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Introduction: RNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non-small-cell lung cancer (NSCLC). In the current 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., Methods: Tissue microarray samples obtained from 218 patients with lung cancer were stained with an anti-PKR antibody and antibodies against 25 biomarkers. Immunohistochemical expression was scored and used for Kaplan-Meier survival analysis. The interaction between PKR and EphA2 in NSCLC cell lines was examined., Results: We found that PKR was associated with EphA2 and that the prognostic information regarding NSCLC provided by the combination of PKR and EphA2 (P/E) was significantly more accurate than that provided by either marker alone. The 5-year overall survival rate in patients with PKR/EphA2 (20%) was significantly lower than that of patients with PKR/EphA2 (74%), patients with PKR/EphA2 (55%), and patients with PKR/EphA2 (55%) (p < 0.0001). We also found that the PKR:EphA2 (P/E) ratio was significantly associated with prognosis (p < 0.0001). Univariate and multivariate Cox analyses revealed that this P/E combination or ratio was an independent predictor of overall survival. In addition, induction of PKR expression reduced EphA2 protein expression levels in NSCLC cell lines., Conclusions: PKR/EphA2 is a significant predictor of prognosis for NSCLC. PKR/EphA2 may be a promising approach to improving screening efficiency and predicting prognosis in patients with NSCLC.

Background: Most patients with nonsmall cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The authors investigated the involvement of insulinlike 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 an NSCLC tissue microarray. The authors 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 mitogen-activated protein kinase/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 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 mutant 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 mutant K-Ras., Conclusions: The mutation status of both EGFR and K-Ras could be a predictive marker of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs., (Copyright © 2012 American Cancer Society.)

Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1alpha pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1alpha. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1alpha and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.

Overexpression of the receptor tyrosine kinase EphA2 occurs in non-small cell lung cancer (NSCLC) and a number of other human cancers. This overexpression correlates with a poor prognosis, smoking, and the presence of Kirsten rat sarcoma (K-Ras) mutations in NSCLC. In other cancers, EphA2 has been implicated in migration and metastasis. To determine if EphA2 can promote NSCLC progression, we examined the relationship of EphA2 with proliferation and migration in cell lines and with metastases in patient tumors. We also examined potential mechanisms involving AKT, Src, focal adhesion kinase, Rho guanosine triphosphatases (GTPase), and extracellular signal-regulated kinase (ERK)-1/2. Knockdown of EphA2 in NSCLC cell lines decreased proliferation (colony size) by 20% to 70% in four of five cell lines (P < 0. 04) and cell migration by 7% to 75% in five of six cell lines (P < 0. 03). ERK1/2 activation correlated with effects on proliferation, and inhibition of ERK1/2 activation also suppressed proliferation. In accordance with the in vitro data, high tumor expression of EphA2 was an independent prognostic factor in time to recurrence (P = 0.057) and time to metastases (P = 0.046) of NSCLC patients. We also examined EphA2 expression in the putative premalignant lung lesion, atypical adenomatous hyperplasia, and the noninvasive bronchioloalveolar component of adenocarcinoma because K-Ras mutations occur in atypical adenomatous hyperplasia and are common in lung adenocarcinomas. Both preinvasive lesion types expressed EphA2, showing its expression in the early pathogenesis of lung adenocarcinoma. Our data suggest that EphA2 may be a promising target for treating and preventing NSCLC.

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling has been implicated in several human neoplasms. However, the role of serum levels of IGFs in lung cancer risk is controversial. We assessed the role of tissue-derived IGFs in lung carcinogenesis. We found that IGF-I and IGF-II levels in bronchial tissue specimens containing high-grade dysplasia were significantly higher than in those containing normal epithelium, hyperplasia, and squamous metaplasia. Derivatives of human bronchial epithelial cell lines with activation mutation in KRAS(V12) or loss of p53 overexpressed IGF-I and IGF-II. The transformed characteristics of these cells were significantly suppressed by inactivation of IGF-IR or inhibition of IGF-I or IGF-II expression but enhanced by overexpression of IGF-IR or exposure to the tobacco carcinogens (TC) 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone and benzo(a)pyrene. We further determined the role of IGF-IR signaling in lung tumorigenesis by determining the antitumor activities of the selective IGF-IR tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine using an in vitro progressive cell system and an in vivo mouse model with a lung-specific IGF-I transgene after exposure to TCs, including 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone plus benzo(a)pyrene. Our results show that airway epithelial cells produce IGFs in an autocrine or paracrine manner, and these IGFs act jointly with TCs to enhance lung carcinogenesis. Furthermore, the use of selective IGF-IR inhibitors may be a rational approach to controlling lung cancer.

