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1.  IGFBP2/FAK pathway is causally associated with dasatinib resistance in non-small Cell Lung Cancer Cells 
Molecular cancer therapeutics  2013;12(12):10.1158/1535-7163.MCT-13-0233.
IGFBP2 expression is increased in various types of cancers, including in a subset of lung cancer patients. Because IGFBP2 is involved in signal transduction of some critical cancer related pathways, we analyzed the association between IGFBP2 and response to pathway-targeted agents in seven human non–small cell lung cancer (NSCLC) cell lines. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) showed that four of the seven NSCLC cell lines analyzed expressed high levels of IGFBP2, while the remaining three had barely detectable IGFBP2. Susceptibilities of those seven cell lines to nine anticancer agents targeting to IGF1R, Src, FAK, MEK, and AKT were determined by dose-dependent cell viability assay. The results showed that high IGFBP2 levels were associated with resistance to dasatinib, and to a lesser degree to sacaratinib, but not to other agents. Ectopic IGFBP2 overexpression or knockdown revealed that changing IGFBP2 expression levels reversed dasatinib susceptibility phenotype, suggesting a causal relationship between IGFBP2 expression and dasatinib resistance. Molecular characterization revealed that FAK activation was associated with increased IGFBP2 expression and partially contributed to IGFBP2-mediated dasatinib resistance. Treatment with a combination of dasatinib and FAK inhibitor led to enhanced antitumor activity in IGFBP2-overexpressing and dasatinib-resistant NSCLC cells in vitro and in vivo. Our results demonstrated that the IGFBP2/FAK pathway is causally associated with dasatinib resistance and may be used as biomarkers for identification of dasatinib responders among lung cancer patients. Simultaneous targeting on Src and FAK will likely improve the therapeutic efficacy of dasatinib for treatment of lung cancer.
doi:10.1158/1535-7163.MCT-13-0233
PMCID: PMC3858413  PMID: 24130049
IGFBP2; FAK; dasatinib; biomarker; chemoresistance; lung cancer
2.  SSTR2-based reporter expression after plasmid-based in vivo gene delivery to Non-Small Cell Lung Cancer 
Molecular imaging  2013;12(7):1-10.
Purpose
Plasmids tend to have much lower expression than viruses. Assessing gene expression after systemic administration of plasmid vectors has not been assessed using SSTR2-based reporters. The purpose of this work was to identify gene expression in non-small cell lung cancer (NSCLC) after systemic liposomal nanoparticle delivery of plasmid containing somatostatin receptor type-2 (SSTR2)-based reporter gene.
Materials and Methods
In vitro, Western blotting was performed after transient transfection with plasmids CMV-SSTR2, CMV-TUSC2-IRES-SSTR2, or CMV-TUSC2. SSTR2 is the reporter and TUSC2 is a therapeutic gene. Mice with A549 NSCLC lung tumors were injected intravenously with CMV-SSTR2, CMV-TUSC2-IRES-SSTR2, or CMV-TUSC2 plasmids in DOTAP:Cholesterol-liposomal nanoparticles. Two days later, mice were injected intravenously with 111In-octreotide. The next day, biodistribution was performed. The experiment was repeated including SPECT-CT imaging. Immunohistochemistry was performed.
Results
In vitro, SSTR2 expression was similar in cells transfected with CMV-SSTR2 or CMV-TUSC2-IRES-SSTR2. TUSC2 expression was similar in cells transfected with CMV-TUSC2 or CMV-TUSC2-SSTR2. Biodistribution demonstrated significantly greater 111In-octreotide uptake in tumors from mice injected with CMV-TUSC2-IRES-SSTR2 or CMV-SSTR2 than control plasmid, CMV-TUSC2 (P<0.05). Gamma-camera and SPECT-CT imaging illustrated SSTR2 expression in tumors in mice injected with CMV-TUSC2-IRES-SSTR2 or CMV-SSTR2 versus background with control plasmid. Immunohistochemistry corresponded with imaging.
Conclusion
SSTR2-based reporter imaging can visualize gene expression in lung tumors after systemic liposomal nanoparticle delivery of plasmid containing SSTR2-based reporter gene or SSTR2 linked to a second therapeutic gene, such as TUSC2.
PMCID: PMC4103180  PMID: 23962694
Somatostatin receptor; reporter; plasmid; non-small cell lung cancer; imaging
3.  Stereotactic Ablative Radiotherapy: A Potentially Curable Approach to Early-Stage Multiple Primary Lung Cancer 
Cancer  2013;119(18):3402-3410.
