Normal Human Tracheobronchial Epithelial Cells Research Articles

Abstract P078: Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer

Abstract Oncogenic KRAS mutation is common in non-small cell lung cancer (NSCLC) and portends poor outcome. Direct KRASG12C inhibitors have clinical activity; however, intrinsic and acquired resistance to these drugs limits their utility. Aurora A Kinase (AURKA), a mitotic cell cycle regulator, has been considered as a key druggable KRAS effector and mediates adaptive resistance to direct KRASG12C inhibitors. We found that AURKA expression correlated with poor outcome of lung cancer patients in caBIG, GEO and TCGA, and its expression was greater in KRAS-mutated NSCLC cells resistant to KRASG12C inhibitor sotorasib (AMG510) compared to normal human tracheobronchial epithelial (NHTBE), lung fibroblast, and KRAS wild-type cells. We have previously demonstrated synergistic antitumor effects for combination AURKA and WEE1 inhibition. Here, we explored a novel combination of AURKA and KRASG12C inhibition in KRAS(G12C)-mutated NSCLC cells intrinsically resistant to the KRASG12C inhibitor, along with a combination of AURKA and WEE1 inhibition in mutant KRAS(G12C) lung cancer cells with acquired resistance to the inhibitor. To overcome resistance to the KRASG12C inhibitor sotorasib, we tested combination treatment with VIC-1911, a newly synthesized selective AURKA inhibitor, with sotorasib in sotorasib-resistant human lung cancer cells. Cooperative screening, Loewe plotting, and clonogenic survival assays were performed to determine synergistic antitumor effects. In addition, we established KRAS(G12C)-mutated human lung cancer cell lines with acquired resistance to sotorasib through the means of escalated incremental dosing or sorting non-quiescent cells after drug exposure. Synergy was further confirmed with cell cycle distribution, phenotypic mitotic catastrophe on confocal microscopy, and induction of apoptosis. Interestingly, cells intrinsically resistant to sotorasib showed profound synergistic suppressive effect on cancer cell survival with addition of VIC-1911 compared to sotorasib-sensitive cells (Loewe synergy scores: NCI-H1792=16.03; HCC44=14.37; NCI-H358=1.48). Further, the combination of AURKA and WEE1 inhibition synergistically induced greater cell death in NCI-H358 lung carcinoma cells harboring acquired resistance to sotorasib, with dramatic induction of Bim. Moreover, the combination of AURKA and WEE1 inhibition resulted in a synergistic tumor control in KRAS/TP53-mutated lung cancer xenograft models. Our findings suggest AURKA activation leads to both intrinsic and acquired resistance to sotorasib in KRAS(G12C)-mutated NSCLC; therefore, addition of AURKA inhibition to sotorasib may be a promising therapeutic approach in NSCLC with intrinsic resistance to direct KRASG12C inhibitors, while the combination of AURKA and WEE1 inhibition merits exploration in acquired resistance to these agents. Citation Format: Jong Woo Lee, Sundong Kim, Cindy Yang, Barbara Burtness. Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P078.

Use of Magnetic Nanoparticles as In Situ Mucus Property Probe

Magnetic nanoparticles (MNPs) are unique in their abilities to penetrate and interact with a wide range of liquid media. Because of their magnetic properties, MNPs can be directed to any area of interest, and interact with core structures deep inside the medium which is normally inaccessible. In this report, we investigate the behavior of MNPs in a specific biological fluid, namely in a mucus layer of air–liquid interface cultured primary normal human tracheobronchial epithelial cells. Using Fokker–Planck algorithm simulations and observing the behavior of MNPs from prior experiments, we found MNPs that are initially less than 100 nm in size, to aggregate into sizes of ~50 μm and to deviate from the expected Fokker–Planck distribution due to the mucus structure. Based on our analysis, human tracheobronchial epithelial (NHTE) cell mucus viscosity ranges from 15 Pa·s to 150 Pa·s. The results not only confirm the possible use of MNPs as a means for medical drug delivery but also underline important consequences of MNP surface modifications.

