Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Identification of lncRNA-associated differential subnetworks in oesophageal squamous cell carcinoma by differential co-expression analysis.

Differential expression analysis has led to the identification of important biomarkers in oesophageal squamous cell carcinoma (ESCC). Despite enormous contributions, it has not harnessed the full potential of gene expression data, such as interactions among genes. Differential co-expression analysis has emerged as an effective tool that complements differential expression analysis to provide better insight of dysregulated mechanisms and indicate key driver genes. Here, we analysed the differential co-expression of lncRNAs and protein-coding genes (PCGs) between normal oesophageal tissue and ESCC tissues, and constructed a lncRNA-PCG differential co-expression network (DCN). DCN was characterized as a scale-free, small-world network with modular organization. Focusing on lncRNAs, a total of 107 differential lncRNA-PCG subnetworks were identified from the DCN by integrating both differential expression and differential co-expression. These differential subnetworks provide a valuable source for revealing lncRNA functions and the associated dysfunctional regulatory networks in ESCC. Their consistent discrimination suggests that they may have important roles in ESCC and could serve as robust subnetwork biomarkers. In addition, two tumour suppressor genes (AL121899.1 and ELMO2), identified in the core modules, were validated by functional experiments. The proposed method can be easily used to investigate differential subnetworks of other molecules in other cancers.

Identifying anticancer peptides by using a generalized chaos game representation.

We generalize chaos game representation (CGR) to higher dimensional spaces while maintaining its bijection, keeping such method sufficiently representative and mathematically rigorous compare to previous attempts. We first state and prove the asymptotic property of CGR and our generalized chaos game representation (GCGR) method. The prediction follows that the dissimilarity of sequences which possess identical subsequences but distinct positions would be lowered exponentially by the length of the identical subsequence; this effect was taking place unbeknownst to researchers. By shining a spotlight on it now, we show the effect fundamentally supports (G)CGR as a similarity measure or feature extraction technique. We develop two feature extraction techniques: GCGR-Centroid and GCGR-Variance. We use the GCGR-Centroid to analyze the similarity between protein sequences by using the datasets 9 ND5, 24 TF and 50 beta-globin proteins. We obtain consistent results compared with previous studies which proves the significance thereof. Finally, by utilizing support vector machines, we train the anticancer peptide prediction model by using both GCGR-Centroid and GCGR-Variance, and achieve a significantly higher prediction performance by employing the 3 well-studied anticancer peptide datasets.

Evolutionary and developmental analysis reveals KANK genes were co-opted for vertebrate vascular development.

Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.

Stressing out over long noncoding RNA.

Genomic studies have revealed that humans possess far fewer protein-encoding genes than originally predicted. These over-estimates were drawn from the inherent developmental and stimuli-responsive complexity found in humans and other mammals, when compared to lower eukaryotic organisms. This left a conceptual void in many cellular networks, as a new class of functional molecules was necessary for "fine-tuning" the basic proteomic machinery. Transcriptomics analyses have determined that the vast majority of the genetic material is transcribed as noncoding RNA, suggesting that these molecules could provide the functional diversity initially sought from proteins. Indeed, as discussed in this review, long noncoding RNAs (lncRNAs), the largest family of noncoding transcripts, have emerged as common regulators of many cellular stressors; including heat shock, metabolic deprivation and DNA damage. These stimuli, while divergent in nature, share some common stress-responsive pathways, notably inhibition of cell proliferation. This role intrinsically makes stress-responsive lncRNA regulators potential tumor suppressor or proto-oncogenic genes. As the list of functional RNA molecules continues to rapidly expand it is becoming increasingly clear that the significance and functionality of this family may someday rival that of proteins. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Methylation biomarkers for pleomorphic lobular breast cancer - a short report.

