In-depth proteomics reveals the characteristic developmental profiles of early lung adenocarcinoma with epidermal growth factor receptor mutation.

INTRODUCTION: Lung adenocarcinoma progresses stepwise from atypical adenomatous hyperplasia to adenocarcinoma in situ (AIS), followed by minimally invasive adenocarcinoma (MIA), and then obvious invasive adenocarcinoma. In this study, we examined the protein expression profiles of early and epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinomas. METHODS: Fifteen cases of small and EGFR mutation-positive adenocarcinomas were collected, including AIS, MIA, and small invasive adenocarcinoma (SIA). We examined their protein expression profiles by tandem mass tag (TMT)-labeling liquid chromatography-mass spectrometry (LC-MS/MS) and compared the results between AIS and MIA versus SIA. The differentially expressed proteins were then verified by Western blot analysis and immunohistochemistry (IHC). The clinicopathological implications of the proteins were also examined by IHC. RESULTS: A total of 4220 proteins were identified by LC-MS/MS analysis. Pathway analysis of the differentially expressed proteins revealed that pathways related to interferon α/ß signaling, glutamate and glutamine metabolism, and gluconeogenesis were upregulated in SIA relative to AIS. Among the 13 differentially expressed proteins, cellular retinoic acid binding protein 2 (CRABP2), delta(24)-sterol reductase (DHCR24), and adenylate kinase 4 (AK4) were expressed significantly more strongly in SIA than in AIS. Patients with high expression of CRABP2, DHCR24, and AK4 showed a significantly poorer outcome than those with low expression. CONCLUSION: In comparison with AIS, SIA shows differences in several different protein expression pathways. Furthermore, CRABP2, DHCR24, and AK4 are useful IHC markers for diagnosis of lung adenocarcinoma invasiveness and may be associated with malignant progression of AIS.

DNA hypomethylation-related overexpression of SFN, GORASP2 and ZYG11A is a novel prognostic biomarker for early stage lung adenocarcinoma.

Although alteration of DNA methylation in advanced cancer has been extensively investigated, few data for early-stage lung adenocarcinoma are available. Here, we compared DNA methylation profiles between adenocarcinoma in situ (AIS) and early invasive adenocarcinoma using the Infinium methylation array to investigate methylation abnormalities causing early progression of adenocarcinomas. We focused on differentially methylated sites which were located in promoter CpG islands or shore regions, and identified 579 hypermethylated sites and 23 hypomethylated sites in early invasive adenocarcinoma relative to AIS and normal lung. These hypermethylated genes were significantly associated with neuronal pathways such as the GABA receptor and serotonin signaling pathways. Among the hypomethylated genes, we found that GORASP2, ZYG11A, and SFN had significantly lower methylation rates at the shore regions and significantly higher protein expression in invasive adenocarcinoma. Moreover, overexpression of those proteins was strongly associated with patient's poor outcome. Despite DNA demethylation at the promoter region might be rare relative to DNA hypermethylation, we identified 2 new genes, GORASP2 and ZYG11A, which show hypomethylation and overexpression in invasive adenocarcinoma, suggesting that they have important functions in tumor cells. These genes may be clinically applicable as prognostic indicators and could be potential novel target molecules for drug development.

Cytoplasmic expression of epithelial cell transforming sequence 2 in lung adenocarcinoma and its implications for malignant progression.

Epithelial cell transforming sequence 2 (ECT2), a guanine nucleotide exchange factor, is predominantly localized in the nucleus of non-transformed cells and functions to regulate cytokinesis. ECT2 is also localized in the cytoplasm of cancer cells. Aberrant cytoplasmic expression of ECT2 is thought to drive tumor growth and invasion. In this study, we investigated the cytoplasmic expression of ECT2 and its prognostic and biological significance in lung adenocarcinoma. Western blotting of cellular fractions from the nucleus and cytoplasm was performed to determine the subcellular localization of ECT2 in lung adenocarcinoma cell lines. The cytoplasmic expression of ECT2 in 167 lung adenocarcinomas was evaluated by immunohistochemistry and its clinical significance was examined using Kaplan-Meier curves and Cox regression analysis. Scraping cytology specimens of 13 fresh lung adenocarcinomas were used to assess the subcellular localization of ECT2 and its phosphorylation at Thr790 (P-ECT2(T790)). We found that ECT2 was localized in both the nucleus and cytoplasm of lung adenocarcinoma cell lines and tumor tissues. Cytoplasmic expression of ECT2 was detected by immunohistochemistry in 83 (50%) of the lung adenocarcinomas, and was found to increase during cancer progression. It was expressed in 30 (29%) small adenocarcinomas ( ≤ 2 cm in diameter) and 53 (82%) advanced adenocarcinomas ( > 2 cm in diameter). Cytoplasmic positivity for ECT2 was associated with a poor outcome in terms of both disease-free and overall survival (both P < 0.001), and was an independent prognostic factor for overall survival (P = 0.025). Immunocytochemical staining for P-ECT2(T790) demonstrated cytoplasmic and membrane positivity in Calu-3 cells and scraping cytology specimens. Positive P-ECT2(T790) staining was correlated with cytoplasmic ECT2 expression in 6 of 13 scraped cytology specimens tested. In conclusion, our findings indicate that cytoplasmic ECT2 expression could promote the malignant progression of lung adenocarcinoma and may represent a potent therapeutic target for patients with lung adenocarcinoma.

