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1.  PILOT STUDY OF GEFITINIB AND FULVESTRANT IN THE TREATMENT OF POST-MENOPAUSAL WOMEN WITH ADVANCED NON-SMALL CELL LUNG CANCER 
SUMMARY
Introduction
Estrogen receptor beta (ERβ) has been detected in NSCLC cell lines and tumor specimens. The ER down-regulator, fulvestrant, blocked estradiol-stimulation of tumor growth and gene transcription in NSCLC preclinical models and showed additive effects with the EGFR inhibitor gefitinib. The safety and tolerability of combination therapy with the EGFR inhibitor, gefitinib, and fulvestrant was explored.
Methods
Post-menopausal women with advanced NSCLC received gefitinib 250 mg po daily and fulvestrant 250 mg IM monthly.
Results
Twenty-two patients were enrolled. Eight patients had adenocarcinoma, 6 NSCLC-NOS, 4 squamous cell, and 4 BAC. Seven patients were never smokers. Eight patients received ≥ 2 lines of prior chemotherapy, 6 received one prior chemotherapy, and 8 were treatment naive. One patient experienced grade 4 dyspnea possibly related to treatment; all other grade 3/4 toxicities were unrelated to treatment. Twenty patients were evaluable for response: 3 PRs were confirmed (response rate of 15%, 95% CI: 5% – 36%). The median PFS, OS, and estimated 1 yr OS were 12 wks (3–112 wks), 38.5 weeks (7–135 wks), and 41% (95% CI 20–62%), respectively. Survival outcomes did not differ by prior lines of therapy. A subset analysis revealed that OS in the 8 patients whose tumors exhibited at least 60% ERβ nuclear IHC staining measured 65.5 weeks, while that of the 5 patients with ERβ staining of less than 60% was 21 weeks. One patient with BAC and a PR had an EGFR L858R mutation in exon 21. There was no correlation between ERβ IHC expression and histology or smoking history.
Conclusions
Combination therapy with gefitinib and fulvestrant in this population was well-tolerated and demonstrated disease activity.
doi:10.1016/j.lungcan.2008.07.002
PMCID: PMC3164240  PMID: 18701186
epidermal growth factor receptor; hormonal treatment; non-small cell lung cancer; sex; age; estrogen receptor
2.  Lung cancer risk prediction using common SNPs located in GWAS-identified susceptibility regions 
Introduction
Genome-wide association studies (GWAS) have consistently identified specific lung cancer susceptibility regions. We evaluated the lung cancer predictive performance of single nucleotide polymorphisms (SNPs) in these regions.
Methods
Lung cancer cases (N=778) and controls (N=1166) were genotyped for 77 SNPs located in GWAS-identified lung cancer susceptibility regions. Variable selection and model development used stepwise logistic regression and decision-tree analysis. In a subset nested in the Pittsburgh Lung Screening Study, change in area under the receiver operator characteristic curve (AUC) and net reclassification improvement (NRI) were used to compare predictions made by risk factor models with and without genetic variables.
Results
Variable selection and model development kept two SNPs in each of three GWAS regions, rs2736100 and rs7727912 in 5p15.33, rs805297 and rs1802127 in 6p21.33, and rs8034191 and rs12440014 in 15q25.1. The ratio of cases to controls was three times higher among subjects with a high-risk genotype in every one as opposed to none of the three GWAS regions (odds ratio 3.14, 95% confidence interval 2.02-4.88, adjusted for sex, age and pack-years). Adding a three-level classified count of GWAS regions with high-risk genotypes to an age and smoking risk factor-only model improved lung cancer prediction by a small amount: AUC 0.725 vs. 0.717 (P=0.056); NRIoverall was 0.052 across low, intermediate, and high 6-year lung cancer risk categories (<3.0%, 3.0% to 4.9%, ≥5.0%).
Conclusions
Specifying genotypes for SNPs in three GWAS-identified susceptibility regions improved lung cancer prediction, but probably by an extent too small to affect disease control practice.
doi:10.1097/JTO.0000000000000666
PMCID: PMC4636453  PMID: 26352532
lung cancer; single nucleotide polymorphism; risk prediction
3.  Co-targeting c-Met and COX-2 leads to enhanced inhibition of lung tumorigenesis in a murine model with heightened airway HGF 
Background
The hepatocyte growth factor (HGF)/c-Met pathway is often dysregulated in non-small cell lung cancer (NSCLC). HGF activation of c-Met induces cyclooxygenase-2 (COX-2), resulting in downstream stimulation by PGE2 of additional pathways. Targeting both c-Met and COX-2 might lead to enhanced anti-tumor effects by blocking signaling upstream and downstream of c-Met.
