Normal Lung Tissue Samples Research Articles

Overexpression of EGFR protein in Bruneian lung cancer patients.

Lung cancer is the leading cause of cancer death in Brunei Darussalam, accounting for almost 20% of the total. The epidermal growth factor receptor (EGFR) is a member of the erbB family of tyrosine kinase receptor proteins, which includes c-erbb2(HER2/neu), erb-B3, and erb-B4. EGFR overexpression is found in a third of all epithelial cancers, often associated with a poor prognosis. Protein expression of EGFR in 27 cases of lung cancer tissue samples and 9 cases of normal lung tissue samples was evaluated using an immunohistochemical approach. The results demonstrated significant increase and overexpression of EGFR in Bruneian lung cancer tissue samples in comparison to normal lung tissue. However, there was no significant relationship between clinicopathologic variables (age and sex) of patients and EGFR protein expression. EGFR is overexpressed in Bruneian lung cancer patient tissue samples in comparison to normal lung tissue samples. This may indicate that EGFR protein over expression plays an important role in the genesis of this type of cancer in Brunei Darussalam.

Expression and significance of myeloid differentiation factor 88 in non-small cell lung carcinoma and normal paracancerous tissues.

We studied the expression level of myeloid differentiation factor 88 (MyD88) in non-small cell lung carcinoma (NSCLC) and normal paracancerous tissues, to determine its relationship with clinical pathological characteristics and prognosis. In total, 82 NSCLC patients who had received surgical treatment in our hospital between September 2008 and December 2013 were selected for this study. Another 82 normal paracancerous lung tissue samples were used as controls. All patients had complete clinical records, and they were followed-up for 5 years. The expression level of MyD88 protein was detected by immunohistochemical assay. The positive expression rate of MyD88 in NSCLC tissues (62.2%) was markedly higher than that in normal tissues (10.9%), and was independent of patient characteristics such as age, gender, pathological pattern, history of smoking, and tumor size (P > 0.05). However, MyD88 expression was significantly correlated with degree of differentiation, clinical staging, and lymphatic metastasis (P < 0.05), and was negatively correlated with prognosis. The 5-year survival rate of patients with positive MyD88 expression was significantly lower than that of patients without positive expression (P < 0.05). MyD88 was expressed at a higher level in NSCLC tissues and was closely associated with poor prognosis. MyD88 may be a novel eligible target for treating NSCLC.

Expression of hepatocyte growth factor and c-Met in non-small-cell lung cancer and association with lymphangiogenesis.

Previous experimental studies have demonstrated that hepatocyte growth factor (HGF) and its receptor c‑Met serve an important function in lymphangiogenesis, but their biological functions in malignant tumors have remained elusive. The present study aimed to investigate the expression patterns of HGF‑α and c‑Met and their association with vascular endothelial growth factor (VEGF)‑C, lymphatic vessel density and lymph node metastasis in non‑small‑cell lung cancer (NSCLC). In the present study, the lymphatic microvessel density (LMVD) and the expression levels of HGF‑α, its receptor c‑Met and VEGF‑C were determined in 113 human NSCLC tissues and 113 normal lung tissue samples, using immunohistochemical staining. As a result, it was determined that the expression levels of HGF‑α, c‑Met and VEGF‑C were significantly higher in NSCLC tissues than those in normal lung tissues (HGF‑α, 67.3 vs. 20.4%, P<0.001; c‑Met, 74.3 vs. 23.0%, P<0.001; and VEGF‑C, 65.5 vs. 23.9%, P<0.001). HGF‑α expression was observed to be significantly associated with that of VEGF‑C (r=0.234, P=0.012) or c‑Met (r=0.648, P<0.001). In addition, there was a positive correlation between the expression levels of VEGF‑C and c‑Met (r=0.224, P=0.017). In NSCLC tissues, the expression of HGF‑α, c‑Met or VEGF‑C was significantly correlated with the LMVD (P=0.045, 0.002 and 0.001, respectively), and lymph node metastasis was more common in HGF‑α, c‑Met or VEGF‑C‑positive groups (P=0.020, 0.020 and 0.009, respectively). In addition, the HGF‑α or VEGF‑C‑positive groups presented shorter survival time periods. In conclusion, the expression of HGF‑α or c‑Met was closely correlated with VEGF‑C, LMVD and metastases of lymph nodes, indicating that HGF‑α, c‑Met and VEGF‑C may perform important and collaborative actions in lymphangiogenesis and lymphatic metastasis of primary NSCLC.

