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author:("HONG, warn KI")
1.  Global assessment of genetic variation influencing response to retinoid chemoprevention in head and neck cancer patients 
Head and neck squamous cell carcinoma (HNSCC) patients are at an increased risk of developing a second primary tumor (SPT) or recurrence following curative treatment. 13-cis-retinoic acid (13-cRA) has been tested in chemoprevention clinical trials but the results have been inconclusive. We genotyped 9,465 SNPs in 450 patients from the Retinoid Head and Neck Second Primary Trial. SNPs were analyzed for associations with SPT/recurrence in patients receiving placebo to identify prognosis markers and further analyzed for effects of 13-cRA in patients with these prognostic loci. Thirteen loci identified a majority subgroup of patients at a high risk of SPT/recurrence and in whom 13-cRA was protective. Patients carrying the common genotype of rs3118570 in the retinoid X receptor (RXRA) were at a 3.33-fold increased risk (95% confidence interval [CI], 1.67–6.67) and represented over 70% of the study population. This locus also identified individuals who received benefit from chemoprevention with a 38% reduced risk (95% CI, 0.43–0.90). Analyses of cumulative effect and potential gene-gene interactions also implicated CDC25C:rs6596428 and JAK2:rs1887427 as two other genetic loci with major roles in prognosis and 13-cRA response. Patients with all three common genotypes had a 76% reduction in SPT/recurrence (95% CI, 0.093–0.64) following 13-cRA chemoprevention. Carriers of these common genotypes constituted a substantial percentage of the study population, indicating that a pharmacogenetics approach could help select patients for 13-cRA chemoprevention. The lack of any alternatives for reducing risk in these patients highlights the need for future clinical trials to prospectively validate our findings.
doi:10.1158/1940-6207.CAPR-10-0125
PMCID: PMC3955084  PMID: 21292633
HNSCC; SPT; single nucleotide polymorphisms; retinoids
2.  The BATTLE to Personalize Lung Cancer Prevention through Reverse Migration 
Agents can enter clinical development for cancer prevention either initially or after previous development for a different indication, such as arthritis, with both approaches consuming many years of development before an agent is fully evaluated for cancer prevention. We propose the following, third approach: Reverse migration, that is, importing agents, targets and study designs to personalize interventions, and concepts developed in advanced cancer to the setting of cancer prevention. Importing these “ready-made” features from therapy will allow reverse migration to streamline preventive-agent development. We recently reported the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial of personalized lung-cancer therapy and now propose the reverse-migration development of personalized lung-cancer prevention based on the BATTLE model.
doi:10.1158/1940-6207.CAPR-11-0232
PMCID: PMC3171137  PMID: 21733820
chemoprevention; personalized; targeted; lung cancer
3.  Elevated PDGFRB gene copy number gain (CNG) is prognostic for improved survival outcomes in resected malignant pleural mesothelioma 
Annals of diagnostic pathology  2014;18(3):140-145.
Background
PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization (FISH) and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome.
Methods
88 archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRβ, p-PDGFRβ) and FISH analysis of PDGFRB gene CNG. Spearman's rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome.
Results
Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in > 10% of tumor cells had lower cytoplasmic p-PDGFRβ (p=0.029), while PDGFRB gene CNG in > 40% of tumor cells had a higher cytoplasmic PDGFRβ (p=0.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics.
PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG > 40% of tumor cells had improved relapse-free survival [HR 0.25 (95% CI 0.09, 0.72), p=0.0096] and improved overall survival [HR 0.32 (95% CI 0.11, 0.89), p=0.029].
Conclusions
PDGFRB CNG > 40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed.
doi:10.1016/j.anndiagpath.2014.02.005
PMCID: PMC4136493  PMID: 24747001
PDGFRB; pleural mesothelioma; copy number gain; prognostic biomarker
4.  A Novel Small-Molecule Inhibitor Targeting CREB-CBP Complex Possesses Anti-Cancer Effects along with Cell Cycle Regulation, Autophagy Suppression and Endoplasmic Reticulum Stress 
PLoS ONE  2015;10(4):e0122628.