Abstract 604: Combination of the Aurora A inhibitor TAS-119 and adavosertib results in synergistic c-Jun-mediated cell death in head and neck and lung cancers

Abstract Head and neck squamous cell carcinomas (HNSCC) account for more than 800,000 cases annually worldwide. Human papillomavirus (HPV)-negative HNSCC mainly harbor disruptive mutations in TP53 and/or CDKN2A tumor suppressor genes, which are associated with refractoriness to treatment as well as poor prognosis and survival. Synthetic lethal approaches hold promise for such patients. Cancers with TP53/CDKN2A mutations lose the G1/S checkpoint, and foster a dependence on the G2/M checkpoint as the primary mechanism to repair the resulting accumulation of genomic instability, thus creating a vulnerability that can potentially be exploited with synthetic lethal targeted therapy. We have previously demonstrated in HPV-negative HNSCC patients that elevation in expression of Aurora kinase A (AURKA), a crucial mitotic regulator, is correlated with worse outcome. Combined inhibition of AURKA and WEE1, a G2/M checkpoint kinase, resulted in a synergistic anti-tumor effect in HNSCC in vitro and in vivo. Upon these findings, we confirmed synergy in testing the more specific AURKA inhibitor, TAS-119 in HNSCC and lung cancers harboring TP53 mutations, and explored mechanisms of cell death. TAS119 and adavosertib combination treatment synergistically suppressed cell viability, clonogenic cell survival, anchorage-independent cell growth in soft agar and oncosphere formation relative to vehicle and single-agent treatment in HNSCC FaDu and CAL27 cells, and lung cancer NCI-H520 and NCI-H358 cells bearing TP53/CDKN2A mutations. Of note, this synergistic anti-tumor effect was not seen in normal human tracheobronchial epithelial cells, predicting a favorable therapeutic index. Furthermore, combination treatment accelerated an accumulation of mitotic catastrophe with mitotic arrest, and led to apoptotic cell death. Mechanistically, combination TAS-119 and adavosertib in HNSCC cells drastically augmented DNA damage and phosphorylation of c-Jun (Ser 63) as demonstrated by kinase array and western blotting, indicating an activation of c-Jun-mediated pro-apoptotic genes. In vivo, this combination demonstrated a synergistic suppressive effect on tumor growth in FaDu xenografts compared with either vehicle or single-agent treatment. Taken together, these results support the promise of further clinical evaluation of AURKA inhibitor in combination with WEE1 inhibitor as a novel and effective treatment for HNSCC and lung cancer patients with TP53/CDKN2A mutations. Citation Format: Jong Woo Lee, Cindy Yang, Barbara Burtness. Combination of the Aurora A inhibitor TAS-119 and adavosertib results in synergistic c-Jun-mediated cell death in head and neck and lung cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 604.

Sorrel Extract Reduces Oxidant Production in Airway Epithelial Cells Exposed to Swine Barn Dust Extract In Vitro.

Exposure to hog barn organic dust contributes to occupational lung diseases, which are mediated by inflammatory and oxidative stress pathways. Isoprostanes—a family of eicosanoids produced by oxidation of phospholipids by oxygen radicals—are biomarkers of pulmonary oxidative stress. Importantly, 8-isoprostane has been implicated as a key biomarker and mediator of oxidative stress because it is a potent pulmonary vasoconstrictor. Antioxidants found in fruits and vegetables hold promise for preventing or reducing effects of oxidative stress-related diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Here, we investigated 8-isoP and oxidant production by organic dust-exposed airway epithelial cells and the inhibitory effects of an extract from calyces of the sorrel plant, Hibiscus sabdariffa, on oxidant-producing pathways. Confluent cultures of normal human tracheobronchial epithelial cells were pretreated or not with 1% sorrel extract prior to 5% dust extract (DE) exposure. Following DE treatments, live cells, cell-free supernatants, or cell extracts were evaluated for the presence of 8-isoprostane, superoxide, hydrogen peroxide, nitric oxide, hydroxyl radical, peroxynitrite, and catalase activity to evaluate sorrel's inhibitory effect on oxidative stress. The well-known radical scavenging antioxidant, N-acetyl cysteine (NAC), was used for comparisons with sorrel. DE exposure augmented the production of all radicals measured including 8-isoprostane (p value < 0.001), which could be inhibited by NAC or sorrel. Among reactive oxygen and nitrogen species generated in response to DE exposure, sorrel had no effect on H2O2 production and NAC had no significant effect on NO· production. The observations reported here suggest a possible role for sorrel in preventing 8-isoprostane and oxidant-mediated stress responses in bronchial epithelial cells exposed to hog barn dust. These findings suggest a potential role for oxidative stress pathways in mediating occupational lung diseases and antioxidants within sorrel and NAC in reducing dust-mediated oxidative stress within the airways of exposed workers.