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

BackgroundGlycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.MethodsThe expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.ResultsExpression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.ConclusionNSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.

Clinicopathological significance and potential drug target of T-cadherin in NSCLC.

BackgroundPrevious studies demonstrate that T-cadherin is a candidate tumor suppressor in several types of human tumors, including non-small cell lung cancer (NSCLC). Lack of protein expression of T-cadherin by hypermethylation has been found to play an important role in lung alveolar differentiation regulation and epithelial tumorigenesis. However, the correlation between T-cadherin hypermethylation and clinicopathological characteristics of NSCLC remains unclear. Here we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of T-cadherin hypermethylation on the incidence of NSCLC and clinicopathological characteristics.MethodsA detailed literature search was carried out for related research publications. Analyses of pooled data were performed. Odds ratio (OR) and hazard ratio (HR) were calculated and summarized, respectively.ResultsFinal analysis of 1,172 NSCLC patients from 15 eligible studies was performed. T-cadherin hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, based on the pooled OR from nine studies including 532 NSCLC and 372 normal lung tissue samples (OR=8.19, 95% confidence interval [CI]=5.41–12.39, P<0.00001). T-cadherin hypermethylation may also be associated with pathological types. The pooled OR was obtained from four studies including 111patients with squamous cell carcinoma and 106 with adenocarcinoma (OR=0.35, 95% CI=0.19–0.66, P=0.001), which indicated that T-cadherin hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. We did not find that T-cadherin hypermethylation was correlated with the sex or smoking status, clinical stages, or epidermal growth factor receptor (EGFR) mutation status. However, T-cadherin hypermethylation was found to be significantly higher in poorly differentiated NSCLC than in moderately and highly differentiated NSCLC, and NSCLC patients with T-cadherin hypermethylation had a lower survival rate than those without T-cadherin hypermethylation.ConclusionThe results of this meta-analysis suggest that T-cadherin hypermethylation is associated with an increased risk and worse survival in NSCLC. T-cadherin hypermethylation, which induces the inactivation of T-cadherin gene, plays an important role in the carcinogenesis, cancer progression, as well as clinical outcome.

Increased MET Gene Copy Number but Not mRNA Level Predicts Postoperative Recurrence in Patients with Non–Small Cell Lung Cancer

The aim of the present study was to investigate the relationship of MET copy number (CN) and MET mRNA expression to other molecular alterations, clinicopathologic characteristics, and survival of patients with resected non–small cell lung cancer. One hundred fifty-one paired surgical samples of tumor and tumor-distant normal lung tissues were analyzed by comparative quantitative polymerase chain reaction (PCR) methods with commercially available assays and the CopyCaller software v. 1.0 for post-PCR data processing (downloadable from www.appliedbiosystems.com). MET copy gain (set as more than 3.0 copies per cell) was found in 18.5% of the samples and occurred more frequently in the adenocarcinomas (ADCs) with an increased epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) CN (P = .001 and .030 for EGFR and HER2, respectively) and in the ADCs with EGFR activating mutations (P = .051) but did not correlate with KRAS dosage or mutational status. MET mRNA level was 1.76-fold higher [95% confidence interval (CI), 1.29-2.40] in the tumor compared to unaffected lung tissue and associated significantly with MET CN (beta coefficient, 1.51; 95% CI, 1.22-1.87; P < .001). In the multivariable analysis, patients diagnosed with ADC with increased MET CN had a significantly higher risk of disease recurrence (hazard ratio, 1.76; 95% CI, 1.20-2.57; P = .004). An increased MET CN in combination with histologic type appears to be a prognostic factor in patients with ADC after a curative surgery.

