Wataru Fujibuchi

NCBI GEO: mining millions of expression profiles—database and tools

The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest fully public repository for high-throughput molecular abundance data, primarily gene expression data. The database has a flexible and open design that allows the submission, storage and retrieval of many data types. These data include microarray-based experiments measuring the abundance of mRNA, genomic DNA and protein molecules, as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology. GEO currently holds over 30 000 submissions representing approximately half a billion individual molecular abundance measurements, for over 100 organisms. Here, we describe recent database developments that facilitate effective mining and visualization of these data. Features are provided to examine data from both experiment- and gene-centric perspectives using user-friendly Web-based interfaces accessible to those without computational or microarray-related analytical expertise. The GEO database is publicly accessible through the World Wide Web at http://www.ncbi.nlm.nih.gov/geo.

Computation with the KEGG pathway database

We introduce and discuss a new computational approach towards prediction and inference of biological functions from genomic sequences by making use of the pathway data in KEGG. Due to its piecewise nature, the current approach of predicting each gene function based on sequence similarity searches often fails to reconstruct cellular functions with all necessary components. The pathway diagram in KEGG, which may be considered a wiring diagram of molecules in biological systems, can be utilised as a reference for functional reconstruction. KEGG also contains binary relations that represent molecular interactions and relations and that can be utilised for computing and comparing pathways.

MiR-126 acts as a tumor suppressor in pancreatic cancer cells via the regulation of ADAM9.

The epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry of metastatic disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entire picture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targeting multiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductal adenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, and human pancreatic cancer cell line to elucidate essential miRNAs which regulate invasive growth of pancreatic cancer cells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reduced expression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to target ADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreatic cancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 3′ untranslated region assay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted in reduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first time that the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells. Mol Cancer Res; 10(1); 3–10. ©2011 AACR.

Network-based de-noising improves prediction from microarray data

BackgroundPrediction of human cell response to anti-cancer drugs (compounds) from microarray data is a challenging problem, due to the noise properties of microarrays as well as the high variance of living cell responses to drugs. Hence there is a strong need for more practical and robust methods than standard methods for real-value prediction.ResultsWe devised an extended version of the off-subspace noise-reduction (de-noising) method [1] to incorporate heterogeneous network data such as sequence similarity or protein-protein interactions into a single framework. Using that method, we first de-noise the gene expression data for training and test data and also the drug-response data for training data. Then we predict the unknown responses of each drug from the de-noised input data. For ascertaining whether de-noising improves prediction or not, we carry out 12-fold cross-validation for assessment of the prediction performance. We use the Pearsons correlation coefficient between the true and predicted response values as the prediction performance. De-noising improves the prediction performance for 65% of drugs. Furthermore, we found that this noise reduction method is robust and effective even when a large amount of artificial noise is added to the input data.ConclusionWe found that our extended off-subspace noise-reduction method combining heterogeneous biological data is successful and quite useful to improve prediction of human cell cancer dru responses from microarray data.

Oil Accumulation by the Oleaginous Diatom Fistulifera solaris as Revealed by the Genome and Transcriptome

F. solaris has an allodiploid genome structure, and activation of lipid accumulation and degradation metabolism pathways at the same time might underlie its simultaneous growth and oil accumulation. Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Up-regulation of MSX2 enhances the malignant phenotype and is associated with twist 1 expression in human pancreatic cancer cells.

MSX2 is thought to be a regulator of organ development and a downstream target of the ras signaling pathway; however, little is known about the role of MSX2 in the development of pancreatic cancers, most of which harbor a K-ras gene mutation. Therefore, we examined whether the presence of MSX2 correlates with the malignant behavior of pancreatic cancer cells. BxPC3 pancreatic cancer cells that stably overexpress MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration, and anchorage-independent cell growth were enhanced in MSX2-expressing cells. Injection of MSX2-expressing cells into the pancreas of nude mice resulted in a significant increase in liver metastases and peritoneal disseminations compared with injection of control cells. Microarray analysis revealed a significant induction of Twist 1 expression in cells that express MSX2. When MSX2 was inactivated in pancreatic cancer cells following transfection with an MSX2-specific small interfering RNA, Twist 1 was down-regulated. Immunohistochemistry of human pancreatic carcinoma tissue revealed that MSX2 was frequently expressed in cancer cells, and that increased expression of MSX2 significantly correlated with higher tumor grade, vascular invasion, and Twist 1 expression. These data indicate that MSX2 plays a crucial role in pancreatic cancer development by inducing changes consistent with epithelial to mesenchymal transition through enhanced expression of Twist 1.

