Phenotype Of Lung Cancer Research Articles

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

IntroductionInherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.MethodsAn isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.ResultsCharacterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.ConclusionOur results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.

Overexpression of Integrin-linked Kinase Promotes Lung Cancer Cell Migration and Invasion via NF-κB-mediated Upregulation of Matrix Metalloproteinase-9

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase which has been implicated in the regulation of various cellular processes. Previously, we have demonstrated that overexpression of ILK correlates with malignant phenotype in non-small cell lung cancer. Furthermore, forced overexpression of ILK promotes lung cancer cell invasion and migration. However, the molecular mechanisms by which ILK enhances the invasive phenotype of lung cancer cells are still not fully understood. In the present study, we found that overexpression of ILK stimulated matrix metalloproteinase-9 (MMP-9) expression and activity in lung cancer cells. ILK-induced cell migration and invasion were significantly inhibited by MMP inhibitor doxycycline as well as by anti-MMP-9 neutralizing antibody. In addition, overexpression of ILK induced phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) subunit p65. Finally, upregulation of MMP-9 was severely abolished by either BAY 11-7028, a specific NF-κB inhibitor, or small interfering RNA targeted to NF-κB p65 in ILK overexpression cells. Taken together, these findings suggest that ILK promotes lung cancer cell migration and invasion via NF-κB-mediated upregulation of MMP-9.

Mutations of the EPHB6 Receptor Tyrosine Kinase Induce a Pro-Metastatic Phenotype in Non-Small Cell Lung Cancer

Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.

P73 Impact of EBUS-TBNA on Modalities For Tissue Acquisition in Patients with Lung Cancer: A Study of 407 Patients: Abstract 73 Table 1

Introduction NICE guidance for the diagnosis and treatment of lung cancer recommends choosing “investigations that give the most information about diagnosis and staging with the least risk to the patient”. [1] EBUS-TBNA is expanding as an important diagnostic modality in lung cancer and provides simultaneous information on lung cancer phenotype, genotype and nodal staging. The impact of the introduction of EBUS-TBNA on the use of diagnostic modalities for tissue acquisition in patients with lung cancer is unknown. Methods A retrospective review of 407 consecutive patients diagnosed with lung cancer at a university teaching hospital in 2007, 2009 and 2011. Data were collected on age, gender, FEV1, performance status, diagnostic modality, and pathological subtype. Patients where only a clinical diagnosis was made (n=21) were excluded. Data were analysed using the SPSS version 17 (Chicago, IL, USA). For comparison between categorical variables, Chi-square or Fisher’s exact test were used as appropriate. All reported p -values are two-tailed, and are considered statistically significant when p Results 386 patients were included in the analysis. The mean (SD) age (years) and FEV1 (L/min) were 69 (12) and 1.81 (0.80) for 2007, 67 (10) and 1.81 (0.79) for 2009 and 68 (12) and 2.07 (0.68) for 2011 respectively. In 2007, 2009 and 2011 57.9%, 51.4% and 62.7% were males. The results on diagnostic modalities, performance status, histological subtype and staging are listed in Table 1. Comparing 2007 to 2011 there has been a significant reduction in standard bronchoscopy (P=0.0001), CT guided biopsy of peripheral lesions (P=0.0008) and mediastinoscopy (P=0.0382). The proportion of cases diagnosed by EBUS-TBNA significantly increased from 0% in 2007 to 21.4% in 2009 and 25.4% in 2011 (P Conclusions The use of diagnostic modalities that provide information on diagnosis and staging in a single intervention are increasing. At our hospital, the use of EBUS-TBNA has led to a significant reduction in CT guided biopsies, standard bronchoscopies and mediastinoscopies. These changes in practise have implications for future service provision, training and commissioning. Reference NICE Lung cancer. 2011.http://publications.nice.org.uk/lung-cancer-cg121/key-priorities-for-implementation.

