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1.  EZH2 Protein Expression Associates With the Early Pathogenesis, Tumor Progression and Prognosis of Non-small Cell Lung Carcinoma 
Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non-small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes.
Experimental Design
EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCCs) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status.
EZH2 expression was significantly (P<0.0001) higher in SCCs compared to adenocarcinomas and in brain metastasis relative to matched primary tumors (P=0.0013). EZH2 expression was significantly (P<0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking and higher TNM stage (P=0.02 to P<0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P=0.025; HR 1.54) and overall survival (OS; P=0.0002; HR 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P=0.009; HR 0.51) and OS (P=0.0011; HR 0.45) which was confirmed in the independent set of 91 adenocarcinomas.
In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas.
PMCID: PMC3890101  PMID: 24097870
EZH2; NSCLC; lung adenocarcinoma; lung squamous cell carcinoma; bronchial preneoplasia; brain metastasis; KRAS mutations; EGFR mutations
2.  Prognostic Significance of Combinations of RNA-Dependent Protein Kinase and EphA2 Biomarkers for NSCLC 
RNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non-small cell lung cancer (NSCLC). In the present study, we investigated the correlation between PKR and 25 other biomarkers for NSCLC, identified the markers that could further improve the prognostic significance of PKR, and elucidated the mechanisms of interaction between these markers and PKR.
Tissue microarray samples obtained from 218 lung cancer patients were stained with an anti-PKR antibody and antibodies against 25 biomarkers. Immunohistochemical expression was scored and used for Kaplan-Meier survival analysis. The interaction between PKR and EphA2 in NSCLC cell lines was examined.
We found that PKR was associated with EphA2 and that the prognostic information regarding NSCLC provided by the combination of PKR and EphA2 (P/E) was significantly more accurate than that provided by either marker alone. The 5-year overall survival rate in PKRlow/EphA2high patients (20%) was significantly lower than that of PKRhigh/EphA2low patients (74%), PKRhigh/EphA2high patients (55%), and PKRlow/EphA2low patients (55%) (p< 0.0001). We also found that the PKR:EphA2 (P/E) ratio was significantly associated with prognosis (p< 0.0001). Univariate and multivariate Cox analyses revealed that this P/E combination or ratio was an independent predictor of overall survival. In addition, induction of PKR expression reduced EphA2 protein expression levels in NSCLC cell lines.
PKR/EphA2 is a significant predictor of prognosis for NSCLC. PKR/EphA2 may be a promising approach to improving screening efficiency and predicting prognosis in NSCLC patients.
PMCID: PMC3573252  PMID: 23370317
PKR; EphA2; Biomarker; Lung cancer
3.  The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors 
PLoS ONE  2013;8(7):e68966.
The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).
PMCID: PMC3729565  PMID: 23935914
4.  G-Protein Coupled Receptor Family C, Group 5, Member A (GPRC5A) Expression Is Decreased in the Adjacent Field and Normal Bronchial Epithelia of Patients with Chronic Obstructive Pulmonary Disease and Non–Small-Cell Lung Cancer 
Understanding oncogenes and tumor suppressor genes expression patterns is essential for characterizing lung cancer pathogenesis. We have previously demonstrated that mGprc5a/hGPRC5A is a lung-specific tumor suppressor evidenced by inflammation-mediated tumorigenesis in Gprc5a-knockout mice. The implication of GPRC5A in human lung cancer pathogenesis, including that associated with inflammatory chronic obstructive pulmonary disease (COPD), a risk factor for the malignancy, remains elusive.
We sought to examine GPRC5A immunohistochemical expression in histologically normal bronchial epithelia (NBE) from lung disease-free never- and ever-smokers (n = 13 and n = 18, respectively), from COPD patients with (n = 26) and without cancer (n = 24) and in non-small cell lung cancers (NSCLCs) (n = 474). Quantitative assessment of GPRC5A transcript expression in airways (n = 6), adjacent NBEs (n = 29) and corresponding tumors (n = 6) from 6 NSCLC patients was also performed.
GPRC5A immunohistochemical expression was significantly lower in tumors compared to uninvolved NBE (p < 0.0001) and was positively associated with adenocarcinoma histology (p < 0.001). GPRC5A airway expression was highest in lung disease-free NBE, decreased and intermediate in NBE of cancer-free COPD patients (p = 0.004) and further attenuated and lowest in epithelia of COPD patients with adenocarcinoma and SCC (p < 0.0001). Furthermore, GPRC5A mRNA was significantly decreased in NSCLCs and corresponding NBE compared to uninvolved normal lung (p = 0.03).