Lung adenocarcinoma, the most common subtype of lung cancer, is the leading cause of cancer death worldwide. Despite attempts for the treatment of lung cancer which have been accumulating, promising new therapies are still needed. Here, we found that cyclic-AMP response element-binding protein (CREB)-CREB binding protein (CBP) transcription factors complex inhibitor, Naphthol AS-TR phosphate (NASTRp), is a potential therapeutic agent for lung cancer. We show that NASTRp inhibited oncogenic cell properties through cell cycle arrest with concomitant suppression of tumor-promoting autophagy with down-regulations of Atg5-12 and Atg7, and accumulation of p62 in human lung cancer cell lines. In addition, NASTRp induced expression of endoplasmic reticulum stress markers such as DDIT3/CHOP, and led to apoptosis along with Bim induction. These findings suggest that transcription factor/co-activator complex, CREB-CBP, can be a potential therapeutic target and its inhibition could be a novel therapeutic strategy for lung cancer.
doi:10.1371/journal.pone.0122628
PMCID: PMC4405579  PMID: 25897662
5.  Transcriptomic Architecture of the Adjacent Airway Field Cancerization in Non–Small Cell Lung Cancer 
Background
Earlier work identified specific tumor-promoting abnormalities that are shared between lung cancers and adjacent normal bronchial epithelia. We sought to characterize the yet unknown global molecular and adjacent airway field cancerization (FC) in early-stage non–small cell lung cancer (NSCLC).
Methods
Whole-transcriptome expression profiling of resected early-stage (I–IIIA) NSCLC specimens (n = 20) with matched tumors, multiple cytologically controlled normal airways with varying distances from tumors, and uninvolved normal lung tissues (n = 194 samples) was performed using the Affymetrix Human Gene 1.0 ST platform. Mixed-effects models were used to identify differentially expressed genes among groups. Ordinal regression analysis was performed to characterize site-dependent airway expression profiles. All statistical tests were two-sided, except where noted.
Results
We identified differentially expressed gene features (n = 1661) between NSCLCs and airways compared with normal lung tissues, a subset of which (n = 299), after gene set enrichment analysis, statistically significantly (P < .001) distinguished large airways in lung cancer patients from airways in cancer-free smokers. In addition, we identified genes (n = 422) statistically significantly and progressively differentially expressed in airways by distance from tumors that were found to be congruently modulated between NSCLCs and normal lung tissues. Furthermore, LAPTM4B, with statistically significantly increased expression (P < .05) in airways with shorter distance from tumors, was upregulated in human immortalized cells compared with normal bronchial epithelial cells (P < .001) and promoted anchorage-dependent and -independent lung cancer cell growth.
Conclusions
The adjacent airway FC comprises both site-independent profiles as well as gradient and localized airway expression patterns. Profiling of the airway FC may provide new insights into NSCLC oncogenesis and molecular tools for detection of the disease.
doi:10.1093/jnci/dju004
PMCID: PMC3982778  PMID: 24563515
6.  Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer* 
The Journal of Biological Chemistry  2015;290(12):7345-7359.
Background: EphA5 is a functional target in lung cancer, the most common cause of tumor-related death in mankind.
Results: EphA5 regulates cell cycle checkpoints and DNA damage repair induced by ionizing radiation.
Conclusion: EphA5 is a novel regulator of DNA damage repair with clinical implications.
Significance: EphA5 may serve as a novel biomarker of radioresistance and a candidate target for therapeutic intervention in human lung cancer.
Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.
doi:10.1074/jbc.M114.630525
PMCID: PMC4367244  PMID: 25623065
Cell Cycle; DNA Damage; DNA Damage Response; DNA Repair; Monoclonal Antibody; Receptor Tyrosine Kinase; Ionizing Radiation
7.  Cyclin D1 and Cancer Development in Laryngeal Premalignancy Patients 
In a previous trial, we found that combined 13-cis retinoic acid (13-cRA), interferon-α and α-tocopherol more effectively reversed advanced premalignant lesions of the larynx than of the oral cavity and that cyclin D1 (CD1)G/A870 single nucleotide polymorphism correlated with cancer risk. We conducted the present trial primarily to confirm the clinical activity of the combination in advanced laryngeal premalignancy and to confirm and extend our findings on CD1, both genotype and protein expression, in association with cancer risk in this setting. Twenty-seven moderate-to-severe laryngeal dysplasia patients underwent induction with combined 13-cRA daily, α-interferon twice weekly, and α-tocopherol daily for one year; 14 non-progressing patients then were randomized to maintenance fenretinide or placebo for two years. During induction, 2 patients had pathological complete responses, 6 had partial responses (30% overall response rate), and 5 developed laryngeal cancer. There were no significant differences between maintenance fenretinide and placebo in response or cancer rates. Ten patients developed cancer overall. Twenty-four patients were evaluated for the CD1 G/A870 genotype, and 23 for pre- and post-treatment CD1 protein expression. Consistent with our earlier report, shorter cancer-free survival was associated with the CD1 AA/AG genotype (p = 0.05). Extending our earlier work, high CD1 expression was associated with worse cancer-free survival overall (p= 0.04) and within each CD1 genotype group. These findings support CD1 genotype and protein expression as important risk markers for laryngeal cancer and suggest future trials targeting upstream regulators of CD1 transcription.
doi:10.1158/1940-6207.CAPR-08-0111
PMCID: PMC4243520  PMID: 19139013
Premalignant lesions; larynx; biochemoprevention; cyclin D1 genotype; cyclin D1 protein expression
8.  VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy 
Purpose
Investigate the mechanisms of regulation and role associated with EZH2 expression in lung cancer cells.
Experimental Design
We investigated the mechanisms of EZH2 expression associated with the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. Additionally, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients’ clinical characteristics.
Results
In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and HIF-1α, and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. Additionally, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti-VEGFR-2 drug AZD2171.
Conclusion
Our results suggest that VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF-1α and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy.
doi:10.1158/1078-0432.CCR-13-1916
PMCID: PMC4190586  PMID: 24850841
EZH2; NSCLC; VEGF/VEGFR-2 pathway; DZNep
9.  Oral Epithelium as a Surrogate Tissue for Assessing Smoking-Induced Molecular Alterations in Lungs 
Background
Both the lungs and oral cavity are exposed to tobacco carcinogens in smokers. We hypothesized that the oral epithelium undergoes molecular alterations similar to those in lungs and therefore may be used as a surrogate tissue to assess tobacco-induced molecular alterations.
Methods
Promoter methylation of p16 and FHIT genes was analyzed with methylation-specific PCR in 1,774 oral and bronchial brush specimens (baseline and 3 months after intervention) from 127 smokers enrolled in a prospective randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites/total sites per subject) was analyzed blindly.
Results
At baseline, promoter methylation was observed in 23%, 17%, and 35% of the bronchial tissues for p16, FHIT, and either of the two genes, respectively, which were comparable to the 19%, 15%, and 31% observed in the oral tissues. Among the 125 individuals with available data from both oral and bronchial tissues, strong correlations were observed between tissues from the two sites (P<0.0001 for both p16 and FHIT). Among the 39 individuals with oral tissue methylation in either of the two genes, the mean bronchial methylation index was 0.53 (± 0.29) compared with only 0.27 (± 0.26) for the 86 subjects without oral tissue methylation (P<0.0001). Similar correlations were also observed in samples obtained at 3 months after chemopreventive intervention.
