Normal Bronchial Epithelial Cells Research Articles

A transcribed pseudogene ofMYLKpromotes cell proliferation

Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼ 89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5 ± 4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.

Dysregulation of TIM-3–Galectin-9 Pathway in the Cystic Fibrosis Airways

The T-cell Ig and mucin domain-containing molecules (TIMs) have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. It has been reported that proinflammatory cytokine dysregulation and neutrophil-dominated inflammation are the main causes of morbidity in cystic fibrosis (CF). However, the role of TIM receptors in CF has not been investigated. In this study, we demonstrated that TIM-3 is constitutively overexpressed in the human CF airway, suggesting a link between CF transmembrane conductance regulator (CFTR) function and TIM-3 expression. Blockade of CFTR function with the CFTR inhibitor-172 induced an upregulation of TIM-3 and its ligand galectin-9 in normal bronchial epithelial cells. We also established that TIM-3 serves as a functional receptor in bronchial epithelial cells, and physiologically relevant concentrations of galectin-9 induced TIM-3 phosphorylation, resulting in increased IL-8 production. In addition, we have demonstrated that both TIM-3 and galectin-9 undergo rapid proteolytic degradation in the CF lung, primarily because of neutrophil elastase and proteinase-3 activity. Our results suggest a novel intrinsic defect that may contribute to the neutrophil-dominated immune response in the CF airways.

Catalase suppression‐mediated H2O2 accumulation in cancer cells by wogonin effectively blocks tumor necrosis factor‐induced NF‐κB activation and sensitizes apoptosis

Tremendous effort has been made to improve the anticancer value of tumor necrosis factor (TNF). In this study, we show that wogonin, a flavonoid isolated from Huang-Qin (Scutellaria baicalensis), synergistically sensitizes cancer cells derived from the cervix, ovary and lung to TNF-induced apoptosis, which was associated with inhibition of catalase activity and an increase of cellular hydrogen peroxide (H(2)O(2)). Wogonin-induced reactive oxygen species block TNF-induced NF-κB activation through inhibiting phosphorylation on the NF-κB p65 subunit and consequently the DNA binding of NF-κB. In addition, wogonin suppressed the expression of the antiapoptotic factor c-FLIP, which is accompanied with potentiation of TNF-induced caspase 8 activation that initiates apoptosis. Importantly, wogonin did not sensitize normal bronchial epithelial cells to TNF-induced cell death, which was associated with the defect in induction of H(2)O(2). Thus, wogonin specifically sensitizes cancer cells to TNF-induced cytotoxicity through H(2)O(2)-mediated NF-κB suppression and apoptosis activation. Our data provide important insights into the molecular mechanism underlying wogonin's anticancer activity, and suggest this common flavonoid could be used as a TNF adjuvant for cancer therapy.

The Pseudomonas aeruginosa Autoinducer 3O-C12 Homoserine Lactone Provokes Hyperinflammatory Responses from Cystic Fibrosis Airway Epithelial Cells

The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.

Epigenetics Underpinning the Regulation of the CXC (ELR+) Chemokines in Non-Small Cell Lung Cancer

BackgroundAngiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR+) chemokine family are powerful promoters of the angiogenic response.MethodsThe expression of the CXC (ELR+) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines.ResultsOverall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line.ConclusionsCXC (ELR+) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment.

Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom

In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3–20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM—irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.

Mutational and Expressional Analysis of DOK2 Gene in Non-small Cell Lung Cancers

Purpose: Mounting evidence indicates that perturbation of tyrosine phosphorylation is implicated in the development of many human diseases, including cancers. Docking proteins (DOKs) are tyrosine-phosphorylated proteins that negatively regulate tyrosine kinase signaling and they are considered to be tumor suppressors. Deletion and the altered expression of the DOK2 gene have been studied in leukemias and lung cancers. However, the somatic mutation status of the DOK2 gene has not been studied in lung cancers. The aim of this study was to see whether alterations of DOK2 protein expression and somatic mutation of the DOK2 gene are present in human non-small cell lung cancer (NSCLC). Materials and Methods: We analyzed DOK2 somatic mutation in 45 NSCLCs (23 adenocarcinomas (AD) and 22 squamous cell carcinomas (SCC) by single-strand conformation polymorphism (SSCP). We examined the DOK2 protein expression in 45 NSCLCs by immunohistochemistry. Results: SSCP analysis revealed no evidence of somatic mutation in the DNA sequences encoding the DOK2 gene in the 45 NSCLCs. Among the informative cases, 27% and 21% of the ADs and SCCs showed allelic loss in the DOK2 locus, respectively. On the immunohistochemistry, DOK2 protein was expressed in the normal bronchial epithelial cells, while it was lost in 10 (22%) of the NSCLCs. Conclusion: Our data indicates that DOK2 is altered in NSCLC at the expressional level, but not at the mutational level. The data also suggests that loss of the expression of DOK2 might play roles in NSCLC development by possibly altering tyrosine kinase signaling. (J Lung Cancer 2011;10(1):26 �� 31)