Methods
Effects of crizotinib or celecoxib alone or in combination were tested in NSCLC cells in vitro and in mice transgenic for airway expression of human HGF (HGF TG).
Results
Proliferation and invasion of NSCLC cells treated with a combination of crizotinib and celecoxib was significantly lower compared to single treatments. Transgenic mice showed enhanced COX-2 expression localized to preneoplastic areas following exposure to the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), which was not present without carcinogen exposure. This shows that COX-2 activity is present during lung tumor development in a high HGF environment. Following NNK treatment, a significant decrease in the number of lung tumors per animal was observed after 13 week treatments of crizotinib, celecoxib or the combination compared to placebo (P<0.001). With combination treatment, the number of tumors was also significantly lower than single agent treatment (P<0.001). In the resulting lung tumors, P-c-Met, COX-2, PGE2, and P-MAPK were significantly down-modulated by combination treatment compared to single treatment. Expression of the epithelial-mesenchymal transition (EMT) markers E-cadherin and snail were also modulated by combination treatment.
Conclusions
In the presence of high HGF, dual inhibition of c-Met and COX-2 may enhance anti-tumor effects. This combination may have clinical potential in NSCLCs with high HGF/c-Met expression or EMT phenotype.
doi:10.1097/JTO.0000000000000245
PMCID: PMC4134355  PMID: 25057941
HGF; COX-2; c-Met; lung cancer; crizotinib; celecoxib
4.  Epigenetic Repression of CCDC37 and MAP1B Links Chronic Obstructive Pulmonary Disease to Lung Cancer 
Introduction
Lung cancer and chronic obstructive pulmonary disease (COPD) share environmental risk factors. COPD also increases the risk of lung cancer; however, the molecular mechanisms are unclear.
Methods
An epigenome-wide association study of lung tumors and cancer-free lung tissue (CFLT) pairs from non-small cell lung cancer (NSCLC) cases with (n=18) or without (n=17) COPD was conducted using the HumanMethylation450 beadchip (HM450K). COPD-associated methylation of top-ranked genes was confirmed in a larger sample set, independently validated, and their potential as sputum-based biomarkers was investigated.
Results
Methylation of CCDC37 and MAP1B was more prevalent in lung tumors from COPD than non-COPD cases [54/71 (76%) vs. 20/46 (43%), p=0.0013] and [48/71 (68%) vs. 17/46 (37%), p=0.0035], respectively, after adjustment for age, sex, smoking status, and tumor histology. HM450K probes across CCDC37 and MAP1B promoters showed higher methylation in tumors than CFLT with the highest methylation seen in tumors from COPD cases (p<0.05). These results were independently validated using The Cancer Genome Atlas data. CCDC37 methylation was more prevalent in sputum from COPD than non-COPD smokers (p<0.005) from two cohorts. CCDC37 and MAP1B expression was dramatically repressed in tumors and CFLT from COPD than non-COPD cases, p<0.02.
Conclusions
The reduced expression of CCDC37 and MAP1B associated with COPD likely predisposes these genes to methylation that in turn, may contribute to lung cancer.
doi:10.1097/JTO.0000000000000592
PMCID: PMC4511946  PMID: 26200272
EWAS; Methylation; COPD; NSCLC; Sputum
5.  A prospective and retrospective analysis of smoking behavior changes in ever smokers with high risk for lung cancer from New Mexico and Pennsylvania 
Cigarette smoking is the leading preventable cause of death worldwide. The aim of this study is to conduct a prospective and retrospective analysis of smoking behavior changes in the Lovelace Smokers Cohort (LSC) and the Pittsburgh Lung Screening Study cohort (PLuSS). Area under the curve (AUC) for risk models predicting relapse based on demographic, smoking, and relevant clinical variables was 0.93 and 0.79 in LSC and PLuSS, respectively. The models for making a quit attempt had limited prediction ability in both cohorts (AUC≤0.62). We identified an ethnic disparity in adverse smoking behavior change that Hispanic smokers were less likely to make a quit attempt and were more likely to relapse after a quit attempt compared to non-Hispanic Whites. SNPs at 15q25 and 11p14 loci were associated with risk for smoking relapse in the LSC. Rs6495308 at 15q25 has a large difference in minor allele frequency between non-Hispanic Whites and Hispanics (0.46 versus 0.23, P<0.0001) and was associated with risk for ever relapse at same magnitude between the two ethnic groups (OR=1.36, 95% CI=1.10 to 1.67 versus 1.59, 95% CI=1.00 to 2.53, P=0.81). In summary, the risk prediction model established in LSC and PLuSS provided an excellent to outstanding distinguishing for abstainers who will or will not relapse. The ethnic disparity in adverse smoking behavior between Hispanics and non-Hispanic Whites may be at least partially explained by the sequence variants at 15q25 locus that contains multiple nicotine acetylcholine receptors.
