Normal Bronchial Epithelial Cells Research Articles

Titanium dioxide nanoparticles induced reactive oxygen species (ROS) related changes of metabolomics signatures in human normal bronchial epithelial (BEAS-2B) cells

Titanium dioxide often enters the respiratory tract in the form of nano-dust in occupational sites. Metabolomics may be a promising method for studying the toxicology of nano titanium dioxide. The intention of this study was to explore the possible impact of titanium dioxide nanoparticles (TiO2 NPs) on the metabolomics signatures of human normal bronchial epithelial (BEAS-2B) cells and to search for investigate the role of reactive oxygen species (ROS). In this study, BEAS-2B cells were treated by TiO2 NPs (0, 25, 50 and 100 μg/mL) for 48 h. The metabolites extracted from BEAS-2B cells were determined by untargeted metabolomics technique, and the differential metabolites and metabolic pathways were discovered by using multivariate analysis. Intracellular ROS was detected by DCFH-DA probe and flow cytometry method. Machine learning was used to explore the relationship between ROS and metabolomics changes. Totally, seventy-six differential metabolites and the steroid biosynthesis pathway of BEAS-2B cells were observed after treatment of TiO2 NPs. Lipid and lipid-like metabolites were the most significant classes among the metabolite products induced by TiO2 NPs. TiO2 NPs resulted in a dose-dependent increase in intracellular ROS levels, and correlated with most of the differential metabolites. In conclusion, TiO2 NPs increased the level of the oxidative stress, which could contribute to the altered signature represented by lipid metabolism in BEAS-2B cells.

Cytotoxicity and oxidative stress induced by atmospheric mono-nitrophenols in human lung cells

Nitrophenols (NPs) are hazardous pollutants found in various environmental matrices, including ambient fine particulate matter (PM2.5), agricultural residues, rainwater, wildfires, and industrial wastes. This study showed for the first time the effect of three pure nitrophenols and their mixture on human lung cells to provide basic understanding of the NP influence on cell elements and processes. We identified NPs in ambient PM2.5 and secondary organic aerosol (SOA) particles generated from the photooxidation of monocyclic aromatic hydrocarbons in the U.S. EPA smog chamber. We assessed the toxicity of identified NPs and their equimolar mixture in normal bronchial epithelial (BEAS-2B) and alveolar epithelial cancer (A549) lung cell lines. The inhibitory concentration-50 (IC50) values were highest and lowest in BEAS-2B cells treated with 2-nitrophenol (2NP) and 4-nitrophenol (4NP), respectively, at 24 h of exposure. The lactate dehydrogenase (LDH) assay showed that 4NP, the most abundant NP we identified in PM2.5, was the most cytotoxic NP examined in both cell lines. The annexin-V/fluorescein isothiocyanate (FITC) analysis showed that the populations of late apoptotic/necrotic BEAS-2B and A549 cells exposed to 3NP, 4NP, and NP equimolar mixture increased between 24 and 48 h. Cellular reactive oxygen species (ROS) buildup led to cellular death post exposure to 3NP, 4NP and the NP mixtures, while 2NP induced the lowest ROS buildup. An increased mitochondrial ROS signal following NP exposure occurred only in BEAS-2B cells. The tetramethylrhodamine, methyl ester, perchlorate (TMRM) assay showed that exposed cells exhibited collapse of the mitochondrial membrane potential. TMRM signals decreased significantly only in BEAS-2B cells, and most strongly with 4NP exposures. Our results suggest that acute atmospheric exposures to NPs may be toxic at high concentrations, but not at ambient PM2.5 concentrations. Further chronic studies with NP and NP-containing PM2.5 are warranted to assess their contribution to lung pathologies.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called “oncogene-induced senescence (OIS)”, prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called “HBEC3KT”), immortalized with hTERT (“T”) and Cdk4 (“K”), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Antioxidant and Anti-Inflammatory Activity of Combined Phycocyanin and Palmitoylethanolamide in Human Lung and Prostate Epithelial Cells