BACKGROUND
Surgical resection has been the standard treatment for early-stage multiple primary lung cancer (MPLC). However, a significant proportion of patients with MPLC cannot undergo surgery. We explored here the role of stereotactic ablative radiotherapy (SABR) for patients with MPLC.
METHODS
We reviewed MPLC cases treated with SABR (50 Gy in 4 fractions or 70 Gy in 10 fractions) for the second tumor. Four-dimensional CT–based planning/volumetric image-guided treatment was used for all patients. Treatment outcomes/toxicities were analyzed.
RESULTS
For the 101 patients treated with SABR, at a median follow-up interval of 36 months and median overall survival of 46 months, 2-year and 4-year in-field local control rates were 97.4% and 95.7%. 2- and 4-year rates of overall survival (OS) were 73.2% and 47.5% and progression-free survival (PFS) were 67.0% and 58.0%. Patients with metachronous tumors had higher OS and PFS than did patients with synchronous tumors (2-year OS 80.6% metachronous vs. 61.5% synchronous; 4-year OS 52.7% vs. 39.7%; p=0.047; 2-year PFS 84.7% vs. 49.4%; 4-year PFS 75.6% vs. 30.4%; p=0.0001). For patients whose index tumor was treated with surgery or SABR, the incidence of grade ≥3 radiation pneumonitis was 3% (2/71), but this increased to 17% (5/30) for patients whose index tumor was treated with conventional radiotherapy. Other grade ≥3 toxicities included grade 3 chest wall pain (3/101, 3%) and grade 3 skin toxicity (1/101, 1%).
CONCLUSIONS
SABR achieves promising long- term tumor control, survival and could be a potential curative treatment of early-stage MPLC.
doi:10.1002/cncr.28217
PMCID: PMC3775964  PMID: 23798353
multiple primary lung cancer; synchronous tumors; metachronous tumors; stereotactic body radiotherapy; stereotactic ablative radiotherapy
4.  KEAP1-dependent synthetic lethality induced by AKT and TXNRD1 inhibitors in lung cancer 
Cancer research  2013;73(17):10.1158/0008-5472.CAN-13-0712.
Intrinsic resistance to agents targeting phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the major challenges in cancer treatment with such agents. The objective of this study is to identify the genes or pathways that can be targeted to overcome the resistance of non-small cell lung cancer to the AKT inhibitor, MK2206, which is currently being evaluated in phase I and II clinical trials. Using a genome-wide small interfering RNA (siRNA) library screening and biological characterization we identified that inhibition of Thioredoxin Reductase-1 (TXNRD1), one of the key anti-oxidant enzymes, with siRNAs or its inhibitor, Auranofin, sensitized non-small cell lung cancer cells to MK2206 treatment in vitro and in vivo. We found that simultaneous inhibition of TXNRD1 and AKT pathways induced robust reactive oxygen species (ROS) production, which was involved in c-Jun N-terminal Kinase (JNK, MAPK8) activation and cell apoptosis. Furthermore we found that the synthetic lethality interaction between the TXNRD1 and AKT pathways occurred through the KEAP1/NRF2 cellular antioxidant pathway. Lastly, we found that synthetic lethality induced by TXNRD1 and AKT inhibitors relied on wild type KEAP1 function. Our study indicates that targeting the interaction between AKT and TXNRD1 antioxidant pathways with MK2206 and Auranofin, a FDA approved drug, is a rational strategy to treat lung cancer and that KEAP1 mutation status may offer a predicative biomarker for such combination approaches.
doi:10.1158/0008-5472.CAN-13-0712
PMCID: PMC3868367  PMID: 23824739
Synthetic lethality; AKT; TXNRD1; MK2206; KEAP1
5.  EGFR-targeted plasmonic magnetic nanoparticles suppress lung tumor growth by abrogating G2/M cell-cycle arrest and inducing DNA damage 
Background
We have previously demonstrated the epidermal growth factor receptor (EGFR)-targeted hybrid plasmonic magnetic nanoparticles (225-NP) produce a therapeutic effect in human lung cancer cell lines in vitro. In the present study, we investigated the molecular mechanism of 225-NP-mediated antitumor activity both in vitro and in vivo using the EGFR-mutant HCC827 cell line.