PMCID: PMC4913225  PMID: 27335628
Smoking behavior; risk prediction model; ethnicity; single nucleotide polymorphism
6.  NF-κB1 p105 suppresses lung tumorigenesis through the Tpl2 kinase but independently of its NF-κB function 
Oncogene  2015;35(18):2299-2310.
NF-κB is generally believed to be pro-tumorigenic. Here, we report a tumor-suppressive function for NF-κB1, the prototypical member of NF-κB. While NF-κB1 down-regulation is associated with high lung cancer risk in humans and poor patient survival, NF-κB1 deficient mice are more vulnerable to lung tumorigenesis induced by the smoke carcinogen, urethane. Notably, the tumor suppressive function of NF-κB1 is independent of its classical role as an NF-κB factor, but instead through stabilization of the Tpl2 kinase. NF-κB1 deficient tumors exhibit “normal” NF-κB activity, but a decreased protein level of Tpl2. Reconstitution of Tpl2 or the NF-κB1 p105, but not p50 (the processed product of p105), inhibits the tumorigenicity of NF-κB1 deficient lung tumor cells. Remarkably, Tpl2 knockout mice resemble NF-κB1 knockouts in urethane-induced lung tumorigenesis. Mechanistic studies indicate that p105/Tpl2 signaling is required for suppressing urethane-induced lung damage and inflammation, and activating mutations of the K-Ras oncogene. These studies reveal an unexpected, NF-κB-independent but Tpl2-depenednt role of NF-κB1 in lung tumor suppression. These studies also reveal a previously unexplored role of p105/Tpl2 signaling in lung homeostasis.
doi:10.1038/onc.2015.299
PMCID: PMC4548811  PMID: 26300007
lung cancer; NF-κB1; p105; p50; Tpl2
7.  Incidence of head and neck squamous cell carcinoma among subjects at high-risk for lung cancer: results from the Pittsburgh Lung Screening Study 
Cancer  2015;121(9):1431-1435.
Background
Earlier detection and diagnosis of head and neck squamous cell carcinoma (HNSCC) should lead to improved outcomes. However, to date, no effective screening strategy has been identified. We evaluated whether it would be useful to screen subjects targeted for lung cancer screening also for HNSCC.
Methods
Medical records, death certificates, and cancer registry and questionnaire data were used to determine the number of observed incident HNSCC cases in the Pittsburgh Lung Screening Study (PLuSS), a cohort of ≥ 50 year old current and ex-smokers with ≥ 12.5 pack-year smoking history. Expected number of cases was estimated using stratum-specific incidence rates obtained from 2000-2011 SEER data. Standardized incidence ratio (SIR) was calculated to examine the difference between observed and expected number of cases.
Results
Twenty-three (0.64%) of the 3,587 at-risk participants in PLuSS developed HNSCC over in total 32,201 person-years of follow-up. This was significantly higher than expected based on incidence rates obtained from SEER (13.70 cases expected; SIR=1.68, 95% confidence interval: 1.06-2.52). Excess burden of HNSCC in PLuSS was 28.9 cases per 100,000 person-years. Observed incident cases were significantly more often male, started smoking at a younger age, smoked more per day and had more pack-years of smoking than the rest of the PLuSS at-risk participants.
Conclusion
Our results provide a rationale for offering head and neck cancer screening along with CT screening for lung cancer. Randomized controlled trials that assess the effectiveness of adding examination of the head and neck area to lung cancer screening programs are warranted.
doi:10.1002/cncr.29189
PMCID: PMC4409462  PMID: 25559556
head and neck cancer; lung cancer; incidence; high-risk; screening
8.  15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers 
Background:
Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis.
Methods:
A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided.
Results:
Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10–8). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10–9). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin.