Loss of expression of the chromatin remodelling protein SATB1 in lung cancer is an independent prognostic marker

Lung cancer is the leading cause of cancer-related mortality and requires more effective molecular markers of prognosis and therapeutic responsiveness. Special AT-rich binding protein 1 (SATB1) is a global genome organiser that recruits chromatin remodelling proteins to epigenetically regulate hundreds of genes in a tissue-specific manner. SATB1 overexpression is a powerful predictor of poor prognosis in breast cancer, but the significance of SATB1 expression has not been previously evaluated in lung cancer. In a cohort of 285 primary lung cancers, significant loss of SATB1 expression was found in squamous preinvasive lesions ( p p p p = 0.016). Loss of SATB1 was also significantly associated with poor cancer-specific survival in small cell lung cancers (SCLC; HR 14.48, 95%CI 1.3–164.0, p = 0.03), however further investigation of a larger sample is warranted. Treatment of lung cancer cell lines A549 and H520 with the histone deacetylase inhibitor Trichostatin A resulted in up-regulation of SATB1. SATB1 was associated with a decrease in the active chromatin mark acetylated histone H3K9 and an increase in the repressive polycomb mark, trimethylated H3K27 in the SCC cell line NCI-H520 relative to the normal bronchial epithelial cell line NL20. This is the first study showing that SATB1 expression is lost in early preinvasive squamous lesions in the lung and that loss of SATB1 is associated with poor prognosis in lung SCC. Loss of SATB1 expression may be a result of epigenetic silencing caused by histone modifications.

S36 Check novel in vivo real time imaging of the bronchial mucosa using an endo-cytoscopy

<h3>Objectives</h3> We investigated the capabilities of an Endo-Cytoscopy system (ECS) that enables microscopic imaging of the tracheobronchial tree during bronchoscopy, including normal bronchial epithelium, dysplastic mucosa and hilar lung cancer. <h3>Methods</h3> The newly developed integrated-type ECS for the bronchoscope has a built-in two imaging system with a conventional mode and a high-power endocytoscopic mode. ECS has a high magnification of 570×. Thirty-seven patients including 9 hilar lung cancer, 6 abnormal sputum cytology, 19 squamous dysplasia, and 3 after photodynamic therapy were entered into the study and underwent white light, narrow band imaging and autofluorescence imaging bronchoscopy. Both the abnormal area of interest and surrounding normal bronchial mucosa were stained with 0.5% methylene blue and examined with ECS. Histological examinations with haematoxylin and eosin stain were performed using the biopsied specimens. The ECS imaging was analysed and correlated with the corresponding histological examination. <h3>Results</h3> ECS imaging could distinguish between different types of bronchial epithelium including normal bronchial mucosa, squamous dysplasia, and hilar lung cancer. Squamous dysplasia and hilar lung cancer were predictive with sensitivity of 85.7% (12/14) and 90.9% (10/11) and specificity of 100% (12/12), respectively. These ECS images corresponded well conventional histology. <h3>Conclusion</h3> ECS was useful for the discrimination between normal bronchial epithelial cells and dysplastic cells or malignant cells during bronchoscopy in real time. This novel technology has an excellent potential to provide <i>in vivo</i> diagnosis during bronchoscopic examinations.

MicroRNA‐206 Is Associated With Invasion and Metastasis of Lung Cancer

MicroRNAs are novel small noncoding RNA molecules that regulate gene expression at the post-transcriptional level. Compelling evidence reveals that there is a causative link between microRNAs deregulation and cancer development and progression. The present study aims to explore the function of miR-206 in the proliferation, apoptosis, motility, and invasion of nonsmall cell lung cancer. Using real-time PCR, we detected the miR-206 expression of normal lung tissues, tumor tissues, human normal bronchial epithelial cell line, and six lung cancer cell lines (LCCLs). Then, we evaluated the role of miR-206 in cell proliferation, apoptosis, and invasion using Cell Counting Kit-8 assay, Annexin-V/FITC assay, wound healing, and Transwell assay in LCCLs. As a result, miR-206 expression level was lower in high metastasis tumors and 95D than low metastasis tumors and normal lung tissues as well as other LCCLs. After miR-206 was upregulated in LCCLs, cell proliferation was notably attenuated and apoptosis was significantly increased. Furthermore, overexpression of miR-206 inhibited migration and invasion of lung cancer cells. In conclusion, our data suggest that expression level of miR-206 was inversely correlated with metastatic potential of lung cancer.

Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer

Background Minichromosome maintenance (MCM) proteins 2–7 form a complex essential for the initiation of DNA replication. In the process to screen expression changes related to growth suppression of non-small cell lung cancer (NSCLC) cells by a cJun dominant-negative mutant, we found that reduced expression of MCM4 was correlated with this growth suppression. Method We determined the relevance of MCM4 in proliferation of NSCLC by downregulating its expression with small-interfering RNA in three NSCLC cell lines. We then immunohistochemically analyzed MCM4 expression in 156 surgically resected NSCLCs to correlate clinicopathologic characteristics. Results MCM4 downregulation reduced proliferation in two cell lines. MCM4 expression was higher in cancer cells than in adjacent normal bronchial epithelial cells ( p < 0.001). High MCM4 expression was correlated with male gender, heavy smoking, poorer differentiation and non-adenocarcinoma histology ( p < 0.001, respectively). High MCM4 expression was also correlated with proliferation markers, Ki-67 and cyclin E expression ( p < 0.001, respectively). MCM4 expression was not associated with survival. Conclusion MCM4 may play an essential role in the proliferation of some NSCLC cells. Taken together with higher expression in NSCLCs and its correlation with clinicopathologic characteristics such as non-adenocarcinoma histology, MCM4 may have potential as a therapeutic target in certain population with NSCLCs.

In vivo microscopic imaging of the bronchial mucosa using an endo-cytoscopy system

Objectives We investigated the capabilities of an endo-cytoscopy system (ECS) that enables microscopic imaging of the tracheobronchial tree during bronchoscopy, including normal bronchial epithelium, dysplastic mucosa and squamous cell carcinoma. Methods The newly developed ECS has a 3.2 mm diameter that can be passed through the 4.2 mm working channel of a mother endoscope for insertion of the ECS. It has a high magnification of 570× on a 17 in. video monitor. Twenty-two patients (7 squamous cell carcinoma, 11 squamous dysplasia and 4 after PDT therapies) were underwent white light, NBI light and AFI bronchoscopy. Both abnormal areas of interest and normal bronchial mucosa were stained with 0.5% methylene blue and examined with ECS at high magnification (570×). Histological examinations using haematoxylin and eosin staining were made of biopsied specimens. Analyzed ECS images were compared with the corresponding histological examinations. Results In normal bronchial mucosa, ciliated columnar epithelial cells were visible. In bronchial squamous dysplasia, superficial cells with abundant cytoplasm were arranged regularly. In squamous cell carcinoma, large, polymorphic tumor cells showed increased cellular densities with irregular stratified patterns. These ECS images corresponded well with the light-microscopic examination of conventional histology. Conclusion ECS was useful for the discrimination between normal bronchial epithelial cells and dysplastic cells or malignant cells during bronchoscopy in real time. This novel technology has an excellent potential to provide in vivo diagnosis during bronchoscopic examinations.

Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma

Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes--adenocarcinoma (AC) and squamous cell carcinoma (SqCC)--respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome. We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage. This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy. Please see later in the article for the Editors' Summary.

Gene expression differences in current versus former smokers lung squamous preneoplasia.