MUC18 regulates IL-13-mediated airway inflammatory response

To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice. Primary normal human tracheobronchial epithelial (HTBE) cells, wild-type (WT) and Muc18 knockout (KO) mice, and mouse tracheal epithelial cells (mTECs) were utilized. Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10ng/mL) for 24h. Mice were intranasally instilled with 500ng of IL-13 for 3days. mTECs were treated with IL-13 (10ng/mL) for 3days. PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials. MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183±44 vs. 380±59pg/mL, p<0.05), while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95±3 vs. 58±3pg/mL, p<0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25±3 vs. 35±3%, p=0.0565). These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g., IL-13) milieu.

Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

Abstract C172: Concomitant disruption of WEE1 and AurKA synergistically enhances anti-cancer effects in p53 mutated head and neck squamous cell carcinoma

Abstract Head and neck cancer is a devastating disease which annually affects over half a million patients worldwide. Squamous cell carcinoma of the head and neck (SCCHN), which accounts for approximately 90% of all head and neck cancers, often harbors a disruptive mutation of the TP53 gene. Cells harboring TP53 mutations evade cisplatin-induced cellular senescence with cell cycle arrest in the G2 phase, allowing for DNA repair. Wee1 and Aurora Kinase A (AurKA) are crucial regulators of the mitotic cell cycle including G2/M phases and are significantly activated in SCCHN harboring TP53 mutations. Therefore, we hypothesized that a combination therapy aimed at these synthetic lethal targets, Wee1 and AurKA, would enhance anti-cancer effects and cisplatin sensitivity due to abrogation of G2 arrest concomitant with defects in tumor cell mitotic machinery, which is indeed caused by cisplatin. We tested this hypothesis using assays of cell viability, colony formation, anchorage independent cell growth, and finally a three-dimensional oncosphere formation assay in vitro. The combination index (CI) was calculated according to the Chou-Talalay method (CI&amp;lt;0.8, synergistic; 0.8&amp;lt;CI&amp;lt;1.2, additive; CI&amp;gt;1.2, antagonistic). The cell viability assay indicated that the combination of the Wee1 inhibitor MK-1775 with the AurKA inhibitor MLN8237 exhibited striking synergistic inhibitory effects on the cell growth of SCCHN cell lines including FaDu (TP53 mutation, CI = 0.397004), Detroit562 (TP53 mutation, CI = 0.202677) and UNC-7 (TP53 WT, CI = 0.587106). Consistent with the cell viability data we obtained, we also confirmed this synergistic effect under anchorage dependent and independent cell growth conditions in FaDu and UNC-7 cell lines. Interestingly, this combination dramatically reduced the numbers of a stem cell-like oncosphere formation, showing a more effective combination in comparison to MK-1775 with cisplatin (inhibitory effect (% Vehicle): MK-1775+MLN8238 vs MK-1775+Cisplatin; FaDu = 74% vs 69%; UNC-7 = 43% vs 36%). Moreover, all spheroids exposed to the combination of MK-1775 with MLN8237 did not survive following further incubation under normal cell culture conditions. Induction of apoptosis, as shown gradually increased PARP cleavage at Asp214-Gly215 in FaDu and UNC-7 cells, was demonstrated. Furthermore, this synergistic synthetic lethal effect was not seen in normal human tracheobronchial epithelial (NHTBE) cells in a three-dimensional organotypic (air-liquid interface culture condition) culture system, predicting low toxicity. These results suggest that SCCHN cells, including high-risk TP53 mutations, are sensitive to the combination of MK-1775 and MLN8237 in vitro, thereby supporting the preclinical and further clinical evaluation of MK-1775 in combination with MLN8237 for the treatment of patients with TP53 inactivated HNSCC. Citation Format: Jong Woo Lee, Janaki Parameswaran, Ja Seok Koo, Barbara Burtness. Concomitant disruption of WEE1 and AurKA synergistically enhances anti-cancer effects in p53 mutated head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C172.