Overexpression of integrin-linked kinase correlates with malignant phenotype in non-small cell lung cancer and promotes lung cancer cell invasion and migration via regulating epithelial–mesenchymal transition (EMT)-related genes

Integrin-linked kinase (ILK), a member of the serine/threonine kinases, has been implicated in oncogenesis and progression of human cancers. The aim of this study was to characterize the role of ILK in lung cancer aggressiveness and the underlying molecular mechanisms. ILK protein expression was assessed by immunohistochemistry in a cohort of non-small cell lung cancer (NSCLC) patients, and a series of in vitro assays was conducted to elucidate the function of ILK in lung cancer. Overexpression of ILK protein was detected in 30.6% (33/108) of primary NSCLC tissues and correlated with the TNM stage (P=0.001) and lymph node metastasis (P=0.033). Ectopic overexpression of ILK in lung cancer cells promoted cell migration and invasion in vitro, and led to the acquisition of epithelial–mesenchymal transition (EMT) phenotype, as evidenced by the spindle-like morphology, down-regulation of E-cadherin, and up-regulation of vimentin, fibronectin, Snail and Slug. In addition, the down-regulation of E-cadherin induced by ILK was significantly reversed by nuclear factor-κB (NF-κB) inhibitor BAY 11-7028 and small interfering RNA (siRNA) targeting NF-κB p65, suggesting a role of the NF-κB signaling pathway in ILK-induced EMT. Overall, our results suggest that ILK promotes lung cancer cell migration and invasion through the induction of EMT process.

Expression of ezrin correlates with malignant phenotype of lung cancer, and in vitro knockdown of ezrin reverses the aggressive biological behavior of lung cancer cells

Ezrin, one of the ezrin-radixin-moesin proteins, is involved in the formation of cell membrane processes such as lamellipodia and filopodia and acts as a membrane-cytoskeleton linker. Its aberrant expression correlates with development and progression of several human cancers. However, the expression of ezrin and its role in lung cancer are currently unknown. In this study, we performed ezrin small interfering RNA transfection in two lung cancer cell lines and examined the effects on malignant phenotypes in cancer cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and chamber transwell assays. Ezrin knockdown significantly reduced the proliferation, migration, and invasion of lung cancer cells in vitro. To address the possible mechanisms, we evaluated the expression of adhesion molecules E-cadherin and β-catenin by Western blot and reverse transcriptase-polymerase chain reaction analyses. The results demonstrated that downregulation of ezrin reduced β-catenin and increased E-cadherin at the protein level but had no effects on their mRNA levels, suggesting posttranscriptional regulation of these two adhesion molecules. Immunofluorescence assays revealed that ezrin knockdown restored membranous expression of E-cadherin and decreased cytoplasmic β-catenin in lung cancer cells. In addition, ezrin expression was immunohistochemically evaluated on 135 normal and 183 lung cancer tissues. The expression of ezrin was significantly higher in cancer samples than paired autologous normal lung tissues. In normal bronchial epithelium, ezrin was mainly localized on the apical membrane, while in lung cancers and metastatic foci, ezrin was primarily distributed in cytoplasm. Among lung cancer tissues, expression of ezrin was higher in the invasive front of primary lesions and the highest in lymphatic metastasis. Statistical analysis demonstrated that ezrin expression correlated significantly with lymphatic metastasis and advanced TNM stage. Our data suggest that ezrin may play a crucial role in governing the biological behavior of lung cancer.

Abstract 3444: Exon 4 of osteopontin: A unique target to reduce malignant behavior in non-small cell lung cancer