10575 Background: The study of the molecular biology of the lung carcinogenesis would help finding biomarkers for early diagnosis and targets for chemoprevention. The present study aim to characterize genomic profiles between current and formers smokers in lung squamous carcinogenesis. Methods: 122 bronchial biopsies (67/55 from current/former smokers) at successive stages of lung squamous carcinogenesis (normal, hyperplasia, metaplasia, mild, moderate and severe dysplasia, carcinoma in situ and invasive carcinoma) obtained by fluorescence bronchoscopy in 74 high risk individuals (40/34 current/former smokers) were analyzed by gene expression microarrays (Mascaux, J Thorac Oncol 2009 4(9) S282). Results: 740 genes were significantly differentially expressed (FDR 0.05) between biopsies from current versus former smokers in bronchial pre-invasive lesions. The most significantly associated pathways was metabolism of xenobiotic by cytochrome CP450 eg CYP1A1, CYP2A6, ALDH1L1, but genes related to cancer pathways were also found, ie metallopeptidases, IGF binding proteins, cell growth and proliferation (ie EGF), or apoptosis (ie survivin). Our findings in lung preneoplasia were consistent by gene set enrichement analyses. (GSEA) with data previously reported on normal bronchial epithelial cells (Spira, PNAS 2004 101(27) 10143). Association between gene expression differences observed between the early steps of lung carcinogenesis in our study and between current and former smokers in normal bronchial epithelial cells in Spira study were analysed by GSEA. Some associations were observed: genes down-regulated in current smokers were also down-regulated in transition to squamous metaplasia including cancer genes like eg BCL2, FGF2, AKT3 and eicosanoid signaling eg PTGDR, PLA2G5. Conclusions: Genes discriminating current and former smokers during lung carcinogenesis include genes involved in metabolism of xenobiotic by cytochrome CP450, cell proliferation and apoptosis. Cigarette smoke exposure effects on normal epithelial cells and on preneoplasia from different data sets are related. Genes related to current smoking like eicosanoid signaling genes are involved in the early steps of carcinogenesis. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Bristol-Myers Squibb/ImClone, Boehringer Ingelheim, Lilly, Merck Serono, OSI/Genentech/ Roche, Pfizer, Syndax AstraZeneca, Biogen Idec, Genentech, Genmab, Merck, OSI, Syndax, Ventana-Roche Abbott Laboratories

Abstract 1592: The role of T-oligo and its associated proteins in mediating anticancer responses in melanoma and lung cancer

Abstract Telomeres are tandem repeats of the DNA sequence TTAGGG. The 3’ end of each telomere consists of a single stranded overhang that is normally concealed in a loop structure at the chromosome ends. We postulate that exposure of this sequence upon telomere disruption induces anticancer responses such as apoptosis and senescence. An 11-base oligonucleotide homologous to the telomere 3’ overhang sequence, (T-oligo) is thought to mimic exposure of the telomere overhang. T-oligo decreased NSCLC viability in six cell lines by 40-50% and induced a 3 fold increase in apoptosis and 10 fold increase in senescence. T-oligo did not effect cell proliferation or induce apoptosis or senescence in normal bronchial epithelial cells. To investigate proteins that bind to T-oligo which could induce apoptosis and senescence, cancer cells were treated with biotinylated T-oligo or complementary oligo, and T-oligo associated proteins were immunoprecipitated. Further analysis of unique protein bands after gel electrophoresis and mass spectrometry indicated that proteins such as hnRNP C, Purβ, and Msi-1 which are known to interact with the telomere and modulate p53 and pRb were bound to T-oligo. We hypothesize that T-oligo modulates hnRNP C, Purβ, and Msi-1 and induces various anticancer effects. We found in NSCLC cell lines H358 and SW1573 that hnRNP C was upregulated by T-oligo at 18 hrs and Purβ was found to be upregulated at 18 hrs in H358. Similar results were observed in two melanoma cells lines, MU and MM-AN. T-oligo upregulated hnRNP C at 18 hrs and 6 hrs in MM-AN and MU melanoma cells respectively. Additionally, Purβ and Msi-1 were upregulated at 24 and 48 hrs in MM-AN cells treated with T-oligo. To investigate hnRNP Cs involvement in senescence in H358 cells, hnRNP C was downregulated using shRNA in a lentiviral vector. It was found that 7 days after T-oligo treatment, 70% of the cells were positive for senescence associated β-galactosidase (β-gal) compared to 5% positive cells after control shRNA treatment. However, in cells treated with T-oligo after downregulation of hnRNP C only 8% of cells were positive for β-gal indicating that hnRNP C played an important role in mediating senescence. To test the ability of T-oligo as a therapeutic drug, subcutaneous tumors in nude mice were given daily intratumoral injections of 60 nmoles of T-oligo or a complementary oligonucleotide for 6 weeks. SW1573 and H358 tumors treated with T-oligo exhibited a 5.6 and 4.3 fold reduction in tumor size respectively. When tumor sections were further examined for senescence using β-gal, both H358 and SW1573 exhibited strong staining for senescence compared to minimal staining in controls. These results indicate that T-oligo not only reduced tumor size, but also induced senescence in tumor cells. These findings suggest that T-oligo may be a novel molecularly targeted therapy and its associated proteins may be novel cancer targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1592.

