Stages Of Lung Carcinogenesis Research Articles

Abstract 3158: Defining the mechanisms that lead to mutational heterogeneity in the normal respiratory epithelium

Abstract It is well established that tobacco smoking causes lung cancer and this is thought to be the result of a progressive accumulation of genomic mutations induced by tobacco smoke exposure; however, until recently a detailed understanding of the effects of smoke exposure on the genomes of cells in the normal bronchial epithelium was lacking. To address this, we defined the genomic architecture of normal airway basal cells in children and non-smoking, smoking and ex-smoking adults. We discovered two populations of basal cells in individuals with a smoking history: those with high mutational burden, driver gene mutations and tobacco-associated mutational signatures, and those with low mutational burden, similar to basal cells from never smokers and with an absence of mutational signatures associated with tobacco exposure. These cells expand preferentially in ex-smokers, accounting for 20-40% of basal cells compared with 2-5% in current smokers. We hypothesise that these cells are cancer-protective and their expansion reduces the risk of lung cancer development after smoking cessation. Understanding the functional differences between high- and low-mutant airway basal cells and the mechanisms by which some cells resist the accumulation of smoking-induced mutations will be crucial to understanding the earliest stages of lung carcinogenesis. To address this, we performed RNAseq on a subset of whole-genome sequenced airway basal cells. Preliminary data show that expression of markers of stemness is significantly different between high- and low-mutant basal cells. In addition, initial analysis shows that pathways such as carcinogen metabolism and MHC class I antigen presentation are higher in high-mutant basal cells. Further analysis in more patients is ongoing. To complement these data, we expanded whole-genome sequenced basal cell clones in culture and performed a range of assays to assess their progenitor and differentiation capacity. We assessed proliferation, colony-forming efficiency, longevity and differentiation and found no apparent differences between low- and high-mutant basal cells. Initial analysis suggests that carcinogen metabolism and MHC class I antigen presentation may be key pathways in establishing heterogeneity in mutational burden and clonal dynamics in the normal airway epithelium. Additional assays and analysis are ongoing and will be the focus of future research. Citation Format: Kate H. Gowers, Sarah E. Clarke, Ayu Hutami Syaraf, Kenichi Yoshida, Moritz J. Przybilla, Hugh Selway, Adam Pennycuick, Peter J. Campbell, Sam M. Janes. Defining the mechanisms that lead to mutational heterogeneity in the normal respiratory epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3158.

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.

Pathological and Prognostic Indications of the mdig Gene in Human Lung Cancer

Background/Aims:The mineral-dust-induced gene mdig is a lung-cancer-associated oncogene. The focus of this study is to evaluate the expression status of mdig in lung cancer and to assess its influence in predicting the patient’s overall survival.Methods:Using high-density tissue microarrays and clinical samples of synchronous multiple primary lung cancer (SMPLC), we investigated the expression of mdig through immunohistochemistry and utilized the open-access lung cancer patient databases containing genomic and transcriptomic data from the UCSC Xena and TCGA web platforms to determine the prognostic values of mdig expression status among different subtypes of lung cancer.Results:mdig is upregulated in smokers and in lung squamous cell carcinoma. High mdig expression predicted poor overall survival in lung squamous cell carcinoma and female smokers. Among tumor tissues from SMPLC patients, we not only unraveled the highest positive rate of mdig expression, but also revealed a unique cytoplasmic, rather than nuclear localization of mdig protein. Furthermore, by inspecting some pathological but not cancerous lung tissues, we believe that mdig is required for the transformation of non-cancerous lung cells to the fully-fledged cancer cells.Conclusion:These data suggested that mdig is involved in various stages of lung carcinogenesis, possibly through the epigenetic regulation on some critical cancer-associated genes, and increased mdig expression is an important prognostic factor for some types of lung cancer.

