Effect of Akt activation and experimental pharmacological inhibition on responses to neoadjuvant chemoradiotherapy in rectal cancer.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) is one of the preferred initial treatment strategies for locally advanced rectal cancer. Responses are variable, and most patients still require surgery. The aim of this study was to identify molecular mechanisms determining poor response to CRT. METHODS: Global gene expression and pathway enrichment were assessed in pretreatment biopsies from patients with non-metastatic cT2-4 N0-2 rectal cancer within 7 cm of the anal verge. Downstream Akt activation was assessed in an independent set of pretreatment biopsies and in colorectal cancer cell lines using immunohistochemistry and western blot respectively. The radiosensitizing effects of the Akt inhibitor MK2206 were assessed using clonogenic assays and xenografts in immunodeficient mice. RESULTS: A total of 350 differentially expressed genes were identified, of which 123 were upregulated and 199 downregulated in tumours from poor responders. Mitochondrial oxidative phosphorylation (P < 0·001) and phosphatidylinositol signalling pathways (P < 0·050) were identified as significantly enriched pathways among the set of differentially expressed genes. Deregulation of both pathways is known to result in Akt activation, and high immunoexpression of phosphorylated Akt S473 was observed among patients with a poor histological response (tumour regression grade 0-2) to CRT (75 per cent versus 48 per cent in those with a good or complete response; P = 0·016). Akt activation was also confirmed in the radioresistant cell line SW480, and a 50 per cent improvement in sensitivity to CRT was observed in vitro and in vivo when SW480 cells were exposed to the Akt inhibitor MK2206 in combination with radiation and 5-fluorouracil. CONCLUSION: Akt activation is a key event in the response to CRT. Pharmacological inhibition of Akt activation may enhance the effects of CRT. Surgical relevance Organ preservation is an attractive alternative in rectal cancer management following neoadjuvant chemoradiotherapy (CRT) to avoid the morbidity of radical surgery. Molecular steps associated with tumour response to CRT may provide a useful tool for the identification of patients who are candidates for no immediate surgery. In this study, tumours resistant to CRT were more likely to have activation of specific genetic pathways that result in phosphorylated Akt (pAkt) activation. Pretreatment biopsy tissues with high immunoexpression of pAkt were more likely to exhibit a poor histological response to CRT. In addition, the introduction of a pAkt inhibitor to cancer cell lines in vitro and in vivo led to a significant improvement in sensitivity to CRT. Identification of pAkt-activated tumours may thus allow the identification of poor responders to CRT. In addition, the concomitant use of pAkt inhibitors to increase sensitivity to CRT in patients with rectal cancer may constitute an interesting strategy for increasing the chance of a complete response to treatment and organ preservation.

Towards a global cancer knowledge network: dissecting the current international cancer genomic sequencing landscape.

BACKGROUND: While next generation sequencing has enhanced our understanding of the biological basis of malignancy, current knowledge on global practices for sequencing cancer samples is limited. To address this deficiency, we developed a survey to provide a snapshot of current sequencing activities globally, identify barriers to data sharing and use this information to develop sustainable solutions for the cancer research community. METHODS: A multi-item survey was conducted assessing demographics, clinical data collection, genomic platforms, privacy/ethics concerns, funding sources and data sharing barriers for sequencing initiatives globally. Additionally, respondents were asked as to provide the primary intent of their initiative (clinical diagnostic, research or combination). RESULTS: Of 107 initiatives invited to participate, 59 responded (response rate = 55%). Whole exome sequencing (P = 0.03) and whole genome sequencing (P = 0.01) were utilized less frequently in clinical diagnostic than in research initiatives. Procedures to identify cancer-specific variants were heterogeneous, with bioinformatics pipelines employing different mutation calling/variant annotation algorithms. Measurement of treatment efficacy varied amongst initiatives, with time on treatment (57%) and RECIST (53%) being the most common; however, other parameters were also employed. Whilst 72% of initiatives indicated data sharing, its scope varied, with a number of restrictions in place (e.g. transfer of raw data). The largest perceived barriers to data harmonization were the lack of financial support (P < 0.01) and bioinformatics concerns (e.g. lack of interoperability) (P = 0.02). Capturing clinical data was more likely to be perceived as a barrier to data sharing by larger initiatives than by smaller initiatives (P = 0.01). CONCLUSIONS: These results identify the main barriers, as perceived by the cancer sequencing community, to effective sharing of cancer genomic and clinical data. They highlight the need for greater harmonization of technical, ethical and data capture processes in cancer sample sequencing worldwide, in order to support effective and responsible data sharing for the benefit of patients.