Methods
The growth inhibitory effect of 225-NP on lung tumor cells was determined by cell viability and cell-cycle analysis. Protein expression related to autophagy, apoptosis, and DNA-damage were determined by Western blotting and immunofluorescence. An in vivo efficacy study was conducted using a human lung tumor xenograft mouse model.
Results
The 225-NP treatment markedly reduced tumor cell viability at 72 hours compared with the cell viability in control treatment groups. Cell-cycle analysis showed the percentage of cells in the G2/M phase was reduced when treated with 225-NP, with a concomitant increase in the number of cells in Sub-G1 phase, indicative of cell death. Western blotting showed LC3B and PARP cleavage, indicating 225-NP-treatment activated both autophagy- and apoptosis-mediated cell death. The 225-NP strongly induced γH2AX and phosphorylated histone H3, markers indicative of DNA damage and mitosis, respectively. Additionally, significant γH2AX foci formation was observed in 225-NP-treated cells compared with control treatment groups, suggesting 225-NP induced cell death by triggering DNA damage. The 225-NP-mediated DNA damage involved abrogation of the G2/M checkpoint by inhibiting BRCA1, Chk1, and phospho-Cdc2/CDK1 protein expression. In vivo therapy studies showed 225-NP treatment reduced EGFR phosphorylation, increased γH2AX foci, and induced tumor cell apoptosis, resulting in suppression of tumor growth.
Conclusion
The 225-NP treatment induces DNA damage and abrogates G2/M phase of the cell cycle, leading to cellular apoptosis and suppression of lung tumor growth both in vitro and in vivo. Our findings provide a rationale for combining 225-NP with other DNA-damaging agents for achieving enhanced anticancer activity.
doi:10.2147/IJN.S65990
PMCID: PMC4134185  PMID: 25143731
lung cancer; epidermal growth factor receptor; autophagy
6.  Pathway-based serum microRNA profiling and survival in patients with advanced-stage non-small cell lung cancer 
Cancer research  2013;73(15):4801-4809.
This study was designed to identify TGF-β signaling pathway-related serum microRNAs (miRNAs) as predictors of survival in advanced non-small cell lung cancer (NSCLC). Serum samples from 391 patients with advanced NSCLC were collected prior to treatment. Global miRNA microarray expression profiling based on sera from four patients with good survival (>24 months) and four patients with poor survival (<6 months) was used to identify 140 highly expressed serum miRNAs, among which 35 miRNAs had binding sites within the 3’-untranslated regions of a panel of 11 genes in the TGF-β signaling pathway and were assayed by quantitative RT-PCR for their associations with survival in a training (n=192) and testing set (n=191). Out of the 35 miRNAs, survival analysis using Cox regression model identified 17 miRNAs significantly associated with 2-year patient survival. MiR-16 exhibited the most statistically significant association: high expression of miR-16 was associated with a significantly better survival (adjusted hazard ratio = 0.4, 95% confidence interval: 0.3–0.5). A combined 17-miRNA risk score was created that was able to identify patients at the highest risk of death. Those with a high risk score had a 2.5-fold increased risk of death compared to those with a low risk score (95% CI=1.8–3.4, P=1.1×10−7). This increase in risk of death was corresponding to an 7.8 month decrease in median survival time (P=9.5×10−14). Our results suggest that serum miRNAs could serve as predictors of survival for advanced NSCLC.
doi:10.1158/0008-5472.CAN-12-3273
PMCID: PMC3760306  PMID: 23774211
serum miRNA; TGF-β; survival; NSCLC
7.  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
8.  Does the Timing of Esophagectomy after Neoadjuvant Chemoradiation Affect Outcome? 
The Annals of thoracic surgery  2011;93(1):207-213.
Purpose
After neoadjuvant chemoradiation (CXRT) for esophageal cancer, surgery has traditionally been recommended to be performed within 8 weeks. However, surgery is often delayed for various reasons. Data from other cancers suggests that delaying surgery may increase the pathologic complete response rate. However, there are theoretical concerns that waiting longer after radiation may lead to a more difficult operation and more complications. The optimal timing of esophagectomy after CXRT is unknown.
Methods
From a prospective database, we analyzed 266 patients with resected esophageal cancer who were treated with neoadjuvant CXRT from 2002–2008. Salvage resections were excluded from this analysis. We compared patients who had surgery within 8 weeks of CXRT and those who had surgery after 8 weeks. We used multivariable analysis to determine whether increased interval between chemoradiation and surgery was independently associated with perioperative complication, pathologic response, or overall survival.