Abstract INTRODUCTION: Osteopontin (OPN) is a ubiquitous protein associated with a wide range of normal and pathologic functions. It is a central regulator of malignant phenotype in non-small cell lung cancer (NSCLC). OPNa, the full length isoform, is disproportionately expressed in NSCLC tumors and cell lines, and increases malignant behavior when over expressed. OPNc, the shortest isoform, does not induce malignant properties when expressed in NSCLC. The only structural difference between OPNa and OPNc is the transcription of exon 4. The hypothesis of this project is that a small interfering peptide to OPN exon 4 will reduce in vitro malignant activity in NSCLC. METHODS: A 16-amino acid peptide which mimics the central sequence in OPN exon 4 and a scrambled sham were constructed and florescent labeled. Three NSCLC cell lines, H358 (low), A549 (moderate), and H460 (high endogenous OPN) were used in binding, proliferation, and invasion assays in presence of exon 4 mimic or sham. Cisplatin sensitivity was performed with peptides in A549. RESULTS: There was florescent evidence for cell surface binding of the exon 4 mimic in all of the NSCLC cell lines without binding of the sham. The exon 4 mimic significantly decreased in vitro invasion and proliferation in the A549 and H460 cell lines, with a lesser effect in H358 with low endogenous OPN. (Table) The exon 4 mimic increased cisplatin sensitivity, decreasing cell viability 2-fold compared to sham and control at cisplatin concentrations 0.1-0.8 uM/ml. CONCLUSIONS: Exon 4 of OPN has not previously been identified as a regulatory region essential for OPN's effect on malignant behavior. We have evidence for an un-described cell surface receptor to this region. A small mimicking peptide to OPN exon 4 significantly decreased in vitro malignant behavior, suggesting interference with important regulatory pathways. This novel binding site could serve as an ideal for target for antibody or small molecule therapies in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3444. doi:1538-7445.AM2012-3444

Abstract B39: Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

Abstract microRNA expression is frequently altered in human cancers. Epigenetic silencing, especially DNA methylation, is one of many mechanisms of microRNA suppression in cancer. To search for epigenetically silenced microRNAs in non-small-cell lung cancer (NSCLC), we mapped human microRNAs on autosomal chromosomes and selected in silico 55 microRNAs that met one of the following criteria: (i) microRNAs within CpG islands, (ii) microRNAs within 1 kbp downstream of CpG islands, and (iii) microRNAs within gene introns whose promoters have CpG islands. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 microRNAs. Fourteen microRNAs (mir-375, mir-196b, mir-126, mir-34b, mir-127, mir-203, mir-148a, mir-181c, mir-30e, mir-449a, mir-340, mir-486, mir-483, mir-139) were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. Among the 14 microRNAs, seven (mir-126, mir-34b, mir-203, mir-30e, mir-449a, mir-486, mir-139) were frequently suppressed in primary NSCLCs. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, while mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. A significant enrichment of H3K27me3 was observed in lung cancer cell lines with decreased mir-126 expression, suggesting the involvement of a polycomb complex in the regulation of EGFL7 and mir-126. The overexpression of mir-34b and mir-126 by plasmid vectors decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these microRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41% and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

Abstract B27: Integrative analysis identifies GAS41 as a novel oncogene in NSCLC, localizing to the 12q15 amplicon

Abstract Background: The application of a multidimensional integrated analysis of recurring genomic and expression alterations can identify new insights into the molecular mechanisms involved in the pathogenesis of NSCLC. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also new molecular markers and therapeutic targets. Using this approach, we identified the chromosomal region at 12q13-15, and more specifically Glioma amplified sequence 41 (GAS41) to be frequently amplified and overexpressed in NSCLC. A putative transcription factor, amplification of GAS41 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma. While the oncogene MDM2 has long been believed to be the driver of this amplicon, we hypothesize GAS41 is an oncogene capable of driving selection of the 12q15 amplicon, and not merely a passenger event. Methods: An integrative genomics approach, examining 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) profiled for copy number and gene expression alterations was used to identify novel candidate oncogenes in NSCLC. Recurrent DNA amplifications were identified using the GISTIC algorithm and integrated with gene expression data to identify genes frequently amplified and overexpressed. Genes were classified as overexpressed if the fold change between tumor and matched non-malignant tissues was greater than 2 fold. The functional significance of GAS41 was assessed by lentiviral knockdown and ectopic overexpression in lung cancer cell lines with and without GAS41 amplification, and human bronchial epithelial cells respectively. In vitro assays measuring proliferation, anchorage independent growth, senescence and apoptosis were used to assess the phenotypic effect of gene dosage manipulation. Survival analysis was performed using the Cox regression model for multiple independent cohorts. Results: GAS41 is gained or amplified in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC (26%) and SqCC (24%). Although frequently co-amplified with MDM2, amplification of GAS41 was observed to occur in the absence of MDM2 amplification. Overexpression of GAS41 in human bronchial epithelial cells abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence only in lung cancer cell lines with amplification. Western blotting revealed increased p21, cleaved PARP and reduced levels of phospho-p53 in knockdown lines as compared to empty vector controls, suggesting GAS41 is implicated in the regulation of the p21-p53 pathway. Consistent with in vitro results, patients expressing high levels of GAS41 displayed poorer outcomes compared to those with lower levels of GAS41. Conclusions: Our findings reveal GAS41 as a candidate oncogene frequently amplified and overexpressed in NSCLC, both in the presence and absence of MDM2 amplification. Gene dosage manipulation resulted in distinct phenotypic changes characteristic of oncogenes, and thus implicate amplification of GAS41 as a novel mechanism of NSCLC tumorigenesis.