Conclusions
The oral epithelium may be used as a surrogate tissue to assess tobacco-induced molecular damage in lungs, which has an important implication in conducting biomarker-based lung cancer prevention trials.
doi:10.1158/1940-6207.CAPR-08-0058
PMCID: PMC4183362  PMID: 19138934
10.  Impact of smoking cessation on global gene expression in the bronchial epithelium of chronic smokers 
Cigarette smoke is the major cause of lung cancer and can interact in complex ways with drugs for lung cancer prevention or therapy. Molecular genetic research promises to elucidate the biologic mechanisms underlying divergent drug effects in smokers versus non-smokers and to help in developing new approaches for controlling lung cancer. The present study compared global gene expression profiles (determined via Affymetrix microarray measurements in bronchial epithelial cells) between chronic smokers, former smokers, and never smokers. Smoking effects on global gene expression were determined from a combined analysis of three independent datasets. Differential expression between current and never smokers occurred in 591 of the 13,902 genes measured on the microarrays (P < 0.01 and >2 fold change; pooled data)—a profound effect. In contrast, differential expression between current and former smokers occurred in only 145 of the measured genes (P < 0.01 and >2 fold change; pooled data). Nine of these 145 genes showed consistent and significant changes in each of the three datasets (P < 0.01 and >2 fold change), with 8 being down-regulated in former smokers. Seven of the 8 down-regulated genes, including CYP1B1 and 3 AKR genes, influence the metabolism of carcinogens and/or therapeutic/chemopreventive agents. Our data comparing former and current smokers allowed us to pinpoint the genes involved in smoking–drug interactions in lung cancer prevention and therapy. These findings have important implications for developing new targeted and dosing approaches for prevention and therapy in the lung and other sites, highlighting the importance of monitoring smoking status in patients receiving oncologic drug interventions.
doi:10.1158/1940-6207.CAPR-07-0017
PMCID: PMC4181408  PMID: 19138944
11.  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
12.  CXCR2 expression in tumor cells is a poor prognostic factor and promotes invasion and metastasis in lung adenocarcinoma 
Cancer research  2012;73(2):571-582.
CXCR2 in non-small cell lung cancer (NSCLC) has been studied mainly in stromal cells and is known to increase tumor inflammation and angiogenesis. Here, we examined the prognostic importance of CXCR2 in NSCLC and the role of CXCR2 and its ligands in lung cancer cells. The effect of CXCR2 expression on tumor cells was studied using stable knockdown clones derived from a murine KRAS/p53-mutant lung adenocarcinoma cell line with high metastatic potential and an orthotopic syngeneic mouse model and in vitro using a CXCR2 small molecule antagonist (SB225002). CXCR2 protein expression was analyzed in tumor cells from 262 NSCLC. Gene expression profiles for CXCR2 and its ligands (CXCR2 axis) were analyzed in 52 human NSCLC cell lines and 442 human lung adenocarcinomas. Methylation of CXCR2 axis promoters was determined in 70 human NSCLC cell lines. Invasion and metastasis were decreased in CXCR2 knockdown clones in vitro and in vivo. SB225002 decreased invasion in vitro. In lung adenocarcinomas, CXCR2 expression in tumor cells was associated with smoking and poor prognosis. CXCR2 axis gene expression profiles in human NSCLC cell lines and lung adenocarcinomas defined a cluster driven by CXCL5 and associated with smoking, poor prognosis and RAS pathway activation. Expression of CXCL5 was regulated by promoter methylation. The CXCR2 axis may be an important target in smoking-related lung adenocarcinoma.
doi:10.1158/0008-5472.CAN-12-0263
PMCID: PMC3548940  PMID: 23204236
lung cancer; prognosis; metastasis; CXCR2; chemokine
13.  Characterizing the molecular spatial and temporal field of injury in early stage smoker non-small cell lung cancer patients after definitive surgery by expression profiling 
Gene expression alterations in response to cigarette smoke have been characterized in normal-appearing bronchial epithelium of healthy smokers and it has been suggested that adjacent histologically normal tissue display tumor-associated molecular abnormalities. We sought to delineate the spatial and temporal molecular lung field of injury in smoker early stage non-small cell lung cancer (NSCLC) patients (n=19) who were accrued into a surveillance clinical trial for annual follow-up and bronchoscopies within one year after definitive surgery. Bronchial brushings and biopsies were obtained from six different sites in the lung at the time of inclusion in the study and at 12, 24 and 36 months after the first time point. Affymetrix Human Gene 1.0 ST arrays were used for whole-transcript expression profiling of airways (n=391). Microarray analysis identified gene features (n=1165) that were non-uniform by site and differentially expressed between airways adjacent to tumors relative to more distant samples as well as those (n=1395) that were significantly altered with time up to three years. In addition, gene-interaction networks mediated by PI3K and ERK1/2 were modulated in adjacent compared to contralateral airways and the latter network with time. Furthermore, phosphorylated AKT and ERK1/2 immunohistochemical expression were significantly increased with time (nuclear pAKT, p=0.03; cytoplasmic pAKT, p<0.0001; pERK1/2, p=0.02) and elevated in adjacent compared to more distant airways (nuclear pAKT, p=0.04; pERK1/2, p=0.03). This study highlights spatial and temporal cancer-associated expression alterations in the molecular field of injury of early stage NSCLC patients after definitive surgery that warrant further validation in independent studies.