Conclusions:
A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.
doi:10.1093/jnci/djv035
PMCID: PMC4555640  PMID: 25713168
9.  RET Rearrangements in Lung Adenocarcinoma and Radiation 
Background
RET rearrangement, a hallmark of radiation-induced thyroid cancer, has been reported to occur in 1% of lung adenocarcinoma patients. Patients with this rearrangement tend to be younger and never smokers, raising a possibility of other causes, such as radiation. We hypothesized that RET chromosomal rearrangement may represent a genetic mechanism of radiation-induced lung cancer.
Methods
Two hundred forty-five consecutive primary lung adenocarcinomas without history of radiation and 38 lung adenocarcinoma patients with a history of therapeutic radiation for breast carcinoma or mediastinal Hodkgin lymphoma were tested for RET rearrangement by fluorescence in situ hybridization. Human lung adenocarcinoma cells (201T) were subjected to γ radiation and tested for RET gene fusions by reverse transcriptase-polymerase chain reaction and Southern blot hybridization.
Results
We identified one case with RET rearrangement in the group without history of radiation (1 of 240; 0.4%) and two cases in the group with history of radiation (2 of 37; 5.4%; P=0.0436). Both these patients were women, who were former smokers with a history of breast carcinoma treated with surgery and radiation. Furthermore, we found that RET fusions could be directly induced in 201T human lung cells by exposure to 1 Gy of γ radiation. All fusions identified were between RET and KIF5B genes, and no RET fusions to CCDC6 or NCOA4 genes, characteristic for thyroid cancer, were identified in the irradiated lung cells.
Conclusion
RET fusions may represent a genetic mechanism of radiation-induced lung adenocarcinoma.
doi:10.1097/JTO.0000000000000015
PMCID: PMC4836180  PMID: 24346100
Lung adenocarcinoma; RET proto oncogene; Radiation
10.  Kinin B2 Receptor Mediates Induction of Cyclooxygenase-2 and Is Over-Expressed In Head and Neck Squamous Cell Carcinomas 
Molecular cancer research : MCR  2008;6(12):1946-1956.
Bradykinin (BK) has been shown to promote growth and migration of head and neck squamous cell carcinoma (HNSCC) cells via epidermal growth factor receptor (EGFR) transactivation. It has also been reported that BK can cause the induction of cyclooxygenase-2 (COX-2), a pro-tumorigenic enzyme, via the MAPK (Mitogen-Activated Protein Kinase) pathway in human airway cells. To determine whether COX-2 is up-regulated by BK in HNSCC, the current study investigated BK- induced EGFR transactivation, MAPK activation, and cyclooxygenase-2 (COX-2) expression in human HNSCC cells. BK induced a concentration- and time-dependent induction of COX-2 protein in HNSCC, which was preceded by phosphorylation of EGFR and MAPK. These effects were abolished by the B2 receptor (B2R) antagonist Hoe 140 but not the B1 receptor (B1R) antagonist, Lys-[Leu8]des-Arg9-BK. COX-2 induction was accompanied by increased release of PGE2. No effect of a B1R agonist (des-Arg9-BK) on p-MAPK or COX-2 expression was observed. B2R protein was found to be expressed in all four head and neck cell lines tested. Immunohistochemical analysis and immunoblot analysis revealed that B2R, but not B1R, was significantly over-expressed in HNSCC tumors compared to levels in normal mucosa from the same patient. In HNSCC cells, the BK-induced expression of COX-2 was inhibited by the EGFR kinase inhibitor gefitinib or mitogen activated protein kinase kinases (MEK) inhibitors (PD98059 or U0126). These results suggest that EGFR and MAPK are required for COX-2 induction by BK. Up-regulation of the B2R in head and neck cancers suggests this pathway is involved in HNSCC tumorigenesis.
doi:10.1158/1541-7786.MCR-07-2197
PMCID: PMC3575100  PMID: 19074839
Bradykinin; B2 receptor; EGFR; MAPK; cyclooxygenase-2; head and neck cancer
11.  Expression of PAM50 Genes in Lung Cancer: Evidence that Interactions between Hormone Receptors and HER2/HER3 Contribute to Poor Outcome12 
Neoplasia (New York, N.Y.)  2015;17(11):817-825.