BACKGROUND: Pleomorphic invasive lobular cancer (pleomorphic ILC) is a rare variant of ILC that is characterized by a classic ILC-like growth pattern combined with an infiltrative ductal cancer (IDC)-like high nuclear atypicality. There is an ongoing discussion whether pleomorphic ILC is a dedifferentiated form of ILC or in origin an IDC with a secondary loss of cohesion. Since gene promoter hypermethylation is an early event in breast carcinogenesis and thus may provide information on tumor progression, we set out to compare the methylation patterns of pleomorphic ILC, classic ILC and IDC. In addition, we aimed at analyzing the methylation status of pleomorphic ILC. METHODS: We performed promoter methylation profiling of 24 established and putative tumor suppressor genes by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis in 20 classical ILC, 16 pleomorphic ILC and 20 IDC cases. RESULTS: We found that pleomorphic ILC showed relatively low TP73 and MLH1 methylation levels and relatively high RASSF1A methylation levels compared to classic ILC. Compared to IDC, pleomorphic ILC showed relatively low MLH1 and BRCA1 methylation levels. Hierarchical cluster analysis revealed a similar methylation pattern for pleomorphic ILC and IDC, while the methylation pattern of classic ILC was different. CONCLUSION: This is the first report to identify TP73, RASSF1A, MLH1 and BRCA1 as possible biomarkers to distinguish pleomorphic ILC from classic ILC and IDC.

Focal Cortical Dysplasia.

Focal cortical dysplasias are common malformations of cerebral cortical development and are highly associated with medically intractable epilepsy. They have been classified into neuropathological subtypes (type Ia, Ib, IIa, IIb, and III) based on the severity of cytoarchitectural disruption--tangential or radial dispersion, or loss of laminar structure--and the presence of unique cells types such as cytomegalic neurons or balloon cells. Most focal cortical dysplasias can be identified on neuroimaging and many require resective epilepsy surgery to cure refractory seizures. The pathogenesis of focal cortical dysplasias remains to be defined, although there is recent evidence to suggest that focal cortical dysplasias arise from de novo somatic mutations occurring during brain development. Some focal cortical dysplasia subtypes show a link to the mammalian target of rapamycin signaling cascade; this has now extended to other cortical malformations, including hemimegalencephaly.

Dependence receptors and colorectal cancer.

The research on colorectal cancer (CRC) biology has been leading the oncology field since the early 1990s. The search for genetic alterations has allowed the identification of the main tumour suppressors or oncogenes. Recent work obtained in CRC has unexpectedly proposed the existence of novel category of tumour suppressors, the so-called 'dependence receptors'. These transmembrane receptors behave as Dr Jekyll and Mr Hyde with two opposite sides: they induce a positive signalling (survival, proliferation, differentiation) in presence of their ligand, but are not inactive in the absence of their ligand and rather trigger apoptosis when unbound. This trait confers them a conditional tumour suppressor activity: they eliminate cells that grow abnormally in an environment offering a limited quantity of ligand. This review will describe how receptors such as deleted in colorectal carcinoma (DCC), uncoordinated 5 (UNC5), rearranged during transfection (RET) or TrkC constrain CRC progression and how this dependence receptor paradigm may open up therapeutical perspectives.

Evidence for the coevolution of axon guidance molecule Netrin and its receptor Frazzled.

Coevolution of a ligand and its receptor is critical for maintaining their function in different species, but how ligand and its receptor coevolve is poorly understood. The axon guidance molecule Netrin and its receptor Frazzled (Fra) are useful to study the mechanisms of ligand-receptor coevolution. Here, we have applied codon substitution models to identify positive selection of the netrin and fra genes. The sites under positive selection in netrin and fra were detected in same lineage, such as nematode, dipteran, hymenopteran, hemichordate, and teleost. Several amino acid residues that are under positive selection were identified in the interaction domains. Here we provide evidence that positive selection is essential for the coevolution of Netrin and Fra during central nervous system evolution.

ATM activation in the presence of oxidative stress.

The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H2O2. The active ATM in this case is a disulfide-crosslinked dimer containing 2 or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSBs is inhibited in the presence of H2O2 because oxidation blocks the ability of MRN to bind to DNA. However, ATM activation via direct oxidation by H2O2 complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSBs and oxidative stress.

Ancient origin of animal U-box ubiquitin ligases.

BACKGROUND: The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described. RESULTS: Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans (UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4) were already present in the ancestor of all current metazoans and the seventh (WDSUB1) is found in placozoans, cnidarians and bilaterians. The fact that only 4-5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families (RBR, HECT) of ubiquitin ligases. CONCLUSIONS: Finding that the patterns of emergence, diversification and loss of three unrelated families of ubiquitin ligases (RBR, HECT and U-box) are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system.

