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1.  Characterizing the cancer genome in lung adenocarcinoma 
Nature  2007;450(7171):893-898.
Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.
doi:10.1038/nature06358
PMCID: PMC2538683  PMID: 17982442
2.  Combining COPD with Clinical, Pathological and Demographic Information Refines Prognosis and Treatment Response Prediction of Non-Small Cell Lung Cancer 
PLoS ONE  2014;9(6):e100994.
Background
Accurate assessment of a patient’s risk of recurrence and treatment response is an important prerequisite of personalized therapy in lung cancer. This study extends a previously described non-small cell lung cancer prognostic model by the addition of chemotherapy and co-morbidities through the use of linked SEER-Medicare data.
Methodology/Principal Findings
Data on 34,203 lung adenocarcinoma and 26,967 squamous cell lung carcinoma patients were used to determine the contribution of Chronic Obstructive Pulmonary Disease (COPD) to prognostication in 30 treatment combinations. A Cox model including COPD was estimated on 1,000 bootstrap samples, with the resulting model assessed on ROC, Brier Score, Harrell’s C, and Nagelkerke’s R2 metrics in order to evaluate improvements in prognostication over a model without COPD. The addition of COPD to the model incorporating cancer stage, age, gender, race, and tumor grade was shown to improve prognostication in multiple patient groups. For lung adenocarcinoma patients, there was an improvement on the prognostication in the overall patient population and in patients without receiving chemotherapy, including those receiving surgery only. For squamous cell carcinoma, an improvement on prognostication was seen in both the overall patient population and in patients receiving multiple types of chemotherapy. COPD condition was able to stratify patients receiving the same treatments into significantly (log-rank p<0.05) different prognostic groups, independent of cancer stage.
Conclusion/Significance
Combining patient information on COPD, cancer stage, age, gender, race, and tumor grade could improve prognostication and prediction of treatment response in individual non-small cell lung cancer patients. This model enables refined prognosis and estimation of clinical outcome of comprehensive treatment regimens, providing a useful tool for personalized clinical decision-making.
doi:10.1371/journal.pone.0100994
PMCID: PMC4072724  PMID: 24967586
3.  Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations 
Molecular cancer research : MCR  2013;11(6):638-650.
We used CDK4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, MYC) and followed the stepwise transformation of HBECs to full malignancy. This model demonstrated that: 1) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; 2) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; 3) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 4) high levels of KRASV12 are required for full malignant transformation of HBECs, however prior loss of p53 function is required to prevent oncogene-induced senescence; 5) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 6) growth of parental HBECs in serum-containing medium induces differentiation while growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); 7) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; 8) an mRNA signature derived by comparing tumorigenic vs. non-tumorigenic clones was predictive of outcome in lung cancer patients. Collectively, our findings demonstrate this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.
doi:10.1158/1541-7786.MCR-12-0634-T
PMCID: PMC3687022  PMID: 23449933
p53; KRAS; c-MYC; immortalized human bronchial epithelial cell; in vitro transformation model of lung cancer; epithelial mesenchymal transition
5.  Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer 
Scientific Reports  2014;4:4480.
Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.
doi:10.1038/srep04480
PMCID: PMC3967199  PMID: 24670678
6.  MicroRNA-Related Genetic Variants Associated with Clinical Outcomes in Early Stage Non-Small Cell Lung Cancer Patients 
Cancer research  2013;73(6):1867-1875.
Given the density of single nucleotide polymorphisms (SNPs) in the human genome and the sensitivity of single nucleotide changes in microRNA (miRNA) functionality and processing, we asked whether polymorphisms within miRNA processing pathways and binding sites may influence non-small cell lung cancer (NSCLC) patients’ prognosis. We genotyped 240 miRNA-related SNPs in 535 stage I and II NSCLC patients to determine associations with overall recurrence and survival, as well as effect in specific treatment subgroups. After correcting for multiple comparisons, the G allele of FZD4:rs713065 displayed a significant association with decreased risk of death in surgery-only patients (HR:0.46, 95%CI:0.32-0.65). DROSHA:rs6886834 variant A allele (HR:6.38, 95%CI:2.49-16.31) remained significant for increased risk of recurrence in the overall and surgery-only populations, respectively. FAS:rs2234978 G allele remained significantly associated with survival in all patients (HR:0.59, 95%CI:0.44-0.77), while borderline significant in subgroups (surgery only: HR:0.59, 95%CI:0.42-0.84; surgery plus chemo: HR:0.19, 95%CI:0.07-0.46). Luciferase assays demonstrated that the FAS SNP created a miR-651 functional binding site. Survival tree analysis was performed to classify patients into distinct risk subgroups based on their risk genotype combinations. These results indicate that miRNA-related polymorphisms may be associated with NSCLC patients’ clinical outcomes through altered miRNA regulation of target genes.
doi:10.1158/0008-5472.CAN-12-0873
PMCID: PMC3602350  PMID: 23378343
NSCLC; recurrence; overall survival; early stage; miRNA; binding site; single nucleotide polymorphism
7.  GWAS Meets TCGA to Illuminate Mechanisms of Cancer Predisposition 
Cell  2013;152(3):10.1016/j.cell.2013.01.027.
Genome-wide association studies (GWASs) have unraveled a large number of cancer risk alleles. Understanding how these allelic variants predispose to disease is a major bottleneck confronting translational application. In this issue, Li and colleagues combine GWASs with The Cancer Genome Atlas (TCGA) to disambiguate the contributions of germline and somatic variants to tumorigenic gene expression programs. They find that close to half of the known risk alleles for estrogen receptor (ER)-positive breast cancer are expression quantitative trait loci (eQTLs) acting upon major determinants of gene expression in tumors.
doi:10.1016/j.cell.2013.01.027
PMCID: PMC3813952  PMID: 23374335
8.  The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma 
Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.
doi:10.1002/ijc.27598
PMCID: PMC3479344  PMID: 22510946
apoptosis; BMAL1; mesothelioma; targeted therapy; mitotic catastrophe
9.  Comparison between concurrent and sequential chemoradiation for non-small cell lung cancer in vitro 
Oncology Letters  2013;7(2):307-310.
Current practice guidelines recommend the combination of chemotherapy and thoracic radiation for locally advanced non-small cell lung cancer (NSCLC). Previous meta-analyses have shown that concurrent chemoradiation (CCRT) may be superior to sequential chemoradiation (SCRT). However, few previous in vitro studies have analyzed these two treatment schedules. In the current study, four lung cancer cell lines harboring wild-type epidermal growth factor receptor, comprising two squamous and two non-squamous cell lines, were used. The IC10 concentrations of three platinum-based regimens were combined with radiation treatment. Cells were irradiated at 0, 2, 4, 6 and 8 Gy using a 137Cs irradiator concurrently or sequentially. Surviving fractions (SFs) were plotted as a function of the radiation dose. In A549 cells, only the docetaxel (Doc) and carboplatin (Carbo) combination showed a significant radiosensitizing effect with CCRT treatment. For the other three cell lines, no difference was identified in the SFs between CCRT and SCRT. An in vitro method of comparing CCRT with SCRT was established using lung cancer cell lines. Overall, no significant difference was detected in the radiosensitizing effect of the two treatment schedules, with the exception of the A549 cell lines treated with Doc/Carbo.
doi:10.3892/ol.2013.1707
PMCID: PMC3881197  PMID: 24396437
chemotherapy; radiation; concurrent; sequential; carcinoma; non-small cell lung cancer; cell line
10.  Genome-wide association study of genetic predictors of overall survival for non-small cell lung cancer in never smokers 
Cancer research  2013;73(13):4028-4038.
To identify the genetic factors that influence overall survival in never smokers who have non-small cell lung cancer (NSCLC), we performed a consistency meta-analysis study utilizing genome-wide association approaches for overall survival in 327 never smoker NSCLC patients from the MD Anderson Cancer Center and 293 cases from the Mayo Clinic. We then performed a two-pronged validation of the top 25 variants that included additional validation in 1,256 NSCLC patients from Taiwan and assessment of expression quantitative trait loci (eQTL) and differential expression of genes surrounding the top loci in 70 tumors and matched normal tissues. A total of 94 loci were significant for overall survival in both MD Anderson and Mayo studies in the consistency meta-analysis phase, with the top 25 variants reaching a p-value of 10−6. Two variants of these 25 were also significant in the Taiwanese population: rs6901416 (HR:1.44, 95%CI:1.01-2.06) and rs10766739 (HR:1.23, 95%CI:1.00-1.51). These loci resulted in a reduction in median survival time of at least 8 and 5 months in three populations, respectively. An additional six variants (rs4237904, rs7976914, rs4970833, rs954785, rs485411, and rs10906104) were validated through eQTL analysis that identified significant correlations with expression levels of six genes (LEMD3, TMBIM, ATXN7L2, SHE, ITIH2, and NUDT5, respectively) in normal lung tissue. These genes were also significantly differentially expressed between the tumor and normal lung. These findings identify several novel, candidate prognostic markers for NSCLC in never smokers, with eQTL analysis suggesting a potential biological mechanism for a subset of these observed associations.
doi:10.1158/0008-5472.CAN-12-4033
PMCID: PMC3719971  PMID: 23704207
11.  Statin Use and Risk of Lung Cancer: a Meta-Analysis of Observational Studies and Randomized Controlled Trials 
PLoS ONE  2013;8(10):e77950.
Clinical studies have shown that statin use may alter the risk of lung cancer. However, these studies yielded different results. To quantify the association between statin use and risk of lung cancer, we performed a detailed meta-analysis. A literature search was carried out using MEDLINE, EMBASE and COCHRANE database between January 1966 and November 2012. Before meta-analysis, between-study heterogeneity and publication bias were assessed using adequate statistical tests. Fixed-effect and random-effect models were used to calculate the pooled relative risks (RR) and corresponding 95% confidence intervals (CIs). Subgroup analyses, sensitivity analysis and cumulative meta-analysis were also performed. A total of 20 (five randomized controlled trials, eight cohorts, and seven case–control) studies contributed to the analysis. Pooled results indicated a non-significant decrease of total lung cancer risk among all statin users (RR = 0.89, 95% CI [0.78, 1.02]). Further, long-term statin use did not significantly decrease the risk of total lung cancer (RR = 0.80, 95% CI [0.39 , 1.64]). In our subgroup analyses, the results were not substantially affected by study design, participant ethnicity, or confounder adjustment. Furthermore, sensitivity analysis confirmed the stability of results. The findings of this meta-analysis suggested that there was no significant association between statin use and risk of lung cancer. More studies, especially randomized controlled trials and high quality cohort studies are warranted to confirm this association.
doi:10.1371/journal.pone.0077950
PMCID: PMC3808274  PMID: 24205041
12.  Systems Approaches to Modeling Chronic Mucosal Inflammation 
BioMed Research International  2013;2013:505864.
The respiratory mucosa is a major coordinator of the inflammatory response in chronic airway diseases, including asthma and chronic obstructive pulmonary disease (COPD). Signals produced by the chronic inflammatory process induce epithelial mesenchymal transition (EMT) that dramatically alters the epithelial cell phenotype. The effects of EMT on epigenetic reprogramming and the activation of transcriptional networks are known, its effects on the innate inflammatory response are underexplored. We used a multiplex gene expression profiling platform to investigate the perturbations of the innate pathways induced by TGFβ in a primary airway epithelial cell model of EMT. EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF-κB pathways. Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF-κB2 is required to reduce the noncanonical pathway coupling interval. Experiments using amantadine confirmed the prediction that TRAF-1 and NF-κB2/p100 production is mediated by an IRES-dependent mechanism. These data indicate that the epigenetic changes produced by EMT induce dynamic state changes of the innate signaling pathway. Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.
doi:10.1155/2013/505864
PMCID: PMC3818818  PMID: 24228254
13.  The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner 
PLoS ONE  2013;8(10):e77067.
TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.
doi:10.1371/journal.pone.0077067
PMCID: PMC3798310  PMID: 24146957
14.  Molecular biology of lung cancer 
Journal of Thoracic Disease  2013;5(Suppl 5):S479-S490.
Lung cancers are characterised by abundant genetic diversity with relatively few recurrent mutations occurring at high frequency. However, the genetic alterations often affect a common group of oncogenic signalling pathways. There have been vast improvements in our understanding of the molecular biology that underpins lung cancer in recent years and this has led to a revolution in the diagnosis and treatment of lung adenocarcinomas (ADC) based on the genotype of an individual’s tumour. New technologies are identifying key and potentially targetable genetic aberrations not only in adenocarcinoma but also in squamous cell carcinoma (SCC) of the lung. Lung cancer mutations have been identified in v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), BRAF and the parallel phosphatidylinositol 3-kinase (PI3K) pathway oncogenes and more recently in MEK and HER2 while structural rearrangements in ALK, ROS1 and possibly rearranged during transfection (RET) provide new therapeutic targets. Amplification is another mechanism of activation of oncogenes such as MET in adenocarcinoma, fibroblastgrowth factor receptor 1 (FGFR1) and discoidin domain receptor 2 (DDR2) in SCC. Intriguingly, many of these genetic alternations are associated with smoking status and with particular racial and gender differences, which may provide insight into the mechanisms of carcinogenesis and role of host factors in lung cancer development and progression. The role of tumour suppressor genes is increasingly recognised with aberrations reported in TP53, PTEN, RB1, LKB11 and p16/CDKN2A. Identification of biologically significant genetic alterations in lung cancer that lead to activation of oncogenes and inactivation of tumour suppressor genes has the potential to provide further therapeutic opportunities. It is hoped that these discoveries may make a major contribution to improving outcome for patients with this poor prognosis disease.
doi:10.3978/j.issn.2072-1439.2013.08.03
PMCID: PMC3804875  PMID: 24163741
Lung cancer; mutation; molecular pathology; oncogene; tumour suppressor gene
15.  Probe mapping across multiple microarray platforms 
Briefings in Bioinformatics  2011;13(5):547-554.
Access to gene expression data has become increasingly common in recent years; however, analysis has become more difficult as it is often desirable to integrate data from different platforms. Probe mapping across microarray platforms is the first and most crucial step for data integration. In this article, we systematically review and compare different approaches to map probes across seven platforms from different vendors: U95A, U133A and U133 Plus 2.0 from Affymetrix, Inc.; HT-12 v1, HT-12v2 and HT-12v3 from Illumina, Inc.; and 4112A from Agilent, Inc. We use a unique data set, which contains 56 lung cancer cell line samples—each of which has been measured by two different microarray platforms—to evaluate the consistency of expression measurement across platforms using different approaches. Based on the evaluation from the empirical data set, the BLAST alignment of the probe sequences to a recent revision of the Transcriptome generated better results than using annotations provided by Vendors or from Bioconductor's Annotate package. However, a combination of all three methods (deemed the ‘Consensus Annotation’) yielded the most consistent expression measurement across platforms. To facilitate data integration across microarray platforms for the research community, we develop a user-friendly web-based tool, an API and an R package to map data across different microarray platforms from Affymetrix, Illumina and Agilent. Information on all three can be found at http://qbrc.swmed.edu/software/probemapper/.
doi:10.1093/bib/bbr076
PMCID: PMC3431719  PMID: 22199380
microarray; gene expression; probe; integrated analysis; probe mapping
16.  Global evaluation of Eph receptors and ephrins in lung adenocarcinomas identifies EphA4 as an inhibitor of cell migration and invasion 
Molecular cancer therapeutics  2012;11(9):2021-2032.
The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. Our aim was to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma versus normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were performed in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer versus non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, likely by affecting cancer cell migration and invasion.
doi:10.1158/1535-7163.MCT-12-0030
PMCID: PMC3438283  PMID: 22807579
non-small cell lung cancer; adenocarcinoma; Eph receptor; ephrin; prognosis
17.  Molecular Biology of Lung Cancer: Clinical Implications 
Clinics in Chest Medicine  2011;32(4):703-740.
doi:10.1016/j.ccm.2011.08.003
PMCID: PMC3367865  PMID: 22054881
18.  E2F Inhibition Synergizes with Paclitaxel in Lung Cancer Cell Lines 
PLoS ONE  2014;9(5):e96357.
The CDK/Rb/E2F pathway is commonly disrupted in lung cancer, and thus, it is predicted that blocking the E2F pathway would have therapeutic potential. To test this hypothesis, we have examined the activity of HLM006474 (a small molecule pan-E2F inhibitor) in lung cancer cell lines as a single agent and in combination with other compounds. HLM006474 reduces the viability of both SCLC and NSCLC lines with a biological IC50 that varies between 15 and 75 µM, but with no significant difference between the groups. Combination of HLM006474 with cisplatin and gemcitabine demonstrate little synergy; however, HLM006474 synergizes with paclitaxel. Surprisingly, we discovered that brief treatment of cells with HLM006474 led to an increase of E2F3 protein levels (due to de-repression of these promoter sites). Since paclitaxel sensitivity has been shown to correlate with E2F3 levels, we hypothesized that HLM006474 synergy with paclitaxel may be mediated by transient induction of E2F3. To test this, H1299 cells were depleted of E2F3a and E2F3b with siRNA and treated with paclitaxel. Assays of proliferation showed that both siRNAs significantly reduced paclitaxel sensitivity, as expected. Taken together, these results suggest that HLM006474 may have efficacy in lung cancer and may be useful in combination with taxanes.
doi:10.1371/journal.pone.0096357
PMCID: PMC4022639  PMID: 24831239
19.  How Do We Safely Get People to Stop Smoking? 
Nicotine replacement therapy (NRT) is a valuable, proven, and U.S. Food and Drug Administration–approved tool for smoking cessation. However, the discoveries of functional nicotinic acetylcholine receptors (nAChR) on lung epithelial and cancer cells and of nAChR polymorphisms associated with lung cancer risk, in addition to a large number of preclinical studies indicating that nicotine may promote or facilitate cancer development and growth, have prompted concern that NRT, although important for smoking cessation, may actually augment lung carcinogenesis. Therefore, it is of great public health interest that two independent studies reported in this issue of the journal (Murphy and colleagues, beginning on page 1752, and Maier and colleagues, beginning on page 1743) showed that nicotine given in drinking water at a dose to achieve blood concentrations in mice similar to those achieved in people receiving NRT did not enhance lung carcinogenesis or tumor growth in several mouse models of lung cancer. Effective non-nicotine alternatives to NRT, such as varenicline and bupropion, are also available and perhaps better than NRT for smoking cessation therapy. In the near future, nicotine vaccines will likely be added to the smoking cessation armamentarium. However, the normal and pathophysiologic role of nicotine, nAChRs, and the signaling pathways they activate in lung epithelial cells and lung cancer still requires elucidation.
doi:10.1158/1940-6207.CAPR-11-0449
PMCID: PMC3372398  PMID: 22052339
20.  Distinct transcriptome profiles identified in normal human bronchial epithelial cells after exposure to γ-rays and different elemental particles of high Z and energy 
BMC Genomics  2013;14:372.
Background
Ionizing radiation composed of accelerated ions of high atomic number (Z) and energy (HZE) deposits energy and creates damage in cells in a discrete manner as compared to the random deposition of energy and damage seen with low energy radiations such as γ- or x-rays. Such radiations can be highly effective at cell killing, transformation, and oncogenesis, all of which are concerns for the manned space program and for the burgeoning field of HZE particle radiotherapy for cancer. Furthermore, there are differences in the extent to which cells or tissues respond to such exposures that may be unrelated to absorbed dose. Therefore, we asked whether the energy deposition patterns produced by different radiation types would cause different molecular responses. We performed transcriptome profiling using human bronchial epithelial cells (HBECs) after exposure to γ-rays and to two different HZE particles (28Si and 56Fe) with different energy transfer properties to characterize the molecular response to HZE particles and γ-rays as a function of dose, energy deposition pattern, and time post-irradiation.
Results
Clonogenic assay indicated that the relative biological effectiveness (RBE) for 56Fe was 3.91 and for 28Si was 1.38 at 34% cell survival. Unsupervised clustering analysis of gene expression segregated samples according to the radiation species followed by the time after irradiation, whereas dose was not a significant parameter for segregation of radiation response. While a subset of genes associated with p53-signaling, such as CDKN1A, TRIM22 and BTG2 showed very similar responses to all radiation qualities, distinct expression changes were associated with the different radiation species. Gene enrichment analysis categorized the differentially expressed genes into functional groups related to cell death and cell cycle regulation for all radiation types, while gene pathway analysis revealed that the pro-inflammatory Acute Phase Response Signaling was specifically induced after HZE particle irradiation. A 73 gene signature capable of predicting with 96% accuracy the radiation species to which cells were exposed, was developed.
Conclusions
These data suggest that the molecular response to the radiation species used here is a function of the energy deposition characteristics of the radiation species. This novel molecular response to HZE particles may have implications for radiotherapy including particle selection for therapy and risk for second cancers, risk for cancers from diagnostic radiation exposures, as well as NASA’s efforts to develop more accurate lung cancer risk estimates for astronaut safety. Lastly, irrespective of the source of radiation, the gene expression changes observed set the stage for functional studies of initiation or progression of radiation-induced lung carcinogenesis.
doi:10.1186/1471-2164-14-372
PMCID: PMC3680091  PMID: 23724988
Gene expression; HZE particles; Ionizing radiation; Human bronchial epithelial cells
21.  Gene Expression Profile and Toxic Effects in Human Bronchial Epithelial Cells Exposed to Zearalenone 
PLoS ONE  2014;9(5):e96404.
Zearalenone (ZEA), a mycoestrogen produced by Fusarium fungal species, is mainly found in cereal crops such as maize, wheat and barley. Although ZEA has been reported to be present in air, little is known about the health risk or the molecular basis of action when lung cells are exposed to ZEA. As ZEA has a similar structure to estrogen, its potential risk as an endocrine disrupting chemical (EDC) has thus aroused both environmental and public health concerns. The purpose of this study is to identify the responses and underlying molecular changes that occur when human bronchial epithelial BEAS-2B cells are exposed to ZEA. Differential gene expression profiles were identified in cells that were treated with 40 µM ZEA for 6 h and 24 h by high-throughput microarray analysis using Affymetrix Human Gene 2.0 GeneChip. The array results showed that after ZEA treatment, 262 genes at 6 h and 1073 genes at 24 h were invovled in the differential regulation. Pathway analysis revealed that diverse cellular processes were affected when lung cells were exposed to ZEA resulting in impaired response to DNA damage, cell cycle arrest, down-regulation of inflammatory responses and alterations of epigenetic marks. Results of further experiments indicated that 40 µM ZEA decreased cell viability, induced apoptosis and promoted reactive oxygen species (ROS) generation in a time-dependent manner. Immuno-suppressive effects of ZEA were further revealed through the suppression of lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1β). Interestingly, the level of global DNA methylation was markedly decreased after 24 h exposure to ZEA. Collectively, these observations suggested that a broad range of toxic effects are elicited by ZEA. Particularly, ROS may play a pivotal role in ZEA-induced cell death. These adverse effects observed in lung cells suggest that exposure to ZEA may increase susceptibility of lung cells to diseases and required further investigations.
doi:10.1371/journal.pone.0096404
PMCID: PMC4008614  PMID: 24788721
22.  High Incidence of Ischemic Stroke Occurrence in Irradiated Lung Cancer Patients: A Population-Based Surgical Cohort Study 
PLoS ONE  2014;9(4):e94377.
Background and Purpose
A high risk of stroke occurrence has been reported in several types of irradiated cancer patients. However, clinical data are lacking in irradiated lung cancer patients. The present study intended to explore a risk level of ischemic stroke occurrence in irradiated lung cancer patients.
Methods
A nationwide population-based database obtained from the Taiwan National Health Insurance was analyzed. Between 2003 and 2006, we recruited 560 resected lung cancer patients into two study groups: surgery-plus-irradiation (n = 112) and surgery-alone (n = 448). Patients treated with chemotherapy were excluded. Propensity score match was used for pairing cases with a ratio of 1∶4. Two-year ischemic-stroke-free survival was defined as the primary endpoint.
Results
Three observations supported a high risk of ischemic stroke occurrence in patients with postoperative irradiation when compared with those patients with surgery alone: first, a high incidence per 1,000 person-year (22.3 versus 11.2, 1.99 folds); second, a low two-year ischemic-stroke-free survival rate (92.2% versus 98.1%, P = 0.019); and third, a high adjusted hazard ratio (HR, 4.19; 95% CI, 1.44–12.22; P = 0.009). More notably, the highest risk of ischemic stroke occurrence was found in irradiated patients who had diabetes mellitus (HR, 34.74; 95% CI, 6.35->100; P<0.0001).
Conclusions
A high incidence of ischemic stroke was observed in irradiated lung cancer patients, especially in those with diabetes mellitus. For these patients, close clinical surveillance and strict diabetes control should be considered. Further studies to define detail biological mechanisms are encouraged.
doi:10.1371/journal.pone.0094377
PMCID: PMC3978039  PMID: 24709837
23.  Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome 
Background
Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non–small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting.
Methods
We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan–Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided.
Results
Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor–dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers.
Conclusions
Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.
doi:10.1093/jnci/djr523
PMCID: PMC3274509  PMID: 22247021
24.  SMAC mimetic (JP1201) sensitizes non-small cell lung cancers to multiple chemotherapy agents in an IAP dependent but TNFα independent manner 
Cancer Research  2011;71(24):7640-7648.
Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are inhibited by the second mitocondrial activator of caspases (SMAC). Previously, a small subset of TNFα-expressing non-small cell lung cancers (NSCLCs) was found to be sensitive to SMAC mimetics alone. In this study we determined if a SMAC mimetic (JP1201) could sensitize non-responsive NSCLC cell lines to standard chemotherapy. We found that JP1201 sensitized NSCLCs to doxorubicin, erlotinib, gemcitabine, paclitaxel, vinorelbine, and the combination of carboplatin with paclitaxel in a synergistic manner at clinically achievable drug concentrations. Sensitization did not occur with platinum alone. Furthermore, sensitization was specific for tumor compared to normal lung epithelial cells, increased in NSCLCs harvested after chemotherapy treatment, and did not induce TNFα secretion. Sensitization also was enhanced in vivo with increased tumor inhibition and increased survival of mice carrying xenografts. These effects were accompanied by caspase 3, 4, and 9 activation, indicating that both mitochondrial and ER stress-induced apoptotic pathways are activated by the combination of vinorelbine and JP1201. Chemotherapies that induce cell death through the mitochondrial pathway required only inhibition of XIAP for sensitization, while chemotherapies that induce cell death through multiple apoptotic pathways required inhibition of cIAP1, cIAP2, and XIAP. Therefore, the data suggest that IAP-targeted therapy using a SMAC mimetic provides a new therapeutic strategy for synergistic sensitization of NSCLCs to standard chemotherapy agents, which appears to occur independently of TNFα secretion.
doi:10.1158/0008-5472.CAN-10-3947
PMCID: PMC3382117  PMID: 22049529
non-small cell lung cancer; smac mimetic; vinorelbine; gemcitabine; IAPs
25.  Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion 
Loss of terminal cell differentiation promotes tumorigenesis. 15-LOX-1 contributes to terminal cell differentiation in normal cells. The mechanistic significance of 15-LOX-1 expression loss in human cancers to terminal cell differentiation suppression is unknown. In a screen of 128 cancer cell lines representing more than 20 types of human cancer, we found that 15-LOX-1 mRNA expression levels were markedly lower than levels in terminally differentiated cells. Relative expression levels of 15-LOX-1 (relative to the level in terminally differentiated primary normal human-derived bronchial epithelial cells) were lower in 79% of the screened cancer cell lines than relative expression levels of p16 (INK4A), which promotes terminal cell differentiation and is considered one of the most commonly lost tumor suppressor genes in cancer cells. 15-LOX-1 was expressed during terminal differentiation in three-dimensional air-liquid interface cultures, and 15-LOX-1 expression and terminal differentiation occurred in immortalized non-transformed bronchial epithelial but not lung cancer cell lines. 15-LOX-1 expression levels were lower in human tumors than paired normal lung epithelia. Short hairpin RNA-mediated downregulation of 15-LOX-1 in Caco-2 cells blocked enterocyte-like differentiation, disrupted tight junction formation, and blocked E-cadherin and ZO-1 localization to the cell wall membrane. 15-LOX-1 episomal expression in Caco-2 and HT-29 colon cancer cells induced differentiation. Our findings indicate that 15-LOX-1 downregulation in cancer cells is an important mechanism for terminal cell differentiation dysregulation and support the potential therapeutic utility of 15-LOX-1 re-expression to inhibit tumorigenesis.
doi:10.1158/1940-6207.CAPR-10-0280
PMCID: PMC3232310  PMID: 21881028
15-lipoxygenase-1; terminal cell differentiation; tumorigenesis

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