A genetic cluster of patients with variant xeroderma pigmentosum with two different founder mutations.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). OBJECTIVES: To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. METHODS: Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. RESULTS: Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. CONCLUSIONS: This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe.

Intratumoral heterogeneity of ADAM23 promotes tumor growth and metastasis through LGI4 and nitric oxide signals.

Intratumoral heterogeneity (ITH) represents an obstacle for cancer diagnosis and treatment, but little is known about its functional role in cancer progression. The A Desintegrin And Metalloproteinase 23 (ADAM23) gene is epigenetically silenced in different types of tumors, and silencing is often associated with advanced disease and metastasis. Here, we show that invasive breast tumors exhibit significant ADAM23-ITH and that this heterogeneity is critical for tumor growth and metastasis. We demonstrate that while loss of ADAM23 expression enhances invasion, it causes a severe proliferative deficiency and is not itself sufficient to trigger metastasis. Rather, we observed that, in ADAM23-heterotypic environments, ADAM23-negative cells promote tumor growth and metastasis by enhancing the proliferation and invasion of adjacent A23-positive cells through the production of LGI4 (Leucine-rich Glioma Inactivated 4) and nitric oxide (NO). Ablation of LGI4 and NO in A23-negative cells significantly attenuates A23-positive cell proliferation and invasion. Our work denotes a driving role of ADAM23-ITH during disease progression, shifting the malignant phenotype from the cellular to the tissue level. Our findings also provide insights for therapeutic intervention, enforcing the need to ascertain ITH to improve cancer diagnosis and therapy.

Papel da proteína Mx1 na resposta a interferons e no processo neoplásico / Role of Mx1 protein in the response to interferons and neoplastic process

A proteína Mx1 é codificada por um gene induzido por interferon e compartilha a organização de seus domínios, a capacidade de homo-oligomerização e associação com membranas com as grandes dinaminas GTPases. A proteína Mx1 está envolvida na resposta contra um grande número de vírus de RNA, como aqueles pertencentes à família Buniavírus e o vírus influenza. Curiosamente, o gene MX1 foi encontrado como silenciado por metilação em diversos processos neoplásicos, incluindo carcinomas de cabeça e pescoço de células escamosas. Neste cenário, o silenciamento gênico de MX1 está associado à imortalização de uma série de linhagens celulares neoplásicas. Assim, Mx1 se destaca como uma das principais proteínas envolvidas nas respostas imunes induzidas por interferon e também desempenha um importante papel no controle do ciclo celular. Aqui discutimos os aspectos funcionais da proteína Mx1 abordando sua atividade antiviral, organização estrutural, envolvimento com neoplasias e, principalmente, os aspectos funcionais obtidos pela determinação de seus parceiros celulares.

The Mx1 protein is encoded by an interferon-induced gene and shares domain organization, homo-oligomerization capacity and membrane association with the large dynamin-like GTPases. The Mx1 protein is involved in the response to a large number of RNA viruses, such as the bunyavirus family and the influenza virus. Interestingly, it has also been found as a methylation-silenced gene in several types of neoplasm, including head and neck squamous cell carcinoma. In this scenario, MX1 gene silencing is associated with immortalization in several neoplastic cell lines. Thus, Mx1 stands out as one of the key proteins involved in interferon-induced immune response and also plays an important role in cell cycle control. Here we discuss some of the functions of the Mx1 protein, including its antiviral activity, protein folding and involvement in neoplasia, as well as those revealed by investigating its cellular partners.

Bioinformatics construction of the human cell surfaceome.