Abstract 1002: Immune microenvironment features of lung adenomatous premalignancy in a Chinese cohort

Abstract Introduction: The widespread of implementation of computed tomography (CT) has resulted in a drastic increase in the detection of small pulmonary nodules presenting as ground glass opacities (GGOs). Many of these GGOs are atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and sometimes invasive adenocarcinoma (IAC). The concept of a continuum between the precursor lesions AAH and AIS to MIA and frankly invasive IAC is backed by a wealth of recent data showing a gradual decrease in overall survival from 100% for AAH, AIS and MIA to moderately lower rates for IAC. A growing body of evidence suggests that the immune response is a significant determinant of cancer progression. However, little is known about the immune microenvironment features in the early stages of lung carcinogenesis. Methods: A total of 40 tumor tissue samples including 10 AAH, 10 AIS, 10 MIA and 10 early IAC from 40 patients who underwent surgical resection of pulmonary nodules at Yancheng No.1 People's Hospital, China were included. Immunohistochemistry (IHC) staining for the expression of CD3, CD4, CD8 and programmed death ligand 1 (PD-L1) to assess the role of T-cell immune surveillance in these pre- and early lung cancers were performed. Results: We observed CD3, CD4, CD8 and PD-L1 positivity in AAH, AIS, MIA and IAC, suggesting that immunoediting has started before the invasion. Higher expressions of CD3, CD4, CD8 and PD-L1 in IAC implying tumor invasion. Conclusions: Our preliminary results of the immune subgroups provide evidence for immune recognition in pulmonary premalignancy and are critical for understanding tumorigenesis and the development of effective preventive and interception strategies. Associations with genomic landscape is underway. Citation Format: Liting Guo, Weiwei Shao. Immune microenvironment features of lung adenomatous premalignancy in a Chinese cohort [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 1002.

Abstract 1419: Characterization of the evolution of immune response in lung squamous carcinogenesis

Abstract Molecular profiling of from pre-invasive bronchial lesions, and particularly the evolution of immune response, may identify promising new biomarkers for early detection and targets for chemoprevention/treatment of lung cancer. mRNA expression was analyzed (Agilent microarrays) from fresh frozen human bronchial biopsies (N = 122, 77 patients) at successive morphological stages of lung squamous carcinogenesis. Modules of co-expressed genes were identified among the stage associated genes, selected using a linear mixed-effects model adjusting for smoking status, gender, and history of cancer as fixed effects and patient as a random effect. Cell type specific signatures were applied to the genes co-expressed in these modules in order to identify the immune response signal through the stages of lung carcinogenesis. Genes expression alterations were grouped into 4 successive molecular steps. Based on their behavior across the 4 steps, 8 modules of genes with different patterns were observed: gene modules primarily up-regulated among the early or late steps, up-regulated in a linearly across the 4 steps, as well as down- then up-regulated (biphasic). Genes signing the presence of activating CD8 and CD4 T cells, as well as some memory B cells were progressively and linearly up-regulated from metaplasia through all stages of carcinogenesis. At the later stages, the transition to high-grade dysplasia, the most significant gene expression changes included immune/inflammatory response genes. At this stage a very strong adaptive immune response is noted involving T cells - mainly Th1 and T regulators - and dendritic cells. In addition, immunosuppressive signals, corresponding to negative co-inhibitory molecules, also significantly appears at the high-grade dysplasia stage. The adaptive CD4/CD8 related immune response starts from the earlier stages (metaplasia) of lung carcinogenesis. The significant modification of inflammatory/immune response genes in association with high-grade lesions suggests, at this critical stage of carcinogenesis, a major role of the surrounding microenvironment with an adaptive immune response, driven by Th1 and Treg cells, and but also, before tumor invasion across the basal membrane, a tumor versus host immunosuppressive response. Citation Format: Celine Mascaux, Mihaela Angelova, Jennifer Beane, Kahkeshan Hijazi, Geraldine Antoine, Karen Willard Gallo, Vincent Ninane, Arsène Burny, Jean-Paul Sculier, Avrum Spira, Jérôme Galon. Characterization of the evolution of immune response in lung squamous carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1419.

