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1.  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
2.  Comprehensive biomarker analysis and final efficacy results of sorafenib in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial 
Purpose
To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE program.
Patients and Methods
Patients with previously treated non-small–cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).
Results
105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for 8-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 (95% confidence interval [CI], 2.04-3.58) and 8.48 months (95% CI, 5.78-10.97), respectively. Eight-week DCR was higher in patients with wt-EGFR than patients with EGFR mutation (P=0.012), and in patients with EGFR gene copy number gain (FISH positive) versus patients FISH negative (P=0.048). In wt-EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS versus 1.84 months in low SSS, P=0.026) but not with 8-week DCR. Increased expression of fibroblast growth factor-1, NF-kB and hypoxia pathways were identified potential drivers of sorafenib resistance.
Conclusion
Sorafenib demonstrates clinical activity in NSCLC, especially with wt-EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. ClinicalTrials.gov: NCT00411671.
doi:10.1158/1078-0432.CCR-12-1818
PMCID: PMC3905243  PMID: 24166906
multikinase inhibitor; non–small cell lung cancer; sorafenib; biomarkers; targeted treatment
3.  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
4.  VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy 
Purpose
Investigate the mechanisms of regulation and role associated with EZH2 expression in lung cancer cells.
Experimental Design
We investigated the mechanisms of EZH2 expression associated with the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. Additionally, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients’ clinical characteristics.
Results
In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and HIF-1α, and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. Additionally, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti-VEGFR-2 drug AZD2171.
Conclusion
Our results suggest that VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF-1α and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy.
doi:10.1158/1078-0432.CCR-13-1916
PMCID: PMC4190586  PMID: 24850841
EZH2; NSCLC; VEGF/VEGFR-2 pathway; DZNep
5.  Nonlinear Quantitative Radiation Sensitivity Prediction Model Based on NCI-60 Cancer Cell Lines 
The Scientific World Journal  2014;2014:903602.
We proposed a nonlinear model to perform a novel quantitative radiation sensitivity prediction. We used the NCI-60 panel, which consists of nine different cancer types, as the platform to train our model. Important radiation therapy (RT) related genes were selected by significance analysis of microarrays (SAM). Orthogonal latent variables (LVs) were then extracted by the partial least squares (PLS) method as the new compressive input variables. Finally, support vector machine (SVM) regression model was trained with these LVs to predict the SF2 (the surviving fraction of cells after a radiation dose of 2 Gy γ-ray) values of the cell lines. Comparison with the published results showed significant improvement of the new method in various ways: (a) reducing the root mean square error (RMSE) of the radiation sensitivity prediction model from 0.20 to 0.011; and (b) improving prediction accuracy from 62% to 91%. To test the predictive performance of the gene signature, three different types of cancer patient datasets were used. Survival analysis across these different types of cancer patients strongly confirmed the clinical potential utility of the signature genes as a general prognosis platform. The gene regulatory network analysis identified six hub genes that are involved in canonical cancer pathways.
doi:10.1155/2014/903602
PMCID: PMC4083270  PMID: 25032244
6.  27-Hydroxycholesterol Promotes Cell-autonomous ER-positive Breast Cancer Growth 
Cell reports  2013;5(3):637-645.
Summary
To date estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells and it stimulates cell-autonomous, ER-dependent and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally-modulated, non-aromatized ER ligand that promotes ER+ breast tumor growth.
doi:10.1016/j.celrep.2013.10.006
PMCID: PMC3950897  PMID: 24210818
7.  An epithelial-mesenchymal transition (EMT) gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance 
Purpose
EMT has been associated with metastatic spread and EGFR inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using NSCLC cell lines and patients treated in the BATTLE study.
Methods
We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from NSCLC patients. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination) study, and potential therapeutic targets associated with EMT were identified.
Results
Compared with epithelial cells, mesenchymal cells demonstrated significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend towards greater sensitivity to the Axl inhibitor SGI-7079, while the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In NSCLC patients, the EMT signature predicted 8-week disease control in patients receiving erlotinib, but not other therapies.
Conclusion
We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype
doi:10.1158/1078-0432.CCR-12-1558
PMCID: PMC3567921  PMID: 23091115
lung cancer; EMT; EGFR inhibition; PI3K inhibition; Axl
8.  Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1 
Cancer discovery  2012;2(9):798-811.
Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLC and NSCLC. SCLC demonstrated lower levels of several receptor tyrosine kinases and decreased activation of PI3K and Ras/MEK pathways, but significantly increased levels of E2F1-regulated factors including EZH2, thymidylate synthase, apoptosis mediators, and DNA repair proteins. Additionally, poly (ADP-ribose) polymerase 1 (PARP1), a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLC. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than NSCLC, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy.
doi:10.1158/2159-8290.CD-12-0112
PMCID: PMC3567922  PMID: 22961666
9.  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
10.  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
11.  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
12.  Antitumor activity of a novel STAT3 inhibitor and redox modulator in non-small cell lung cancer cells☆ 
Biochemical pharmacology  2012;83(10):1456-1464.
NSC-743380 is a novel STAT3 inhibitor that suppresses the growth of several NCI-60 cancer cell lines derived from different tissues and induces regression of xenograft tumors in vivo at various doses. To evaluate the antitumor activity of NSC-743380 in lung cancer cells, we analyzed the susceptibility of 50 NSCLC cell lines to this compound using cell viability assay. About 32% (16 of 50) of these cell lines were highly susceptible to this compound, with a 50% inhibitory concentration (IC50) of <1 µM. In mechanistic studies, the increased numbers of apoptotic cells as well as increased PARP cleavage showed that cytotoxic effects correlate with apoptosis induction. Treatment with NSC-743380 inhibited transcription factor STAT3 activation and induced ROS production in sensitive human lung cancer cell lines but not in resistant cells. Blocking ROS generation with the antioxidant NDGA dramatically abolished NSC-743380-induced growth suppression and apoptosis, but had minimal effect on NSC-743380-induced STAT3 inhibition, suggesting that STAT3 inhibition is not caused by ROS production. Interestingly, knockdown of STAT3 with use of shSTAT3 induced ROS generation and suppressed tumor cell growth. Moreover, scavenging ROS induced by STAT3 inhibition also diminished antitumor activity of STAT3 inhibition. In vivo administration of NSC-743380 suppressed tumor growth and p-STAT3 in lung tumors. Our results indicate that NSC-743380 is a potent anticancer agent for lung cancer and that its apoptotic effects in lung cancer cells are mediated by induction of ROS through STAT3 inhibition.
doi:10.1016/j.bcp.2012.02.010
PMCID: PMC3391570  PMID: 22387047
Drug development; Lung Cancer; STAT3; Reactive oxygen species
13.  The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA 
Cancer Research  2012;72(9):2275-2284.
The ubiquitin-like SUMO proteins covalently modify protein substrates and regulate their functional properties. In a broad spectrum of cancers, the tumor suppressor PML undergoes ubiquitin-mediated degradation primed by CK2 phosphorylation. Here we report that the SUMO E3-ligase inhibitor PIAS1 regulates oncogenic signaling through its ability to sumoylate PML and the PML-RARA oncoprotein of acute promyelocytic leukemia (APL). PIAS1-mediated SUMOylation of PML promoted CK2 interaction and ubiquitin/proteasome-mediated degradation of PML, attenuating its tumor suppressor functions. In addition, PIAS1-mediated SUMOylation of PML-RARA was essential for induction of its degradation by arsenic trioxide, an effective APL treatment. Moreover, PIAS1 suppression abrogated the ability of arsenic trioxide to trigger apoptosis in APL cells. Lastly, PIAS1 was also essential for PML degradation in non-small cell lung cancer cells, and PML and PIAS1 were inversely correlated in NSCLC cell lines and primary specimens. Together, our findings reveal novel roles for PIAS1 and the SUMOylation machinery in regulating oncogenic networks and the response to leukemia therapy.
doi:10.1158/0008-5472.CAN-11-3159
PMCID: PMC3342450  PMID: 22406621
14.  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
15.  Robust Gene Expression Signature from Formalin-Fixed Paraffin-Embedded Samples Predicts Prognosis of Non-Small-Cell Lung Cancer Patients 
Purpose
The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes.
Experimental Design
We randomly selected 100 Non-Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We micro dissected tumor area from FFPE specimens, and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray data sets were used to validate the prognosis model.