Inflammation involving the innate and adaptive immune systems is a normal response to infection; however, when allowed to continue unchecked, inflammation may result in several pathologies. Natural molecules with antioxidant properties can target the key players of inflammation and exert beneficial health effects. In this study, human normal bronchial (Beas-2B) and prostate (HPrEpiC) epithelial cell lines were exposed to infectious stimulation and treated with phycocyanin (PC) and palmitoylethanolamide (PEA), with the aim of demonstrating the enhanced antioxidant and anti-inflammatory properties of the combination. The cotreatment protected from cytotoxicity and greatly abated both the production of radical oxygen species (ROS) and the transcription of several inflammatory cytokines. Oxidative stress and inflammation were curtailed by affecting three main pathways: (1) inhibition of cyclooxygenase-2 enzyme and consequent decrease of signaling generating ROS; (2) increased synthesis of glutathione and therefore strengthening of the natural antioxidant defenses of the cells; (3) decreased infection-driven mitochondrial respiratory burst which generates oxidative stress. Based on the mounting interest in using nutraceuticals as adjuvants in the clinical practice, the present study unveils new mechanisms of action and enhanced efficacy of PC and PEA, supporting the possible exploitation of this combination in human disorders.

C,O‐Chelated organotin(IV) derivatives as potential anticancer agents: Synthesis, characterization, and cytotoxic activity

AbstractOrganotin(IV) chemistry is nowadays in a continuous expansion due to the biological and medicinal potential found for some of these species. Within this study, the cytotoxic activity of several organotin(IV) compounds was investigated on two lung cancer cell lines (H522 and SK‐MES‐1) and on a normal lung cell line in order to have an overview of the toxicity and the selectivity of these derivatives. Moreover, the synthetic protocols, as well as the structural particularities of the novel organotin(IV) species, are also discussed. Hydrolysis of [2‐{(CH2O)2CR}C6H4]2SnPh2[R = H (1), Me (2)] withp‐toluenesulfonic acid as a proton source afford the expected derivatives [2‐(O═CR)C6H4]2SnPh2[R = H (3), Me (4)]. When hydrochloric acid is used in these reactions, a chlorine‐phenyl exchange took place in addition to the removal of the acetal fragments and the following products [2‐(O═CR)C6H4]2SnPhCl [R = H (5), Me (6)] were isolated. Treatment of5with 2 equiv of 3‐aminomethylpyridine in neat affords the imino(aryl)tin compound 2‐(3′‐PyCH2N═CH)C6H4]2SnPhCl (7). The reaction between6and potassium nicotinate allows the isolation of the organotin(IV) carboxylate [2‐{O═C (CH3)}C6H4]2SnPh[O(O)CC5H4N‐3] (8). Compounds1–8were characterized by multinuclear NMR spectroscopy in solution, mass spectrometry, and IR spectroscopy, and their molecular structures were confirmed by X‐ray diffraction analysis. Compounds1–8were investigated for their antitumoral effects onHomo sapienslung cancer cell lines (H522 and SK‐MES‐1) and on a normal bronchial epithelial cell line, BEAS‐2B.

SIX1 Predicts Poor Prognosis and Facilitates the Progression of Non-small Lung Cancer via Activating the Notch Signaling Pathway.

Background: Many transcription factors involved in embryonic development and reactivated in tumors are considered potential prognostic biomarkers and novel therapeutic targets in various cancers. Sine oculis homeobox homolog 1 (SIX1), a developmentally restricted transcriptional regulator, plays a critical role during tumor initiation and development. However, the prognostic value and biological function of SIX1 in non-small cell lung cancer (NSCLC) remain unclear.Methods: Bioinformatic analyses were conducted to investigate the expression of SIX1 in cancer and adjacent normal tissues of NSCLC and further explore the correlations between SIX1 expression and clinical outcomes. Western blotting and RT-PCR analysis were performed to detect of SIX1 expression level in NSCLC cell lines and normal bronchial epithelial cell. EdU, CCK-8, clonal formation assay, wound healing and transwell assay were performed to explore the effects of gain- or loss-of-function of SIX1 on cellular proliferation, migration and invasion in vitro. Gene set enrichment analysis (GSEA) was used to identify the potential signaling pathways involved in SIX1 mediated biological function and the correlation was confirmed by western blotting and RT-PCR analysis. In vivo experiment was conducted to further validate the tumor-promoting effects of SIX1.Results: Bioinformatic analysis indicated that SIX1 was markedly upregulated in NSCLC tissues of and positively correlated with poor prognosis of patients with NSCLC. Ectopic expression of SIX1 facilitated proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells. On the contrary, knocking down SIX1 exhibited the opposite effects. Mechanistic studies suggested that SIX1 activated the Notch pathway to promote the malignant biological behaviors of NSCLC, which could be reversed by inhibiting the Notch signaling with γ-secretase inhibitor.Conclusions: SIX1 could facilitate multiple malignant biological behaviors by activating the Notch signaling pathway and function as a promising prognostic biomarker.