Abstract 2352: Mapping the airway-wide molecular field of injury in smokers with lung cancer

Abstract Lung cancer mortality is the leading cause of cancer death in the United States in part because diagnosis occurs after regional or distant metastasis of the disease. Identifying effective early detection biomarkers is crucial for improving lung cancer clinical management. Moreover, molecular biomarkers for early disease detection may provide insight into the molecular pathways associated with disease development and progression. Our lab has shown that smoking-induced gene expression alterations are mirrored in the epithelia of the mainstem bronchus, buccal and nasal cavity. We have additionally demonstrated that gene-expression profiles in cytologically normal mainstem bronchial epithelium can serve as an early diagnostic biomarker for lung cancer. Here we expand on our previous work by spatially mapping the molecular field of injury throughout the entire respiratory tract in smokers with lung cancer. Using Affymetrix Gene ST 2.0 arrays, we profiled genome-wide gene-expression in 1) lung lesions and adjacent normal lung obtained from smokers undergoing surgical resection, 2) epithelial brushings obtained at intraoperative bronchoscopy from the nasal epithelium, main carina and ipsilateral and contralateral proximal and distal bronchi (relative to the location of the resected lung lesion), and 3) epithelial brushings obtained at lobectomy from sub-segmental bronchus (adjacent to tumor). Linear modeling approaches comparing the airways and tumors of patients with cancer to those with benign lung disease were used to explore relationships in cancer-specific gene-expression alterations across sites within the respiratory tract. We found that genes upregulated in the small airways leading to the tumor were enriched in genes upregulated in the mainstem bronchus and main carina of smokers with lung cancer. In addition, genes upregulated in the bronchus and main carina of smokers with lung cancer showed enrichment among cancer associated genes elevated in the nose. Furthermore, a linear mixed effects model uncovered genes and pathways which change in expression in a gradient-like manner as distance from the tumor increases. Our findings suggest that the molecular field of injury encompasses airway-wide alterations throughout the entire respiratory tract of smokers with lung cancer as well as gradient profiles that change with respect to proximity of the nearby tumor. These molecular alterations may ultimately serve as early detection biomarkers for lung cancer and provide new insights into early stages of lung carcinogenesis. Citation Format: Rebecca Kusko, Christina Anderlind, Gerald Wang, Sherry Zhang, W. Dean Wallace, Tonya Wasler, Michael Ebright, Melinda M. Garcia, Rosana Eisenberg, Gina Lee, Gang Liu, David Elashoff, Neda Kalhor, Cesar Moran, Reza Mehran, Junya Fujimoto, Pierre P. Massion, Steven Dubinett, Ignacio Wistuba, Marc Lenburg, Humam Kadara, Avrum Spira. Mapping the airway-wide molecular field of injury in smokers with lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2352. doi:10.1158/1538-7445.AM2014-2352

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

The adherens junction protein p120-catenin (p120ctn) shuttles between E-cadherin-bound and cytoplasmic pools to regulate E-cadherin/catenin complex stability and cell migration, respectively. When released from the adherens junction, p120ctn promotes cell migration through modulation of the Rho GTPases Rac1, Cdc42, and RhoA. Accordingly, the down-regulation and cytoplasmic mislocalization of p120ctn has been reported in all subtypes of lung cancers and is associated with grave prognosis. Previously, we reported that cigarette smoke induced cytoplasmic translocation of p120ctn and cell migration, but the underlying mechanism was unclear. Using primary human bronchial epithelial cells exposed to smoke-concentrated medium (Smk), we observed the translocation of Rac1 and Cdc42, but not RhoA, to the leading edge of polarized and migrating human bronchial epithelial cells. Rac1 and Cdc42 were robustly activated by smoke, whereas RhoA was inhibited. Accordingly, siRNA knockdown of Rac1 or Cdc42 completely abolished Smk-induced cell migration, whereas knockdown of RhoA had no effect. p120ctn/Rac1 double knockdown completely abolished Smk-induced cell migration, whereas p120ctn/Cdc42 double knockdown did not. These data suggested that Rac1 and Cdc42 coactivation was essential to smoke-promoted cell migration in the presence of p120ctn, whereas migration proceeded via Rac1 alone in the absence of p120ctn. Thus, Rac1 may provide an omnipotent therapeutic target in reversing cell migration during the early (intact p120ctn) and late (loss of p120ctn) stages of lung carcinogenesis.