Our findings highlight decreased GPRC5A expression in the field cancerization of NSCLC, including that associated with lung inflammation. Assessment of the use of GPRC5A expression as a risk factor for NSCLC development in COPD patients is warranted.
PMCID: PMC3622592  PMID: 23154545
Field cancerization; Chronic obstructive pulmonary disease; Non–small-cell lung cancer; g-protein coupled receptor family C; group 5; member A; gene expression
5.  Robust Gene Expression Signature from Formalin-Fixed Paraffin-Embedded Samples Predicts Prognosis of Non-Small-Cell Lung Cancer Patients 
The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes.
Experimental Design
We randomly selected 100 Non-Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We micro dissected tumor area from FFPE specimens, and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray data sets were used to validate the prognosis model.
This study demonstrated that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes, can be used to refine the prognosis for stage I lung cancer patients and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature.
We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings.
PMCID: PMC3166982  PMID: 21742808
Lung Cancer Prognosis; Gene Expression Signature; Formalin Fixed Paraffin Embedded Samples
6.  Histologic Patterns and Molecular Characteristics of Lung Adenocarcinoma Associated With Clinical Outcome 
Cancer  2011;118(11):2889-2899.
Lung adenocarcinoma is histologically heterogeneous and has 5 distinct histologic growth patterns: lepidic, acinar, papillary, micropapillary, and solid. To date, there is no consensus regarding the clinical utility of these patterns.
The authors performed a detailed semiquantitative assessment of histologic patterns of 240 lung adenocarcinomas and determined the association with patients’ clinicopathologic features, including recurrence-free survival (RFS) and overall survival (OS) rates. In a subset of tumors, expression levels of 2 prognostic molecular markers were evaluated: thyroid transcription factor-1 (TTF-1) (n = 218) and a panel of 5 proteins (referred as the FILM signature index) (n = 185).
Four mutually exclusive tumor histology pattern groups were identified: 1) any solid (38%), 2) any papillary but no solid (14%), 3) lepidic and acinar but no solid or papillary (30%), and 4) acinar only (18%). Patients in group 3 had a higher RFS rate than patients in group 1 (hazard ratio [HR], 0.4510; P = .0165) and group 2 (HR, 0.4253; P = .0425). Solid pattern tumors (group 1) were associated with a lower OS rate than nonsolid pattern tumors (all stages: HR; 1.665; P = .0144; stages I and II: HR, 2.157; P = .008). In the patients who had tumors with a nonsolid pattern, high TTF-1 expression was associated significantly with higher RFS (HR, 0.994; P = .0017) and OS (HR, 0.996; P = .0276) rates in all stages, and a high FILM signature index score was associated with lower RFS and OS rates in all stages (RFS: HR, 1.343; P = .0192; OS: HR, 1.371; P = .0156) and in stages I and II (RFS: HR, 1.419; P = .0095; OS: HR, 1.315; P = .0422).
The presence of a solid histologic pattern was identified as a marker of unfavorable prognosis in patients with primary lung adenocarcinoma. High TTF-1 expression and low FILM signature index scores were associated with a better prognosis for patients who had tumors with a nonsolid pattern.
PMCID: PMC3369269  PMID: 22020674
histologic patterns; lung adenocarcinoma; thyroid transcription factor 1; prognostic signature
7.  Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development 
PLoS ONE  2012;7(5):e37775.
For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.
PMCID: PMC3357406  PMID: 22629454
8.  ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion 
The Journal of Clinical Investigation  2012;122(5):1869-1880.
In contrast to the well-studied classic MAPKs, such as ERK1/2, little is known concerning the regulation and substrates of the atypical MAPK ERK3 signaling cascade and its function in cancer progression. Here, we report that ERK3 interacted with and phosphorylated steroid receptor coactivator 3 (SRC-3), an oncogenic protein overexpressed in multiple human cancers at serine 857 (S857). This ERK3-mediated phosphorylation at S857 was essential for interaction of SRC-3 with the ETS transcription factor PEA3, which promotes upregulation of MMP gene expression and proinvasive activity in lung cancer cells. Importantly, knockdown of ERK3 or SRC-3 inhibited the ability of lung cancer cells to invade and form tumors in the lung in a xenograft mouse model. In addition, ERK3 was found to be highly upregulated in human lung carcinomas. Our study identifies a previously unknown role for ERK3 in promoting lung cancer cell invasiveness by phosphorylating SRC-3 and regulating SRC-3 proinvasive activity by site-specific phosphorylation. As such, ERK3 protein kinase may be an attractive target for therapeutic treatment of invasive lung cancer.