Correlation and clinical significance of methyltransferases SETDB1 and SPG20 methylation in lung adenocarcinoma

Objective: The incidence of primary lung adenocarcinoma is increasing year by year in worldwide, and it has surpassed squamous cell carcinoma to become the most common pathological type of lung cancer. Advances in medical technology have made the overall survival rate of lung cancer patients improve, but it is still very low. In the study, we investigated the methyltransferase SETDB1 expression and the methylation level of SPG20, and analyzed the correlation and its clinical significance in the pathogenesis of lung adenocarcinoma. Methods: 60 lung adenocarcinoma and normal lung tissues were selected.The expression of SPG20 and SETDB1 mRNA was examined by RT-qPCR; Protein expression of SPG20 and SETDB1 was determined by immunohistochemistry(SP) and immune protein imprinting(Western blot);Methylation level of SPG20 gene was determined by pyrosequencing. Correlation between SETDB1 and SPG20 methylation levels and both and clinical case characteristics was analysed by statistics. Western blot detection of SETDB1 and SPG20 protein expression in lung adenocarcinoma cells and normal bronchial mucosal epithelial cells.The small interfering RNA of SETDB1 was transfected into lung adenocarcinoma A549 cells by liposome-mediated method, the mRNA and protein expression levels of SETDB1 were detected by RT-qPCR and Wesrern blot, and the methylation level of SPG20 gene was detected by pyrosequencing. Results:Relative expression of SPG20 mRNA was significantly lower in lung adenocarcinoma tissue than in normal lung tissue, while relative SETDB1 mRNA expression was higher in cancer tissue than in normal lung tissue. SETDB1 protein expression was significantly higher in lung adenocarcinoma tissue than in normal lung tissue, while SPG20 protein showed lower expression in cancer tissue than in normal lung tissue. The methylation rate of the SPG20 gene was significantly higher in cancerous tissues than in normal lung tissue, the difference was statistically significant. SPG20 methylation in cancer tissues showed a correlation with SETDB1 and showed a positive correlation; SPG20 methylation and SETDB1 are closely related with TNM stage, tissue differentiation, and lymph node metastasis in lung adenocarcinoma. The vitro experiments showed that SETDB1 was highly expressed, while SPG20 was lowIn lung adenocarcinoma cells, SETDB1 was low and SPG20 was high in normal bronchial epithelial cells. RNA interference with SETDB1 could significantly reduce the mRNA and protein expression of SETDB1 in A549 cells, the methylation rate of SPG20 gene was significantly decreased, and the protein expression was increased. Conclusion:There is a significant correlation between methyltransferases SETDB1 and SPG20 methylation in lung adenocarcinoma, and high SETDB1 expression may be the upstream molecule of SPG20 methylation, working together in promoting the occurrence and development of lung adenocarcinoma.

Silybin has therapeutic efficacy against non-small cell lung cancer through targeting of Skp2

Silybin (SB), a natural flavonoid isolated from Silybum marianum, has been used to treat hepatic fibrosis in clinical settings and as a dietary supplement, because of its hepatoprotective potential. Numerous studies have shown that SB also exerts promising anticancer effects; however, the anticancer targets of SB and the underlying mechanism were unclear. Herein, we found that SB significantly inhibited the proliferation of non-small cell lung cancer without causing cytotoxicity toward normal Beas-2B bronchial epithelial cells. Mechanistically, SB binds the F-box protein Skp2 and disrupts Skp1-Skp2 interaction, thereby decreasing Skp2 protein levels, inducing accumulation of Skp2 substrates, and leading to G1-phase cell-cycle arrest and the suppression of cell migration. In lung orthotopic xenografts, SB also significantly decreased Skp2 expression and increased p27/Kip1 protein levels. SB administration inhibited tumor growth and metastasis in lung tissue, thus prolonging survival time in mice without causing obvious toxicity. Thus, SB is a potential Skp2-targeting agent that warrants further clinical investigation.