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.

Unique microRNA‐profiles in EGFR ‐mutated lung adenocarcinomas

The findings of mutations and the development of targeted therapies have improved lung cancer management. Still, the prognosis remains poor, and we need to know more about the genetic and epigenetic alterations in lung cancer. MicroRNAs are involved in crucial biological processes like carcinogenesis by regulating gene expression at the post-transcriptional level. In this project, we have studied the microRNA expression of lung adenocarcinomas and corresponding normal lung tissue and correlated the expression with clinical data and EGFR- and KRAS-mutational status. Agilent microarrays have been used, examining microRNA expression in 154 surgically resected lung adenocarcinomas and 20 corresponding normal lung tissue samples. Findings were confirmed by RT-qPCR in the same cohort and in an independent cohort of 103 lung cancer patients. EGFR and KRAS mutation analyses were also performed. 129 microRNAs were significantly differentially expressed in lung adenocarcinomas compared with normal lung tissue, and 17 microRNAs were differentially expressed between EGFR-mutated and EGFR wildtype tumors. We identified microRNAs associated with time to progression. We have identified several aberrantly expressed microRNAs that discriminate lung adenocarcinomas from normal lung tissue, and hence may be potential biomarkers for early detection. We have found microRNAs that are differentially expressed between EGFR-mutated and EGFR wildtype lung adenocarcinomas, suggesting that microRNAs can be used as molecular biomarkers in classification. We hypothesize that microRNA expression can be used as biomarkers for clinical course.

MACC1 overexpression predicts a poor prognosis for non-small cell lung cancer

The expression of metastasis-associated in colon cancer-1 (MACC1) in non-small cell lung cancer (NSCLC) and its association with pathological characteristics and prognosis for NSCLC patients were investigated retrospectively. The expression of MACC1 was evaluated through immunohistochemical staining of tissue microarrays from 180 samples of resected lung cancer tissues and adjacent normal lung tissues. MACC1 protein and mRNA expression were also examined from lung cancer cell lines with different metastatic potentials, 28 pairs of samples of resected fresh non-small cell lung cancer tissues, and adjacent normal lung tissues. Immunohistochemical staining of tissue microarrays showed that MACC1 was located in the cytoplasm. In addition, the expression of MACC1 protein in NSCLC was significantly higher compared to adjacent normal tissues (P < 0.001). The expression of MACC1 was positively associated with differentiation grade (P = 0.020), postoperative pathological TNM stage (P = 0.033), and lymph node metastasis (P = 0.028). Disease-free survival (DFS) and overall survival (OS) for the high MACC1 expression group were lower than the low expression group; univariate and multivariate regression analyses showed that MACC1 was an independent prognostic indicator for DFS (HR 3.124, P = 0.01) and OS (HR 2.905, P = 0.01) in NSCLC patients. The expression of MACC1 protein and mRNA was also upregulated in highly metastatic human lung cancer. In conclusion, the overexpression of MACC1 protein and mRNA may represent a potentially useful biomarker for the prognosis of NSCLC patients and might be involved in progression of NSCLC.

Epigenetic regulation of ANKRD18B in lung cancer

The identification of the key genetic and epigenetic changes underlying lung carcinogenesis would aid effective early diagnosis and targeted therapies for lung cancer. In this study, we screened a novel hypermethylated gene ankyrin repeat domain 18B (ANKRD18B), to determine whether it is regulated by DNA methylation and clarify its biological and clinical implications in lung cancer. Methylation status and expression level were analyzed by methylation-specific PCR, bisulfite genomic sequencing, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We detected ANKRD18B hypermethylation in 52 of 98 (53.1%) primary lung cancer tissues and in nine of 10 (90%) cell lines, whereas no methylation was seen in 10 normal lung tissue samples. ANKRD18B methylation was more frequently observed in patients with poor differentiation (P < 0.05). Notably, 62 pairs of samples from patients whose tumor tissue showed hypermethylation of ANKRD18B exhibited the same aberrant methylation in 72.7% and 69.7% of their corresponding plasma and sputum samples, respectively; whereas no hypermethylation of ANKRD18B was detected in the sputum and plasma from patients whose tumor sample lacked this alteration. In addition, ANKRD18B mRNA expression was significantly decreased or silenced in lung cancer tissues and cell lines associated with hypermethylation of the ANKRD18B region. Demethylation agent 5-aza-2'-deoxycytidine significantly increased ANKRD18B mRNA expression in lung cancer cell lines. Furthermore, overexpression of ANKRD18B suppressed lung cancer cell growth. These results suggest that the expression of ANKRD18B is regulated by CpG island hypermethylation in lung cancer. Our findings confirm the importance of the identification of new markers of epigenetic dysregulation in cancer.