Genetic and Epigenetic Inactivation of Extracellular Superoxide Dismutase Promotes an Invasive Phenotype in Human Lung Cancer by Disrupting ECM Homeostasis

Extracellular superoxide dismutase (EcSOD) is an important superoxide scavenger in the lung in which its loss, sequence variation, or abnormal expression contributes to lung diseases; however, the role of EcSOD in lung cancer has yet to be studied. We hypothesized that EcSOD loss could affect malignant progression in lung, and could be either genetic or epigenetic in nature. To test this, we analyzed EcSOD expression, gene copy number, promoter methylation, and chromatin accessibility in normal lung and carcinoma cells. We found that normal airway epithelial cells expressed abundant EcSOD and had an unmethylated promoter, whereas EcSOD-negative lung cancer cells displayed aberrant promoter hypermethylation and decreased chromatin accessibility. 5-aza-dC induced EcSOD suggesting that cytosine methylation was causal, in part, to silencing. In 48/50 lung tumors, EcSOD mRNA was significantly lower as early as stage I, and the EcSOD promoter was hypermethylated in 8/10 (80%) adenocarcinomas compared with 0/5 normal lung samples. In addition, 20% of the tumors showed loss of heterozygosity (LOH) of EcSOD. Reexpression of EcSOD attenuated the malignant phenotype of lung carcinoma cells by significantly decreasing invasion and survival. Finally, EcSOD decreased heparanase and syndecan-1 mRNAs in part by reducing NF-κB. By contrast, MnSOD and CuZnSOD showed no significant changes in lung tumors and had no effect on heparanase expression. Taken together, the loss of EcSOD expression is unique among the superoxide dismutases in lung cancer and is the result of EcSOD promoter methylation and LOH, suggesting that its early loss may contribute to ECM remodeling and malignant progression.

Are Anaplastic Lymphoma Kinase Gene Rearrangements in Non-small Cell Lung Cancer Prognostic, Predictive, or Both?