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40 per cent

Coordinated, network-based research as a strategic component of science in Brazil

Scientific research plays a fundamental role in the health and development of any society, since all technological advances depend ultimately on scientific discovery and the generation of wealth is intricately dependent on technological advance. Due to their importance, science and technology generally occupy important places in the hierarchical structure of developed societies, and they receive considerable public and private investment. Publicly funded science is almost entirely devoted to discovery, and it is administered and structured in a very similar way throughout the world. Particularly in the biological sciences, this structure, which is very much centered on the individual scientist and his own hypothesis-based investigations, may not be the best suited for either discovery in the context of complex biological systems, or for the efficient advancement of fundamental knowledge into practical utility. The adoption of other organizational paradigms, which permit a more coordinated and interactive research structure, may provide important opportunities to accelerate the scientific process and further enhance its relevance and contribution to society. The key alternative is a structure that incorporates larger organizational units to tackle larger and more complex problems. One example of such a unit is the research network. Brazil has utilized such networks to great effect in genome sequencing projects, demonstrating their relevance to the Brazilian research community and opening the possibility of their wider utility in the future

Morphological aspects of muscle regeneration in the Nile tilapia (Oreochromis niloticus).

The aim of our study was to analyze the morphological events in the skeletal muscle of the Nile tilapia (Oreochromis niloticus) after a traumatic lesion. Thirty-two fish were used, on which a small longitudinal incision was made in the muscle. The fish were sacrificed after 7, 14, 21, and 42 days and muscle samples were collected from the lesion and processed for morphological analysis. Muscle regeneration in the tilapia occurred gradually through the analyzed period, possibly due to the proliferation and differentiation of myosatellite cells, which were more morphologically evident 7 and 14 days after lesion.

An international database and integrated analysis tools for the study of cancer gene expression.

Researchers working collaboratively in Brazil and the United States have assembled an International Database of Cancer Gene Expression. Several strategies have been employed to generate gene expression data including expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and open reading-frame expressed sequence tags (ORESTES). The database contains six million gene tags that reflect the gene expression profiles in a wide variety of cancerous tissues and their normal counterparts. All sequences are deposited in the public databases, GenBank and SAGEmap. A suite of informatics tools was designed to facilitate in silico analysis of the gene expression datasets and are available through the NCI Cancer Genome Anatomy Project web site (http://cgap.nci.nih.gov).

Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12.

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed

Collagen XVIII, containing an endogenous inhibitor of angiogenesis and tumor growth, plays a critical role in the maintenance of retinal structure and in neural tube closure (Knobloch syndrome).

Knobloch syndrome (KS) is an autosomal recessive disorder defined by the occurrence of high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. The KS causative gene had been assigned to a 4.3 cM interval at 21q22.3 by linkage analysis of a large consanguineous Brazilian family. We reconstructed the haplotypes of this family with ten additional markers (five were novel) and narrowed the candidate interval to a region of <245 kb, which contains 24 expressed sequence tags, the KIAA0958 gene and the 5' end of the COL18A1 gene. We identified a homozygous mutation at the AG consensus acceptor splice site of COL18A1 intron 1 exclusively among the 12 KS patients, which was not found among 140 control chromosomes. This mutation predicts the creation of a stop codon in exon 4 and therefore the truncation of the alpha1(XVIII) collagen short form, which was expressed in human adult retina. These findings provide evidence that KS is caused by mutations in COL18A1 which, therefore, has a major role in determining the retinal structure as well as in the closure of the neural tube. Therefore, we show for the first time that the absence of a collagen isoform impairs embryonic cell proliferation and/or migration as a primary or secondary effect.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Evolution and the inevitability of human cancer.

Natural selection, which is absolutely dependent on genetic differences between individuals, is the process by which life has evolved on this planet. Genetic variability is ultimately depended on the occurrence of new mutations in the germ-line of species. The rate at which this occurs appears not to be arbitrary or dependent on chance external events. Rather the available evidence suggests that it is highly controlled and determined by endogenous processes. However, the body does not have separate mechanisms for controlling mutation frequency in the germinal and somatic lineages and the selective process described inevitably has also led to somatic cells being subject to mutation accumulation. Indeed, since mutation frequency increases exponentially with time, the human somatic mutation frequency at approximately 80 years of age in epithelial tissues appears to be more than 10-fold higher than in the human germline. This normal but highly elevated somatic mutation frequency is sufficient to account for the complex multi-step process of human tumorigenesis even in the absence of the effects of major external mutagens or rare transitions to even more elevated mutation frequencies. Thus, scrutiny of the apparently disparate biological phenomena of evolution and tumorigenesis leads to the postulate that they are in fact two interdependent manifestations of the same underlying process and that given an evolutionary process dependent on mutation accumulation then cancer in long lived organisms is an inevitable consequence.