Epigenetic silencing of Aristaless‐like homeobox‐4, a potential tumor suppressor gene associated with lung cancer

Using genome-wide methylation screening, we found Aristaless-like homeobox-4 (ALX4) preferentially methylated in lung cancer. ALX4 is a putative transcription factor that belongs to the family of paired-class homeoproteins involved in epithelial development. However, the role of ALX4 in tumorigenesis remains largely unclear. Here, we analyzed its epigenetic regulation, biological functions and related molecular mechanisms in lung cancer. CpG island methylation and expression of ALX4 were evaluated by methylation-specific polymerase chain reaction (PCR), bisulfite genomic sequencing, reverse-transcription PCR and Western blotting. ALX4 functions were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. ALX4 hypermethylation was detected in 55% (54/98) of primary lung cancers compared to none (0/20) of the normal lung tissue samples tested (p < 0.01). ALX4 was readily expressed in normal lung tissues with an unmethylated status, but downregulated or silenced in 90% (9/10) of lung cancer cell lines with a hypermethylation status. Demethylation experiments further confirmed that loss of ALX4 expression was regulated by CpG island hypermethylation. Re-expression of ALX4 in lung cancer cell lines suppressed cell viability, colony formation and migration, whereas it induced apoptosis and G1/S arrest and restrained the tumorigenicity in nude mice. These effects were associated with upregulation of proapoptotic proteins caspase-7, -8 and -9, and downregulation of Bcl-2. On the other hand, knockdown of ALX4 expression by siRNA increased cell viability and proliferation, whereas it inhibited apoptosis and cell cycle arrest. In conclusion, our results suggest that ALX4 is a novel putative tumor suppressor with epigenetic silencing in lung carcinogenesis.

PBK/TOPK expression in non‐small‐cell lung cancer: its correlation and prognostic significance with Ki67 and p53 expression

The aim of this study was to evaluate the prevalence of PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase) expression, and explore the prognostic significance of PBK/TOPK expression alone and in combination with Ki67 and p53 expression in non-small-cell lung cancer (NSCLC). We detected PBK/TOPK expression in 30 samples of normal lung tissue, 32 lymph node metastases and 279 primary non-small-cell lung cancers by immunohistochemistry, and analysed the correlation of PBK/TOPK expression with Ki67 and p53 expression in primary tumour tissues. The results showed that PBK/TOPK expression was higher in lymph node metastases (75%) than in primary tumours (44.8%) and normal lung tissues (0%). PBK/TOPK expression was associated with histological type, lymph node metastasis, and TNM stage, and was positively correlated with Ki67 and p53 expression in NSCLC. Univariate and multivariate survival analyses showed that PBK/TOPK expression was significantly associated with an unfavourable prognosis in NSCLC. The prognosis of patients with tumours positive for both PBK/TOPK expression and Ki67 or p53 expression was also significantly unfavourable. PBK/TOPK expression is positively correlated with Ki67 and p53 expression, and can be used as an independent prognostic factor in NSCLC.