Results
150 patients were resected within 8 weeks and 116 were resected greater than 8 weeks after completing CXRT. Mean length of operation, intraoperative blood loss, anastomotic leak rate, and perioperative complication rate were similar for the two groups. Pathologic complete response rate and overall survival were also similar for the two groups (p=NS). In multivariable analysis, timing of surgery was not an independent predictor of perioperative complication, pathologic complete response, or overall survival.
Conclusion
The timing of esophagectomy after neoadjuvant CXRT is not associated with perioperative complication, pathologic response, or overall survival. It may be reasonable to delay esophagectomy beyond 8 weeks for patients who have not yet recovered from chemoradiation.
doi:10.1016/j.athoracsur.2011.05.021
PMCID: PMC4041623  PMID: 21962263
Adjuvant/neoadjuvant therapy; Esophageal Cancer; Radiation Therapy; Esophageal Surgery
9.  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
10.  Prognostic Significance of Combinations of RNA-Dependent Protein Kinase and EphA2 Biomarkers for NSCLC 
Introduction
RNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non-small cell lung cancer (NSCLC). In the present study, we investigated the correlation between PKR and 25 other biomarkers for NSCLC, identified the markers that could further improve the prognostic significance of PKR, and elucidated the mechanisms of interaction between these markers and PKR.
Methords
Tissue microarray samples obtained from 218 lung cancer patients were stained with an anti-PKR antibody and antibodies against 25 biomarkers. Immunohistochemical expression was scored and used for Kaplan-Meier survival analysis. The interaction between PKR and EphA2 in NSCLC cell lines was examined.
Results
We found that PKR was associated with EphA2 and that the prognostic information regarding NSCLC provided by the combination of PKR and EphA2 (P/E) was significantly more accurate than that provided by either marker alone. The 5-year overall survival rate in PKRlow/EphA2high patients (20%) was significantly lower than that of PKRhigh/EphA2low patients (74%), PKRhigh/EphA2high patients (55%), and PKRlow/EphA2low patients (55%) (p< 0.0001). We also found that the PKR:EphA2 (P/E) ratio was significantly associated with prognosis (p< 0.0001). Univariate and multivariate Cox analyses revealed that this P/E combination or ratio was an independent predictor of overall survival. In addition, induction of PKR expression reduced EphA2 protein expression levels in NSCLC cell lines.
Conclusions
PKR/EphA2 is a significant predictor of prognosis for NSCLC. PKR/EphA2 may be a promising approach to improving screening efficiency and predicting prognosis in NSCLC patients.
doi:10.1097/JTO.0b013e318282def7
PMCID: PMC3573252  PMID: 23370317
PKR; EphA2; Biomarker; Lung cancer
11.  Ionizing radiation-induced γ-H2AX activity in whole blood culture and the risk of lung cancer 
Background
Phenotypic biomarkers of DNA damage repair may enhance cancer risk prediction. The γ-H2AX formed at the sites of double strands break (DSB) after ionizing radiation (IR) is a specific marker of DNA damage.
Methods
In an ongoing case-control study, the baseline and IR-induced γ-H2AX levels in peripheral blood lymphocytes (PBLs) from frequency-matched 306 untreated lung cancer patients and 306 controls were measured by a laser scanning cytometer-based immunocytochemical method. The ratio of IR-induced γ-H2AX level to the baseline was used to evaluate inter-individual variation of DSB damage response and to assess the risk of lung cancer by using unconditional multivariable logistic regression with adjustment of age, sex, ethnicity, smoking status, family history of lung cancer, dust exposure and emphysema.
Results
The mean γ-H2AX ratio was significantly higher in cases than controls (1.46±0.14 vs. 1.41±0.12, P < 0.001). Dichotomized at the median in controls, high γ-H2AX ratio was significantly associated with increased risk of lung cancer (OR = 2.43, 95% CI: 1.66–3.56). There was also a significant dose-response relationship between γ-H2AX ratio and lung cancer risk in quartile analysis. Analysis of joint effects with other epidemiological risk factors revealed elevated risk with increasing number of risk factors.
Conclusion
γ-H2AX activity as shown by measuring DSB damage in IR-irradiated PBLs may be a novel phenotypic marker of lung cancer risk.