Abstract 2094: GSK-3 is a novel target of CREB and GSK-3-CREB signaling participates in cell survival in lung cancer

Abstract The transcription factor cyclic AMP-response element-binding protein (CREB) regulates diverse cellular processes, including cell differentiation, proliferation, survival, glucose metabolism, immune regulation, and synaptic plasticity associated with memory. Previously, we showed that CREB is critical on the regulation of mucous differentiation of normal human tracheobronchial epithelial (NHTBE) cells. In addition, the overexpression of CREB is associated with negative prognosis of patients with non-small cell lung cancer (NSCLC). Further studies showed that the knockdown of CREB suppresses the growth of lung cancer cells. It has been demonstrated that genes regulated by CREB have been reported to be involved in the suppression of apoptosis, induce cell proliferation, inflammation and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. In this study, we identified GSK-3 as one of the CREB target genes critical for the growth and survival of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3 and the direct binding of CREB on the promoter of GSK-3 was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3 expression in lung cancer. Inhibition of GSK-3 suppressed cell proliferation, colony formation and tumor growth. Although GSK-3 has been studied as a tumor suppressor in certain types of cancer, there is increasing evidence that GSK-3 plays an oncogenic role in various human cancers. Here, for the first time, we demonstrated that GSK-3 is regulated by CREB in lung cancer and is required for the survival of lung cancer cells. These findings implicate CREB-GSK-3 axis as a novel therapeutic target for lung cancer treatment. Citation Format: Sin-Aye Park, Jong Woo Lee, Roy S. Herbst, Jaseok Peter Koo. GSK-3 is a novel target of CREB and GSK-3-CREB signaling participates in cell survival in lung cancer. [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 2094. doi:10.1158/1538-7445.AM2015-2094

Magnetic Nanoparticle Delivery System for Mucus Layer Penetration

Excessive mucus production is a common and significant problem for several prominent human lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In these diseases, the mucus barrier poses a significant challenge to drug delivery, leading to increased healthcare cost and poor quality of life for patients. One method of overcoming structural and fluid resistance of mucus is by utilizing a targeted delivery method of magnetic nanoparticles (NPs) to diseased epithelial cells. However, previous experimental studies showed no mucus penetration using superparamagnetic iron oxide (Fe3O4) FeNPs. In our experiments we generated a field gradient that is an order of magnitude stronger than that in previously applied FeNP delivery systems. In our delivery system, FeNPs successfully penetrated the approximately 100 μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial cells indicating the potential of magnetic nanoparticles for targeted drug delivery to the airway mucosal surface.

Prevention of Bronchial Hyperplasia by EGFR Pathway Inhibitors in an Organotypic Culture Model

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. This study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured by using the 3-dimensional (3D) organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands, EGF, TGF-α, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MAP/ERK kinase (MEK) inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion by using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we showed that bronchial hyperplasia can be modeled in vitro by using primary NHTBE cells maintained in a 3D organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role.