The prognostic significance of N-myc downregulated gene 1 in lung adenocarcinoma.

It has been reported that N-myc downstream regulated gene 1 (NDRG1) is related to the prognosis of non-small cell lung cancer (NSCLC), and associated with c-Myc degradation in NSCLC cell lines. However, the relationship of NDRG1 to prognosis or c-Myc expression in lung adenocarcinoma has not been well clarified. The present study was designed to investigate the prognostic significance of NDRG1 and/or c-Myc expression in lung adenocarcinoma using immunohistochemistry with a tissue microarray. We examined 184 lung adenocarcinomas and observed low expression of NDRG1 in adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA), whereas high expression of NDRG1 was seen in invasive adenocarcinoma. Each of the clinicopathological features except age was significantly correlated with NDRG1 expression. Kaplan-Meier curves indicated that high expression of NDRG1 was significantly correlated with poor prognosis in comparison with low expression (log-rank, P < 0.001). Univariate and multivariate analyses indicated that vascular invasion (P = 0.012), lymphatic permeation (P = 0.038), and NDRG1 expression (P = 0.026) were independent prognostic factors. Expression of NDRG1 and positivity for c-Myc were significantly correlated (P = 0.005). These findings indicate that NDRG1 expression is associated with both prognosis and c-Myc expression in lung adenocarcinoma.

miR-3941: A novel microRNA that controls IGBP1 expression and is associated with malignant progression of lung adenocarcinoma.

Immunoglobulin (CD79a) binding protein 1 (IGBP1) is universally overexpressed in lung adenocarcinoma and exerts an anti-apoptotic effect by binding to PP2Ac. However, the molecular mechanism of IGBP1 overexpression is still unclear. In the present study, we used a microRNA (miRNA) array and TargetScan Human software to detect IGBP1-related miRNAs that regulate IGBP1 expression. The miRNA array analysis revealed more than 100 miRNAs that are dysregulated in early invasive adenocarcinoma. On the other hand, in silico analysis using TargetScan Human revealed 79 miRNAs that are associated with IGBP1 protein expression. Among the miRNAs selected by miRNA array analysis, six (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941) were also included among those selected by TargetScan analysis. Real-time reverse transcription PCR (real-time RT-PCR) showed that the six microRNAs were downregulated in invasive adenocarcinoma (IGBP1+) relative to adjacent normal lung tissue (IGBP1-). Among these microRNAs, only miR-34b and miR-3941 depressed luciferase activity by targeting 3'UTR-IGBP1 in the luciferase vector. We transfected miR-34b and miR-3941 into lung adenocarcinoma cell lines (A549, PC-9), and both of them suppressed IGBP1 expression and cell proliferation. Moreover, the transfected miR-34b and miR-3941 induced apoptosis of a lung adenocarcinoma cell line, similarly to the effect of siIGBP1 RNA. As well as miR-34b, we found that miR-3941 targeted IGBP1 specifically and was able to exclusively downregulate IGBP1 expression. These findings indicate that suppression of miR-3941 has an important role in the progression of lung adenocarcinoma at an early stage.

Drebrin: A new oncofetal biomarker associated with prognosis of lung adenocarcinoma.