Yin Yang Gene Expression Ratio Signature for Lung Cancer Prognosis

Many studies have established gene expression-based prognostic signatures for lung cancer. All of these signatures were built from training data sets by learning the correlation of gene expression with the patients' survival time. They require all new sample data to be normalized to the training data, ultimately resulting in common problems of low reproducibility and impracticality. To overcome these problems, we propose a new signature model which does not involve data training. We hypothesize that the imbalance of two opposing effects in lung cancer cells, represented by Yin and Yang genes, determines a patient’s prognosis. We selected the Yin and Yang genes by comparing expression data from normal lung and lung cancer tissue samples using both unsupervised clustering and pathways analyses. We calculated the Yin and Yang gene expression mean ratio (YMR) as patient risk scores. Thirty-one Yin and thirty-two Yang genes were identified and selected for the signature development. In normal lung tissues, the YMR is less than 1.0; in lung cancer cases, the YMR is greater than 1.0. The YMR was tested for lung cancer prognosis prediction in four independent data sets and it significantly stratified patients into high- and low-risk survival groups (p = 0.02, HR = 2.72; p = 0.01, HR = 2.70; p = 0.007, HR = 2.73; p = 0.005, HR = 2.63). It also showed prediction of the chemotherapy outcomes for stage II & III. In multivariate analysis, the YMR risk factor was more successful at predicting clinical outcomes than other commonly used clinical factors, with the exception of tumor stage. The YMR can be measured in an individual patient in the clinic independent of gene expression platform. This study provided a novel insight into the biology of lung cancer and shed light on the clinical applicability.

Mitochondrial Genome Microsatellite Instability and Copy Number Alteration in Lung Carcinomas

Mitochondrial DNA (mtDNA) is considered a hotspot of mutations in various tumors. However, the relationship between microsatellite instability (MSI) and mtDNA copy number alterations in lung cancer has yet to be fully clarifieds. In the current study, we investigated the copy number and MSI of mitochondrial genome in lung carcinomas, as well as their significance for cancer development. The copy number and MSI of mtDNA in 37 matched lung carcinoma/adjacent histological normal lung tissue samples were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays for sequence variation, followed by sequence analysis and fluorogenic 5'-nuclease real-time PCR. Student's t test and linear regression analyses were employed to analyze the association between mtDNA copy number alterations and mitochondrial MSI (mtMSI). The mean copy number of mtDNA in lung carcinoma tissue samples was significantly lower than that of the adjacent histologically normal lung tissue samples (p < 0.001). mtMSI was detected in 32.4% (12/37) of lung carcinoma samples. The average copy number of mtDNA in lung carcinoma samples containing mtMSI was significantly lower than that in the other lung carcinoma samples (P < 0.05). Results suggest that mtMSI may be an early and important event in the progression of lung carcinogenesis, particularly in association with variation in mtDNA copy number.

Abstract 5364: DNA methylation profiles at precancerous stage cluster lung adenocarcinomas into subclusters associated with carcinogenetic pathway, clinicopathological aggressiveness and patient outcome.

Abstract Background: We have reported DNA methylation alterations in non-cancerous lung tissue obtained from lung cancer patients. These previous data drew our attention to DNA methylation alterations at precancerous stage of lung adenocarcinoma (LADC). However, the impact of DNA methylation alterations at precancerous stage on the characteristics of established LADCs has been unclear. The purpose of this study is to clarify the significance of DNA methylation profiles during lung carcinogenesis. Methods: Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue (C), 139 samples of non-cancerous lung tissue (N) obtained from patients with LADCs, and the corresponding 139 samples of tumorous tissue (T) as a learning cohort. As a validation cohort, the 50 paired samples of N and the corresponding T were obtained from patients with primary LADCs. Results: 3,621 probes showed significant differences in DNA methylation levels between the 36 C and 139 N samples in the learning cohort, suggesting that N was at precancerous stage with DNA methylation alterations. Unsupervised hierarchical clustering using DNA methylation levels of N on 26,447 autosomal probes subclustered 139 patients of the learning cohort into Cluster A (n=32), B (n=35) and C (n=72). Most of patients in Cluster A were heavy smokers and frequently showed severe pleural anthracosis which mainly reflects smoking history. LADCs in Cluster A were locally invasive tumors which develop in emphysematous lung tissue associated with inflammation. Most of patients in Cluster B were non-smokers and LADCs in Cluster B showed less aggressive features. Most of patients in Cluster C were light smokers and LADCs in Cluster C showed more frequent lymph node metastasis and higher Tumor-Node-Metastasis (TNM) stages. The cancer-free and overall survival rates of patients in Cluster C were significantly lower than those of Cluster B. We indetified 21, 26 and 35 probes as markers which characterize Clusters A, B and C, respectively. In the validation cohort, DNA methylation levels of Cluster A, B and C marker probes were significantly correlated with pleural anthracosis, never-smoking history, and lymph node metastases and higher TNM stages, respectively, i.e. correlation between DNA methylation profiles and each clinicopathological parameter were validated in the validation cohort. Conclusion: DNA methylation profiles of Clusters A and C at precancerous stage may be established by the effects of smoking through or not thorough inflammation, respectively, whereas those of Cluster B may be associated with carcinogenesis in non-smokers. DNA methylation profiles at precancerous stage may underlie distinct pathways of lung carcinogenesis and determine tumor aggressiveness and patient outcome. Citation Format: Takashi Sato, Eri Arai, Takashi Kohno, Koji Tsuta, Shun-ichi Watanabe, Kenzo Soejima, Tomoko Betsuyaku, Yae Kanai. DNA methylation profiles at precancerous stage cluster lung adenocarcinomas into subclusters associated with carcinogenetic pathway, clinicopathological aggressiveness and patient outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5364. doi:10.1158/1538-7445.AM2013-5364