doi:10.1158/1940-6207.CAPR-12-0290
PMCID: PMC3774536  PMID: 23087048
Early stage NSCLC; gene expression profiling; lung airway epithelium; chemoprevention
14.  EGFR and K-Ras Mutations and Resistance of Lung Cancer to IGF-1R Tyrosine Kinase Inhibitors 
Cancer  2012;118(16):3993-4003.
Background
Most patients with non–small cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs.
Methods
Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in a NSCLC tissue microarray. We analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations.
Results
pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with wt EGFR and wt K-Ras but not in those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing wt K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mut K-Ras.
Conclusions
The mutation status of both EGFR and K-Ras could be predictive markers of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.
doi:10.1002/cncr.26656
PMCID: PMC3674414  PMID: 22359227
EGFR; K-Ras; IGF-1R; lung cancer; TKI
15.  Genetic variants in the PI3K/PTEN/AKT/MTOR pathway predict head and neck cancer patient second primary tumor/recurrence risk and response to retinoid chemoprevention 
Clinical Cancer Research  2012;18(13):3705-3713.
Purpose
The development of second primary tumors (SPT) or recurrence alters prognosis for curatively-treated head and neck squamous cell carcinoma (HNSCC) patients. 13-cis-retnoic acid (13-cRA) has been tested as a chemoprevention agent in clinical trials with mixed results. Therefore, we investigated if genetic variants in the PI3K/PTEN/AKT/MTOR pathway could serve as biomarkers to identify which patients are at high risk of an SPT/recurrence while also predicting response to 13-cRA chemoprevention.
Experimental Design
A total of 137 pathway SNPs were genotyped in 440 patients from the Retinoid Head and Neck Second Primary Trial and assessed for SPT/recurrence risk and response to 13-cRA. Risk models were created based on epidemiology, clinical, and genetic data.
Results
Twenty-two genetic loci were associated with increased SPT/recurrence risk with six also being associated with a significant benefit following chemoprevention. Combined analysis of these high-risk/high-benefit loci identified a significant (P = 1.54×10−4) dose-response relationship for SPT/recurrence risk, with patients carrying 4–5 high-risk genotypes having a 3.76-fold (95%CI:1.87–7.57) increase in risk in the placebo group (n=215). Patients carrying 4–5 high-risk loci showed the most benefit from 13-cRA chemoprevention with a 73% reduction in SPT/recurrence (95%CI:0.13–0.58) compared to those with the same number of high-risk genotypes who were randomized to receive placebo. Incorporation of these loci into a risk model significantly improved the discriminatory ability over models with epidemiology, clinical, and previously identified genetic variables.
Conclusions
These results demonstrate that loci within this important pathway could identify individuals with a high-risk/high-benefit profile and are a step towards personalized chemoprevention for HNSCC patients.
doi:10.1158/1078-0432.CCR-11-3271
PMCID: PMC3404728  PMID: 22577058
16.  Phase II study of TAS-106 in patients with platinum-failure recurrent or metastatic head and neck cancer and nasopharyngeal cancer 
Cancer Medicine  2013;2(3):351-359.