Results
This study demonstrated that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes, can be used to refine the prognosis for stage I lung cancer patients and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature.
Conclusions
We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings.
doi:10.1158/1078-0432.CCR-11-0196
PMCID: PMC3166982  PMID: 21742808
Lung Cancer Prognosis; Gene Expression Signature; Formalin Fixed Paraffin Embedded Samples
16.  Increased VEGFR-2 Gene Copy Is Associated With Chemoresistance and Shorter Survival in Patients with Non-small Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy 
Cancer research  2011;71(16):5512-5521.
Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.
doi:10.1158/0008-5472.CAN-10-2614
PMCID: PMC3159530  PMID: 21724587
17.  STAT3 mediates resistance to MEK inhibitor through microRNA miR-17 
Cancer Research  2011;71(10):3658-3668.
AZD6244 is a small molecule inhibitor of the MEK kinase pathway currently in clinical trials. However, the mechanisms mediating intrinsic resistance to MEK inhibition are not fully characterized. To define molecular mechanisms of MEK inhibitor resistance, we analyzed responses of 38 lung cancer cell lines following AZD6244 treatment and their genome-wide gene expression profiles and identified a panel of genes correlated with sensitivity or resistance to AZD6244 treatment. In particular, Ingenuity pathway analysis revealed that activation of the STAT3 pathway was associated with MEK inhibitor resistance. Inhibition of this pathway by JSI-124, a STAT3-specific small molecule inhibitor, or with STAT3-specific siRNA sensitized lung cancer cells to AZD6244 and induced apoptosis. Moreover, combining a STAT3 inhibitor with AZD6244 induced expression of BIM and polyADP-ribose polymerase (PARP) cleavage, whereas activation of the STAT3 pathway inhibited BIM expression and elicited resistance to MEK inhibitors. We found that the STAT3-regulated microRNA miR-17 played a critical role in MEK inhibitor resistance, such that miR-17 inhibition sensitized resistant cells to AZD6244 by inducing BIM and PARP cleavage. Together, these results indicated that STAT3-mediated overexpression of miR-17 blocked BIM expression and caused resistance to AZD6244. Our findings suggest novel approaches to overcome resistance to MEK inhibitors by combining AZD6244 with STAT3 or miR-17 inhibitors.
doi:10.1158/0008-5472.CAN-10-3647
PMCID: PMC3392199  PMID: 21444672
Gene expression profiling; MEK inhibitor resistance; AZD6244; STAT3 pathway; miR-17
18.  Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells 
Cancer Science  2011;102(8):1493-1500.
Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27Kip1 upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.
doi:10.1111/j.1349-7006.2011.01973.x
PMCID: PMC3381954  PMID: 21535318
19.  Research Resource: Diagnostic and Therapeutic Potential of Nuclear Receptor Expression in Lung Cancer 
Molecular Endocrinology  2012;26(8):1443-1454.
Lung cancer is the leading cause of cancer-related death. Despite a number of studies that have provided prognostic biomarkers for lung cancer, a paucity of reliable markers and therapeutic targets exist to diagnose and treat this aggressive disease. In this study we investigated the potential of nuclear receptors (NRs), many of which are well-established drug targets, as therapeutic markers in lung cancer. Using quantitative real-time PCR, we analyzed the expression of the 48 members of the NR superfamily in a human panel of 55 normal and lung cancer cell lines. Unsupervised cluster analysis of the NR expression profile segregated normal from tumor cell lines and grouped lung cancers according to type (i.e. small vs. non-small cell lung cancers). Moreover, we found that the NR signature was 79% accurate in diagnosing lung cancer incidence in smokers (n = 129). Finally, the evaluation of a subset of NRs (androgen receptor, estrogen receptor, vitamin D receptor, and peroxisome proliferator-activated receptor-γ) demonstrated the therapeutic potential of using NR expression to predict ligand-dependent growth responses in individual lung cancer cells. Preclinical evaluation of one of these receptors (peroxisome proliferator activated receptor-γ) in mouse xenografts confirmed that ligand-dependent inhibitory growth responses in lung cancer can be predicted based on a tumor's receptor expression status. Taken together, this study establishes NRs as theragnostic markers for predicting lung cancer incidence and further strengthens their potential as therapeutic targets for individualized treatment.
