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.

Heteroduplex formation and S1 digestion for mapping alternative splicing sites.

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

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.

PCR-assisted contig extension: stepwise strategy for bacterial genome closure.

Finishing is rate limiting for genome projects, and improvements in the efficiency of complete genome-sequence compilation will require improved protocols for gap closure. Here we report a novel approach for extending shotgun contigs and closing gaps that we termed PCR-assisted contig extension (PACE). PACE depends on the capture of rare mismatched interactions that occur between arbitrary primers and template DNA of unknown sequence, even under highly stringent conditions, by means of elevated PCR-cycle repetition and the use of specific anchoring primers corresponding to adjacent regions of known sequence. Using PACE, we have generated extensions with an average of 1 kb from all contigs generated from the shotgun sequencing of a 5-Mb genome, which closed the majority of gaps with a single round of experimentation. This included the generation of multiple extensions for contigs that terminated in one of the eight copies of the rRNA operon. We calculate that the switch from shotgun sequencing to PACE should occur between 5- and 8-fold genome coverage for maximum benefit and minimum overall cost. PACE is a robust and straightforward strategy that should simplify the finishing phase of bacterial genome projects.

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).

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

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

Directed gap closure in large-scale sequencing projects.

A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. By use of the Xylella fastidiosa genome as a test system, we present here an approach to generate, in a directed manner, sequence information from those gaps. We suggest using the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridization procedure similar to representational difference analysis (RDA). The resulting sequence information can be used to screen selectively other clone resources or serve directly for gap closure.

Definition of the gene content of the human genome: the need for deep experimental verification.

Based on the analysis of the drafts of the human genome sequence, it is being speculated that our species may possess an unexpectedly low number of genes. The quality of the drafts, the impossibility of accurate gene prediction and the lack of sufficient transcript sequence data, however, render such speculations very premature. The complexity of human gene structure requires additional and extensive experimental verification of transcripts that may result in major revisions of these early estimates of the number of human genes.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

Mapping analysis of the Xylella fastidiosa genome.

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.