OBJECTIVES: With the aim of searching for novel oncofetal tumor biomarkers of lung adenocarcinoma other than carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), we developed a strategy involving monoclonal antibodies generated from embryonic tissue of miniature swine. MATERIALS AND METHODS: Using immunohistochemistry, we selected suitable hybridoma clones that were reactive against swine fetal lung but not adult lung using tissue microarray loading of human normal lung, lung cancer, and fetal and adult swine tissues. RESULTS: The selected clones included several that were uniquely reactive against both swine fetal lung and human lung adenocarcinoma, and protein microarray revealed that the antigen they recognized was "drebrin" (DBN1). We then examined the association between the pattern of drebrin expression and the clinicopathological characteristics of lung adenocarcinoma using surgically resected samples of human lung adenocarcinoma. Two hundred formalin-fixed and paraffin-embedded tumor samples were immunostained for drebrin using clone B246, one of the clones that were reactive against drebrin. The cases were divided into those with strong (n=85) and weak (n=115) drebrin expression. In terms of disease-free survival, cases showing strong drebrin expression had a significantly poorer prognosis than those with weak drebrin expression (p=0.033). CONCLUSION: The present findings indicate that "drebrin" is a unique oncofetal protein that can be applied as a new biomarker of lung adenocarcinoma.

Methylation of MGMT and ADAMTS14 in normal colon mucosa: biomarkers of a field defect for cancerization preferentially targeting elder African-Americans.

Somatic hypermethylation of the O6-methylguanine-DNA methyltransferase gene (MGMT) was previously associated with G > A transition mutations in KRAS and TP53 in colorectal cancer (CRC). We tested the association of MGMT methylation with G > A mutations in KRAS and TP53 in 261 CRCs. Sixteen cases, with and without MGMT hypermethylation, were further analyzed by exome sequencing. No significant association of MGMT methylation with G > A mutations in KRAS, TP53 or in the whole exome was found (p > 0.5 in all comparisons). The result was validated by in silico comparison with 302 CRCs from The Cancer Genome Atlas (TCGA) consortium dataset. Transcriptional silencing associated with hypermethylation and stratified into monoallelic and biallelic. We also found a significant clustering (p = 0.001) of aberrant hypermethylation of MGMT and the matrix metalloproteinase gene ADAMTS14 in normal colonic mucosa of CRC patients. This suggested the existence of an epigenetic field defect for cancerization disrupting the methylation patterns of several loci, including MGMT or ADAMTS14, that may lead to predictive biomarkers for CRC. Methylation of these loci in normal mucosa was more frequent in elder (p = 0.001) patients, and particularly in African Americans (p = 1 × 10-5), thus providing a possible mechanistic link between somatic epigenetic alterations and CRC racial disparities in North America.

Genetics supersedes epigenetics in colon cancer phenotype.

A CpG island DNA methylator phenotype has been postulated to explain silencing of the hMLH1 DNA mismatch repair gene in cancer of the microsatellite mutator phenotype. To evaluate this model, we analyzed methylation in CpG islands from six mutator and suppressor genes, and thirty random genomic sites, in a panel of colorectal cancers. Tumor-specific somatic hypermethylation was a widespread age-dependent process that followed a normal Gaussian distribution. Because there was no discontinuity in methylation rate, our results challenge the methylator phenotype hypothesis and its hypothetical pathological underlying defect. We also show that the mutator phenotype dominates over the gradual accumulation of DNA hypermethylation in determining the genotypic features that govern the phenotypic peculiarities of colon cancer of the mutator pathway.

Low mutation incidence in polymorphic noncoding short mononucleotide repeats in gastrointestinal cancer of the microsatellite mutator phenotype pathway.

Frameshifts in short mononucleotide tracts (SMT) in genes, such as TGFbetaRII and BAX, are common in gastrointestinal tumors of the microsatellite mutator phenotype (MMP). The significance of less common mutations has been recently challenged because frequencies as high as 50% were reported in some noncoding SMTs in MMP colon cancer cell lines (L. Zhang, et al., Cancer Res., 61: 3801-3805, 2001). We did not confirm these findings after examining >50 MMP gastrointestinal cancers for mutations in eight SMT loci with the highest reported frequencies. In three of these loci, no clonal mutations were detected, and they were infrequent (2.9-6.7%) in the other five. Length polymorphisms are frequent (25.7-43.9%) in one-half of these SMTs, suggesting an explanation for the discrepancy. Because of the peculiar features of MMP tumors, low prevalence of mutations in cancer genes may not be a disqualifying criterion for their functionality.