels increased following exposure to SHP099. To determine whether SHP-2 controlled expression at the transcriptional level, mRNA quantification by qRT-PCR displayed increased levels of PD-L1 mRNA following SHP099 treatment. Together, these results suggest that SHP-2 regulates PD-L1 expression in KRAS-active NSCLC at the level of PD-L1 gene expression. It is presently unclear whether SHP-2 also has a role in controlling protein turnover. Using SHP-2 mutant constructs that mimic a constitutively active phosphatase, we hope to gain further insight into the roles of both SHP-2 catalytic and scaffolding functionality on PD-L1 expression. Citation Format: Keller Toral. The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1773.

Plasma miRNA-23a and miRNA-451 as candidate biomarkers for early diagnosis of nonsmall cell lung cancer: a case-control study

To study the value of plasma miRNA23-a and miRNA-451 as potential biomarkers for early diagnosis of non-small cell lung cancer (NSCLC). Fifty patients with NSCLC and 50 healthy control subjects were recruited for testing the plasma levels of miRNA23-a and miRNA-451 and their expression levels in the tumor tissues using qRT-PCR. The correlations of the plasma levels of miRNA23-a and miRNA-451 with their expressions in the tumor tissues were analyzed. The diagnostic power of CEA, miRNA23-a and miRNA-451 for NSCLC was evaluated using the receiver-operating characteristics (ROC) curves and the area under the ROC curves (AUC). In the NSCLC cell line A549, we tested the effect of inhibition of miRNA-23a and miRNA-451 on the expression levels of SPRY2 and MIF mRNA using qRT-PCR. The expression levels of miRNA-23a and miRNA-451 in NSCLC tissues was significantly associated with smoking, tumor size, lymph node metastasis and TNM stage (P < 0.05). Compared with those in the control group, miRNA-23a level was significantly increased while miRNA-451 was significantly down-regulated in the tumor tissues and plasma of NSCLC patients. The plasma levels of miRNA-23a and miRNA-45 were strongly correlated with their expression levels in the tumor tissues. ROC analysis showed that for the diagnosis of NSCLC, the AUC, sensitivity and specificity of either miRNA-23a or miRNA-451 were significantly higher than those of CEA (P < 0.05). The combination of miRNA23-a and miRNA-451 markedly improved the AUC (0.900), sensitivity (78%) and specificity (86%) for the diagnosis. In A549 cells, inhibition of miRNA23-a and miRNA-451 resulted in significantly increased expression levels of SPRY2 mRNA and MIF mRNA, respectively. miRNA-23a and miRNA-451 can be used as potential biomarkers for early diagnosis of NSCLC, and their combined detection can be more effective for the diagnosis.

SFRP2 modulates non‑small cell lung cancer A549cell apoptosis and metastasis by regulating mitochondrial fission via Wnt pathways.

The secreted frizzled‑related protein 2 (SFRP2) has been reported to inhibit non‑small cell lung cancer (NSCLC) cell survival and metastasis; however, the underlying mechanisms are yet to be fully determined. The present study focused on mitochondrial fission and the Wnt signaling pathway. The results demonstrated that SFRP2 was downregulated in the NSCLC cell line A549 compared with in a normal pulmonary epithelial cell line using western blotting, reverse transcription‑quantitative PCR and immunofluorescence. Subsequently, it was demonstrated that SFRP2 overexpression promoted the apoptosis, and inhibited the proliferation and metastasis of A549cells using MTT assays, TUNEL staining and 5‑ethynyl‑2'‑deoxyuridine labeling. At the molecular level, the overexpression of SFRP2 in A549cells led to the activation of mitochondrial fission by inhibiting the Wnt signal pathway. Excessive mitochondrial fission induced low ATP generation, impaired mitochondrial respiratory function, induced mitochondrial potential depolarization, and increased mitochondrial permeability transition pore opening, and imbalances in pro‑ and antiapoptotic protein expression. Furthermore, mitochondrial fission was involved in the inhibition of A549cell proliferation and metastasis. Thus, SFRP2 may inhibit the survival and metastasis of NSCLC cells via the Wnt/mitochondrial fission pathway.

Hsa_circ_0001946 Inhibits Lung Cancer Progression and Mediates Cisplatin Sensitivity in Non-small Cell Lung Cancer via the Nucleotide Excision Repair Signaling Pathway.