Alteration of serum miR-206 and miR-133b is associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The alteration of microRNA (miRNA) expression plays an important role in chemical carcinogenesis. Presently, few reports have been published that concern the significance of circulating miRNAs in lung carcinogenesis induced by environmental carcinogens. The purpose of this study was to identify serum miRNAs that could be associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Male F344 rats were systemically administered with NNK. The rat serum differential expression profiles of miRNAs were analyzed by small RNA solexa sequencing. Using quantitative real-time PCR, the differentially expressed serum miRNAs were identified in each individual rat. Serum miR-206 and miR-133b were selected for further identification in rat serum at different stages of lung carcinogenesis; we detected the levels of serum miR-206 and miR-133b in lung cancer tissues induced by NNK. NNK causes significant alteration of serum miRNA expression. Compared to the control group, serum miR-206 and miR-133b were significantly up-regulated in the early stage of NNK-induced lung carcinogenesis. miR-206 and miR-133b exhibited low-expression in lung cancer tissues. Our results demonstrate that lung carcinogen NNK exposure changes the expression of serum miRNAs. Serum miR-206 and miR-133b could be associated with lung carcinogenesis induced by NNK.

A Critical Role for Macrophages in Promotion of Urethane-Induced Lung Carcinogenesis

Macrophages have established roles in tumor growth and metastasis, but information about their role in lung tumor promotion is limited. To assess the role of macrophages in lung tumorigenesis, we developed a method of minimally invasive, long-term macrophage depletion by repetitive intratracheal instillation of liposomal clodronate. Compared with controls treated with repetitive doses of PBS-containing liposomes, long-term macrophage depletion resulted in a marked reduction in tumor number and size at 4 mo after a single i.p. injection of the carcinogen urethane. After urethane treatment, lung macrophages developed increased M1 macrophage marker expression during the first 2-3 wk, followed by increased M2 marker expression by week 6. Using a strategy to reduce alveolar macrophages during tumor initiation and early promotion stages (weeks 1-2) or during late promotion and progression stages (weeks 4-16), we found significantly fewer and smaller lung tumors in both groups compared with controls. Late-stage macrophage depletion reduced VEGF expression and impaired vascular growth in tumors. In contrast, early-stage depletion of alveolar macrophages impaired urethane-induced NF-κB activation in the lungs and reduced the development of premalignant atypical adenomatous hyperplasia lesions at 6 wk after urethane injection. Together, these studies elucidate an important role for macrophages in lung tumor promotion and indicate that these cells have distinct roles during different stages of lung carcinogenesis.

Molecular analysis of DNA repair gene methylation and protein expression during chemical-induced rat lung carcinogenesis

A defective ratio between DNA damage and repair may result in the occurrence of a malignant phenotype. Previous studies have found that many genetic alterations in DNA repair genes occur frequently in lung cancer. However, the epigenetic mechanisms underlying this tumorigenesis are not clear. Herein, we have used a chemical-induced rat lung carcinogenesis model to study the evolution of methylation alterations of DNA repair genes BRCA1, ERCC1, XRCC1, and MLH1. Methylation-specific PCR and immunohistochemistry were used to analyze gene methylation status and protein expression during the progression of lung carcinogenesis. Promoter hypermethylation of BRCA1 was only detected in three samples of infiltrating carcinoma. CpG island hypermethylation of ERCC1, XRCC1, and MLH1 was found to increase gradually throughout lung carcinogenesis progression. Both the prevalence of at least one methylated gene and the average number of methylated genes were heightened in squamous metaplasia and dysplasia compared with normal tissue and hyperplasia, and was further increased in carcinoma in situ (CIS) and infiltrating carcinoma. Immunohistochemical analysis showed that BRCA1 and MLH1 protein expression decreased progressively during the stages of lung carcinogenesis, whereas ERCC1 and XRCC1 expression were only found in later stages. Although methylation levels were elevated for ERCC1 and XRCC1 during carcinogenesis, an inverse correlation with protein expression was found only for BRCA1 and MLH1. These results suggest that a continuous accumulation of DNA repair gene hypermethylation and the consequent protein alterations might be a vital molecular mechanism during the process of multistep chemical-induced rat lung carcinogenesis.