Abstract 4466: Integrative study of dysregulated genes in translocated ALK-positive non-small cell lung cancer and personalized targeted therapy resistance.

Abstract Background: Recent studies revealed that ALK (anaplastic lymphoma kinase) 2p23 translocation, most commonly fused with echinoderm microtubule-associated protein-like 4 (EML4), is a novel driver oncogene in about 5% of non-small cell lung cancer (NSCLC). ALK 2p23 fusion (ALK+) can be diagnosed by dual color break-part ALK FISH assay to inform personalized targeted therapy using the ALK inhibitor crizotinib. However, the underlying mechanisms of ALK oncogenic signaling and more importantly ALK inhibitor crizotinib resistance are not well understood. Materials and Methods: To understand the mechanism of oncogenic ALK signaling, an ALK+ NSCLC microarray dataset was retrieved from NCBI-GEO data repository (GSE31210) and analyzed using data-mining bioinformatics approaches. This dataset includes the gene expression profiles of 11 ALK+ NSCLC and 20 normal lung tissue samples. Statistical analysis was carried out to identify altered genes in ALK+ NSCLC and Ingenuity Pathway Analysis (IPA) software was deployed to uncover their network interactions. To study the resistance mechanism of the ALK+ H3122 human NSCLC cell line against the specific ALK inhibitor TAE684, real-time polymerase chain reaction (qRT-PCR), cell viability assay and immunoblotting were performed using standard techniques. Results: We identified a set of 1,656 genes that were significantly altered in ALK+ NSCLC, consisting of 662 upregulated and 994 downregulated. The principal component analysis (PCA) based on the top 51 of these dysregulated genes evidently separated ALK+ and normal lung tissue samples, implicating that these genes could represent potential genomic signature of ALK+ NSCLC. The IPA analysis revealed 35 canonical pathways linked to ALK signaling, including beta-adrenergic and angiopoietin signaling pathways. The results of qRT-PCR, cell viability assay and immunoblotting showed that in translocated ALK+ H3122 human NSCLC cell line, HGF (hepatocyte growth factor) overexpression could activate ALK downstream signaling via activation of MET receptor kinase pathway, thus potentially bypassing the oncogenic ALK signaling under ALK inhibitor TAE684 treatment. Hence, alternative direct activation of ALK downstream signaling molecules via MET/HGF axis activation may potentially contribute to ALK inhibitor drug resistance. Conclusion: Using bioinformatics data-mining, we have identified dysregulated genes and pathways in ALK+ NSCLC, many of which are novel biomarkers not previously reported. We also verified the relevance and potential key role of MET/HGF signaling axis in ALK inhibitor resistance. Taken together, our results highlighted the cross-talk between ALK and MET signaling in NSCLC and shed new insights into possible pitfalls of potential specific ALK targeting inhibitor drugs. Citation Format: Ivy Shi, Lihong Yin, Patrick C. Ma. Integrative study of dysregulated genes in translocated ALK-positive non-small cell lung cancer and personalized targeted therapy resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2013-4466

Abstract 5315: Interactions between LRIG proteins and LMO7 and the expression of LMO7 in human lung cancer.