Many aspects of the clinical and pathological phenotype of non-small cell lung cancer (NSCLC) harboring rearrangements in the anaplastic lymphoma kinase (ALK) gene have been characterized following the description of the EML4-ALK fusion gene in NSCLC in 2007.1,2 These tumors most frequently occur in younger, never or light smokers with adenocarcinoma, are almost always mutually exclusive with EGFR or KRAS mutations, and demonstrate exquisite sensitivity to treatment with the ALK inhibitor crizotinib.3 However, there are few published data regarding the natural history and clinical outcomes of ALK-positive NSCLC. Two reports in this issue of the Journal of Thoracic Oncology, conducted in different patient populations using different methods to detect ALK rearrangements, provide apparently conflicting conclusions about the prognostic significance of ALK gene rearrangements in NSCLC and raise questions about the optimal method to detect ALK gene rearrangements in clinical samples. In the first study, Wu et al. performed a retrospective analysis using reverse transcription polymerase chain reaction (RT-PCR) to detect ALK gene rearrangements in cell pellets derived from malignant pleural effusions in 116 EGFR wild-type, Taiwanese patients with stage IIIB/IV NSCLC. Thirty-nine patients (34%) were found to have ALK gene rearrangements and, in contrast to other studies, these patients did not differ from ALK-negative patients with respect to age or smoking history. Wu et al. observed better median survival in the ALK-positive patients compared with the EGFR wild-type, ALK-negative patients (14.7 versus 10.3 months, p 0.009). In the second study, Yang et al. screened 300 never smokers from the Mayo Clinic Lung Cancer Cohort with a two-staged process involving immunohistochemistry (IHC) using the commercially available monoclonal ALK1 antibody (Dako, Capinteria, Ca) followed by fluorescence in situ hybridization (FISH) using break-apart probes (Vysis, Des Plaines, IL). In this cohort of predominantly early-stage tumors (191/300 stage I/II, 62/300 stage II, and 47/300 stage IV), 22 patients (8.2%) were determined to be ALK-positive by FISH. No overall survival data are presented, but using a composite end point of 5-year progression-free survival (PFS) and recurrence-free survival, adjusted for stage and treatment modality, Yang et al. found inferior outcomes in the ALK-positive cohort, with a twofold increase in the risk of experiencing disease progression or recurrence within 5 years in ALK-positive compared with ALK-negative patients. How do these articles with apparently contradictory findings fit in with what is known about clinical outcomes in this patient population? Of note, assessment of the predictive and prognostic significance of ALK gene rearrangements in NSCLC is limited by the fact that, with the exception of one prospective, single-arm study,3 the published data are limited to relatively small retrospective studies. Furthermore, the possibility of ascertainment bias cannot be excluded because of potential differences in the populations

Genome structure‐based screening identified epigenetically silenced microRNA associated with invasiveness in non‐small‐cell lung cancer

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

347 INVITED Cancer Proteomics – From low Resolution to High Resolution to Study Lung Cancer Phenotype

347 INVITED Cancer Proteomics – From low Resolution to High Resolution to Study Lung Cancer Phenotype

Overexpression of Frat1 correlates with malignant phenotype and advanced stage in human non-small cell lung cancer

Frat1 has been reported to be overexpressed in several human malignant tumors, including esophageal squamous, cervical, breast, and ovarian carcinoma, but the role of Frat1 in lung cancer is unknown. Our purpose is to investigate the expression of Frat1 and its correlation with clinicopathologic features and prognosis in lung cancer patients. Immunohistochemistry was performed on 137 cases of non-small cell lung cancer (NSCLC), including 78 cases with clinical follow-up, and Western blot and reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to detect the protein and mRNA expression levels in 30 NSCLC and autologous matched normal tissues. In addition, lung cancer cell line A549 was transfected with Frat1-siRNA plasmids and Matrigel invasive assay was carried out to study the function of Frat1 in cancer cell invasiveness. The results showed that Frat1 was expressed in 85 (62.04%) cases of NSCLC by immunohistochemistry, while all 30 specimens of normal lung tissues were negative. Western blot and RT-PCR results for Frat1 mRNA were in agreement with immunohistochemical findings. Of interest, the expression of Frat1 was strongly correlated with tumor differentiation, TNM stage, and lymph node metastasis (P < 0.05). The Kaplan-Meier survival analysis demonstrated that the cases with Frat1 expression had significantly shorter survival than those without Frat1 (P < 0.001). In addition, down-regulation of Frat1 expression reduced the invasive ability in the A549 cell line, further supporting the idea that Frat1 may play a crucial role in carcinogenesis, tumor invasiveness and dissemination in human lung cancer.