TAS-106, a RNA polymerase inhibitor, was studied in solid tumors with potential clinical benefit and reasonable tolerability. We conducted a multicenter, international phase II trial of TAS-106 in salvage metastatic or recurrent head and neck squamous cell cancer (HNSCC) and nasopharyngeal cancer (NPC) patients. TAS-106 monotherapy was given at 6.5 mg/m2 over 24-h continuous infusion every 3 weeks. Translational studies for blood and tissue were included. Twenty-seven enrolled patients experienced the most common drug-related adverse events of neutropenia, fatigue, non-neutropenic fever, injection site reaction, and skin rash/dermatitis. The greater than or equal to grade 3 adverse events included neutropenia (14.8%), febrile neutropenia (7.4%), pneumonia (7.4%), and peripheral neuropathy (3.7%). The overall response rate was 0% in both subgroups; five HNSCC patients had stable disease (median duration 99 days) and four NPC patients had stable disease (median duration of 92.5 days). Median progression-free survival (PFS) for HNSCC patients was 52 days (95% CI 43.0–99.0 days) and 48 days (95% CI 41.0–83.0 days) for NPC. Median overall survival (OS) for HNSCC patients was 175 days (95% CI 92.0–234.0 days) and 280 days (95% CI 107.0–462.0 days) for NPC. The TAS-106 plasma levels were equivalent between Asian and Caucasian patients. There was no significant correlation of tumor UCK2 protein expression levels to TAS-106 efficacy. TAS-106 was reasonably tolerated in patients with platinum-failure HNSCC and NPC. The administration schedule of 24-h continuous infusion prevented neurologic toxicity, but had myelosuppression as its main toxicity. There was no anti-tumor efficacy seen with TAS-106 monotherapy. Future studies will focus on TAS-106 combinations and mechanisms of drug resistance.
doi:10.1002/cam4.79
PMCID: PMC3699847  PMID: 23930212
Head and neck squamous cell carcinoma; nasopharyngeal cancer; TAS-106
17.  Prognostic impact of Insulin Receptor Expression on Survival of Patients with Nonf-Small Cell Lung Cancer 
Cancer  2011;118(9):2454-2465.
Background
The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with non-small cell lung cancer (NSCLC).
Methods
A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays.
Results
The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC) whereas cytoplasmic IGF-1R score was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (RFS) (3.8 vs. 3.3 years; 3.8 vs. 2.0 years, respectively), but similar overall survival (OS). Patients with high expression levels of IR and IGF-1R had shorter RFS and OS compared to those with low levels of IR and/or IGF-1R expression. Finally, a multivariate analysis revealed the impact of IR, but not of IGF-1R, as an independent predictive marker of NSCLC survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001 – 1.010], HR for RFS, 1.005 (95% CI, 1.001 – 1.009), when IR score was tested as a continuous variable.
Conclusions
Overexpression of IR predicts a poor survival among patients with NSCLC, especially those with SCC. These results might serve as future guidance to the clinical trials involving IR or IGR-1R targeting agents.
doi:10.1002/cncr.26492
PMCID: PMC3298843  PMID: 21952750
Carcinoma; Non-Small-Cell Lung; Receptor; Insulin; Receptor; IGF Type 1; Prognosis; Survival
18.  Dual inhibition of Tumor Energy Pathway by 2-deoxy glucose and metformin Is Effective Against a Broad Spectrum of Preclinical Cancer Models 
Molecular cancer therapeutics  2011;10(12):2350-2362.
Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics.The AMP-activated kinase (AMPK) pathway appears dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxy glucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro anti-tumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy demonstrated in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) while metformin compromises OXPHOS. Importantly, forced energy restoration with methylpyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors.