Diarylheptanoid glycosides from Zingiber officinale peel and their anti-apoptotic activity

Four new diarylheptanoid glycosides (1–4), (1S,3R,5S)-2-(4-hydroxy-3- methoxyphenyl)-6-[2-(4-hydroxyphenyl)ethyl]-tetrahydropyran-4-ol-4’-O-β-D-glucopyranoside (1), (1S,3R,5S)-2-(4,5-dihydroxy-3-methoxyphenyl)-6-[2-(4-hydroxyphenyl) ethyl]-tetrahydropyran-4-ol-4’-O-β-D-glucopyranoside (2), (1S,3R,5S)-2-(4-hydroxy- 3,5-dimethoxyphenyl)-6-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-tetrahydropyran-4-ol-4’-O-β-D-glucopyranoside (3), and (1R,3R,5R)-2-(4-hydroxy-3,5-dimethoxyphenyl)- 6-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-tetrahydropyran-4-ol-3-O-β-D-glucopyranoside (4) were isolated from the 50% ethanol extract of Zingiber officinale peel. The structures of the isolated compounds were determined by HR-ESI-MS and extensive spectroscopic techniques (UV, IR, 1D-NMR, and 2D-NMR). Compounds 1–4 significantly increased the survival rate of human normal lung bronchial epithelial cells (BEAS-2B) induced by lipopolysaccharide (LPS) at the concentration of 10 μM.

Fibulin 2 Is Hypermethylated and Suppresses Tumor Cell Proliferation through Inhibition of Cell Adhesion and Extracellular Matrix Genes in Non-Small Cell Lung Cancer.

Fibulins (FBLNs), interacting with cell adhesion receptors and extracellular matrix (ECM) components, play multiple roles in ECM structures and tissue functions. Abnormal expression of FBLN2, one of the fibulin family members, contributes to tumor initiation and development. However, the function of FBLN2 in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In this study, we found that FBLN2 was downregulated in 9 out of 11 lung cancer cell lines compared to normal bronchial epithelial cells, which was associated with DNA hypermethylation. Primary lung squamous cell carcinoma expressed significantly more FBLN2 protein compared to adenocarcinoma (p = 0.047). Ectopic expression of FBLN2 led to decreased cell proliferation, migration and invasion, accompanied by inactivated MAPK/ERK and AKT/mTOR pathways, while FBLN2 siRNA knockdown resulted in an opposite biological behaviour in NSCLC cells. Additionally, overexpression of FBLN2 led to dysregulation of cell adhesion molecules, ECM markers and a panel of lysate/exosome-derived-microRNAs, which are involved in cell adhesion and ECM remodelling. Taken together, our data indicate that FBLN2 is methylated and exerts a tumor suppressor function through modulation of MAPK/ERK and AKT pathways and regulation of cell adhesion and ECM genes. Moreover, FBLN2 might be a potential biomarker for the sub-classification of NSCLC.

Ovarian carcinoma immunoreactive antigen domain 2 controls mitochondrial apoptosis in lung adenocarcinoma.

Ovarian carcinoma immunoreactive antigen domain 2 (OCIAD2) has been reported to show significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, and its abnormal expression is associated with poorer prognosis of the patients. However, the cellular function of OCIAD2 in this tumor remains poorly understood. In the present study, we first validated that OCIAD2 showed higher expression in human lung adenocarcinoma tissues or cell lines than in normal lung tissue or immortalized normal bronchial epithelial cells. OCIAD2 was localized predominantly at the mitochondrial membrane in lung adenocarcinoma cells. Interestingly, suppression of OCIAD2 led to loss of mitochondrial structure and a reduction in the number of mitochondria. Moreover, OCIAD2 suppression led to downregulation of cellular growth, proliferation, migration, and invasion, and upregulation of mitochondria‐related apoptosis. We also showed that OCIAD2 suppression induced a decrease in mitochondrial membrane potential and release of cytochrome c. Transcriptional profiling using RNA sequencing revealed a total of 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827, and PC9). Pathway enrichment analysis of those genes demonstrated significant enrichment in apoptotic signaling or endoplasmic reticulum (ER) stress pathways. Our data suggest that OCIAD2 inhibits the mitochondria‐initiated apoptosis and thus promotes the survival of lung cancer cells. Therefore, OCIAD2 may be an effective target for treatment of lung adenocarcinoma.