Subcellular proteomics revealed the epithelial–mesenchymal transition phenotype in lung cancer

This article was originally published in Proteomics 2011, 11, 429–439, DOI 10.1002/pmic.200900819

Abstract SY12-01: Stem cell approaches dissect lung cancer biology

Abstract Lung cancer is the major cause of death from cancer worldwide. The five-year survival rate for the majority of lung cancer patients is only 16% because their tumors are refractory to chemotherapeutics or quickly become resistant to therapeutic response. Also contributing to morbidity, most lung cancer patients already have advanced, metastatic disease at the time of diagnosis or develop metastases after surgical removal of early-stage primary lung tumors. The cancer stem cell (CSC) hypothesis is an attractive explanation for tumor growth, recurrence after treatment, and metastasis in some cancers, yet the precise role of CSCs in lung tumors has not been established. Since successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor, it is crucial to characterize the cells that are able to propagate a tumor in vivo. We refer to these functionally important cancer cells as tumor-propagating cells (TPCs). Importantly, understanding the identity and molecular nature of TPCs will provide new opportunities for early detection, treatment and prevention of metastatic disease, whether or not they fit the CSC model in each type of cancer. My laboratory recently showed that murine lung adenocarcinomas, the most prevalent lung cancer subtype, contain a TPC population. In mice bearing tumors initiated with oncogenic Kras and deletion of p53, the cell surface marker Sca1 identifies cancer cells with stem cell properties. In contrast, TPCs are Sca1-negative in lung tumors that express a mutant EGFR. Thus, the predominant driving oncogenotype is an important determinant in the identification of TPC phenotype in lung cancer. This finding suggests that treatments directed against cancer stem cells should be targeted to subsets of patients grouped according to their oncogenotype. We have recently obtained global gene expression data from the Sca1-sorted populations of murine lung tumor cells, as well as their normal wild-type counterparts, to uncover the molecular pathways responsible for tumor propagation in lung adenocarcinoma. I will also discuss new avenues we have taken to understand the connections between TPCs and metastasis in mouse models as well as translating the importance of our findings in human lung cancer cells and patient samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY12-01. doi:10.1158/1538-7445.AM2011-SY12-01

Abstract 3419: Defining the role of ZEB1 in the molecular pathogenesis of lung cancer

Abstract Background: Epithelial-to-mesenchymal transition (EMT) is an important biologic process in cancer resulting in enhanced migration, invasiveness, and therapeutic resistance. The transcription factor ZEB1 promotes EMT and tumor metastasis in several human cancers yet its role in the pathogenesis of lung cancer is less well defined. In addition to a large panel of lung cancer lines, our laboratory has developed an isogenic series of human bronchial epithelial cells (HBECs) – immortalized without viral oncoproteins – genetically manipulated with single and multiple oncogenic changes common in lung cancer (sh-p53, KRASV12, mutant EGFR, and c-MYC), providing an invaluable resource to study the progression of bronchial epithelial cells towards malignancy. Methods and Results: ZEB1 expression was analyzed by qRT-PCR across a panel of 77 non small cell lung cancer (NSCLC) lines to test for a direct correlation with ‘M’ status and indirect correlation with CDH1 expression and ‘E’ status. Genome-wide mRNA expression profiling of the NSCLC cell line cohort using Affymetrix U133 Plus 2.0 arrays was used to identify novel ZEB1-associated genes. Expression of the ephrin receptor EPHA1 significantly inversely correlated with ZEB1 expression. This was confirmed in independent siRNA experiments where knockdown of ZEB1 resulted in an increase in EPHA1 in both NSCLC cell lines and oncogenically transformed HBECs. To characterize the role of ZEB1 in the oncogenic progression of bronchial epithelial cells, ZEB1 was analyzed across our series of genetically manipulated HBECs. Expression of ZEB1 was generally absent, but was highly up-regulated with the combination of p53-knockdown, KRASV12, and c-MYC over-expression. This onco-genetic combination led to the most tumorigenic phenotype in vivo accompanied with a switch from ‘E’ to ‘M’ status. The functional importance of ZEB1 was seen with siRNA knockdown of ZEB1 resulting in a significant decrease in soft agar colonies, transwell invasiveness, and motility suggesting ZEB1 is a crucial driver of tumorigenicity in sh-p53 + KRASV12 + c-MYC transformed HBECs. To confirm the oncogenic role of ZEB1 in lung cancer, shRNA-mediated knockdown of ZEB1 was performed in several NSCLC cell lines, leading to a significant decrease in soft agar colonies, transwell invasiveness, motility, and 3D growth. Subcutaneous injection into NOD/SCID mice found inhibition of tumor growth in shZEB1 knockdown lines. Finally, loss of ZEB1 expression in NSCLCs and transformed HBECs resulted in a decrease in a “cancer stem cell” like population detected by ALDH activity. Conclusion: These findings involving mechanistic studies in NSCLC lines and oncogenically manipulated HBECs demonstrate the prominent role of ZEB1 function in EPHA1 expression, EMT, tumorigenicity, and the invasive phenotype of lung cancer, supporting the important role of ZEB1 in the pathogenesis of lung cancer making it an important new therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2011-3419