Purpose: To determine the frequency of estrogen receptor alpha and beta and progesterone receptor protein immunohistochemical expression in a large set of non-small cell lung carcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports., Experimental Design: Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors alpha and beta and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients' clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status., Results: Estrogen receptors alpha and beta were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for alpha and 42-56% for beta; cytoplasm: <1-42% for alpha and 20-98% for beta) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor alpha nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor alpha expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor alpha expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor beta and progesterone receptor but not estrogen receptor alpha expressed in the normal epithelium adjacent to lung adenocarcinomas., Conclusions: Estrogen receptor alpha and beta expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor alpha expression correlates with EGFR mutations.

Purpose: Up-regulation of the receptor tyrosine kinase EphA2 has been shown in several epithelial cancers. Epidermal growth factor receptor (EGFR) and K-Ras have been reported to regulate EphA2 in several in vitro models, but this regulation has never been examined in tumors from patients. Because of the established importance of EGFR and K-Ras mutations in non-small cell lung cancer (NSCLC), we investigated the relationship between these mutations and EphA2 in this cancer type. The significance of EphA2 expression was further examined by testing for correlation with other clinical parameters., Experimental Design: EphA2 expression was analyzed by immunohistochemistry in tissue microarray format using surgically resected NSCLC specimens (n = 279). EGFR and K-Ras mutation status was determined for most specimens. The correlation between EphA2 expression and EGFR or K-Ras mutation status was examined, along with several clinicopathologic variables of the tumors. The effects of increasing EGFR and K-Ras activity on EphA2 expression and activity were examined in two cell lines., Results: EphA2 expression was detected in >90% of tumor samples. Expression of EphA2 was positively correlated with activated EGFR but not with EGFR mutations. EphA2 expression was increased in patients harboring K-Ras mutations. EphA2 expression was positively correlated with a history of smoking, and high EphA2 scores predicted poorer progression-free and overall survivals., Conclusions: EphA2 expression in NSCLC is associated with K-Ras mutations, EGFR activation, smoking history, and poor prognosis. EphA2 expression is up-regulated in the context of EGFR or K-Ras activation. The potential of EphA2 as a therapeutic target for NSCLC should be further investigated.

Epithelial-to-mesenchymal transition is a process in which cells undergo a developmental switch from an epithelial to a mesenchymal phenotype. We investigated the role of this phenomenon in the pathogenesis and progression of adenocarcinoma and squamous cell carcinoma of the lung. Archived tissue from primary tumors (n=325), brain metastases (n=48) and adjacent bronchial epithelial specimens (n=192) were analyzed for immunohistochemical expression by image analysis of E-cadherin, N-cadherin, integrin-alpha v beta 6, vimentin, and matrix metalloproteinase-9. The findings were compared with the patients' clinicopathologic features. High expression of the epithelial-to-mesenchymal transition phenotype (low E-cadherin and high N-cadherin, integrin-alpha v beta 6, vimentin, and matrix metalloproteinase-9) was found in most lung tumors examined, and the expression pattern varied according to the tumor histologic type. Low E-cadherin membrane and high N-cadherin cytoplasmic expression were significantly more common in squamous cell carcinoma than in adenocarcinoma (P=0.002 and 0.005, respectively). Dysplastic lesions had significantly lower expression of the epithelial-to-mesenchymal transition phenotype than the squamous cell carcinomas, and integrin-alpha v beta 6 membrane expression increased stepwise according to the histopathologic severity. Brain metastases had decreased epithelial-to-mesenchymal transition expression compared with primary tumors. Brain metastases had significantly lower integrin-alpha v beta 6 membrane (P=0.04), N-cadherin membrane, and cytoplasm (P<0.0002) expression than the primary tumors. The epithelial-to-mesenchymal transition phenotype is commonly expressed in primary squamous cell carcinoma and adenocarcinoma of the lung; this expression occurs early in the pathogenesis of squamous cell carcinoma. Brain metastases showed characteristics of reversed mesenchymal-to-epithelial transition. Our findings suggest that epithelial-to-mesenchymal transition is a potential target for lung cancer chemoprevention and therapy.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In Kras(LA1) mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from Kras(LA1) mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in Kras(LA1) mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC.