Modulatory role of PACAP and VIP on HIFs expression in lung adenocarcinoma

Lung adenocarcinoma is the most frequent form of non-small cell lung cancer. Inside the tumor mass, uncontrolled cell proliferation generates hypoxic areas leading to activation of hypoxia-inducible factors (HIFs) responsible for neovascularization and tumor metastasis. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two neuropeptides widely distributed in respiratory organs. Previous studies have demonstrated that these peptides interfere with hypoxic pathways in various diseases, including tumors. However, their modulatory role in HIFs expression in lung adenocarcinomas has not yet been evaluated. In the present paper, we detected the expression profile of PACAP, VIP and related receptors in healthy and adenocarcinoma human lung tissue. To characterize peptides’ modulatory effects on HIFs expression, we also exposed A549 lung adenocarcinoma cells and human normal bronchial epithelial BEAS-2B cells to microenvironmental hypoxia by treating them with deferoxamine (DFX). The results showed that PACAP and VIP significantly reduced HIF-1α and HIF-2α levels in both cell lines following hypoxic stress. The HIF-3α expression profile was related to cellular phenotype as it was lower in BEAS-2B and higher in A549 cells under low oxygen tension. In lung adenocarcinoma cells, peptide treatment restored HIF-3 α expression to control levels. These results suggest that endogenous PACAP and VIP exert controversial roles in cellular hypoxic microenvironments depending on the pathophysiological conditions of the lung tissue.

TU4. COMPREHENSIVE ANALYSIS OF OMICS DATA IDENTIFIED ADHD RELEVANT GENE NETWORKS

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3′UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation.

Gigantol inhibits cell proliferation and induces apoptosis by regulating DEK in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a common type of cancer, with a mortality of >80% worldwide. Gigantol is a bibenzyl compound that displays anticancer activity. The aim of the present study was to determine the biological activity of gigantol in NSCLC and to elucidate the underlying molecular mechanism of its action. The expression of DEK proto-oncogene (DEK) was measured in NSCLC tissues and cell lines by reverse transcription-quantitative PCR (RT-qPCR). The results suggested that DEK levels were significantly increased in NSCLC tissues and cell lines compared with adjacent non-tumor tissues and BEAS-2B normal bronchial epithelial cells, respectively. A549 cells were exposed to a series of gigantol concentrations (0, 25, 50 and 100 µM) and transfected with DEK small interfering RNA. The results of cell viability measured by MTT assay indicated that gigantol significantly decreased cell viability. Additionally, cell proliferation was assessed by CCK-8 and apoptosis was measured by flow cytometry. In comparison with the control group, gigantol treatment inhibited cell proliferation and promoted apoptosis, whereas DEK knockdown increased gigantol-induced suppression of proliferation and acceleration of apoptosis. Additionally, DEK overexpression reversed gigantol-induced effects on proliferation and apoptosis. Moreover, compared with the control group, gigantol treatment decreased Ki-67 and Bcl-2 expression levels, increased Bax expression levels and inactivated the Wnt/β-catenin signaling pathway, as assessed by RT-qPCR and/or western blot. DEK knockdown further increased gigantol-induced effects, but DEK overexpression reversed gigantol-induced effects. To conclude, the results of the present study suggested that gigantol inhibited cell proliferation and induced apoptosis by decreasing Ki-67 and Bcl-2 expression, increasing Bax expression and activating the Wnt/β-catenin signaling pathway by regulating DEK. The present study indicated the therapeutic potential of gigantol in patients with NSCLC. In addition, DEK may serve as a novel therapeutic target to enhance the effects of gigantol treatment.