Disabled-2 and Axin are concurrently colocalized and underexpressed in lung cancers

Disabled-2 expression is reduced in many cancers, suggesting that it is a potential tumor suppressor protein. To elucidate the role of Disabled-2 in lung cancer, we examined the expression of Disabled-2, the Disabled-2-binding protein Axin, and DNA methyltransferase-1 in lung cancer tissues and corresponding normal lung tissues using immunohistochemistry and Western blots. We also determined the subcellular localization of Axin and Disabled-2 in A549 cells using confocal immunofluorescence. Disabled-2 expression was significantly reduced in lung cancers and was colocalized and coexpressed with Axin (correlation coefficient = 0.321, P < .001 for cytoplasmic expression; correlation coefficient = 0.393, P < .001 for nuclear expression). Reduced nuclear Disabled-2 expression was correlated with the differentiation (P = .048) and TNM stage (P = .048) of the tumor. The cytoplasmic expression of Axin was also correlated with differentiation (P = .042), whereas the nuclear expression of Axin was correlated with both histologic type (P = .001) and TNM stage (P < .001) of lung cancers. Expression of DNA methyltransferase-1 was negatively correlated with the cytoplasmic expression of Axin (correlation coefficient = -0.244, P = .012) but positively correlated with thehistologic type (P = .004), differentiation (P = .036), TNM stage (P = .044), and lymphatic metastasis (P = .011). Expressions of Disabled-2 and Axin were concurrently reduced and correlated with the malignant phenotype of lung cancers. Enhanced expression of DNA methyltransferase-1 correlated with the reduced expression of Axin and could be a marker for lung cancer development and progression.

Proteasomal non‐catalytic subunit PSMD2 as a potential therapeutic target in association with various clinicopathologic features in lung adenocarcinomas

We previously identified PSMD2, a subunit of the 19S regulatory complex of proteasomes, as a constituent of a signature associated with the acquisition of metastatic phenotype and poor prognosis in lung cancers. In the present study, we found that knockdown of PSMD2 decreased proteasome activity, and induced growth inhibition and apoptosis in lung cancer cell lines. These effects of siRNA-mediated PSMD2 inhibition were associated with changes in the balance between phosphorylated AKT and p38, as well as with induction of p21. In addition, patients with higher PSMD2 expression had poorer prognosis and a small fraction of lung cancer specimens carried increased copies of PSMD2. Notably, our findings clearly illustrate that lung adenocarcinomas can be divided into two groups; those with and without general upregulation of proteasome pathway genes including PSMD2. This general upregulation was significantly more prevalent in the non-terminal respiratory unit (non-TRU)-type, a recently proposed genetically and clinicopathologically relevant expression profile-defined classification of adenocarcinomas (P < 0.001 by Fisher's exact test). Patients with adenocarcinomas with general upregulation had significantly shorter survival after potentially curative resection (P = 0.0001 by log-rank test) independent of disease stage, as shown by multivariate Cox regression analysis. Our results suggest that PSMD2 may be a good molecular target candidate and that other co-regulated proteasome pathway genes and/or their common regulator(s) might also be potential targets, warranting future study including elucidation of the underlying common regulatory mechanism.

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.