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Introduction: Surgical procedure is the treatment of choice in early stage I lung adenocarcinoma. However, a considerable number of patients experience recurrence within the first 2 years after complete resection. Suitable prognostic biomarkers that identify patients at high risk of recurrence (who may probably benefit from adjuvant treatment) are still not available. This study aimed at identifying methylation markers for early recurrence that may become important tools for the development of new treatment modalities. Methods: Genome-wide DNA methylation profiling was performed on 30 stage I lung adenocarcinomas, comparing 14 patients with early metastatic recurrence with 16 patients with a long-term relapse-free survival period using methylated-CpG-immunoprecipitation followed by high-throughput next-generation sequencing. The differentially methylated regions between the two subgroups were validated for their prognostic value in two independent cohorts using the MassCLEAVE assay, a high-resolution quantitative methylation analysis. Results: Unsupervised clustering of patients in the discovery cohort on the basis of differentially methylated regions identified patients with shorter relapse-free survival (hazard ratio: 2.23; 95% confidence interval: 0.66-7.53; p = 0.03). In two validation cohorts, promoter hypermethylation of the long noncoding RNA PLUT was significantly associated with shorter relapse-free survival (hazard ratio: 0.54; 95% confidence interval: 0.31-0.93; p < 0.026) and could be reported as an independent prognostic factor in the multivariate Cox regression analysis. Conclusions: Promoter hypermethylation of the long noncoding RNA PLUT is predictive in patients with early stage I adenocarcinoma at high risk for early recurrence. Further studies are needed to validate its role in carcinogenesis and its use as a biomarker to facilitate patient selection and risk stratification.

Current diagnosis of renal cancer consists of histopathologic examination of tissue sections and classification into tumor stages and grades of malignancy. Until recently, molecular differences between tumor types were largely unknown. To examine such differences, we did gene expression measurements of 112 renal cell carcinoma and normal kidney samples on renal cell carcinoma–specific cDNA microarrays containing 4,207 genes and expressed sequence tags. The gene expression patterns showed deregulation of complete biological pathways in the tumors. Many of the molecular changes corresponded well to the histopathologic tumor types, and a set of 80 genes was sufficient to classify tumors with a very low error rate. Distinct gene expression signatures were associated with chromosomal abnormalities of tumor cells, metastasis formation, and patient survival. The data highlight the benefit of microarrays to detect novel tumor classes and to identify genes that are associated with patient variables and tumor properties.

OBJECTIVES: The therapeutic scheme for non-small cell lung cancer (NSCLC) patients can be improved if adapted to the individual response. For example, 60-70% of adenocarcinoma patients show response to EGFR-tyrosine kinase inhibitors in the presence of mutated EGFR. We searched for additional target molecules involved in the action of the EGFR-tyrosine kinase inhibitor erlotinib in the absence of EGFR mutations, which might be suitable for combinatorial therapy approaches. MATERIALS AND METHODS: Erlotinib-response associated proteins were investigated in patient-derived NSCLC mouse xenografts by reverse-phase protein array technology (RPPA) and Western blotting. A combinatorial treatment approach was carried out in NSCLC cell lines and H1299 mouse xenografts, and subsequently analyzed for consequences in cell growth and signal transduction. RESULTS: AMP-activated protein kinase (AMPK) expression was increased in erlotinib responders before and after treatment. In a combinatorial approach, activation of AMPK by A-769662 and erlotinib treatment showed a synergistic effect in cell growth reduction and apoptosis activation in H1299 cells compared to the single drugs. AMPK pathway analyses revealed an effective inhibition of mTOR signaling by drug combination. In H1299 xenografts, the tumor size was significantly decreased after combinatorial treatment. CONCLUSION: Our results suggest that AMPK activation status affects response to erlotinib in distinct lung tumor models.

Zielsetzung: Pulmonale Adenokarzinome der neu definierten Subtypen: lepidisch, azinär, papillär, mikropapillär und solide weisen charakteristische Wachstumseigenschaften und damit auch unterschiedliche klinische Überlebensprognosen auf. Bei Patienten mit lepidischen Adenokarzinomen[for full text, please go to the a.m. URL], Gemeinsame Jahrestagung der Deutschen, Österreichischen und Schweizerischen Gesellschaft für Thoraxchirurgie

Semaphorins and their receptors, plexins, have emerged as key regulators of various aspects of neuronal development. In contrast to the Plexin-A family, the cellular functions of Plexin-B family proteins in developing neurons are only poorly understood. An activation of Plexin-B1 via its ligand, semaphorin 4D (Sema4D), produces an acute collapse of axonal growth cones in hippocampal and retinal neurons over the early stages of neurite outgrowth. However, the functional role of Sema4D-Plexin-B interactions over subsequent stages of neurite development, differentiation and maturation has not been characterized. Here we addressed this question using morphogenetic assays and time-lapse imaging on developing rat hippocampal neurons as a model system. Interestingly, Sema4D treatment over several hours was observed to promote branching and complexity in hippocampal neurons via the activation of Plexin-B1. The activation of receptor tyrosine kinases and the Rho kinase following Sema4D treatment was found to control dendritic and axonal morphogenesis by differentially regulating branching and extension. Phosphoinositide-3-kinase, but not extracellular signal-regulated kinase 1/2, was observed to be important for the stimulatory effects of Sema4D on dendritic branching. Furthermore, we observed that the mammalian target of rapamycin is activated downstream of Plexin-B1 and contributes to Sema4D-induced effects on dendritic branching. In contrast, glycogen synthase kinase-3 beta, another effector of phosphoinositide-3-kinase signalling, was not involved. Thus, our results show that Sema4D-Plexin-B interactions modulate dendritic and axonal arborizations of developing neurons by co-ordinated and concerted activation of diverse signalling pathways.

Functional gamma-aminobutyric acid(B) (GABA(B)) receptors assemble from two subunits, GABA(B(1)) and GABA(B(2).) This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABA(B(1)) and GABA(B(2)) for G-protein coupling. We generated a series of C-terminal mutants of GABA(B(1)) and GABA(B(2)) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABA(B(1)) functioned only in the context of specific amino acids, which constitute part of the GABA(B(1)) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABA(B(2).) In HEK293 cells, we observed that the two well-known GABA(B) receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABA(B(1)) and GABA(B(2)) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABA(B) receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABA(B) receptor may determine its constitutive activity, which could be a therapeutic target for inverse agonists.