Abstract 2350: xCT overexpression induces metabolic reprogramming, epigenetic alterations, and DNA damage in precancerous airway epithelial cells

Abstract Metabolic reprogramming and genomic instability are two hallmarks of cancer known to drive tumorigenesis. While each has been investigated individually, there is a growing interest in their interplay during early cancer development. High expression of the cystine/glutamate antiporter SLC7A11 (xCT) is frequently observed in lung cancer. Our laboratory previously demonstrated that overexpressing xCT in normal bronchial airway epithelial cells induced metabolic reprogramming and caused cells to display precancerous properties, such as increased proliferation and colony formation in soft agar. In this study, we further investigated the effects of xCT overexpression in the airway epithelium with the goal of elucidating the role of xCT in influencing cancer-driving hallmarks and the interplay of these hallmarks during early tumorigenesis. We hypothesized that xCT overexpression-induced metabolic reprogramming leads to epigenetic changes through the perturbation of epigenetic-related metabolite levels. Furthermore, we posited that this altered epigenome changes the expression levels of key genes involved in genome stability maintenance, ultimately inducing genomic instability. Utilizing a LC-MS/MS-based metabolomic analysis, we observed significantly altered levels of 224 different metabolites in our xCT overexpressing cells, including metabolites involved in epigenetic processes such as S-adenosylhomocysteine and acetyl-CoA. We found that xCT overexpression alters several global histone modifications, increasing H3K9me3 and H3K9ac by 3.2 (P = 0.02) and 3.9-fold (P = 0.02) respectively. We also showed that xCT overexpression increased global DNA methylation by 56% (P = 0.04). Additionally, RNA-seq revealed significant alterations (logFC>2 or <0.5, pAdj<0.05) in the expression of over 100 genes, including a significant decrease in the expression of certain tumor suppressor genes such as TRIM29, and a significant increase in the expression of certain proto-oncogenes, such as CCND1. Finally, we discovered that xCT overexpression induces DNA damage, increasing the percentage of damaged DNA in comet tails in a comet assay by 15.1% (P = 0.002). Taken together, xCT overexpression in normal bronchial airway epithelial cells induces metabolic reprogramming, alters the epigenetic landscape and gene expression, and causes higher levels of DNA damage. Future experiments elucidating causal links between these processes, including ChIP-seq and WGBS are underway. This study provides a promising foundation for the connections between xCT-induced metabolic reprogramming, epigenetic alterations, and genomic instability, an imperative step in elucidating an effective, potentially multifaceted approach to preventing lung cancer development. Citation Format: Dalton Hill, Jamshedur Rahman, Christien Kluwe, Jamey Young, Emily Hodges, Pierre P. Massion. xCT overexpression induces metabolic reprogramming, epigenetic alterations, and DNA damage in precancerous airway epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2350.

Abstract 2434: Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis

Abstract Introduction: The Hippo pathway effector YAP contributes to carcinogenesis by directing stem cell activity, cell proliferation and cell survival signals. In the lung, aberrant nuclear YAP activity promotes bronchial epithelial growth, which in animal models can drive pathology that resembles lung premalignant lesions (PMLs). However, the molecular mechanisms by which YAP contributes to aberrant lung epithelial pathology are poorly understood. In this project, we investigated the DNA binding pattern and gene expression program regulated by YAP and associated TEAD and TP63 transcriptional factors in primary normal human bronchial epithelial cells (HBECs) and lung cancer cell lines. Methods: RNA-seq experiments were conducted using HBEC, H2170 and SW900 cells transfected with siRNA targeting YAP, TEADs or TP63, and expression profiles were compared to corresponded controls to derive gene signatures. In parallel, ChIP-seq experiments were carried out using antibodies specific for the respective proteins to identify genome binding peaks. Direct target genes were inferred with Binding and Expression Target Analysis (BETA) using gene signature and peak profile of each factor in each cell line respectively. The metagene score for target genes in longitudinally collected endobronchial biopsies from patients with PMLs was calculated using GSVA and the association with PML phenotypes was tested with a mixed-effects model adjusting for batch and patient as random effect (GSE109743). Results: Chromatin binding analyses revealed that YAP co-occupies genomic regions with TEAD and TP63 in normal bronchial epithelial cells aligning with H3K27ac chromatin modifications. Integrating genomic binding and gene expression profiling, we identified large overlaps in target genes directly regulated by YAP, TEAD and TP63 in HBECs, demonstrating cooperation between these factors in the control of bronchial epithelial transcription. YAP-TEAD-TP63 induced targets in HBECs are enriched for genes involved in cell proliferation and extracellular matrix alteration, while repressed targets are enriched for genes involved in cell fate specification. Notably, cooperatively regulated genes are strongly associated with gene expression profile identified in progressive PML, and in particular with genes linked to early immune responses that are observed with PML pathology progression. Similar analyses of YAP, TEAD and TP63 in lung cancer cells revealed an altered transcriptional program for YAP with a divergence of YAP-TEAD away from TP63, suggesting contextual changes in YAP activity with cancer progression. Conclusion: We found that YAP, TP63 and TEADs co-regulate lung epithelial gene expression related to PML pathology progression. These findings provide insight into potential YAP function in the early stages of lung carcinogenesis and offer potential new avenues to develop lung cancer interception strategies. Citation Format: Boting Ning, Andrew Tilston-Lunel, Julia Hicks-Berthet, Sarah A. Mazzilli, Jennifer E. Beane, Xaralabos Varelas, Marc E. Lenburg. Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2434.