doi:10.1210/me.2011-1382
PMCID: PMC3404298  PMID: 22700587
20.  Oncogenic KRAS-induced interleukin-8 overexpression promotes cell growth and migration and contributes to aggressive phenotypes of non-small cell lung cancer 
The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.
doi:10.1002/ijc.26164
PMCID: PMC3374723  PMID: 21544811
non-small cell lung cancer; KRAS; interleukin-8; molecular target
21.  Functional Characterization of CLPTM1L as a Lung Cancer Risk Candidate Gene in the 5p15.33 Locus 
PLoS ONE  2012;7(6):e36116.
Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.
doi:10.1371/journal.pone.0036116
PMCID: PMC3366984  PMID: 22675468
22.  Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer 
Oncogene  2009;29(10):1421-1430.
DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover novel such alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adaptor protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to EGF-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contributes to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlighting a role of adapter proteins as primary genetic drivers of tumorigenesis.
doi:10.1038/onc.2009.437
PMCID: PMC3320568  PMID: 19966867
CRKL; lung cancer; DNA amplification; genomic profiling; adapter protein
23.  Cells lacking IκB kinase α (IKKα) demonstrate nuclear cyclin D1 overexpression and a neoplastic phenotype: Role of IKKα as a Tumor Suppressor 
Molecular cancer research : MCR  2011;9(3):341-349.
IKKα and IKKβ, catalytic subunits of IκB kinase (IKK) complex are involved in activation of NF-κB and in mediating a variety of other biological functions. Though these proteins have a high sequence homology, IKKα exhibits different functional characteristics as compared to IKKβ. Earlier, we have demonstrated that cyclin D1 is over-expressed and predominantly localized in the nucleus of IKKα−/− cells, indicating that IKKα regulates turnover and sub-cellular distribution of cyclin D1, which is mediated by IKKα-induced phosphorylation of cyclin D1. Since cyclin D nuclear localization is implicated in tumor development, we examined whether the absence of IKKα leads to tumor development as well. In the current study, we demonstrate that IKKα plays a critical role in tumorigenesis. Though IKKα−/− MEF cells demonstrate a slower anchorage-dependent growth, they are clonogenic in soft agar. These cells are tumorigenic in nude mice. Microarray analysis of IKKα−/− cells indicates a differential expression of genes involved in proliferation and apoptosis. Further, analysis of microarray data of human lung cancer cell lines revealed decreased IKKα RNA expression level as compared to cell lines derived from normal bronchial epithelium. These results suggest that IKKα may function as a tumor suppressor gene. Absence of IKKα may induce tumorigenicity by nuclear localization of cyclin D1 and modulating the expression of genes involved in neoplastic transformation.
doi:10.1158/1541-7786.MCR-10-0248
PMCID: PMC3072035  PMID: 21317297
24.  Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy 
Molecular cancer therapeutics  2011;10(2):336-346.
Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
doi:10.1158/1535-7163.MCT-10-0750
PMCID: PMC3061393  PMID: 21306997
25.  Comparing Statistical Methods for Constructing Large Scale Gene Networks 
PLoS ONE  2012;7(1):e29348.
The gene regulatory network (GRN) reveals the regulatory relationships among genes and can provide a systematic understanding of molecular mechanisms underlying biological processes. The importance of computer simulations in understanding cellular processes is now widely accepted; a variety of algorithms have been developed to study these biological networks. The goal of this study is to provide a comprehensive evaluation and a practical guide to aid in choosing statistical methods for constructing large scale GRNs. Using both simulation studies and a real application in E. coli data, we compare different methods in terms of sensitivity and specificity in identifying the true connections and the hub genes, the ease of use, and computational speed. Our results show that these algorithms performed reasonably well, and each method has its own advantages: (1) GeneNet, WGCNA (Weighted Correlation Network Analysis), and ARACNE (Algorithm for the Reconstruction of Accurate Cellular Networks) performed well in constructing the global network structure; (2) GeneNet and SPACE (Sparse PArtial Correlation Estimation) performed well in identifying a few connections with high specificity.
doi:10.1371/journal.pone.0029348
PMCID: PMC3260142  PMID: 22272232

Results 1-25 (46)