Abstract 160: Altered levels of circulating microRNAs in the plasma of non-small cell lung cancer patients: Potential as biomarker

Abstract The development of molecular markers for lung cancer could offset the deficiencies of currently available early detection and staging approaches. To this end, blood markers are particularly attractive since samples are obtained in a minimally invasive way at any stage of lung carcinogenesis. In this study we compared the levels of 10 microRNAs (miR-21, miR-31, miR-155, miR-147, miR-218, miR-377, miR-551b*, miR-569, miR-1283, and miR-1468) in the blood plasma of 27 non-small-cell lung cancer (NSCLC) patients and 38 healthy controls, using quantitative reverse transcription- polymerase chain reaction (qRT-PCR), to determine if these miRNAs might have a diagnostic value for NSCLC. The results of the area under the receiver operating characteristic curve (ROC), which measures the ability of the test to correctly classify those with and without the disease, were 0.98, 1.00, 0.8, and 0.98 for miR-21, miR-155, miR-377 and miR-1468, respectively, indicating that plasma levels of miR-21, miR-155, miR-377 and miR-1468 are powerful discriminators between patients and controls. At the optimum cutoff point for the cycle threshold (CT) value of the four miRNAs, the sensitivities of miR-21, miR-155, miR-377 and miR-1468 were 92.5%, 100%, 84%, and 100%, respectively; the corresponding values for the test specificity were 100%, 100%, 88%, and 100%, respectively. These results indicate the great potential of miR-21, miR-155, miR-377 and miR-1468 as diagnostic markers for NSCLC but need to be proved in prospective studies with larger samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2011-160

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2′-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.

Abstract PL02-03: Insights from a new mouse model into the role of inflammation at early stages of lung carcinogenesis

Abstract PL02-03: Insights from a new mouse model into the role of inflammation at early stages of lung carcinogenesis

HLungDB: an integrated database of human lung cancer research

The human lung cancer database (HLungDB) is a database with the integration of the lung cancer-related genes, proteins and miRNAs together with the corresponding clinical information. The main purpose of this platform is to establish a network of lung cancer-related molecules and to facilitate the mechanistic study of lung carcinogenesis. The entries describing the relationships between molecules and human lung cancer in the current release were extracted manually from literatures. Currently, we have collected 2585 genes and 212 miRNA with the experimental evidences involved in the different stages of lung carcinogenesis through text mining. Furthermore, we have incorporated the results from analysis of transcription factor-binding motifs, the promoters and the SNP sites for each gene. Since epigenetic alterations also play an important role in lung carcinogenesis, genes with epigenetic regulation were also included. We hope HLungDB will enrich our knowledge about lung cancer biology and eventually lead to the development of novel therapeutic strategies. HLungDB can be freely accessed at http://www.megabionet.org/bio/hlung.