Abstract The Leucine-rich repeats and immunoglobulin-like domains proteins (LRIG) 1, 2 and 3 are integral membrane proteins that have been implicated in human cancer. We and others have shown that LRIG1 is a negative regulator of receptor tyrosine kinase (RTK) signaling. LRIG1 is down regulated in many tumors; the expression of LRIG1 is often associated with long patient survival; and LRIG1 has been shown to be a tumor suppressor in the mouse intestine. Cytoplasmic expression of LRIG2 is associated with a poor survival for oligodendroglioma patients and perinuclear expression of LRIG3 in astrocytic tumors is associated with a better survival for the patients. LRIG3 is involved in the formation of the inner ear during embryology and a study suggests that LRIG3 is a serum biomarker for NSCLC. To further elucidate the functions of the LRIG proteins we performed a yeast two hybrid (YTH) screen in which we identified LMO7 and LIMCH1 as interaction partners to LRIG3. LMO7 is a cytoskeletal binding protein and functions as a transcription factor. A study in which LMO7 was deleted in mice showed that these mice developed spontaneous lung cancers, especially adenocarcinomas, at high age. Using laser confocal microscopy we found that LRIG3 and LMO7 co-localized in cytoskeletal like structures, the border of the cell and at the perinuclear region. To analyze the possible interaction between LRIG1, 2 and 3 and LMO7, we used the proximity ligation assay (PLA) technique. Endogenously expressed LRIG1, 2 and 3 all appeared to interact with endogenous LMO7 in all cell lines tested. LMO7 down regulation by siRNA resulted in fewer apparent LMO7-LRIG interactions, confirming the specificity of the PLA assays. Using QRT-RTPCR we found that LMO7 mRNA was highly expressed in small intestine, lung and colon. Comparison of the mRNA level of LMO7 in normal human lung and a panel of different human lung tumors showed a dramatic decrease of LMO7 mRNA expression in the human lung tumors. Analysis of the cancer genome atlas (TCGA) database indicated that LMO7 gene expression was high in human normal colorectal and lung tissues, and the expression of LMO7 was dramatically decreased in malignant colorectal and lung tissues. To further study LMO7 in lung cancer, we stained a tissue microarray with 364 lung cancer tissue samples and 17 normal human lung tissue samples for LMO7. LMO7 was rarely expressed in the human lung tumors. Multivariate Cox regression analysis indicated that LMO7 was a prognostic factor in human lung cancer (p = 0.013). In summary LMO7 interacted with all three LRIG proteins, the expression of LMO7 was decreased in malignant colorectal and lung tissues compared to normal tissues and LMO7 appeared to be a prognostic factor in human lung cancer. Citation Format: Terese Karlsson, Samuel Kvarnbrink, Camilla Holmlund, Johan Botling, Patrick Micke, Mikael Johansson, Roger Henriksson, Håkan Hedman. Interactions between LRIG proteins and LMO7 and the expression of LMO7 in human lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2013-5315

Abstract 1962: Differentially expressed microRNAs in adenocarcinomas of the lung and tumor-adjacent normal lung tissue.

