The potential of new tumor endothelium-specific markers for the development of antivascular therapy.

Angiogenesis is a hallmark of solid tumors, and disruption of tumor vasculature is an active anticancer therapy in some cases. Several proteins expressed on the surface of tumor endothelium have been identified during the last decade. However, due to the expression in both physiological and tumor angiogenesis, only a few targets have been developed for clinical therapeutics. By thorough SAGE analysis of mouse endothelial cells isolated from various normal resting tissues, regenerating liver, and liver-metastasized tumor, Seaman and colleagues in this issue of Cancer Cell have demonstrated organ-specific endothelial markers, physiological angiogenesis endothelial markers, and tumor endothelial markers and revealed striking differences between physiological and pathological angiogenesis

How the vertebrates were made: selective pruning of a double-duplicated genome

Vertebrates are the result of an ancient double duplication of the genome. A new study published in BMC Biology explores the selective retention of genes after this event, finding an extensive enrichment of signaling proteins and transcription factors. Analysis of their expression patterns, interactions and subsequent history reflect the forces that drove their evolution, and with it the evolution of vertebrate complexity

2R and remodeling of vertebrate signal transduction engine

<p>Abstract</p> <p><b>Background</b></p> <p>Whole genome duplication (WGD) is a special case of gene duplication, observed rarely in animals, whereby all genes duplicate simultaneously through polyploidisation. Two rounds of WGD (2R-WGD) occurred at the base of vertebrates, giving rise to an enormous wave of genetic novelty, but a systematic analysis of functional consequences of this event has not yet been performed.</p> <p><b>Results</b></p> <p>We show that 2R-WGD affected an overwhelming majority (74%) of signalling genes, in particular developmental pathways involving receptor tyrosine kinases, Wnt and transforming growth factor-β ligands, G protein-coupled receptors and the apoptosis pathway. 2R-retained genes, in contrast to tandem duplicates, were enriched in protein interaction domains and multifunctional signalling modules of Ras and mitogen-activated protein kinase cascades. 2R-WGD had a fundamental impact on the cell-cycle machinery, redefined molecular building blocks of the neuronal synapse, and was formative for vertebrate brains. We investigated 2R-associated nodes in the context of the human signalling network, as well as in an inferred ancestral pre-2R (AP2R) network, and found that hubs (particularly involving negative regulation) were preferentially retained, with high connectivity driving retention. Finally, microarrays and proteomics demonstrated a trend for gradual paralog expression divergence independent of the duplication mechanism, but inferred ancestral expression states suggested preferential subfunctionalisation among 2R-ohnologs (2ROs).</p> <p><b>Conclusions</b></p> <p>The 2R event left an indelible imprint on vertebrate signalling and the cell cycle. We show that 2R-WGD preferentially retained genes are associated with higher organismal complexity (for example, locomotion, nervous system, morphogenesis), while genes associated with basic cellular functions (for example, translation, replication, splicing, recombination; with the notable exception of cell cycle) tended to be excluded. 2R-WGD set the stage for the emergence of key vertebrate functional novelties (such as complex brains, circulatory system, heart, bone, cartilage, musculature and adipose tissue). A full explanation of the impact of 2R on evolution, function and the flow of information in vertebrate signalling networks is likely to have practical consequences for regenerative medicine, stem cell therapies and cancer treatment.</p

Pseudo–Messenger RNA: Phantoms of the Transcriptome

The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo–messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo–messenger RNAs (approximately half of which are transposon-associated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level

The antiangiogenic agent ZD4190 prevents tumour outgrowth in a model of minimal residual carcinoma in deep tissues

BACKGROUND: Tumour cells may persist at the operative site after seemingly adequate surgery. Radiotherapy is often given in an attempt to prevent repopulation, but this modality cannot be relied upon to prevent locoregional recurrence. An alternative strategy is to take advantage of the requirement of tumour cells to develop an independent blood supply and block this process to prevent recurrence. METHODS: In this study, we evaluate the effect of the angiogenesis inhibitor, ZD4190, using a rodent model of residual carcinoma in deep tissues, mimicking the clinical scenario where low numbers of malignant cells persist at the operative site. RESULTS: The tumour burden that could be eliminated was dependent on the site where the cells were implanted. Immediate treatment with ZD4190 prevented outgrowth of up to 2.5 x 10(5) cells in the rectus muscle and 1 x 10(5) in the gastrocnemius, whereas control animals developed large tumours. When more than 2.5 x 10(6) cells were implanted into the rectus or 1 x 10(6) into the gastrocnemius and treatment was maintained for 3 weeks, the carcinomas that developed in ZD4190-treated animals showed a reduced microvessel density and increased necrosis when compared with the vehicle-treated controls, but an infiltrative growth pattern was common. CONCLUSION: These findings suggest that antiangiogenic agents have a role to play in preventing outgrowth of residual carcinoma and are likely to be most effective when the tumour burden is minimal