Non–small cell lung cancers (NSCLCs) frequently express estrogen receptor (ER) β, and estrogen signaling is active in many lung tumors. We investigated the ability of genes contained in the prediction analysis of microarray 50 (PAM50) breast cancer risk predictor gene signature to provide prognostic information in NSCLC. Supervised principal component analysis of mRNA expression data was used to evaluate the ability of the PAM50 panel to provide prognostic information in a stage I NSCLC cohort, in an all-stage NSCLC cohort, and in The Cancer Genome Atlas data. Immunohistochemistry was used to determine status of ERβ and other proteins in lung tumor tissue. Associations with prognosis were observed in the stage I cohort. Cross-validation identified seven genes that, when analyzed together, consistently showed survival associations. In pathway analysis, the seven-gene panel described one network containing the ER and progesterone receptor, as well as human epidermal growth factor receptor (HER)2/HER3 and neuregulin-1. NSCLC cases also showed a significant association between ERβ and HER2 protein expression. Cases positive for HER2 expression were more likely to express HER3, and ERβ-positive cases were less likely to be both HER2 and HER3 negative. Prognostic ability of genes in the PAM50 panel was verified in an ERβ-positive cohort representing all NSCLC stages. In The Cancer Genome Atlas data sets, the PAM50 gene set was prognostic in both adenocarcinoma and squamous cell carcinoma, whereas the seven-gene panel was prognostic only in squamous cell carcinoma. Genes in the PAM50 panel, including those linking ER and HER2, identify lung cancer patients at risk for poor outcome, especially among ERβ-positive cases and squamous cell carcinoma.
doi:10.1016/j.neo.2015.11.002
PMCID: PMC4681883  PMID: 26678909
NSCLC, non–small cell lung cancer; ER, estrogen receptor; PGR, progesterone receptor; HR, hazard ratio; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; FFPE, formalin-fixed, paraffin embedded; PCA, principal component analysis; DFS, disease-free survival; PFS, progression-free survival; OS, overall survival; TCGA, The Cancer Genome Atlas; PAM50, prediction analysis of microarray 50 gene set; IHC, immunohistochemistry; IPA, Ingenuity Pathway Analysis
12.  Dual blockade of EGFR and c-Met abrogates redundant signaling and proliferation in head and neck carcinoma cells 
Purpose
Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.
Experimental Design
Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with anti-tumor responses to inhibitors of these pathways.
Results
Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, transforming growth factor-alpha (TGF-α), were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also demonstrated that TGF-α can induce phosphorylation of c-Met over 6-fold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared to single agent treatment in vivo.
Conclusions
Together these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.
doi:10.1158/1078-0432.CCR-10-3339
PMCID: PMC3138116  PMID: 21622718
13.  HGF-independent Potentiation of EGFR Action by c-Met 
Oncogene  2011;30(33):3625-3635.
The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant Epidermal Growth Factor Receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors is minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met which begins at 8 h continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of MAPK or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of HGF or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pre-treatment with pan-SFK inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared to control, further, confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft anti-tumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.
doi:10.1038/onc.2011.84
PMCID: PMC3126872  PMID: 21423210
c-Met; c-Src; EGFR; cross-talk
14.  Combined analysis of estrogen receptor β–1 and progesterone receptor expression identifies lung cancer patients with poor outcome 
Purpose
Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.
Experimental Design
Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β–1 ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR).
Results
Tumors expressed higher levels of ERβ compared to matched normal lung, while the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS) (HR=1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP) (HR=1.59). After adjusting for stage, age, sex and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR=2.64) and on TTP (HR=6.02). Further effects on OS were observed when EGFR expression was included (HR=5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR and high total PR had shorter OS than patients with the opposite pattern (HR= 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody.
Conclusions
Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ–1 /low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers.
doi:10.1158/1078-0432.CCR-10-0992
PMCID: PMC3064257  PMID: 21062926
estrogen receptor; progesterone receptor; aromatase; epidermal growth factor receptor; lung cancer
15.  Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma 
BMC Cancer  2015;15:649.
Background
ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.
Methods
We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.
Results
No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.
Conclusions
Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1649-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1649-3
PMCID: PMC4595318  PMID: 26438152
ATM; ATR; Fanconi anemia; Lung carcinoma
16.  Matrix Metalloproteinase-19 Promotes Metastatic Behavior In Vitro and Is Associated with Increased Mortality in Non–Small Cell Lung Cancer 
Rationale: Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects.
Objectives: To test the hypothesis that MMP19 plays a significant role in the development and progression of non–small cell lung cancer (NSCLC).
Methods: We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells.