The N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation.

The RASSF (Ras-association domain family) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. The present review will summarize what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins with the classical RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members or imposters in the RASSF family.

The netrin protein family.

The name netrin is derived from the Sanskrit Netr, meaning 'guide'. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down's syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.

[Structure and functions of isoforms of polyfunctional tumoral suppressor PML].

Polyfunctional protein PML contributes significantly in vital activity of cell. 11 isoforms of PML differ from one another by C-terminal domain. In spite of intensive research into the protein, the role of the isoforms in cellular processes remains obscure. In addition, the literature contains various names of the isoforms. The goal of this work was to review the structure of the PML gene, variants of alternative splicing of mPNA, and domain organization of corresponding protein forms. The PML isoforms were classified and functional specificity of each PML isoform was characterized: contribution to gene transcription, contribution to cell apoptosis, cell growth, immune response, formation of nuclear bodies.

Reviewing the current classification of inhibitor of growth family proteins.

Inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors for more than a decade. Recent emerging results using siRNA and knockout mice are expanding the previous understanding of this protein family. The results of ING1 knockout mouse experiments revealed that ING1 has a protective effect on apoptosis. Our recent results showed that ING2 is overexpressed in colorectal cancer, and induces colon cancer cell invasion through an MMP13-dependent pathway. Knockdown of ING2 by siRNA induces premature senescence in normal human fibroblast cells, and apoptosis or cell cycle arrest in various adherent cancer cells. Taken together, these results suggest that ING2 may also have roles in cancer progression and/or malignant transformation under some conditions. Additionally, knockdown of ING4 and ING5 by siRNA shows an inhibitory effect on the transition from G(2)/M to G(1) phase and DNA replication, respectively, suggesting that these proteins may play roles during cell proliferation in some context. ING family proteins may play dual roles, similar to transforming growth factor-beta, which has tumor suppressor-like functions in normal epithelium and also oncogenic functions in invasive metastatic cancers. In the present article, we briefly review ING history and propose a possible interpretation of discrepancies between past and recent data.

The ING gene family in the regulation of cell growth and tumorigenesis.

The five members of the inhibitor of growth (ING) gene family have garnered significant interest due to their putative roles as tumor suppressors. However, the precise role(s) of these ING proteins in regulating cell growth and tumorigenesis remains uncertain. Biochemical and molecular biological analysis has revealed that all ING members encode a PHD finger motif proposed to bind methylated histones and phosphoinosital, and all ING proteins have been found as components of large chromatin remodeling complexes that also include histone acetyl transferase (HAT) and histone deacetylase (HDAC) enzymes, suggesting a role for ING proteins in regulating gene transcription. Additionally, the results of forced overexpression studies performed in tissue culture have indicated that several of the ING proteins can interact with the p53 tumor suppressor protein and/or the nuclear factor-kappa B (NF-kappaB) protein complex. As these ING-associated proteins play well-established roles in numerous cell processes, including DNA repair, cell growth and survival, inflammation, and tumor suppression, several models have been proposed that ING proteins act as key regulators of cell growth not only through their ability to modify gene transcription but also through their ability to alter p53 and NF-kappaB activity. However, these models have yet to be substantiated by in vivo experimentation. This review summarizes what is currently known about the biological functions of the five ING genes based upon in vitro experiments and recent mouse modeling efforts, and will highlight the potential impact of INGs on the development of cancer.

Biochemical and functional evidence of p53 homology is inconsistent with molecular phylogenetics for distant sequences.

