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.

Objective To construct a stringently controlled expression system for exogenous gene of interest in eukaryotic cells. Methods A new expression system was established by fusing tetracycline-inducible elements,bacterial artificial chromosome(BAC)and Gateway technology and then tested. Results The fusion protein β-catenin-ERα encoded by gene of interest was tested by luciferase assay and Western blot,and its expression was tightly controlled by tetracycline or deoxycycline.In the final expression vector BAC E11-IGR-β-catenin-ERα,the expression of EGFP was stable,which could be an effective marker for FACS selection. Conclusion This system could be used as an efficient tool for the expression of exogenous gene of interest in eukaryotic cells in a stringently-controlled manner.

Objective To explore the relationship of erythropoietin-producing hepatocyte B6(EPHB6) receptor tyrosine kinase expression with the distant metastasis of cancer cell in early stage non-small cell lung cancer(NSCLC) patients,and the mechanism of inactivation of EPHB6 expression.Methods The expression levels of EPHB6 were compared among normal lung tissue(n=9),NSCLC without metastasis(n=39),and NSCLC with metastasis(n=39).EPHB6 expression levels in matched tumor-normal pairs from 24 NSCLC patients were analyzed.Correlation of EPHB6 methylation levels with EPHB6 mRNA expression was analyzed.Results Compared with matched normal lung tissue,mRNA and protein expression levels of EPHB6 were both significantly reduced in NSCLC.Decreased EPHB6 expression level was associated with an increased risk of metastasis in NSCLC patients.Loss of EPHB6 expression was correlated with hypermethylation.EPHB6 expression was induced by 5-Aza-2′-deoxycytidine treatment in a NSCLC cell line.Conclusion The results demonstrate that EPHB6 is frequently silenced by hypermethylation of its promoter in NSCLC.

Authors' Oncology a 2Center of Fourth Mili of Viscera 4Departme

Purpose: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non–small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. Experimental Design: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. Results: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. Conclusions: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC. Clin Cancer Res; 16(8); 2275–83. ©2010 AACR.

Network architecture is a brain-organizational motif present across spatial scales from cell assemblies to distributed systems. Structural pathology in some neurodegenerative disorders selectively afflicts a subset of functional networks, motivating the network degeneration hypothesis (NDH). Recent evidence suggests that structural pathology recapitulating physiology may be a general property of neuropsychiatric disorders. To test this possibility, we compared functional and structural network meta-analyses drawing upon the BrainMap database. The functional meta-analysis included results from >7,000 experiments of subjects performing >100 task paradigms; the structural meta-analysis included >2,000 experiments of patients with >40 brain disorders. Structure-function network concordance was high: 68% of networks matched (pFWE<0.01), confirming the broader scope of NDH. This correspondence persisted across higher model orders. A positive linear association between disease and behavioral entropy (p=0.0006;R-2=0.53) suggests nodal stress as a common mechanism. Corroborating this interpretation with independent data, we show that metabolic 'cost' significantly differs along this transdiagnostic/multimodal gradient. Peter Fox & Thomas Vanasse et al. compare atrophy patterns in 40+ brain disorders with healthy task-activation networks across 100+ behavioral domains. Their analyses demonstrate strong spatial correspondence between disease-related structural coalterations and functional networks, an observation that greatly extends the scope of the network-degeneration hypothesis. Further, the metabolic cost of network hubs (nodal stress) is implicated as a common mechanism across a wide range of brain disorders.

Decreased expression levels of EPHB6, a member of the receptor tyrosine kinases (RTKs), are associated with an increased risk of metastasis development in early stage non-small cell lung cancer (NSCLC). However, the signaling properties of the kinase-defective EPHB6 receptor are not well-understood. Here, we show that expression of EPHB6 in A549 lung adenocarcinoma cells led to phosphorylation of the MAP kinase ERK. Conversely, siRNA based knockdown of EPHB6 reversed ERK phosphorylation. Intriguingly, EPHB6-induced phosphorylation of ERK was uncoupled by activation of the Elk-1 transcriptional factor. These analyses suggest that kinase defective EPHB6 can lead to MAPK activation.

Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non-small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC.Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2.Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control sh RNA (P = 0.021).Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Objective:To observe the mitochondria gene expression in human liver cancer cell line SMMC-7721.Methods: Four pairs of primers for multiple PCR were designed according to the sequence of human mtDNA with primer premier 5.0 bio-soft.The primers were then used to amplify D-loop region and the gene ND6,ATPase8-ATPase6 and 16sRNA.The expression of D-loop region and the 3 genes of mtDNA in SMMC-7721 was measured by RT-PCR,and the results were compared with those in normal human liver cells(L0_(2)).Results: Compared with those in L0_(2)cells,the expression of the D-loop region,ND6,ATPase8-ATPase6 and 16sRNA subunits in SMMC-7721 cells was increased,and the expression of 16sRNA subunit remained unchanged.Meanwhile, there was difference in the transcription of H-strand and L-strand of a certain gene.Conclusion: The mtDNA-encoded polypeptides are enzyme complex subunits of the oxidative phosphorylation compound,and their aberrant expression harms the oxidative phosphorylation capacity of cells and leads to decreased synthesis of ATP,which may be one of the reasons for glycolysis of SMMC-7721 cells.