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1.  RNAi-induced synthetic lethality in cancer therapy 
Cancer biology & therapy  2009;8(23):2314-2316.
PMCID: PMC4512945  PMID: 19949302
synthetical lethality; RNA interference; in vivo delivery; oncogenic addiction; library screening; target therapy; drug resistance
2.  Genetic variation in the TNF/TRAF2/ASK1/p38 kinase signaling pathway as markers for postoperative pulmonary complications in lung cancer patients 
Scientific Reports  2015;5:12068.
Post-operative pulmonary complications are the most common morbidity associated with lung resection in non-small cell lung cancer (NSCLC) patients. The TNF/TRAF2/ASK1/p38 kinase pathway is activated by stress stimuli and inflammatory signals. We hypothesized that genetic polymorphisms within this pathway may contribute to risk of complications. In this case-only study, we genotyped 173 germline genetic variants in a discovery population of 264 NSCLC patients who underwent a lobectomy followed by genotyping of the top variants in a replication population of 264 patients. Complications data was obtained from a prospective database at MD Anderson. MAP2K4:rs12452497 was significantly associated with a decreased risk in both phases, resulting in a 40% reduction in the pooled population (95% CI:0.43–0.83, P = 0.0018). In total, seven variants were significant for risk in the pooled analysis. Gene-based analysis supported the involvement of TRAF2, MAP2K4, and MAP3K5 as mediating complications risk and a highly significant trend was identified between the number of risk genotypes and complications risk (P = 1.63 × 10−8). An inverse relationship was observed between association with clinical outcomes and complications for two variants. These results implicate the TNF/TRAF2/ASK1/p38 kinase pathway in modulating risk of pulmonary complications following lobectomy and may be useful biomarkers to identify patients at high risk.
doi:10.1038/srep12068
PMCID: PMC4499815  PMID: 26165383
3.  Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy 
PLoS ONE  2015;10(6):e0123967.
Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.
doi:10.1371/journal.pone.0123967
PMCID: PMC4460038  PMID: 26053020
4.  Genetic variants of the Wnt signaling pathway as predictors of recurrence and survival in early-stage non-small cell lung cancer patients 
Carcinogenesis  2014;35(6):1284-1291.
Summary
NSCLC is often a lethal disease but potentially curative at early stages. In this study, we showed that genetic variants in the developmental and stem cell-related Wnt signaling pathway may influence disease recurrence and survival in early-stage NSCLC patients.
Early-stage non-small cell lung cancer (NSCLC) is potentially curative. Nevertheless, many patients will show disease recurrence after curative treatment. The Wnt signaling pathway is a developmental and stem cell pathway that plays an important role in tumorigenesis and may affect cancer progression. We hypothesize that genetic variants of the Wnt pathway may influence clinical outcome in early-stage NSCLC patients. We genotyped 441 functional and tagging single nucleotide polymorphisms (SNPs) from 54 genes of the Wnt pathway in 535 early-stage NSCLC patients treated with curative intent therapy including surgery and chemotherapy. For validation, 4 top SNPs were genotyped in 301 early-stage NSCLC patients from the Mayo Clinic. Cox proportional hazard model and combined SNP analyses were performed to identify significant SNPs correlated with recurrence-free and overall survival. Results from discovery group showed a total of 40 SNPs in 20 genes correlated with disease recurrence (P < 0.05). After correction for multiple comparisons, rs2536182 near Wnt16 remained significant (q < 0.1), which was validated in the replication population. Thirty-nine SNPs in 16 genes correlated with overall survival (P < 0.05) in the discovery group, and seven remained significant after multiple comparisons were considered (q < 0.1). In patients receiving surgery-only treatment, rs10898563 of FZD4 gene was associated with both recurrence-free and overall survival. Joint SNP analyses identified predictive markers for recurrence stratified by treatment. Our findings suggest inherited genetic variation in the Wnt signaling pathway may contribute to variable clinical outcomes for patients with early-stage NSCLC.
doi:10.1093/carcin/bgu034
PMCID: PMC4043238  PMID: 24517998
5.  RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer 
Oncotarget  2015;6(13):11114-11124.
We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expresson of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinaseT172 [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation.
PMCID: PMC4484443  PMID: 25798539
PKR; AMPK; nutrient depletion; lung cancer
6.  Telomere Length in Peripheral Blood Leukocytes and Lung Cancer Risk: A Large Case-Control Study in Caucasians 
Cancer research  2014;74(9):2476-2486.
Telomere dysfunction is a crucial event in malignant transformation and tumorigenesis. Telomere length in peripheral blood leukocytes has been associated with lung cancer risk, but the relationship has remained controversial. In this study, we investigated whether the association might be confounded by study of different histological subtypes of lung cancer. We measured relative telomere lengths in patients in a large case-control study of lung cancer and performed stratified analyses according to the two major histological subtypes (adenocarcinoma [AC] and squamous cell carcinoma [SCC]). Notably, AC patients had longer telomeres than controls, whereas SCC patients had shorter telomeres compared to controls. Long telomeres were associated with increased risk of AC, with the highest risk associated with female sex, younger age (<60 years) and lighter smoking (<30 pack-years). In contrast, long telomeres were protective against SCC, particularly in male patients. Our results extend the concept that telomere length affects risk of lung cancer in a manner that differs with histological subtype.
doi:10.1158/0008-5472.CAN-13-2968
PMCID: PMC4357479  PMID: 24618342
Telomeres; peripheral blood leukocytes; adenocarcinoma; squamous cell carcinoma; lung cancer risk
7.  Expression of sulfotransferase SULT1A1 in cancer cells predicts susceptibility to the novel anticancer agent NSC-743380 
Oncotarget  2014;6(1):345-354.
The small molecule anticancer agent NSC-743380 modulates functions of multiple cancer-related pathways and is highly active in a subset of cancer cell lines in the NCI-60 cell line panel. It also has promising in vivo anticancer activity. However, the mechanisms underlying NSC-743380's selective anticancer activity remain uncharacterized. To determine biomarkers that may be used to identify responders to this novel anticancer agent, we performed correlation analysis on NSC-743380's anticancer activity and the gene expression levels in NCI-60 cell lines and characterized the functions of the top associated genes in NSC-743380–mediated anticancer activity. We found sulfotransferase SULT1A1 is causally associated with NSC-743380's anticancer activity. SULT1A1 was expressed in NSC-743380–sensitive cell lines but was undetectable in resistant cancer cells. Ectopic expression of SULT1A1 in NSC743380 resistant cancer cells dramatically sensitized the resistant cells to NSC-743380. Knockdown of the SULT1A1 in the NSC-743380 sensitive cancer cell line rendered it resistance to NSC-743380. The SULT1A1 protein levels in cell lysates from 18 leukemia cell lines reliably predicted the susceptibility of the cell lines to NSC-743380. Thus, expression of SULT1A1 in cancer cells is required for NSC-743380's anticancer activity and can be used as a biomarker for identification of NSC-743380 responders.
PMCID: PMC4381599  PMID: 25514600
Cancer; drug development; biomarker; sulfotransferase; SULT1A1; personalized therapy
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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