doi:10.1158/1535-7163.MCT-11-0497
PMCID: PMC3237863  PMID: 21992792
Tumor bioenergetics; Targeted therapy; Cancer energy metabolic pathway
19.  Validation of a Novel Statistical Model for Assessing the Synergy of Combined-agent Cancer Chemoprevention 
Lung cancer is the leading cause of cancer death, developing over prolonged periods through genetic and epigenetic changes induced and exacerbated by tobacco exposure. Many epigenetic changes including DNA methylation and histone methylation and acetylation are reversible, and agents that can modulate these aberrations are a potentially effective approach to cancer chemoprevention. Combined epigenetic-targeting agents have gained interest for their potential to increase efficacy and lower toxicity. The present study applied recently developed statistical methods to validate the combined effects of the demethylating agent 5-aza-2-deoxycytidine (5-AZA-CdR, or AZA, or decitabine) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, or vorinostat). This validation compared AZA alone with SAHA alone and with their combinations (at later or earlier time points and in varying doses) for inhibiting the growth of cell lines of an in vitro lung carcinogenesis system. This system comprises isogenic premalignant and malignant cells that are immortalized (earlier premalignant), transformed (later premalignant), and tumorigenic human bronchial epithelial (HBE) cells (immortalized BEAS-2B and its derivatives 1799 [immortalized], 1198 [transformed], and 1170-I [tumorigenic]). AZA alone and SAHA alone produced a limited (< 50%) inhibition of cell growth, whereas combined AZA and SAHA inhibited cell growth more than did either agent alone, reaching 90% inhibition under some conditions. Results of drug-interaction analyses in the Emax model and semiparametric model supported the conclusion that the drug combinations exert synergistic effects, i.e., beyond additivity in the Loewe model. The present results demonstrate the applicability of our novel statistical methodology for quantitatively assessing drug synergy across a wide range of doses of agents with complex dose-response profiles, a methodology with great potential for advancing the development of chemopreventive combinations.
doi:10.1158/1940-6207.CAPR-10-0129
PMCID: PMC3496745  PMID: 20663979
lung cancer; epigenetics; vorinostat; decitabine; premalignant; epithelial cells
20.  Proliferative Changes in the Bronchial Epithelium of Former Smokers Treated With Retinoids 
Background
Retinoids have shown antiproliferative and chemopreventive activity. We analyzed data from a randomized, placebo-controlled chemoprevention trial to determine whether a 3-month treatment with either 9-cis-retinoic acid (RA) or 13-cis-RA and α-tocopherol reduced Ki-67, a proliferation biomarker, in the bronchial epithelium.
Methods
Former smokers (n = 225) were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg), 13-cis-RA (1 mg/kg) and α-tocopherol (1200 IU), or placebo. Bronchoscopic biopsy specimens obtained before and after treatment were immunohistochemically assessed for changes in the Ki-67 proliferative index (i.e., percentage of cells with Ki-67–positive nuclear staining) in the basal and parabasal layers of the bronchial epithelium. Per-subject and per–biopsy site analyses were conducted. Multicovariable analyses, including a mixed-effects model and a generalized estimating equations model, were used to investigate the treatment effect (Ki-67 labeling index and percentage of bronchial epithelial biopsy sites with a Ki-67 index ≥ 5%) with adjustment for multiple covariates, such as smoking history and metaplasia. Coefficient estimates and 95% confidence intervals (CIs) were obtained from the models. All statistical tests were two-sided.
Results
In per-subject analyses, Ki-67 labeling in the basal layer was not changed by any treatment; the percentage of subjects with a high Ki-67 labeling in the parabasal layer dropped statistically significantly after treatment with 13-cis-RA and α-tocopherol treatment (P = .04) compared with placebo, but the drop was not statistically significant after 9-cis-RA treatment (P = .17). A similar effect was observed in the parabasal layer in a per-site analysis; the percentage of sites with high Ki-67 labeling dropped statistically significantly after 9-cis-RA treatment (coefficient estimate = −0.72, 95% CI = −1.24 to −0.20; P = .007) compared with placebo, and after 13-cis-RA and α-tocopherol treatment (coefficient estimate = −0.66, 95% CI = −1.15 to −0.17; P = .008).