CREB Mediates Prostaglandin F2α-Induced MUC5AC Overexpression

Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are PGE(2) and PGF(2alpha). PGE(2)-induced mucin production has been well studied, but the effect of PGF(2alpha) on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF(2alpha) on MUC5AC production, we investigated the signal transduction of PGF(2alpha) associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF(2alpha) induces MUC5AC overproduction via a signaling cascade involving protein kinase C, ERK, p90 ribosomal S6 protein kinase, and CREB. The regulation of PGF(2alpha)-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF(2alpha) receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE(2) and other inflammatory mediators, our findings have important clinical implications for the management of airway mucus hypersecretion.

Cyclic AMP Response Element-Binding Protein Overexpression: A Feature Associated with Negative Prognosis in Never Smokers with Non–Small Cell Lung Cancer

Lung cancer is the leading cause of cancer deaths worldwide. Recent advances in targeted therapies hold promise for the development of new treatments for certain subsets of cancer patients by targeting specific signaling molecule. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of growth of several types of cancers and our recent findings of its importance in normal differentiation of bronchial epithelial cells, we hypothesized that CREB plays an important pathobiologic role in lung carcinogenesis. We conducted this initial study to determine whether the expression and activation status of CREB are altered in non-small cell lung cancer (NSCLC) and of any prognostic importance in NSCLC patients. We found that the expression levels of mRNA and protein of CREB and phosphorylated CREB (p-CREB) were significantly higher in most of the NSCLC cell lines and tumor specimens than in the normal human tracheobronchial epithelial cells and adjacent normal lung tissue, respectively. Analysis of CREB mRNA expression and the CREB gene copy number showed that CREB overexpression occurred mainly at the transcriptional level. Immunohistochemical analysis of tissue microarray slides containing sections of NSCLC specimens obtained from 310 patients showed that a decreased survival duration was significantly associated with overexpression of CREB or p-CREB in never smokers but not in current or former smokers with NSCLC. These are the first reported results illustrating the potential of CREB as a molecular target for the prevention and treatment of NSCLC, especially in never smokers.

Sp1 up-regulates cAMP-response-element-binding protein expression during retinoic acid-induced mucous differentiation of normal human bronchial epithelial cells

CREB [CRE (cAMP-response element)-binding protein] is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA (retinoic acid) rapidly activates CREB without using RARs (RA receptors) or RXRs (retinoid X receptors) in NHTBE cells (normal human tracheobronchial epithelial cells). However, little is known about the role of RA in the physiological regulation of CREB expression in the early mucous differentiation of NHTBE cells. In the present study, we report that RA up-regulates CREB gene expression and that, using 5'-serial deletion promoter analysis and mutagenesis analyses, two Sp1 (specificity protein 1)-binding sites located at nt -217 and -150, which flank the transcription initiation site, are essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nt -119 and -98 contributed to basal promoter activity. Interestingly, RA also up-regulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using siRNA (small interfering RNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA up-regulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in up-regulating human CREB gene expression. This result implies that co-operation of these two transcription factors plays a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells.

Regulation of Mucin Gene Expression by CREB via a Nonclassical Retinoic Acid Signaling Pathway

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Proteomics-Based Identification of Proteins Secreted in Apical Surface Fluid of Squamous Metaplastic Human Tracheobronchial Epithelial Cells Cultured by Three-Dimensional Organotypic Air-Liquid Interface Method

Squamous cell carcinoma in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared with normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified by liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins [SCC antigen 1 (SCCA1), SCC antigen 2 (SCCA2), S100A8, S100A9, Annexin I, and Annexin II] in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens.

Modulation of MUC7 Mucin Expression by Exogenous Factors in Airway Cells In Vitro and In Vivo