Background: Despite great advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC), early diagnosis remains a challenge because patients usually have advanced lung cancer at the time they are diagnosed. The limited efficacy of conventional chemotherapy is another major problem in the treatment of NSCLC. Based on a published set of sequencing data, we find that hsa_circ_0001946 is a circRNA molecule with a significantly different expression level in three cell lines (human normal lung fibroblasts cell line MRC-5, human NSCLC cell line A549, cisplatin-resistant cell line A549/DDP), NSCLC tissues and paired adjacent normal tissues. We believe that hsa_circ_0001946 may have an effect on the progression of NSCLC and its sensitivity to cisplatin.Methods: We focused on investigating the circular RNA, hsa_circ_0001946. RNA interference of hsa_circ_0001946 was carried out in A549 cell lines to determine the effect of reduced hsa_circ_0001946 expression on lung cancer progression and was analyzed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine, clone formation, Hoechst, wound healing, and transwell assays. The nucleotide excision repair (NER) signaling pathway was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, cellular responses to cisplatin were assessed through CCK-8 and flow cytometry assays. Western blot analysis and host-cell reactivation assay were used to determine the effect of hsa_circ_0001946 on NER signaling.Results: In this study, we found that the reduced expression of hsa_circ_0001946 promoted the viability, proliferation, migration, and invasion of NSCLC cells, as well as inhibition of cell apoptosis. Our findings suggest that hsa_circ_0001946 can affect the sensitivity of NSCLC cells to the chemotherapeutic drug cisplatin via modulation of the NER signaling pathway.Conclusions: Our study demonstrated the role of hsa_circ_0001946 in NSCLC pathogenesis, development, and chemosensitivity, and suggests that hsa_circ_0001946 may serve as a novel biomarker for the diagnosis and prediction of platinum-based chemosensitivity in patients with NSCLC.

MiR-875 can regulate the proliferation and apoptosis of non-small cell lung cancer cells via targeting SOCS2.

To investigate the effect of miR-875 on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cancer cell line A549 and the related mechanism. 30 paired tumor tissue and the adjacent tissue were collected. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) has been performed to detect the expression of miR-875 in NSCLC tissues and adjacent normal tissues. Moreover, suppressor of cytokine signaling 2 (SOCS2) has been predicted as a target of miR-875, and Dual-Luciferase reporter assay has been performed to confirm the targeting relationship; furthermore, the expression of SOCS2 in tumor tissue and the adjacent tissue were compared. Next, human NSCLC cell line A549 cells were cultured and transfected with miR-875 inhibitor with or without SOCS2 siRNA, and the proliferation and apoptosis of the cells were evaluated by Cell Counting Kit (CCK-8) and flow cytometry methods. Finally, the relative protein expression of Wnt and β-catenin were analyzed by Western blot analysis. MiR-875 was significantly up-regulated in NSCLC tissues compared with the adjacent tissues. SOCS2 was confirmed as a target of miR-875, and the expression of SOCS2 was markedly decreased in NSCLC tissues. Moreover, the knockdown of miR-875 inhibited the proliferation and promoted the apoptosis of A549 cells, while transfection of SOCS2 siRNA can block miR-875 inhibitor-induced anti-proliferative effects. Finally, the transfection of miR-875 inhibitor decreased the expression of Wnt and β-catenin, and SOCS2 siRNA can reverse the effect. MiR-875 may regulate the proliferation and apoptosis of NSCLC cells via targeting SOCS2, suggesting that miR-875 has the potential to become a therapeutic target for the treatment in NSCLC.

Hydroxysafflor yellow A inhibited lipopolysaccharide-induced non-small cell lung cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT/mTOR and ERK/MAPK signaling pathways.