Impact
γ-H2AX assay is a robust and quantifiable image-based cytometer method that measures mutagen-induced DSB response in PBLs as a potential biomarker in lung cancer risk assessment.
doi:10.1158/1055-9965.EPI-12-0794
PMCID: PMC3601549  PMID: 23300022
Double strands break; γ-H2AX; mutagen sensitivity; lung cancer risk
12.  Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA 
ACS nano  2012;6(9):7681-7691.
The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay.
doi:10.1021/nn301135w
PMCID: PMC3888232  PMID: 22862291
Plasmon; nanoshell; antisense oligonucleotide; siRNA; Gene therapy; controlled drug release; polylysine
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.  Insulin-Like Growth Factor Binding Protein-2 Level Is Increased in Blood of Lung Cancer Patients and Associated with Poor Survival 
PLoS ONE  2013;8(9):e74973.
Background
We recently showed that IGFBP2 is overexpressed in primary lung cancer tissues. This study aims to determine whether IGFBP2 is elevated in blood samples of lung cancer patients and whether its level is associated with clinical outcomes.
Methodology/Principal Findings
Plasma IGFBP2 levels were determined blindly by enzyme-linked immunosorbent assay in 80 lung cancer patients and 80 case-matched healthy controls for comparison. We analyzed blood samples for IGFBP2 levels from an additional 84 patients with lung cancer and then tested for associations between blood IGFBP2 levels and clinical parameters in all 164 lung cancer patients. All statistical tests were two-sided and differences with p<0.05 were considered significant. The mean plasma concentration of IGFBP2 in lung cancer patients was significantly higher than that in healthy controls (388.12±261.00 ng/ml vs 219.30±172.84 ng/ml, p<0.001). IGFBP2 was increased in all types of lung cancer, including adenocarcinoma, squamous cell cancer, and small-cell cancer, regardless of patients’ age, sex, or smoking status. IGFBP2 levels were mildly but significantly associated with tumor size and were significantly higher in stage IV than stage I or III disease. A multivariate analysis showed that lung cancer patients whose blood IGFBP2 was higher than 160.9 ng/ml had a poor survival outcome, with a hazard ratio of 8.76 (95% CI 1.12-68.34, p=0.038 after adjustment for tumor size, pathology, and stage). The median survival time for patients with blood IGFBP2 >160.9 ng/ml is 15.1 months; whereas median survival time was 128.2 months for the patients whose blood IGFBP2 was ≤160.9 ng/ml (p =0.0002).
Conclusions/Significance
Blood IGFBP2 is significantly increased in lung cancer patients. A high circulating level of IGFBP2 is significantly associated with poor survival, suggesting that blood IGFBP2 levels could be a prognostic biomarker for lung cancer.
doi:10.1371/journal.pone.0074973
PMCID: PMC3775736  PMID: 24069370
15.  Relax with CouchDB - Into the non-relational DBMS era of Bioinformatics 
Genomics  2012;100(1):1-7.
With the proliferation of high-throughput technologies, genome-level data analysis has become common in molecular biology. Bioinformaticians are developing extensive resources to annotate and mine biological features from high-throughput data. The underlying database management systems for most bioinformatics software are based on a relational model. Modern non-relational databases offer an alternative that has flexibility, scalability, and a non-rigid design schema. Moreover, with an accelerated development pace, non-relational databases like CouchDB can be ideal tools to construct bioinformatics utilities. We describe CouchDB by presenting three new bioinformatics resources: (a) geneSmash, which collates data from bioinformatics resources and provides automated gene-centric annotations, (b) drugBase, a database of drug-target interactions with a web interface powered by geneSmash, and (c) HapMap-CN, which provides a web interface to query copy number variations from three SNP-chip HapMap datasets. In addition to the web sites, all three systems can be accessed programmatically via web services.
doi:10.1016/j.ygeno.2012.05.006
PMCID: PMC3383915  PMID: 22609849
NoSQL database; copy number variation; drug-target interaction; data integration
16.  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
17.  Genetic Variations in Epigenetic Genes Are Predictors of Recurrence in Stage I or II Non–Small Cell Lung Cancer Patients 
Clinical Cancer Research  2012;18(2):585-592.
Purpose
Early-stage non–small cell lung cancer (NSCLC) is potentially curable, however, many patients develop recurrent disease. Therefore, identification of biomarkers that can be used to predict patient’s risk of recurrence and survival is critical. Genetic polymorphisms or single-nucleotide polymorphisms (SNP) of DNA- and histone-modifying genes, particularly those of O6-methylguanine DNA-methyltransferase (MGMT), have been linked to an increased risk of lung cancer as well as treatment outcomes in other tumors.
Experimental Design
We assessed the association of 165 SNPs in selected epigenetic enzyme genes, DNA methyltransferases, and methyl-CpG–binding proteins with cancer recurrence in 467 patients with stage I or II NSCLC treated with either surgery alone (N = 340) or surgery plus (neo)-adjuvant chemotherapy (N = 127).
Results
We found several SNPs to be strongly correlated with tumor recurrence. We identified 10 SNPs that correlated with the outcome in patients treated with surgery alone but not in patients treated with surgery and adjuvant chemotherapy, which suggested that the addition of platinum-based chemotherapy could reverse the high genetic risk of recurrence. We also identified 10 SNPs that predicted the risk of recurrence in patients treated with surgery plus adjuvant chemotherapy but not in patients treated with surgery alone. The cumulative effect of these SNPs significantly predicted outcomes with P-values of 10−9and 10−6, respectively.
Conclusions
The first set of genotypes may be used as novel predictive biomarkers to identify patients with stage I NSCLC, who could benefit from adjuvant chemotherapy, and the second set of SNPs might predict response to adjuvant chemotherapy.
doi:10.1158/1078-0432.CCR-11-2087
PMCID: PMC3373176  PMID: 22252258
18.  A Prospective Phase 2 Study of Surgery Followed by Chemotherapy and Radiation for Superior Sulcus Tumors 
Cancer  2011;118(2):444-451.
BACKGROUND
The optimal treatment for locally advanced superior sulcus tumors is not clear. The authors report long-term results of a trial examining the safety and efficacy of surgery followed by concurrent chemoradiation therapy for this disease.
METHODS
Thirty-two patients with resectable or marginally resectable superior sulcus tumors at The University of Texas MD Anderson Cancer Center from 1994 to 2010 were enrolled in a prospective trial. Surgery involved segmentectomy or lobectomy with en bloc resection of the involved chest wall and complete nodal staging; radiation therapy (RT) began 14 to 42 days later to a dose of 60 grays (Gy) in 50 1.2-Gy fractions if surgical margins were negative or 64.8 Gy in 54 1.2-Gy fractions if margins were positive. Two cycles of etoposide (50 mg/ m2) and cisplatin (50 mg/m2) were given during RT, and another 3 cycles were given after RT. Eleven patients underwent prophylactic cranial irradiation (PCI).
RESULTS
The protocol completion rate was 78%. Gross total resection was accomplished in all 32 patients; 28% underwent R1 resection. Operative mortality was 0%. The most common surgical complication was postoperative pneumonia (25%). At a median follow-up time of 53.4 months (range, 2–154 months), the 2-year, 5-year, and 10-year rates of locoregional control were 84%, 76%, and 76%; distant metastasis-free survival, 52%, 48%, and 48%; disease-free survival, 49%, 45%, and 45%; and overall survival, 72%, 50%, and 45%, respectively. The brain was the most common site of distant failure (n =5), but no patient who received PCI experienced brain metastasis.
CONCLUSIONS
Surgery followed by postoperative chemoradiation is safe and effective for the treatment of marginally resectable superior sulcus tumors.
doi:10.1002/cncr.26277
PMCID: PMC3465936  PMID: 21713767
Pancoast tumor; lung neoplasms; adjuvant radiotherapy; adjuvant chemotherapy; chemoradiation
19.  Predictors for Locoregional Recurrence for Clinical Stage III-N2 Non-small Cell Lung Cancer with Nodal Downstaging After Induction Chemotherapy and Surgery 
Annals of Surgical Oncology  2012;20(6):1934-1940.
Purpose
Pathologic downstaging following chemotherapy for stage III-N2 NSCLC is a well-known positive prognostic indicator. However, the predictive factors for locoregional recurrence (LRR) in these patients are largely unknown.
Methods
Between 1998 and 2008, 153 patients with clinically or pathologically staged III-N2 NSCLC from two cancer centers in the United States were treated with induction chemotherapy and surgery. All had pathologic N0-1 disease, and none received postoperative radiotherapy. LRR were defined as recurrence at the surgical site, lymph nodes (levels 1–14 including supraclavicular), or both.