Measurements and Main Results: We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial–mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial–mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression.
Conclusions: Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.
doi:10.1164/rccm.201310-1903OC
PMCID: PMC4299607  PMID: 25250855
MMP19; epithelial–mesenchymal transition; metastasis; non–small cell lung cancer; miR-30
17.  HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer 
Purpose
We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.
Experimental Design
Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.
Results
c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.
Conclusions
These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
doi:10.1158/1078-0432.CCR-08-3252
PMCID: PMC3159511  PMID: 19470725
18.  Early Changes in Pulmonary Gene Expression Following Tobacco Exposure Shed Light on the Role of Estrogen Metabolism in Lung Carcinogenesis 
This perspective on Meireles et al. (beginning on p. XXX in this issue of the journal) discusses the increasing evidence for the role of female steroid hormones in lung-cancer development and progression. The novel work of Meireles et al. is the first evidence for the rapid upregulation by tobacco smoke of a key cytochrome P450 gene that can metabolize estrogens such as β-estradiol to potentially carcinogenic catechol and quinine forms, as well as the first evidence for the colocalization of β-estradiol and estrogen receptors in murine airway epithelium. Actions of estrogens that contribute to lung carcinogenesis, especially in the presence of tobacco smoke, may involve both reactive intermediates that damage DNA and steroid hormone-receptor signaling that promotes growth.
doi:10.1158/1940-6207.CAPR-10-0093
PMCID: PMC2881174  PMID: 20515955
19.  Serum biomarker profiles as diagnostic tools in lung cancer 
Background
Computed tomography (CT) scanning has emerged as an effective means of early detection for lung cancer. Despite marked improvement over earlier methodologies, the low level of specificity demonstrated by CT scanning has limited its clinical implementation as a screening tool. A minimally-invasive biomarker-based test that could further characterize CT-positive patients based on risk of malignancy would greatly enhance its clinical efficacy.
Methods
We performed an analysis of 81 serum proteins in 92 patients diagnosed with lung cancer and 172 CT-screened control individuals. We utilize a series of bioinformatics algorithms including Metropolis-Monte Carlo, artificial neural networks, Naïve Bayes, and additive logistic regression to identify multimarker panels capable of discriminating cases from controls with high levels of sensitivity and specificity in distinct training and independent validation sets.
Results
A three-biomarker panel comprised of MIF, prolactin, and thrombospondin identified using the Metropolis-Monte Carlo algorithm provided the best classification with a %Sensitivity/Specificity/Accuracy of 74/90/86 in the training set and 70/93/82 in the validation set. This panel was effective in the classification of control individuals demonstrating suspicious pulmonary nodules and stage I lung cancer patients.
Conclusions
The selected serum biomarker panel demonstrated a high diagnostic utility in the current study and performance characteristics which compare favorably with previous reports. Further advancements may lead to the development of a diagnostic tool useful as an adjunct to CT-scanning.
doi:10.3233/CBM-2012-0229
PMCID: PMC4556364  PMID: 22297547
Lung cancer; NSCLC; diagnosis; serum biomarkers; CT-scanning
20.  Inherited variation in the ATP-binding cassette transporter ABCB1 and survival after chemotherapy for stage III-IV lung cancer 
Background
The ATP-binding cassette transporter gene ABCB1 and the glutathione S-transferase gene GSTP1 code for a multidrug resistance (MDR) protein and for a detoxifying phase II metabolic enzyme, respectively, with substrate specificities that include chemotherapy drugs commonly used to treat lung cancer.
Methods
We genotyped 11 ABCB1 and 8 GSTP1 single nucleotide polymorphisms (SNPs) in 698 white lung cancer patients (all current or former cigarette smokers) and used log-rank test statistics and proportional hazards regression to evaluate associations between SNP genotype and survival.
Results
Using data from all 698 cases, one SNP in ABCB1 (rs2235013) was statistically significantly associated with overall survival (p=0.038, log-rank test). Chemotherapy and stage jointly (p=0.025) significantly modified the association between rs2235013 and survival, with statistically significant (p=0.013, log-rank test) association observed in the subgroup of stage III-IV lung cancer patients who received chemotherapy as part of their first course of treatment (N=160; 93.1% non-small cell). Patients who inherited the minor T allele at ABCB1 rs2235013 experienced better overall and recurrence-free survival [hazard ratio, per minor T allele, (95% confidence interval): 0.66 (0.49-0.90) and 0.55 (0.31-0.95), respectively; adjusted for year of diagnosis, sex, age at diagnosis, cigarette pack years, and stage]. In addition, in the advanced-stage chemotherapy-treated subgroup, four ABCB1 SNPs (rs6949448, rs2235046, rs1128503, and rs10276036) in mutual high linkage disequilibrium with rs2235013 and an independent ABCB1 SNP (rs1045642) showed statistically significant association (p<0.05) with survival.