Conclusions
In per-subject analyses, treatment with 13-cis-RA and α-tocopherol, compared with placebo, was statistically significantly associated with reduced bronchial epithelial cell proliferation; treatment with 9-cis-RA was not. In per-site analyses, statistically significant associations were obtained with both treatments.
doi:10.1093/jnci/djm205
PMCID: PMC3441140  PMID: 17971525
21.  Prevention of Bronchial Hyperplasia by EGFR Pathway Inhibitors in an Organotypic Culture Model 
Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. The current study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured using the 3-dimensional organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands EGF, transforming growth factor-alpha, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MEK inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we demonstrated that bronchial hyperplasia can be modeled in vitro using primary NHTBE cells maintained in a 3-dimensional (3-D) organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role.
doi:10.1158/1940-6207.CAPR-10-0364
PMCID: PMC3151315  PMID: 21505178
NHBE; bronchial hyperplasia; dysplasia; erlotinib; MEK inhibitor
22.  HER Family Receptor Abnormalities in Lung Cancer Brain Metastases and Corresponding Primary Tumors 
Clinical Cancer Research  2009;15(15):4829-4837.
Purpose
To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).
Experimental design
Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.
Results
Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.
Conclusions
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
doi:10.1158/1078-0432.CCR-08-2921
PMCID: PMC3372920  PMID: 19622585
23.  Genome-Wide Association Study of Survival in Non–Small Cell Lung Cancer Patients Receiving Platinum-Based Chemotherapy 
Background
Interindividual variation in genetic background may influence the response to chemotherapy and overall survival for patients with advanced-stage non–small cell lung cancer (NSCLC).
Methods
To identify genetic variants associated with poor overall survival in these patients, we conducted a genome-wide scan of 307 260 single-nucleotide polymorphisms (SNPs) in 327 advanced-stage NSCLC patients who received platinum-based chemotherapy with or without radiation at the University of Texas MD Anderson Cancer Center (the discovery population). A fast-track replication was performed for 315 patients from the Mayo Clinic followed by a second validation at the University of Pittsburgh in 420 patients enrolled in the Spanish Lung Cancer Group PLATAX clinical trial. A pooled analysis combining the Mayo Clinic and PLATAX populations or all three populations was also used to validate the results. We assessed the association of each SNP with overall survival by multivariable Cox proportional hazard regression analysis. All statistical tests were two-sided.
Results
SNP rs1878022 in the chemokine-like receptor 1 (CMKLR1) was statistically significantly associated with poor overall survival in the MD Anderson discovery population (hazard ratio [HR] of death = 1.59, 95% confidence interval [CI] = 1.32 to 1.92, P = 1.42 × 10−6), in the PLATAX clinical trial (HR of death = 1.23, 95% CI = 1.00 to 1.51, P = .05), in the pooled Mayo Clinic and PLATAX validation (HR of death = 1.22, 95% CI = 1.06 to 1.40, P = .005), and in pooled analysis of all three populations (HR of death = 1.33, 95% CI = 1.19 to 1.48, P = 5.13 × 10−7). Carrying a variant genotype of rs10937823 was associated with decreased overall survival (HR of death = 1.82, 95% CI = 1.42 to 2.33, P = 1.73 × 10−6) in the pooled MD Anderson and Mayo Clinic populations but not in the PLATAX trial patient population (HR of death = 0.96, 95% CI = 0.69 to 1.35).
Conclusion
These results have the potential to contribute to the future development of personalized chemotherapy treatments for individual NSCLC patients.
doi:10.1093/jnci/djr075
PMCID: PMC3096796  PMID: 21483023
24.  Abnormalities of the TITF-1 lineage-specific oncogene in NSCLC: Implications in lung cancer pathogenesis and prognosis 
PURPOSE
Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.
EXPERIMENTAL DESIGN
To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.
RESULTS
We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).
CONCLUSIONS
Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.
doi:10.1158/1078-0432.CCR-10-1412
PMCID: PMC3078948  PMID: 21257719
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
25.  Gene expression signature–based prognostic risk score in gastric cancer 
Purpose
Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathological parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment.
Experimental Design
Using microarray technologies, we generated and analyzed gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients.
Results
We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the 6-gene signature in an additional independent cohort.
Conclusions
The risk score derived from the 6-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy.
doi:10.1158/1078-0432.CCR-10-2180
PMCID: PMC3078023  PMID: 21447720

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