The human MUC7 gene encodes a low-molecular-mass mucin that participates in the maintenance of healthy epithelium in the oral cavity, and possibly in respiratory tracts, by promoting the clearance of various bacteria. We examined whether MUC7 gene is expressed in primary normal human tracheobronchial epithelial cells and whether the expression is modulated by exogenous factors. By assessing MUC7 transcripts, we found that the MUC7 gene was induced by culturing the normal human tracheobronchial epithelial cells at the air-liquid interface, in which the cells were well differentiated. When the cells were treated with a panel of cytokines (IL-1beta, IL-4, IL-13, and TNF-alpha), epidermal growth factor, or a bacterial product (Pseudomonas aeruginosa lipopolysaccharide [LPS]), MUC7 transcripts and glycoprotein products were increased 1.7- to 3.2-fold. The effect of LPS on MUC7 gene expression was also studied in the airway tissues of MUC7 gene transgenic mice. In the in vitro cultured trachea and lung explants, the LPS-treated tissues showed over 2-fold increased levels of MUC7 mRNA compared with the untreated specimens. These results were confirmed by in vivo studies using the lungs and tracheas harvested from the transgenic mice irritated by LPS through the tracheal instillation. By immunohistochemistry, MUC7 glycoprotein was localized in tracheal submucosa within the serous cells. Upon LPS stimulation, the overexpressed MUC7 remains confined to the serous glands. In the lungs, MUC7 seems to be expressed within the respiratory epithelium at the level of the bronchioles. Upon stimulation with LPS, it seems to be overexpressed within the same cells and within the stromal tissue.

Endogenous S-Nitrosoglutathione Modifies 5-Lipoxygenase Expression in Airway Epithelial Cells

S-Nitrosoglutathione (GSNO) is an endogenous bronchodilator with several beneficial pulmonary effects. Levels are decreased in the asthmatic airway, and GSNO inhalation has been proposed as an asthma therapy. 5-lipoxygenase (5-LO) is the rate-limiting enzyme in the synthetic pathway for cysteinyl leukotrienes (CysLTs), bronchoconstricting agents that are overproduced in asthma. Here, we have studied the effect of GSNO on the expression of 5-LO in human airway A549 cell lines and in primary normal human tracheobronchial epithelial (NHBE) cells in vitro. GSNO at concentrations of 0.5-1 microM caused a 3- to 6-fold increase in 5-LO expression. However, GSNO at>5 microM significantly inhibited both 5-LO expression and LT production. We also found that airway epithelial cells had gamma-glutamyl transpeptidase (gamma-GT) activity. The effect of 1 microM GSNO on 5-LO expression was prevented by the gamma-GT inhibitor, acivicin, suggesting a convergence of GSNO and CysLT metabolic pathway that may be relevant to asthma. Our data demonstrate that GSNO levels<or=1 microM, likely recapitulating those in the asthmatic airway, increase 5-LO expression, an effect that may increase inflammation and bronchoconstriction. However, GSNO at concentrations>5microM suppresses 5-LO expression. These data suggest that GSNO might inhibit 5-LO expression in the clinical setting.

Nonclassical Action of Retinoic Acid on the Activation of the cAMP Response Element-binding Protein in Normal Human Bronchial Epithelial Cells

Vitamin A (retinol) is essential for normal regulation of cell growth and differentiation. We have shown that the retinol metabolite retinoic acid (RA) induces mucous cell differentiation of normal human tracheobronchial epithelial (NHTBE) cells. However, early biological effects of RA in the differentiation of bronchial epithelia are largely unknown. Here, we showed that RA rapidly activated cAMP response element-binding protein (CREB). However, RA did not use the conventional retinoic acid receptor (RAR)/retinoid X receptor (RXR) to activate CREB. RA activated CREB in NHTBE and H1734 cells in which RARs/RXR were silenced with small interfering RNA (siRNA) targeting RAR/RXR expression or deactivated by antagonist. Inhibition of protein kinase C (PKC) or extracellular regulated kinase (ERK1/2) blocked the RA-mediated activation of CREB. In addition, depletion of p90 ribosomal S6 kinase (RSK) via siRSK1/2 completely abolished the activation, suggesting that PKC, ERK, and RSK are required for the activation. Altogether, this study provides the first evidence that RA rapidly activates CREB transcription factor via PKC, ERK, and RSK in a retinoid receptor-independent manner in normal bronchial epithelial cells. This noncanonical RA signaling pathway may play an important role in mediating early biological effects in the mucociliary differentiation of bronchial epithelia.

15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells

To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a >1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium.