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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.  Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway 
Purpose
Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated.
Experimental Design
A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays.
Results
We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival.
Conclusion
Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs.
doi:10.1158/1078-0432.CCR-13-3292
PMCID: PMC4438754  PMID: 24907115
Lung cancer; cancer stem cells; ALDH1A3; STAT3; Stattic
3.  Epigenetic Inactivation of RASSF1A in Lung and Breast Cancers and Malignant Phenotype Suppression 
Background
The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers.
Methods
We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided.
Results
RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P = .046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity.
Conclusion
RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.
PMCID: PMC4374741  PMID: 11333291
4.  A novel molecular pathway for Snail-dependent, SPARC-mediated invasion in non-small cell lung cancer pathogenesis 
Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to upregulation of Secreted Protein, Acidic and Rich in Cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates TGF-β, ERK1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-βR2 are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulated SPARC expression. Inhibition of MEK phosphorylation downregulated SPARC. MiR-29b is downregulated in Snail overexpressing cell lines, while overexpression of miR-29b inhibited SPARC expression. In addition, miR-29b was upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.
doi:10.1158/1940-6207.CAPR-13-0263
PMCID: PMC3919444  PMID: 24253315
NSCLC; Snail; SPARC; invasion; parallel progression
5.  Loss of miR-125a expression in a model of K-ras-dependent pulmonary premalignancy 
Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)-125a-3p (miR-125a) contributes to the malignant potential of human bronchial epithelial cells (HBECs) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR-125a loss in HBEC K-ras lines and determined that miR-125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR-125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR-125a overexpression significantly reduces K-ras-mediated production of these tumorigenic factors. miR-125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR-125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR-125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR-125a loss in lung carcinogenesis and lay the groundwork for future studies to determine if miR-125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR-125a in K-ras-mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation.
doi:10.1158/1940-6207.CAPR-14-0063
PMCID: PMC4125465  PMID: 24913817
miR-125a; pulmonary premalignancy; molecular markers; PEA3
6.  TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival 
Cancer science  2013;104(2):171-177.
TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7-fold (P < 0.001) in non-small cell lung cancer cell lines (n = 116) compared to normal lung controls (n = 59). In addition, small cell lung cancer cell lines (n = 29) expressed TIM at levels 2.2-fold (P < 0.001) higher than non-small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.
doi:10.1111/cas.12068
PMCID: PMC4454395  PMID: 23173913
7.  Aberrant large tumor suppressor 2 (LATS2) gene expression correlates with EGFR mutation and survival in lung adenocarcinomas 
Background
Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines.
Methods
LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed.
Results
Fifty resected lung AD were included (M:F = 23:27, smokers:non-smokers = 19:31, EGFR mutant:wild-type = 21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR = 0.217; p = 0.003; Overall survival RR = 0.238; p = 0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels.
Conclusions
LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors.
doi:10.1016/j.lungcan.2014.05.025
PMCID: PMC4451109  PMID: 24976335
Lung cancer; Large tumor suppressor 2 gene; Gene expression; Gene silencing; EGFR signaling
8.  Analysis of TP53 Mutation Status in Human Cancer Cell Lines: A Reassessment 
Human mutation  2014;35(6):756-765.
Tumor-derived cell lines play an important role in the investigation of tumor biology and genetics. Across a wide array of studies, they have been tools of choice for the discovery of important genes involved in cancer and for the analysis of the cellular pathways that are impaired by diverse oncogenic events. They are also invaluable for screening novel anticancer drugs. The TP53 protein is a major component of multiple pathways that regulate cellular response to various types of stress. Therefore, TP53 status affects the phenotype of tumor cell lines profoundly and must be carefully ascertained for any experimental project. In the present review, we use the 2014 release of the UMD TP53 database to show that TP53 status is still controversial for numerous cell lines, including some widely used lines from the NCI-60 panel. Our analysis clearly confirms that, despite numerous warnings, the misidentification of cell lines is still present as a silent and neglected issue, and that extreme care must be taken when determining the status of p53, because errors may lead to disastrous experimental interpretations. A novel compendium gathering the TP53 status of 2,500 cell lines has been made available (http://p53.fr). A stand-alone application can be used to browse the database and extract pertinent information on cell lines and associated TP53 mutations. It will be updated regularly to minimize any scientific issues associated with the use of misidentified cell lines (http://p53.fr).
doi:10.1002/humu.22556
PMCID: PMC4451114  PMID: 24700732
TP53; cancer cell line; cross-contamination; misidentification; recommendation
9.  Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer 
Cancer cell  2006;10(1):39-50.
Summary
We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR ligand-dependent signaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in NSCLC and thus offers an excellent opportunity for pharmacological intervention, either alone or in combination with other drugs.
doi:10.1016/j.ccr.2006.05.024
PMCID: PMC4451119  PMID: 16843264
10.  On comparing heterogeneity across biomarkers 
Microscopy reveals complex patterns of cellular heterogeneity that can be biologically informative. However, a limitation of microscopy is that only a small number of biomarkers can typically be monitored simultaneously. Thus, a natural question is whether additional biomarkers provide a deeper characterization of the distribution of cellular states in a population. How much information about a cell’s phenotypic state in one biomarker is gained by knowing its state in another biomarker?
Here, we describe a framework for comparing phenotypic states across biomarkers. Our approach overcomes the current limitation of microscopy by not requiring co-staining biomarkers on the same cells; instead we require staining of biomarkers (possibly separately) on a common collection of phenotypically diverse cell lines.
We evaluate our approach on two image datasets: 33 oncogenically diverse lung cancer cell lines stained with 7 biomarkers, and 49 less diverse subclones of one lung cancer cell line stained with 12 biomarkers. We first validate our method by comparing it to the “gold standard” of co-staining. We then apply our approach to all pairs of biomarkers and use it to identify biomarkers that yield similar patterns of heterogeneity. The results presented in this work suggest that many biomarkers provide redundant information about heterogeneity. Thus, our approach provides a practical guide for selecting independently informative biomarkers and, more generally, will yield insights into both the connectivity of biological networks and the complexity of the state space of biological systems.
doi:10.1002/cyto.a.22599
PMCID: PMC4442742  PMID: 25425168
Heterogeneity; Biomarker Selection; Systems Biology; Microscopy; Bioimage Informatics; Biological Networks; Single-Cell Variability; Information Theory
11.  Ras regulates kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells 
Oncogene  2013;33(47):5457-5466.
We show that expression of the microtubule depolymerizing kinesin KIF2C is induced by transformation of immortalized human bronchial epithelial cells by expression of K-RasG12V and knockdown of p53. Further investigation demonstrates that this is due to the K-Ras/ERK1/2 MAPK pathway, as loss of p53 had little effect on KIF2C expression. In addition to KIF2C, we also found that the related kinesin KIF2A is modestly upregulated in this model system; both proteins are expressed more highly in many lung cancer cell lines compared to normal tissue. As a consequence of their depolymerizing activity, these kinesins increase dynamic instability of microtubules. Depletion of either of these kinesins impairs the ability of cells transformed with mutant K-Ras to migrate and invade matrigel. However, depletion of these kinesins does not reverse the epithelial-mesenchymal transition caused by mutant K-Ras. Our studies indicate that increased expression of microtubule destabilizing factors can occur during oncogenesis to support enhanced migration and invasion of tumor cells.
doi:10.1038/onc.2013.486
PMCID: PMC4025984  PMID: 24240690
12.  Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis 
Oncotarget  2015;6(14):12080-12093.
Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.
PMCID: PMC4494924  PMID: 25900242
Cdk5; retinoblastoma protein; neuroendocrine; medullary thyroid carcinoma; cell cycle
13.  Branching Morphogenesis of Immortalized Human Bronchial Epithelial Cells in Three-Dimensional Culture 
While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases.
doi:10.1016/j.diff.2014.02.003
PMCID: PMC4112006  PMID: 24830354
Fibroblasts; Distal airways; Bronchial epithelial cells; Branching; Differentiation
14.  Aiolos Promotes Anchorage Independence by Silencing p66Shc Transcription in Cancer Cells 
Cancer cell  2014;25(5):575-589.
SUMMARY
Anchorage of tissue cells to their physical environment is an obligate requirement for survival which is lost in mature hematopoietic and in transformed epithelial cells. Here we find that a lymphocyte lineage-restricted transcription factor, Aiolos, is frequently expressed in lung cancers and predicts markedly reduced patient survival. Aiolos decreases expression of a large set of adhesion-related genes, disrupting cell-cell and cell-matrix interactions. Aiolos also reconfigures chromatin structure within the SHC1 gene, causing isoform-specific silencing of the anchorage reporter p66Shc and blocking anoikis in vitro and in vivo. In lung cancer tissues and single cells, p66Shc expression inversely correlates with that of Aiolos. Together, these findings suggest that Aiolos functions as an epigenetic driver of lymphocyte mimicry in metastatic epithelial cancers.
doi:10.1016/j.ccr.2014.03.020
PMCID: PMC4070880  PMID: 24823637
15.  A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets 
Cancer research  2014;74(17):4694-4705.
Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared to other non-tumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications.
doi:10.1158/0008-5472.CAN-13-3725
PMCID: PMC4398029  PMID: 24970476
Cancer/testis antigen; VCX/Y; lung cancer; immunotherapy
16.  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
17.  Radiation-enhanced Lung Cancer Progression in a Transgenic Mouse Model of Lung Cancer is Predictive of Outcomes in Human Lung and Breast Cancer 
Purpose
Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment including the modification of inflammatory responses from anti-tumorigenic to pro-tumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis.
Experimental Design
K-rasLA1 mice were irradiated with various doses and dose regimens and then monitored till death. Microarray analyses were performed using Illumina® BeadChips on whole lung tissue 70 days post-irradiation with a fractionated or acute dose of radiation and compared to age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets.
Results
Radiation exposure accelerates lung cancer progression in the K-rasLA1 lung cancer mouse model with dose fractionation being more permissive for cancer progression. A non-random inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature.
Conclusions
These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by impacting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.
doi:10.1158/1078-0432.CCR-13-2589
PMCID: PMC3961755  PMID: 24486591
Radiation; Lung Cancer; Progression; Mouse models; Genomics
18.  miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer 
Oncogene  2013;33(34):4307-4315.
The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression down-regulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 over-expression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3′UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 over-expression plays an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by down-regulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.
doi:10.1038/onc.2013.381
PMCID: PMC4281941  PMID: 24037530
DAB2; miRNA; miR-93; lung cancer
19.  A Re-evaluation of CD22 Expression by Human Lung Cancer 
Cancer research  2014;74(1):263-271.
CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins.
doi:10.1158/0008-5472.CAN-13-1436
PMCID: PMC3903042  PMID: 24395821
lung cancer; CD22 expression
20.  INTEGRATIVE ANALYSIS OF TWO CELL LINES DERIVED FROM A NON-SMALL-LUNG CANCER PATIENT – A PANOMICS APPROACH 
Cancer cells derived from different stages of tumor progression may exhibit distinct biological properties, as exemplified by the paired lung cancer cell lines H1993 and H2073. While H1993 was derived from chemo-naive metastasized tumor, H2073 originated from the chemo-resistant primary tumor from the same patient and exhibits strikingly different drug response profile. To understand the underlying genetic and epigenetic bases for their biological properties, we investigated these cells using a wide range of large-scale methods including whole genome sequencing, RNA sequencing, SNP array, DNA methylation array, and de novo genome assembly. We conducted an integrative analysis of both cell lines to distinguish between potential driver and passenger alterations. Although many genes are mutated in these cell lines, the combination of DNA- and RNA-based variant information strongly implicates a small number of genes including TP53 and STK11 as likely drivers. Likewise, we found a diverse set of genes differentially expressed between these cell lines, but only a fraction can be attributed to changes in DNA copy number or methylation. This set included the ABC transporter ABCC4, implicated in drug resistance, and the metastasis associated MET oncogene. While the rich data content allowed us to reduce the space of hypotheses that could explain most of the observed biological properties, we also caution there is a lack of statistical power and inherent limitations in such single patient case studies.
PMCID: PMC3940063  PMID: 24297535
21.  Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition 
BMC Genomics  2014;15(1):1079.
Background
DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer.
Results
We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib.
Conclusions
Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1079) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1079
PMCID: PMC4298954  PMID: 25486910
DNA methylation; Epithelial-mesenchymal transition; Erlotinib; Lung cancer
22.  A pan-cancer proteomic perspective on The Cancer Genome Atlas 
Nature communications  2014;5:3887.
Protein levels and function are poorly predicted by genomic and transcriptomic analysis of patient tumors. Therefore, direct study of the functional proteome has the potential to provide a wealth of information that complements and extends genomic, epigenomic and transcriptomic analysis in The Cancer Genome Atlas (TCGA) projects. Here we use reverse-phase protein arrays to analyze 3,467 patient samples from 11 TCGA “Pan-Cancer” diseases, using 181 high-quality antibodies that target 128 total proteins and 53 post-translationally modified proteins. The resultant proteomic data is integrated with genomic and transcriptomic analyses of the same samples to identify commonalities, differences, emergent pathways and network biology within and across tumor lineages. In addition, tissue-specific signals are reduced computationally to enhance biomarker and target discovery spanning multiple tumor lineages. This integrative analysis, with an emphasis on pathways and potentially actionable proteins, provides a framework for determining the prognostic, predictive and therapeutic relevance of the functional proteome.
doi:10.1038/ncomms4887
PMCID: PMC4109726  PMID: 24871328
Proteomics; TCGA; Pan-Cancer; protein expression; protein networks
23.  Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs 
IMPORTANCE
Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials.
OBJECTIVES
To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival.
DESIGN, SETTING, AND PARTICIPANTS
From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival.
INTERVENTIONS
Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies.
MAIN OUTCOMES AND MEASURES
Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival.
RESULTS
From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score–adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006).
CONCLUSIONS AND RELEVANCE
Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival.
doi:10.1001/jama.2014.3741
PMCID: PMC4163053  PMID: 24846037
24.  Proteomic markers of DNA repair and PI3K pathway activation predict response to the PARP inhibitor BMN 673 in small cell lung cancer 
Purpose
SCLC is an aggressive malignancy affecting nearly 30,000 people annually in the United States. We have previously identified elevated PARP1 levels in SCLC and demonstrated in vitro sensitivity to the PARP inhibitors AZD 2281 and AG014699. Here, we evaluate activity of a novel, potent PARP inhibitor, BMN 673, and identify markers of response as a basis for developing predictive markers for clinical application.
Experimental Design
Inhibition of SCLC proliferation by BMN 673 was assayed in vitro and effects on tumor growth were measured in SCLC xenograft models. Protein expression and pathway activation was assessed by reverse phase protein array and western blot analysis. PARP inhibition was confirmed using a PAR ELISA.
Results
We demonstrate striking, single agent activity of BMN 673 in SCLC cell lines and xenografts, with single agent BMN 673 exhibiting in vivo activity similar to cisplatin. Sensitivity to BMN 673 was associated with elevated baseline expression levels of several DNA repair proteins, whereas greater drug resistance was observed in SCLC models with baseline activation of the PI3K/mTOR pathway. Furthermore, we developed and confirmed these data with a novel “DNA repair score” consisting of a group of 17 DNA repair proteins.
Conclusions
Elevated expression of multiple DNA repair proteins, as well as a corresponding “DNA repair protein score,” predict response to BMN 673 in in vitro SCLC models. These observations complement recent work in which PI3K inhibition sensitizes breast cancer models to PARP inhibition, suggesting cooperation between DNA repair and PI3K pathways.
doi:10.1158/1078-0432.CCR-13-1975
PMCID: PMC3882158  PMID: 24077350
PARP1; small cell lung cancer; DNA repair
25.  Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non Small Cell Lung Cancer 
Cell  2013;155(3):10.1016/j.cell.2013.09.041.
SUMMARY
Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occuring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a 7-gene expression signature. Target efficacies were validated in vivo, and mechanism of action studies uncovered new cancer cell biology.
doi:10.1016/j.cell.2013.09.041
PMCID: PMC3836195  PMID: 24243015

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