Abstract 3083: Regulation of novel tumor suppressor gene FUS1 expression through 5’- and 3’-UTRs and in response to DNA damage in human lung cancer cells

Abstract FUS1 is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. FUS1 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous FUS1 protein could be detected in a majority of lung cancer cell lines. Loss or reduction of FUS1 expression was observed in all small cell lung cancer and in more than 70 % of non-small cell lung carcinomas and was associated with significantly worse overall patient survival. However, mechanisms regulating FUS1 protein expression and its inactivation in primary lung cancer cells are largely unknown. In this study, we investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the FUS1 gene in the regulation of FUS1 protein expression and determined changes in FUS1 gene and protein expression in response to oncogenic stress in normal lung and cancer cell lines. We constructed a series of expression plasmids containing the FUS1 coding region with or without the intrinsic UTRs and transfected them into normal lung bronchial epithelial and tumor cells to assess their roles in regulation of FUS1 protein expression. We identified some critical structure elements in FUS1 UTRs that could significantly regulate FUS1 protein expression. We found that two small upstream open-reading frames (uORFs) encoding an 8-amino-acid peptide and another 6-amino-acid peptide, respectively, in the 5’UTR of FUS1 could markedly inhibit the translational initiation of FUS1 protein by interfering with the “scanning” of the ribosome initiation complexes. Several secondary RNA structural elements/motifs on the 3’UTR of FUS1 also showed a significant inhibitory effect on FUS1 protein expression. The 3’UTR-mediated regulatory effect on FUS1 proteins expression was differentially detected in normal lung epithelial and fibroblast cells compared to lung cancer cells. We found that FUS1 expression could be induced or activated in the presence of DNA damaging agents, such as UV irradiation, etoposide, and cisplatin. UV irradiation could induce FUS1 protein and mRNA expression independent of the status of FUS1 UTRs. The FUS1 expression activated by DNA damaging agents such as etoposide or cisplatin facilitated FUS1-induced apoptosis, prevented cells from dividing and accelerated cell death. Our results provide new insight into the molecular mechanisms in the regulation of FUS1 expression in lung cancer cells. (Supported by NIH SPORE P50CA70907 and RO1CA116322 grants) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3083.

Abstract LB-278: BRMS1 promoter methylation correlates with decreased expression of BRMS1 in non-small cell lung cancer

Abstract Background: Breast cancer metastasis suppressor gene-1 (BRMS1) mRNA and protein expression are significantly decreased in non-small cell lung cancer (NSCLC) cell lines and human NSCLC when compared to normal bronchial epithelial cells and adjacent noncancerous tumor tissue, respectively. We hypothesized that one mechanism through which BRMS1 expression was decreased in NSCLC was through BRMS1 promoter methylation. Materials and Methods: Human NSCLC BRMS1 promoter methylation was evaluated using methylation-specific PCR and bisulfite sequencing. The effect of methylation on BRMS1 mRNA and protein expression was assessed following treatment with 5-azacitidine, a demethylating agent in NSCLC cell lines. BRMS1 transcriptional activity was assessed using a human BRMS1-luc reporter gene construct cloned by our group. Student's T-Test and Chi-square tests were used to determine significance between variables of two patient populations separated based on tumor histology (squamous cell or adenocarcinoma) and to evaluate the methylation profile between the two tumor histologies. Immunohistochemical analysis for BRMS1 protein was performed on both tumor and adjacent noncancerous bronchial epithelium. A general linear model tested the association between BRMS1 promoter methylation in human NSCLC and specific clinical and histopathologic variables. Results: The BRMS1 promoter region contains a CpG island that was hypermethylated in all NSCLC cell lines and human tumors. This strongly correlated with reduced BRMS1 promoter activity. NSCLC cell lines treated with 5-azacitidine showed a robust recovery of BRMS1 mRNA and protein levels, suggesting that promoter methylation is involved in BRMS1 transcriptional regulation. Analysis of 40 patient NSCLC samples (20 adenocarcinoma and 20 squamous cell) identified a significant increase in the amount of BRMS1 promoter methylation when comparing tumor specimen to adjacent noncancerous tissue for both tumor histologies (p&amp;lt;0.0001). Additionally, the BRMS1 promoter region was significantly more methylated in squamous cell compared to adenocarcinoma histology (p=0.01). Subsequent immunohistochemical analysis confirmed a correlation between BRMS1 promoter methylation and decreased bronchial epithelium BRMS1 protein expression (p=0.06). Moreover, in the squamous cell histology, pathologic tumor stage was associated with elevated BRMS1 methylation (p=0.04). Conclusions: These studies identify BRMS1 promoter methylation as an important mechanism for silencing BRMS1 expression in NSCLC and highlight the potential clinical relevance of this epigenetic signature with respect to tumor histology and stage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-278.