Evolution of microRNA expression during human bronchial squamous carcinogenesis

MicroRNAs, negative post-transcriptional regulators of gene expression, are involved in cancer. Their role in early bronchial carcinogenesis was analysed in 60 biopsies obtained by fluorescence bronchoscopy (six per stage: normal tissue of nonsmokers, normal normofluorescent and hypofluorescent bronchial tissue of smokers, hyperplasia, metaplasia, mild, moderate and severe dysplasia, in situ carcinoma and invasive squamous cell carcinoma (SQCC)). In total, 69 microRNAs were found to be differentially expressed in the course of bronchial carcinogenesis. Among them, some microRNAs showed a linear evolution of their expression level, such as miR-32 and miR-34c, whose expression progressively decreased from normal bronchial tissues of nonsmokers to SQCC. Others behaved differently at successive stages, such as miR-142-3p or miR-9, or are only altered from a specific stage, such as miR-199a or miR-139. MicroRNAs globally followed a two-step evolution, first decreasing (a reverse of their increase during embryogenesis) during the earliest morphological modifications of bronchial epithelium, and thereafter increasing at later stages of lung carcinogenesis. Moreover, microRNA expression was very efficient for the prediction of the histological classification between low- and high-grade lesions and between in situ and invasive carcinoma. The present data show, for the first time, that microRNAs are involved in bronchial carcinogenesis from the very early steps of this process and, thus, could provide tools for early detection of lung cancer.

Identification of genes expressed differentially in an in vitro human lung carcinogenesis model

Lung cancer is the deadliest among all cancers in the US for both men and women. Early detection of premalignant lesions or tumors appears to be a promising approach to reducing the morbidity and mortality from lung cancer because the survival of early stage lung cancer patients is better than that of patients with advanced cancers. We approached the identification of early biomarkers of human lung carcinogenesis, by combining the use of cDNA array and an in vitro human lung carcinogenesis model that consists of normal (NHBE), immortalized (BEAS-2B and 1799), transformed (1198) and tumorigenic (1170-I) human bronchial epithelial (HBE) cells. We hypothesized that certain genes that are expressed differentially among these cells can serve as both biomarkers for early detection and targets for intervention. Nineteen genes were down-regulated and six were up-regulated in tumorigenic 1170-I HBE cells compared to normal HBE cells (NHBE) using cDNA array. Down- regulated genes encode cell-to-cell and cell-to-matrix adhesion-related proteins, calcium-binding proteins and some enzymes or growth factor-related proteins. The functions of up-regulated gene were more variable. Similar expression of selected genes was found in different non-small cell lung cancer cell lines analyzed by Northern blotting. Furthermore, the differential expression of some genes was also observed by in silico analysis of database of human lung tumors. The differentially expressed genes encode calcium-binding proteins and cell-cell and cell-matrix adhesion proteins. Furthermore, Northern blotting analysis of RNA from different cell lines comprising an in vitro carcinogenesis model including normal, immortalized, transformed, and tumorigenic cells indicated that the expression of subsets of the identified genes changed at different stages of carcinogenesis. These data show that the in vitro carcinogenesis cell system could be useful for discovering potential biomarkers of early and late stages of lung carcinogenesis and targets for chemoprevention.

Mechanisms of the combined effect of asbestos and smoking in the etiology of lung cancer.

The joint effects of exposure to two known lung carcinogens, tobacco smoking and asbestos, are reviewed. The variable pattern of interaction--ranging from supramultiplicative to less than additive--may reflect the fact that both asbestos and smoking are complex carcinogens which can affect more than one stage of lung carcinogenesis. The joint effect of two such agents will depend on the relative magnitude of the effects at each stage. The epidemiologic evidence from studies of insulation workers with high exposures suggests an interaction that approximates the multiplicative model, indicating that each of the two factors has an independent action on the multistage process of carcinogenesis. Very limited information is available on the interaction between these two agents in causing specific histological types of lung cancer. Both tobacco smoke and asbestos fibers can be genotoxic and cytotoxic and cause proliferative lesions in the lungs. Tobacco smoke is known to contain carcinogens that bind to critical genes in DNA (deoxyribonucleic acid) and cause mutations. Asbestos fibers may cause chronic inflammation of the lungs, which releases various cytokines and growth factors, and therefore may provide a possible selective growth advantage for mutated cells.