PMCID: PMC3336992  PMID: 22505454
9.  Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer 
Cancer research  2010;70(16):6639-6648.
Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer, and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in persistence of K14+ cells in the airway epithelium in premalignant lesions. The presence of K14+ cells in non-small cell lung cancer (NSCLC) samples predicted poorer outcomes. This was especially true in smokers where the presence of K14+ cells in NSCLC was predictive of metastasis. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis and this predictive value was strongest in smokers, where it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.
PMCID: PMC2924777  PMID: 20710044
Lung carcinogenesis; dysregulated repair; injury
10.  Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features 
To understand the role of Nrf2 and Keap1 in NSCLC, we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy.
Experimental design
We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients’ clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n=26) or adjuvant platinum-based chemotherapy (n=63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens.
We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P<0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P=0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival (P=0.0139; HR=1.75) in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P=0.0181; HR=2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P=0.0410; HR=3.37).
Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma.
PMCID: PMC2920733  PMID: 20534738
Nrf2; Keap1; NSCLC
11.  Collagen XXIII—a potential biomarker for the detection of primary and recurrent non-small cell lung cancer 
Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer. This study’s purpose was to determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and to assess collagen XXIII’s utility as a biomarker for non small-cell lung cancer (NSCLC).
A multi-cancer tissue microarray (TMA) was used for immunohistochemical examination of collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using TMAs with representative tumor and control lung tissues from NSCLC patients. In addition, NSCLC patient urine samples were analyzed for the presence of collagen XXIII via Western blot.
Collagen XXIII was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in NSCLC subtypes. Collagen XXIII was present in 294 of 333 (88%) lung adenocarcinomas and 97 of 133 (73%) squamous cell carcinomas (SqCC). In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples but only in 15 of 54 (28%) control samples. High collagen XXIII staining intensity correlated with shorter recurrence-free survival in NSCLC patients.
We demonstrate the capability of collagen XXIII as a tissue and urinary biomarker for NSCLC, where positivity in tissue or urine significantly correlates with presence of NSCLC and high staining intensity is a significant recurrence predictor.
Inclusion of collagen XXIII in a tissue or urine-based cancer biomarker panel could inform NSCLC patient treatment decisions.
PMCID: PMC2880394  PMID: 20447926
tissue microarray; fluid biomarker; cancer surveillance and screening
12.  Sex Determining Region Y-Box 2 (SOX2) Is a Potential Cell-Lineage Gene Highly Expressed in the Pathogenesis of Squamous Cell Carcinomas of the Lung 
PLoS ONE  2010;5(2):e9112.
Non-small cell lung cancer (NSCLC) represents the majority (85%) of lung cancers and is comprised mainly of adenocarcinomas and squamous cell carcinomas (SCCs). The sequential pathogenesis of lung adenocarcinomas and SCCs occurs through dissimilar phases as the former tumors typically arise in the lung periphery whereas the latter normally arise near the central airway.
Methodology/Principal Findings
We assessed the expression of SOX2, an embryonic stem cell transcriptional factor that also plays important roles in the proliferation of basal tracheal cells and whose expression is restricted to the main and central airways and bronchioles of the developing and adult mouse lung, in NSCLC by various methodologies. Here, we found that SOX2 mRNA levels, from various published datasets, were significantly elevated in lung SCCs compared to adenocarcinomas (all p<0.001). Moreover, a previously characterized OCT4/SOX2/NANOG signature effectively separated lung SCCs from adenocarcinomas in two independent publicly available datasets which correlated with increased SOX2 mRNA in SCCs. Immunohistochemical analysis of various histological lung tissue specimens demonstrated marked nuclear SOX2 protein expression in all normal bronchial epithelia, alveolar bronchiolization structures and premalignant lesions in SCC development (hyperplasia, dysplasia and carcinoma in situ) and absence of expression in all normal alveoli and atypical adenomatous hyperplasias. Moreover, SOX2 protein expression was greatly higher in lung SCCs compared to adenocarcinomas following analyses in two independent large TMA sets (TMA set I, n = 287; TMA set II, n = 511 both p<0.001). Furthermore, amplification of SOX2 DNA was detected in 20% of lung SCCs tested (n = 40) and in none of the adenocarcinomas (n = 17).
Our findings highlight a cell-lineage gene expression pattern for the stem cell transcriptional factor SOX2 in the pathogenesis of lung SCCs and suggest a differential activation of stem cell-related pathways between squamous cell carcinomas and adenocarcinomas of the lung.
PMCID: PMC2817751  PMID: 20161759

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