Conclusions
Inherited variation in ABCB1 may affect survival specifically in advanced-stage lung cancer patients who receive chemotherapy.
doi:10.1097/JTO.0000000000000262
PMCID: PMC4134100  PMID: 25122423
21.  Estrogen Receptor Signaling in Lung Cancer 
Seminars in oncology  2009;36(6):524.
Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among women in the US also currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small-cell carcinoma of the lung compared to squamous cell carcinoma, and never smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors and aromatase, the enzyme that synthesizes 17β-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Pre-clinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that rise in men as well as women. An additional protein that recognizes 17β-estradiol with high affinity, GPR30, is also expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.
doi:10.1053/j.seminoncol.2009.10.004
PMCID: PMC2818861  PMID: 19995644
22.  Therapeutic targeting of human hepatocyte growth factor with a single neutralizing monoclonal antibody reduces lung tumorigenesis 
Molecular cancer therapeutics  2008;7(7):1913-1922.
The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody (mAb), demonstrated profound inhibition of human HGF-induced P-MAPK induction, wound healing and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were utilized to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), over 4 weeks. Beginning at week 3, intraperitoneal (i.p.) treatment with 100μg L2G7 or isotyped matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7 treated group was significantly lower than in the control group (1.58 versus 3.19, P=0.0005). Proliferative index was decreased by 48% (P=0.013) in tumors from L2G7 treated mice versus 5G8 treated mice while extent of apoptosis was increased in these same tumors by 5–fold (P=0.0013). P-MAPK expression was also significantly decreased by 84% in tumors from L2G7 treated mice versus 5G8 treated mice (P=0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results demonstrate that blocking the HGF/c-Met interaction with a single mAb delivered systemically can have profound inhibitory effects on development of lung tumors.
doi:10.1158/1535-7163.MCT-07-2169
PMCID: PMC2556508  PMID: 18645002
Hepatocyte growth factor; non-small cell lung cancer
23.  Erlotinib, erlotinib-sulindac vs. placebo: a randomized, double-blind, placebo-controlled window trial in operable head and neck cancer 
Purpose
The epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic anti-tumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a non-selective COX inhibitor, vs. placebo.
Experimental Design
Patients with untreated, operable Stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib-sulindac, or placebo. Tumor specimens were collected before and after 7-14 days of treatment. The primary endpoint was change in Ki-67 proliferation index. We hypothesized an ordering effect in Ki-67 reduction: erlotinib-sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX-2 signaling intermediates.
Results
From 2005-2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki-67 was significantly decreased by erlotinib or erlotinib-sulindac (omnibus comparison, two-sided Kruskal-Wallis, p=0.04). Wilcoxon pairwise contrasts confirmed greater Ki-67 effect in both erlotinib groups (erlotinib-sulindac vs. placebo p=0.043; erlobinib vs. placebo, p=0.027). There was a significant trend in ordering of Ki-67 reduction: erlotinib-sulindac > erlotinib > placebo (two-sided exact Jonckheere-Terpstra, p =0.0185). Low baseline pSrc correlated with greater Ki-67 reduction (R2 = .312, p = 0.024).
Conclusions
Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR-COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target.
doi:10.1158/1078-0432.CCR-13-3360
PMCID: PMC4104657  PMID: 24727329
erlotinib; sulindac; phase 0; head and neck cancer; pSrc
24.  Pan-erbB inhibition potentiates BRAF inhibitors for melanoma treatment 
Melanoma research  2014;24(3):207-218.
The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.
doi:10.1097/CMR.0000000000000060
PMCID: PMC4394744  PMID: 24709886
amphiregulin; canertinib; HB-EGF; neuregulin; transforming growth factor-α; vemurafenib
25.  Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells1 
Neoplasia (New York, N.Y.)  2005;7(4):426-431.
Abstract
Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 µM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.
PMCID: PMC1501149  PMID: 15967120
EGFR; GRPR; MAPK; signal transduction; non-small cell lung cancer

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