Abstract 3797: Luteolin induces cytotoxicity in lung cancer cells depending on superoxide-mediated MKP-1 degradation and JNK activation

Abstract Epidemiological studies have suggested luteolin (3′,4′,5,7-tetrahydroxyflavone) as a potential cancer preventive agent. Recently we found that luteolin selectively kills lung cancer but not immortalized human normal bronchial epithelial cells (HBECs). To elucidate the mechanism of luteolin's cancer cell killing activity, we investigated the cytotoxic cellular signaling pathway in lung cancer cells. The results show that luteolin potently induced both apoptosis and necrosis, which was associated with mitochondrion-dependent superoxide accumulation in lung cancer cells. The c-Jun N-terminal kinase (JNK) was substantially activated in luteolin treated cancer cells and luteolin-induced cytotoxicity was effectively blocked when JNK was inhibited. The reactive oxygen species scavengers BHA and NAC, which effectively block luteolin-induced superoxide accumulation, potently suppressed luteolin-induced JNK activation and cytotoxicity, suggesting that luteolin's lung cancer cell killing requires superoxide-mediated JNK activation. Furthermore, JNK activation was tightly associated with a superoxide-dependent rapid reduction of the JNK inactivating phosphatase MKP-1, which was mainly due to destabilizing MKP-1 protein. These results suggest that inhibiting MKP-1 is the main mechanism for luteolin-induced JNK activation. Thus, luteolin activates a novel cytotoxic pathway consisting of superoxide, MKP-1 and JNK in lung cancer cells. Interestingly, luteolin failed in inducing MKP-1 degradation and JNK activation and cell death in the HBECs, although superoxide induction by luteolin was comparable to that in lung cancer cells. Therefore, luteolin-induced cellular signaling in normal and lung cancer cells diverges at JNK activation. Taken together, our results suggest that activation of the speroxide/MKP1/JNK pathway underlies the main mechanism of luteolin's selective ctyotoxicity in lung cancer cells and manipulation of this pathway could be a useful approach for applying luteolin for lung cancer prevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3797.

Differential Expression of Carotenoid Metabolism Related Genes in Human Lung Epithelial Cells

Epidemiologic studies have demonstrated that dietary beta‐carotene and total carotenoids are inversely associated with non‐small cell lung cancer risk. While high‐dose beta‐carotene supplementation for chronic smokers has been linked to increased lung cancer risk in clinical trials, little is known about the carotenoid metabolomic alterations that characterize neoplastic progression. Using two human pulmonary cell lines, we determined the expression of genes regulating carotenoid metabolism in normal bronchial epithelial (BEAS‐2B) and lung adenocarcinoma pulmonary epithelial (A549) cells. We assayed gene pathways including ALDH1A2, ALDH8A1, CMO1, CMO2, Cyp26A1, Cyp26B1, Cyp26C1, LRAT, RHD5, RHD8, RDH11 ~ 14, RHD16 and SDR16C5 by qRT‐PCR. Our results indicate that carotenoid metabolism‐related genes are expressed as different and distinct patterns in BEAS‐2B and A549 cells. BEAS‐2B cells express high levels of CMO2 and LRAT but not CMO1, Cyp26A1 or Cyp26B1, indicating an active lycopene metabolic apparatus. In contrast, A549 cells highly express CMO1, ALDH1A2, Cyp26A1 and Cyp26B1. In A549 cells, we could detect only low levels of CMO2 and no LRAT expression, implying presence of active beta‐carotene metabolism. We further tested this hypothesis by treating BEAS‐2B and A549 cells with lycopene. CMO2 and LRAT expression were increased but there were no effects on CMO1 expression. Finally, immunohistochemistry of NSCLC tissues shows profuse CMO1 staining in the tumors but little in adjacent normal lung. These findings suggest that specific beneficial effects of carotenoid on lung health may be linked to CMO2‐dependence in tissue. A shift to decreased expression of CMO2 and LRAT and increased CMO1 and Cyp26 family genes can alter carotene metabolism may contribute to lung carcinogenesis. (NIH HD42174, Muma Family Endowment)

The effect of N‐acetylcysteine on chloride efflux from airway epithelial cells

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.