Downregulation of microRNA-3646 Through Direct Targeting of F-Box Protein 4 on Interleukin-17-Induced Lung Cancer Cell Migration and Invasion

IL-17 participates in the initiation and growth of malignant cancers, including lung cancer. The aberrant expression of miRNA is also related to tumor growth and metastasis. Studies have confirmed that high expression of miRNA-3646 can boost breast cancer cell invasion and migration, suggesting that miRNA-3646 is a tumor-promoting factor. However, the role of miRNA-3646 in the migration and invasion of IL-17-induced lung cancer cells is unclear. In this study, qRT-PCR was used to determine the level of miRNA-3646. We found that in lung cancer cells, miRNA-3646 levels exceeded those of normal bronchial epithelial 16HBE cells (P < 0.05). The level of miRNA-3646 in NCI-H1299 cells was higher than that in A549, NCI-H446, and SK-MES-1 cells (P < 0.05). After IL-17 treatment, the number of proliferating and migrating lung carcinoma NCI-H1299 cells increased, transport of vimentin increased, and transport of E-cadherin decreased (P < 0.05). After IL-17 treatment, the number of proliferating and migrating lung carcinoma NCI-H1299 cells transfected with miRNA-3646 inhibitor decreased, transport of vimentin decreased, and transport of E-cadherin increased (P < 0.05). FBXO4 siRNA reversed the inhibition of miRNA-3646 on the proliferation and migration of IL-17-induced lung carcinoma NCI-H1299 cells and the transport of E-cadherin and vimentin. Thus, downregulation of miRNA-3646 inhibited IL-17-induced lung carcinoma cell migration and proliferation by directly targeting FBXO4.

PRMT5 Selective Inhibitor Enhances Therapeutic Efficacy of Cisplatin in Lung Cancer Cells.

As a therapeutic approach, epigenetic modifiers have the potential to enhance the efficacy of chemotherapeutic agents. Protein arginine methyltransferase 5 (PRMT5), highly expressed in lung adenocarcinoma, was identified to be involved in tumorigenesis. In the current study, we examined the potential antineoplastic activity of PRMT5 inhibitor, arginine methyltransferase inhibitor 1 (AMI-1), and cisplatin on lung adenocarcinoma. Bioinformatic analyses identified apoptosis, DNA damage, and cell cycle progression as the main PRMT5-associated functional pathways, and survival analysis linked the increased PRMT5 gene expression to worse overall survival in lung adenocarcinoma. Combined AMI-1 and cisplatin treatment significantly reduced cell viability and induced apoptosis. Cell cycle arrest in A549 and DMS 53 cells was evident after AMI-1, and was reinforced after combination treatment. Western blot analysis showed a reduction in demethylation histone 4, a PRMT5- downstream target, after treatment with AMI-1 alone or in combination with cisplatin. While the combination approach tackled lung cancer cell survival, it exhibited cytoprotective abilities on HBEpC (normal epithelial cells). The survival of normal bronchial epithelial cells was not affected by using AMI-1. This study highlights evidence of novel selective antitumor activity of AMI-1 in combination with cisplatin in lung adenocarcinoma cells.

Role of Cigarette Smoke on Angiotensin-Converting Enzyme-2 Protein Membrane Expression in Bronchial Epithelial Cells Using an Air-Liquid Interface Model.