Abstract Introduction: Lung cancer is the most common cause of cancer deaths worldwide. The findings of new mutations and the development of targeted therapies have improved lung cancer management. Still, the prognosis remains poor and we need to know more about the genetic and epigenetic alterations in the tumors to better understand the biology of lung cancer. MicroRNAs are small non-coding RNAs that are involved in crucial biological processes in carcinogenesis by regulating gene expression at the post transcriptional level. In this project we have studied the microRNA expression patterns of lung adenocarcinomas and correspondent tumor-adjacent normal lung tissue and correlated the expression patterns with clinical data and mutational status. Methods: We have examined microRNA expression pattern in tumor tissues from 154 surgically resected lung adenocarcinomas and from 20 tumor-adjacent normal lung tissue samples. The expression of 1205 human microRNAs was conducted using the 60K microRNA microarray from Agilent technology. EGFR and KRAS mutation analyses were also performed. The analysis of differentially expressed microRNAs between groups of samples was done using significance analyses of microarrays (SAM) in the J-express software. We also performed survival analysis using univariate- and multivariate Cox regression analysis. The results are about to be validated by qRT-PCR. Results: Preliminary results show 129 differentially expressed microRNAs in tumor compared to the tumor-adjacent normal lung tissue. EGFR and KRAS mutations were found in 22/152 (14.5%) and 47/137 (34.3%) samples respectively. We have detected 17 microRNAs that are differentially expressed in EGFR mutated tumors compared to EGFR wildtype tumors. Two microRNAs were identified to have a strong association with time to progression in both univariate- and multivariate Cox regression analysis. Discussion: The microRNAs are thought to play an essential role in the development and progression of human malignancies, including lung cancer. We have identified several aberrantly expressed microRNAs that can discriminate lung adenocarcinoma tumor tissue from tumor-adjacent normal lung tissue samples. This can lead to the identification of biomarkers for early detection. 17 microRNAs were differentially expressed between EGFR mutated- and EGFR wt lung adenocarcinomas suggesting that microRNAs can be used as molecular biomarkers for lung cancer classification. We have also identified microRNAs that can be used as prognostic biomarkers. We are now confirming our results with qRT-PCR. We hypothesize that microRNA can be used as biomarkers for classification and clinical course. Citation Format: Maria Bjaanæs, Rita Halvorsen, Steinar Solberg, Lars Jørgensen, Odd-Terje Brustugun, Åslaug Helland. Differentially expressed microRNAs in adenocarcinomas of the lung and tumor-adjacent normal lung tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2013-1962

DNA Methylation Profiles at Precancerous Stages Associated with Recurrence of Lung Adenocarcinoma

The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average β-values based on Infinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (ΔβT–N>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the ADCY5, EVX1, GFRA1, PDE9A, and TBX20 genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2′-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.

Down-regulation of Notch receptor signaling pathway induces caspase-dependent and caspase-independent apoptosis in lung squamous cell carcinoma cells

Notch receptor signaling pathway (NRSP) is increasingly linked to carcinogenesis. Non-small cell lung cancer (NSCLC) appears to actively utilize this conserved developmental pathway. The aims of this study are to determine whether or not Notch 1-4 are overexpressed in NSCLC tissues compared with normal lung tissues and whether inhibiting NRSP could induce caspase-dependent or caspase-independent apoptosis. Immunohistochemistry was used to evaluate the expression of Notch 1-4 in 101 NSCLC tissue samples and 30 normal lung tissue samples. DAPT was used to repress NRSP in SK-MES-1 cells. Apoptosis was determined by Annexin V and PI staining. Cleaved poly ADP-ribose polymerase (PARP) was measured by Western blot; X-linked inhibitor of apoptosis protein (XIAP) and Survivin were assessed by qRT-PCR and Western blot; the release of second mitochondria-derived activator of caspase (Smac) from mitochondria to cytoplasm was evaluated by Western blot; the subcellular locations of endonuclease G (Endo G) and apoptosis inducing factor (AIF) were observed by Western blot and indirect immunofluorescence analysis. (Mech Dev, 98, 2000, 95) Notch 1-4 are up-regulated in NSCLC tissues and Notch 1, 2 are positively correlated with lymph node metastasis, (Proc Natl Acad Sci U S A, 106, 2009, 22293) DAPT treatment could inhibit NRSP and induce apoptosis, with a marked increase in cleaved PARP, decreases in XIAP and Survivin proteins and concomitant release of Smac, EndoG, and AIF from mitochondria, indicating that inhibiting NRSP by DAPT triggers caspase-dependent and caspase-independent apoptosis.