BackgroundChronic inflammation plays a significant role in the occurrence and development of non‐small cell lung cancer (NSCLC). Hydroxysafflor yellow A (HSYA), a chemical compound of the yellow color pigments extracted from the safflower, has been widely used in clinical treatment with positive antioxidation, anti‐inflammation, and antitumor effects. However, the role and underlying mechanisms of HYSA on development and progress in inflammation‐mediated NSCLC are unknown.MethodsCell counting kit‐8, colony formation, EdU, cell apoptosis, wound healing, Transwell migration and invasion, and enzyme‐linked immunosorbent assays; flow cytometry; and Western blotting were conducted using human NSCLC cell lines A549 and H1299.ResultsLipopolysaccharide (LPS) significantly promoted the proliferation and enhanced colony formation of A549 and H1299 cells, while HYSA notably reversed the effects of LPS. HYSA induced apoptosis of LPS‐mediated A549 and H1299 cells in a dose dependent manner; and remarkably suppressed migration, invasion, and epithelial–mesenchymal transition (EMT), significantly regulated production of LPS‐induced inflammation cytokines, and downregulated protein expression of PI3K/Akt/mTOR and ERK/MAPK signaling pathways in LPS‐induced A549 and H1299 cells. Furthermore, PI3K (LY294002) and ERK (SCH772984) inhibitors remarkably inhibited proliferation, migration, invasion, and EMT, and induced apoptosis in LPS‐mediated A549 and H1299 cells. These effects were even more obvious in the presence of HYSA and LY294002 or SCH772984 compared to those of either agent alone.ConclusionHYSA suppressed LPS‐mediated proliferation, migration, invasion, and EMT in A549 and H1299 cells by inhibiting the PI3K/Akt/mTOR and ERK/MAPK signaling pathways, indicating that HYSA may be a potential candidate to treat inflammation‐mediated NSCLC.

Fingolimod sensitizes EGFR wild‑type non‑small cell lung cancer cells to lapatinib or sorafenib and induces cell cycle arrest.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase and mutations in this gene are major drivers of lung cancer development. EGFR tyrosine kinase inhibitors (TKIs) are standard first‑line therapies for patients with advanced non‑small cell lung cancer (NSCLC) with activating EGFR mutations, but are not effective in patients with wild‑type EGFR. In the present study, the cytotoxic effects of various TKIs against EGFR were investigated in wild‑type NSCLC cells as single treatments or in combination with Fingolimod (FTY720), which has been approved for treating multiple sclerosis and has cytotoxic effects against several tumor cell lines. It was found that the combined treatment with TKIs lapatinib (Lap) or sorafenib (Sor) and FTY720 synergistically suppressed the viability of the NSCLC cell lines A549 and H596. Additionally, FTY720 inhibited lysosomal acidification and suppressed autophagy flux. Immunoblotting and reverse transcription‑quantitative polymerase chain reaction showed that FTY720 combined with Lap or Sor, enhanced endoplasmic reticulum (ER) stress loading and cell cycle arrest in A549 cells. The enhancement of ER stress loading and cell cycle arrest induced by combined treatment with Lap or Sor and FTY720, which was associated with the cytotoxicity induced by the combination of these drugs. These findings suggested that FTY720 improved TKI therapy in NSCLC patients with wild‑type EGFR, by sensitizing NSCLC cells to TKIs.

Overexpression of PKMYT1 indicates the poor prognosis and enhances proliferation and tumorigenesis in non-small cell lung cancer via activation of Notch signal pathway.

The protein kinase, membrane‑associated tyrosine/threonine 1 (PKMYT1) has been implicated as an important factor promoting the tumorigenesis of hepatocellular carcinoma and colorectal cancer. The current study was designed to explore the functional role of PKMYT1 in non-small cell lung cancer (NSCLC) cell behaviors and to investigate the possible molecular mechanisms. The expression levels of PKMYT1 in NSCLC tissues and cell lines were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The clinical and prognostic significance of PKMYT1 in 153 cases of NSCLC was determined. We also evaluated the effects of KMYT1 on NSCLC cell proliferation, migration, and invasion in vitro. Western blot was performed to assure whether PKMYT1 affected the Notch signal pathway and MET pathway. We observed that PKMYT1 expression was significantly up-regulated in both NSCLC tissues and cell lines. Higher expression of PKMYT1 was associated with clinical stage and lymph nodes metastasis. Clinical survival assays demonstrated that patients with high PKMYT1 expression had a shorter overall survival time than those with low PKMYT1 expression. Moreover, the multivariate analysis confirmed that increased expression of PKMYT1 was an independent predictor of overall survival. Functionally, knockdown of PKMYT1 in the NSCLC cell lines A549 and H1299 suppressed NSCLC cells proliferation, invasion and migration, and promoted apoptosis. In addition, the down-regulation of PKMYT1 resulted in the inhibition of EMT in NSCLC cells. Further mechanistic studies revealed that when PKMYT1 was silenced, the expression levels of Notch1, p21, and Hes1 were respectively downregulated, suggesting that PKMYT1 could promote the activity of the Notch signal pathway. PKMYT1 plays a significant role in NSCLC aggressiveness and clinical outcome, and may serve as a promising therapeutic target for this disease.