Results
Median follow-up was 39.3 months. Pretreatment N2 status was confirmed pathologically (18.2 %) or by PET/CT (81.8 %). Overall, the 5-year LRR rate was 30.8 % (n = 38), with LRR being the first site of failure in 51 % (22/+99877943). Five-year overall survival for patients with LRR compared with those without was 21 versus 60.1 % (p < 0.001). Using multivariate analysis, significant predictors for LRR were pN1 disease at time of surgery (p < 0.001, HR 3.43, 95 % CI 1.80–6.56) and a trend for squamous histology (p = 0.072, HR 1.93, 95 % CI 0.94–3.98). Five-year LRR rate for pN1 versus pN0 disease was 62 versus 20 %. Neither single versus multistation N2 disease (p = 0.291) nor initial staging technique (p = 0.306) were predictors for LRR. N1 status also was predictive for higher distant recurrence (p = 0.021, HR 1.91, 95 % CI 1.1–3.3) but only trended for poorer survival (p = 0.123, HR 1.48, 95 % CI 0.9–2.44).
Conclusions
LRR remains high in resected stage III-N2 NSCLC patients after induction chemotherapy and nodal downstaging, particularly in patients with persistent N1 disease.
Electronic supplementary material
The online version of this article (doi:10.1245/s10434-012-2800-x) contains supplementary material, which is available to authorized users.
doi:10.1245/s10434-012-2800-x
PMCID: PMC3656229  PMID: 23263700
20.  CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity 
Oncogene  2003;22(4):615-626.
The CACNA2D2 gene, a new subunit of the Ca2+-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca2+ signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca2+ contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca2+ were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca2+ contents.
doi:10.1038/sj.onc.1206134
PMCID: PMC3484891  PMID: 12555074
tumor suppressor genes; apoptosis; calcium channel proteins; human chromosome 3p21.3; lung cancer
21.  Expression of Several Genes in the Human Chromosome 3p21.3 Homozygous Deletion Region by an Adenovirus Vector Results in Tumor Suppressor Activities in Vitro and in Vivo1 
Cancer research  2002;62(9):2715-2720.
A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.
PMCID: PMC3478680  PMID: 11980673
22.  The Role of Consolidation Therapy for Stage III Non-Small Cell Lung Cancer With Persistent N2 Disease After Induction Chemotherapy 
The Annals of thoracic surgery  2012;94(3):914-920.
Background
Persistent pathologic mediastinal nodal involvement after induction chemotherapy and surgical resection is a negative prognostic factor for stage III-N2 non-small cell lung cancer patients. This population has high rates of local-regional failure and distant failure, yet the effectiveness of additional therapies is not clear. We assessed the role of consolidative therapies (postoperative radiation therapy and chemotherapy) for such patients.
Methods
In all, 179 patients with stage III-N2 non-small cell lung cancer at MD Anderson Cancer Center were treated with induction chemotherapy followed by surgery from 1998 through 2008; 61 patients in this cohort had persistent, pathologically confirmed, mediastinal nodal disease, and were treated with postoperative radiation therapy. Local-regional failure was defined as recurrence at the surgical site or lymph nodes (levels 1 to 14, including supraclavicular), or both. Overall survival was calculated using the Kaplan-Meier method, and survival outcomes were assessed by log rank tests. Univariate and multivariate Cox proportional hazards models were used to identify factors influencing local-regional failure, distant failure, and overall survival.
Results
All patients received postoperative radiation therapy after surgery, but approximately 25% of the patients also received additional chemotherapy: 9 (15%) with concurrent chemotherapy, 4 (7%) received adjuvant sequential chemotherapy, and 2 (3%) received both. Multivariate analysis indicated that additional postoperative chemotherapy significantly reduced distant failure (hazard ratio 0.183, 95% confidence interval: 0.052 to 0.649, p = 0.009) and improved overall survival (hazard ratio 0.233, 95% confidence interval: 0.089 to 0.612, p = 0.003). However, additional postoperative chemotherapy had no affect on local-regional failure.
Conclusions
Aggressive consolidative therapies may improve outcomes for patients with persistent N2 disease after induction chemotherapy and surgery.
doi:10.1016/j.athoracsur.2012.04.088
PMCID: PMC3468148  PMID: 22819472
23.  Histopathologic Response Criteria Predict Survival of Patients with Resected Lung Cancer After Neoadjuvant Chemotherapy 
Introduction
We evaluated the ability of histopathologic response criteria to predict overall survival (OS) and disease-free survival (DFS) in patients with surgically resected non-small cell lung cancer (NSCLC) treated with or without neoadjuvant chemotherapy.