R-Smad Competition Controls Activin Receptor Output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, “BMP-specific” Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma

Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression

<p>Abstract</p> <p>Background</p> <p>SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of <it>SLIT-ROBO </it>genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified <it>SLIT-ROBO </it>transcripts in HCC cell lines, and in normal and tumor tissues from liver.</p> <p>Methods</p> <p>Expression of <it>SLIT-ROBO </it>family members was quantified by real-time qRT-PCR in 14 HCC cell lines, 8 normal and 35 tumor tissues from the liver. ANOVA and Pearson's correlation analyses were performed in R environment, and different clinicopathological subgroups were pairwise compared in Minitab. Gene expression matrices of cell lines and tissues were analyzed by Mantel's association test.</p> <p>Results</p> <p>Genewise hierarchical clustering revealed two subgroups with coordinate expression pattern in both the HCC cell lines and tissues: <it>ROBO1</it>, <it>ROBO2</it>, <it>SLIT1 </it>in one cluster, and <it>ROBO4</it>, <it>SLIT2</it>, <it>SLIT3 </it>in the other, respectively. Moreover, <it>SLIT-ROBO </it>expression predicted <it>AFP</it>-dependent subgrouping of HCC cell lines, but not that of liver tissues. <it>ROBO1 </it>and <it>ROBO2 </it>were significantly up-regulated, whereas <it>SLIT3 </it>was significantly down-regulated in cell lines with high-<it>AFP </it>background. When compared to normal liver tissue, <it>ROBO1 </it>was found to be significantly overexpressed, while <it>ROBO4 </it>was down-regulated in HCC. We also observed that <it>ROBO1 </it>and <it>SLIT2 </it>differentiated histopathological subgroups of liver tissues depending on both tumor staging and differentiation status. However, <it>ROBO4 </it>could discriminate poorly differentiated HCC from other subgroups.</p> <p>Conclusion</p> <p>The present study is the first in comprehensive and quantitative evaluation of <it>SLIT-ROBO </it>family gene expression in HCC, and suggests that the expression of <it>SLIT-ROBO </it>genes is regulated in hepatocarcinogenesis. Our results implicate that <it>SLIT-ROBO </it>transcription profile is bi-modular in nature, and that each module shows intrinsic variability. We also provide quantitative evidence for potential use of <it>ROBO1</it>, <it>ROBO4 </it>and <it>SLIT2 </it>for prediction of tumor stage and differentiation status.</p

Using Paleogenomics to Study the Evolution of Gene Families: Origin and Duplication History of the Relaxin Family Hormones and Their Receptors

Recent progress in the analysis of whole genome sequencing data has resulted in the emergence of paleogenomics, a field devoted to the reconstruction of ancestral genomes. Ancestral karyotype reconstructions have been used primarily to illustrate the dynamic nature of genome evolution. In this paper, we demonstrate how they can also be used to study individual gene families by examining the evolutionary history of relaxin hormones (RLN/INSL) and relaxin family peptide receptors (RXFP). Relaxin family hormones are members of the insulin superfamily, and are implicated in the regulation of a variety of primarily reproductive and neuroendocrine processes. Their receptors are G-protein coupled receptors (GPCR's) and include members of two distinct evolutionary groups, an unusual characteristic. Although several studies have tried to elucidate the origins of the relaxin peptide family, the evolutionary origin of their receptors and the mechanisms driving the diversification of the RLN/INSL-RXFP signaling systems in non-placental vertebrates has remained elusive. Here we show that the numerous vertebrate RLN/INSL and RXFP genes are products of an ancestral receptor-ligand system that originally consisted of three genes, two of which apparently trace their origins to invertebrates. Subsequently, diversification of the system was driven primarily by whole genome duplications (WGD, 2R and 3R) followed by almost complete retention of the ligand duplicates in most vertebrates but massive loss of receptor genes in tetrapods. Interestingly, the majority of 3R duplicates retained in teleosts are potentially involved in neuroendocrine regulation. Furthermore, we infer that the ancestral AncRxfp3/4 receptor may have been syntenically linked to the AncRln-like ligand in the pre-2R genome, and show that syntenic linkages among ligands and receptors have changed dynamically in different lineages. This study ultimately shows the broad utility, with some caveats, of incorporating paleogenomics data into understanding the evolution of gene families