Purpose: FUS1, a novel tumor-suppressor gene located in the chromosome 3p21.3 region, may play an important role in lung cancer development. Currently, FUS1-expressing nanoparticles have been developed for treating patients with lung cancer. However, the expression of Fus1 protein has not been examined in a large series of lung cancers and their sequential preneoplastic lesions., Experimental Design: Using tissue microarrays, we examined Fus1 immunohistochemical expression in 281 non-small cell lung carcinoma (NSCLC) and 22 small cell lung carcinoma tissue specimens and correlated the findings with patients' clinicopathologic features. To investigate the expression of Fus1 in the early sequential pathogenesis of NSCLC, we studied Fus1 expression in 211 histologically normal and mildly abnormal bronchial epithelia, and 118 bronchial and alveolar preneoplastic lesions obtained from patients with lung cancer., Results: Loss and reduction of expression was detected in 82% of NSCLCs and 100% of small cell lung carcinomas. In NSCLCs, loss of Fus1 immunohistochemical expression was associated with significantly worse overall survival. Bronchial squamous metaplastic and dysplastic lesions expressed significantly lower levels of Fus1 compared with normal (P = 0.014 and 0.047, respectively) and hyperplastic (P = 0.013 and 0.028, respectively) epithelia., Conclusions: Our findings show a high frequency of Fus1 protein loss and reduction of expression in lung cancer, and suggests that this reduction may play an important role in the early pathogenesis of lung squamous cell carcinoma. These findings support the concept that FUS1 gene and Fus1 protein abnormalities could be used to develop new strategies for molecular cancer therapy for a significant subset of lung tumors.

The development of new modes of diagnosis and targeted therapy for lung cancer is dependent on the identification of unique cell surface features on cancer cells and isolation of reagents that bind with high affinity and specificity to these biomarkers. We recently isolated a 20-mer peptide which binds to the lung adenocarcinoma cell line, H2009, from a phage-displayed peptide library. We show here that the cellular receptor for this peptide, TP H2009.1, is the uniquely expressed integrin, alphavbeta6, and the peptide binding to lung cancer cell lines correlates to integrin expression. The peptide is able to mediate cell-specific uptake of a fluorescent nanoparticle via this receptor. Expression of alphavbeta6 was assessed on 311 human lung cancer samples. The expression of this integrin is widespread in early-stage nonsmall cell lung carcinoma (NSCLC). Log-rank test and Cox regression analyses show that expression of this integrin is significantly associated with poor patient outcome. Preferential expression is observed in the tumors compared with the surrounding normal lung tissue. Our data indicate that alphavbeta6 is a prognostic biomarker for NSCLC and may serve as a receptor for targeted therapies. Thus, cell-specific peptides isolated from phage biopanning can be used for the discovery of cell surface biomarkers, emphasizing the utility of peptide libraries to probe the surface of a cell.

The discovery that mutation of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene occurs in a subset of lung cancers and predicts for sensitivity to tyrosine kinase inhibitors has generated enormous interest and immediately led to intense basic, translational, and clinical research in many laboratories around the globe. All these findings have led to the identification of a subset of lung cancers with relatively distinct molecular, pathologic, and clinical features that demonstrate response to targeted therapy. Currently, the best marker to predict response and better survival for EGFR tyrosine kinase inhibitors, such as EGFR mutation, gene increased copy number, and protein immunohistochemical expression, is still controversial. Importantly, the findings of EGFR abnormalities in lung cancer have supported the notion that different molecular mechanisms and pathways are involved in the pathogenesis of lung cancer arising in never and ever smokers.