Methods
Tissue specimens from 358 patients with NSCLC were evaluated by pathologists blinded to the patient treatment and outcome. The surgical specimens were reviewed for various histopathologic features in the tumor including percentage of residual viable tumor cells, necrosis, and fibrosis. The relationship between the histopathologic findings and OS was assessed.
Results
The percentage of residual viable tumor cells and surgical pathologic stage were associated with OS and DFS in 192 patients with NSCLC receiving neoadjuvant chemotherapy in multivariate analysis (p = 0.005 and p = 0.01, respectively). There was no association of OS or DFS with percentage of viable tumor cells in 166 patients with NSCLC who did not receive neoadjuvant chemotherapy (p = 0.31 and p = 0.45, respectively). Long-term OS and DFS were significantly prolonged in patients who had ≤10% viable tumor compared with patients with >10% viable tumor cells (5 years OS, 85% versus 40%, p < 0.0001 and 5 years DFS, 78% versus 35%, p < 0.001).
Conclusion
The percentages of residual viable tumor cells predict OS and DFS in patients with resected NSCLC after neoadjuvant chemotherapy even when controlled for pathologic stage. Histopathologic assessment of resected specimens after neoadjuvant chemotherapy could potentially have a role in addition to pathologic stage in assessing prognosis, chemotherapy response, and the need for additional adjuvant therapies.
doi:10.1097/JTO.0b013e318247504a
PMCID: PMC3465940  PMID: 22481232
Lung cancer; Neoadjuvant chemotherapy; Histopathology
24.  Variations in the Vascular Endothelial Growth Factor Pathway Predict Pulmonary Complications 
The Annals of thoracic surgery  2012;94(4):1079-1085.
Background
Clinical factors predicting pulmonary complications after lung resection have been well described, whereas the role of genetics is unknown. The vascular endothelial growth factor (VEGF) signaling pathway has been linked to acute lung injury. We hypothesized that genetic variations in this pathway may be associated with postoperative pulmonary complications after lung resection.
Methods
One hundred ninety-six single nucleotide polymorphisms (SNPs) in 17 genes in the VEGF pathway were genotyped in a discovery set of 264 patients and a replication set of 264 patients who underwent lobectomy for lung cancer. Multivariable analysis adjusting for baseline clinical factors was used to identify SNPs associated with pulmonary complications. Cumulative and classification and regression tree (CART) analyses were used to further stratify risk groups.
Results
The overall number of pulmonary complications was 164/528 (31%). The effects of 6 SNPs were consistent in the discovery and replication sets (pooled p value < 0.05). The rs9319425 SNP in the VEGF receptor gene FLT1 resulted in a 1.50-fold increased risk (1.15–1.96; p = 0.003). A cumulative effect for the number of risk genotypes and complications was also evident (p < 0.01). Patients carrying 5 risk genotypes had a 5.76-fold increase in risk (2.73–12.16; p = 4.44 × 10−6). Regression tree analysis identified potential gene-gene interactions between FLT1:rs9319425 and RAF1:rs713178. The addition of the 6 SNPs to the clinical model increased the area under the receiver operating characteristic curve by 6.8%.
Conclusions
Genetic variations in the VEGF pathway are associated with risk of pulmonary complications after lobectomy. This may offer insight into the underlying biological mechanisms of pulmonary complications.
doi:10.1016/j.athoracsur.2012.05.048
PMCID: PMC3466075  PMID: 22795057
25.  Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer specific cytotoxicity and tumor growth delay 
Purpose
Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment and novel therapies are therefore in high demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug
Experimental design
The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter Insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the non-viral nanoparticle, DOTAP:Cholesterol for transgene delivery.
Results
INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene and prodrug Ganciclovir (GCV). The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared to control treated xenografts.
Conclusions
The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to demonstrate an anti-tumor effect from the delivery of suicide gene therapeutics for SCLC in vivo.
doi:10.1158/1078-0432.CCR-09-3057
PMCID: PMC3457699  PMID: 20371678
gene therapy; transcriptional targeting; Insulinoma-associated 1 promoter; small cell lung cancer; suicide genes

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