The tumor suppressor p53 is mutated in approximately 50% of all human cancer cases worldwide. It is commonly assumed that the phylogenetic history of this important tumor suppressor has been thoroughly studied; however, few detailed studies of the entire extended p53 protein family have been reported, and none comprehensively and simultaneously consider functional, molecular, and phylogenetic data. Herein we examine a diverse collection of reported p53-like protein sequences, including representatives from the arthropods, nematodes, and protists, with the goal of answering several important questions. First, what evidence supports these highly divergent proteins being true homologues to the p53 family? Second, is the inferred overall family phylogeny concordant with known structures and functions? Third, does the extended p53 family possess recognizable conserved sites outside of the within-chordate, highly-conserved DNA-binding domain? Our study shows that the biochemical and functional evidence of p53 homology for nematodes, arthropods, and protists is inconsistent with their implied phylogenetic relationship within the overall family. Although these divergent sequences are always reported as functionally similar to human p53, our results confirm and extend the hypothesis that p63 is a far more appropriate protein for comparison. Within these divergent sequences, we find minimal conservation within the DNA-binding domain, and no conservation elsewhere. Taken together, our findings suggest that these sequences are not bona fide homologues of the extended p53 family and provide baseline criteria for the future identification and characterization of distant p53-family homologues.

The Crohn's disease susceptibility gene DLG5 as a member of the CARD interaction network.

Discs large homolog 5 (DLG5), a member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, has been associated with Crohn's disease (CD), but its role in the pathogenesis of this inflammatory bowel disease is disputed. Here, we used sequence comparisons and phylogenies to analyse the DLG5 gene and its protein product. We identified a 5' exon, which codes for an N-terminal caspase recruitment domain (CARD) and experimentally confirmed its expression in colonic tissue. DLG5 shares this new domain with nucleotide-binding oligomerisation domain containing 2 (NOD2); the first CD susceptibility factor identified in genetic studies. An extensive phylogenetic analysis redefines the family organisation of the MAGUK proteins: DLG5 is closely related to CARD10, CARD11 and CARD14, CARD-containing proteins which initiate pro-inflammatory NFkappaB signalling, but not to DLG1-4, previously considered the closest related proteins. Therefore, we suggest renaming DLG5 to correctly annotate the gene in its phylogenetic and functional context. Our study provides evidence that the scaffolding protein DLG5 belongs to the CARD protein family. Thus, DLG5 likely acts in the regulation of NFkB activation or caspase activation as part of host defence mechanisms. As there is substantial crosstalk between CARD-mediated pathways, both CD susceptibility genes, NOD2 and DLG5, may interact functionally to contribute to CD risk.

The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module.

The tumor suppressor CYLD antagonizes NF-kappaB and JNK signaling by disassembly of Lys63-linked ubiquitin chains synthesized in response to cytokine stimulation. Here we describe the crystal structure of the CYLD USP domain, revealing a distinctive architecture that provides molecular insights into its specificity toward Lys63-linked polyubiquitin. We identify regions of the USP domain responsible for this specificity and demonstrate endodeubiquitinase activity toward such chains. Pathogenic truncations of the CYLD C terminus, associated with the hypertrophic skin tumor cylindromatosis, disrupt the USP domain, accounting for loss of CYLD catalytic activity. A small zinc-binding B box domain, similar in structure to other crossbrace Zn-binding folds--including the RING domain found in E3 ubiquitin ligases--is inserted within the globular core of the USP domain. Biochemical and functional characterization of the B box suggests a role as a protein-interaction module that contributes to determining the subcellular localization of CYLD.

Crystal structure of human ribosomal protein L10 core domain reveals eukaryote-specific motifs in addition to the conserved fold.

A phylogenetically conserved ribosomal protein L16p/L10e organizes the architecture of the aminoacyl tRNA binding site on the large ribosomal subunit. Eukaryotic L10 also exhibits a variety of cellular activities, and, in particular, human L10 is known as a putative tumor suppressor, QM. We have determined the 2.5-A crystal structure of the human L10 core domain that corresponds to residues 34-182 of the full-length 214 amino acids. Its two-layered alpha+beta architecture is significantly similar to those of the archaeal and bacterial homologues, substantiating a high degree of structural conservation across the three phylogenetic domains. A cation-binding pocket formed between alpha2 and beta 6 is similar to that of the archaeal L10 protein but appears to be better ordered. Previously reported L10 mutations that cause defects in the yeast ribosome are clustered around this pocket, indicating that its integrity is crucial for its role in L10 function. Characteristic interactions among Arg90-Trp171-Arg139 guide the C-terminal part outside of the central fold, implying that the eukaryote-specific C-terminal extension localizes on the outer side of the ribosome.