Prevalence studies of current smoking, among hospitalized COVID-19 patients, demonstrated an unexpectedly low prevalence among patients with COVID-19. The aim of the present study was to evaluate the effect of smoke from cigarettes on ACE-2 in bronchial epithelial cells. Normal bronchial epithelial cells (H292) were exposed to smoke by an air-liquid-interface (ALI) system and ACE-2 membrane protein expression was evaluated after 24 h from exposure. Our transcriptomics data analysis showed a significant selective reduction of membrane ACE-2 expression (about 25%) following smoking exposure. Interestingly, we observed a positive direct correlation between ACE-2 reduction and nicotine delivery. Furthermore, by stratifying GSE52237 as a function of ACE-2 gene expression levels, we highlighted 1,012 genes related to ACE-2 in smokers and 855 in non-smokers. Furthermore, we showed that 161 genes involved in the endocytosis process were highlighted using the online pathway tool KEGG. Finally, 11 genes were in common between the ACE-2 pathway in smokers and the genes regulated during endocytosis, while 12 genes with non-smokers. Interestingly, six in non-smokers and four genes in smokers were closely involved during the viral internalization process. Our data may offer a pharmaceutical role of nicotine as potential treatment option in COVID-19.

Construction of a Prognostic Immune-Related LncRNA Risk Model for Lung Adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) originates mainly from the mucous epithelium and glandular epithelium of the bronchi. It is the most common pathologic subtype of non-small cell lung cancer (NSCLC). At present, there is still a lack of clear criteria to predict the efficacy of immunotherapy. The 5-year survival rate for LUAD patients remains low.MethodsAll data were downloaded from The Cancer Genome Atlas (TCGA) database. We used Gene Set Enrichment Analysis (GSEA) database to obtain immune-related mRNAs. Immune-related lncRNAs were acquired by using the correlation test of the immune-related genes with R version 3.6.3 (Pearson correlation coefficient cor = 0.5, P < 0.05). The TCGA-LUAD dataset was divided into the testing set and the training set randomly. Based on the training set to perform univariate and multivariate Cox regression analyses, we screened prognostic immune-related lncRNAs and given a risk score to each sample. Samples were divided into the high-risk group and the low-risk group according to the median risk score. By the combination of Kaplan–Meier (KM) survival curve, the receiver operating characteristic (ROC) (AUC) curve, the independent risk factor analysis, and the clinical data of the samples, we assessed the accuracy of the risk model. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the differentially expressed mRNAs between the high-risk group and the low-risk group. The differentially expressed genes related to immune response between two risk groups were analyzed to evaluate the role of the model in predicting the efficacy and effects of immunotherapy. In order to explain the internal mechanism of the risk model in predicting the efficacy of immunotherapy, we analyzed the differentially expressed genes related to epithelial-mesenchymal transition (EMT) between two risk groups. We extracted RNA from normal bronchial epithelial cell and LUAD cells and verified the expression level of lncRNAs in the risk model by a quantitative real-time polymerase chain reaction (qRT-PCR) test. We compared our risk model with other published prognostic signatures with data from an independent cohort. We transfected LUAD cell with siRNA-LINC0253. Western blot analysis was performed to observed change of EMT-related marker in protein level.ResultsThrough univariate Cox regression analysis, 24 immune-related lncRNAs were found to be strongly associated with the survival of the TCGA-LUAD dataset. Utilizing multivariate Cox regression analysis, 10 lncRNAs were selected to establish the risk model. The K-M survival curves and the ROC (AUC) curves proved that the risk model has a fine predictive effect. The GO enrichment analysis indicated that the effect of the differentially expressed genes between high-risk and low-risk groups is mainly involved in immune response and intercellular interaction. The KEGG enrichment analysis indicated that the differentially expressed genes between high-risk and low-risk groups are mainly involved in endocytosis and the MAPK signaling pathway. The expression of genes related to the efficacy of immunotherapy was significantly different between the two groups. A qRT-PCR test verified the expression level of lncRNAs in LUAD cells in the risk model. The AUC of ROC of 5 years in the independent validation dataset showed that this model had superior accuracy. Western blot analysis verified the change of EMT-related marker in protein level.ConclusionThe immune lncRNA risk model established by us could better predict the prognosis of patients with LUAD.