Inhibitor of apoptosis protein family as diagnostic markers and therapeutic targets of colorectal cancer

The apoptosis and antiapoptotic signaling pathways are important for regulating carcinogenesis and cancer progression, and for determining prognosis. Molecules involved in apoptosis represent potential cancer diagnostic markers and therapeutic targets. The inhibitor of apoptosis protein (IAP) family includes several important molecules involved in apoptosis that might represent such targets. Increasing evidence has demonstrated that the IAP family of proteins is integral for antiapoptotic and nuclear factor-κB signal transduction, and enhanced expression of IAPs contributes to colon carcinogenesis and its poor prognosis, as well as to drug resistance of tumors. X-linked IAP, cIAP1, cIAP2, and survivin are prognostic markers of colorectal cancer, and survivin and cIAP2 are also utilized to predict the effect of anticancer treatment in colorectal cancer patients. Novel therapies such as YM155 and LY2181308 targeting survivin, AEG35156 and phenoxodiol targeting X-linked IAP, AT-406 as a Smac mimetic, and survivin peptides are currently being evaluated in clinical trials. This report reviews the involvement of the IAP family in colorectal adenocarcinoma in order to summarize the role of the IAP family members as diagnostic and therapeutic targets, and to provide an overview of the future course of research in this area.

THE SIZE DIFFERENCES AMONG MAMMALIAN INTRONS ARE DUE TO THE ACCUMULATION OF SMALL DELETIONS

In order to investigate the molecular mechanisms that alter intron size, we conducted an extensive interspecies comparison of homologous introns among three mammalian groups: human, artiodactyls, and rodents. The size differences of introns were statistically significant among all three groups (longest intron was for human and shortest for rodents), and appear to be due to the accumulation of small deletions, according to the separate count of insertion and deletion frequencies. The distribution of intron size differences also has a shape similar to that for the distribution of insertion/deletion sizes found in pseudogenes. It is suggested that introns are selectively neutral to small‐scale changes of the genome size, which inherently contain the bias of favoring short deletions against short insertions.

Down‐regulation of cIAP2 enhances 5‐FU sensitivity through the apoptotic pathway in human colon cancer cells

Currently 5‐fluorouracil (5‐FU) plays a central role in the chemotherapeutic regimens for colorectal cancers and thus it is important to understand the mechanisms that determine 5‐FU sensitivity. The expression profiles of human colon cancer cell line DLD‐1, its 5‐FU‐resistant subclone DLD‐1/FU and a futher 21 types of colon cancer cell lines were compared to identify the novel genes defining the sensitivity to 5‐FU and to estimate which population of genes is responsible for 5‐FU sensitivity. In the hierarchical clustering, DLD‐1 and DLD‐1/FU were most closely clustered despite over 100 times difference in their 50% inhibitory concentration of 5‐FU. In DLD‐1/FU, the population of genes differentially expressed compared to DLD‐1 was limited to 3.3%, although it ranged from 4.8% to 24.0% in the other 21 cell lines, thus indicating that the difference of 5‐FU sensitivity was defined by a limited number of genes. Next, the role of the cellular inhibitor of apoptosis 2 (cIAP2) gene, which was up‐regulated in DLD‐1/FU, was investigated for 5‐FU resistance using RNA interference. The down‐regulation of cIAP2 efficiently enhanced 5‐FU sensitivity, the activation of caspase 3/7 and apoptosis under exposure to 5‐FU. The immunohistochemistry of cIAP2 in cancer and corresponding normal tissues from colorectal cancer patients in stage III revealed that cIAP2 was more frequently expressed in cancer tissues than in normal tissues, and cIAP2‐positive patients had a trend toward early recurrence after fluorouracil‐based chemotherapy. Although the association between drug sensitivity and the IAP family in colorectal cancer has not yet been discussed, cIAP2 may therefore play an important role as a target therapy in colorectal cancer. (Cancer Sci 2009; 100: 903–913)

Splice isoforms as therapeutic targets for colorectal cancer

Alternative pre-mRNA splicing allows exons of pre-mRNA to be spliced in different arrangements to produce functionally distinct mRNAs. More than 95% of human genes encode splice isoforms, some of which exert antagonistic functions. Recent studies revealed that alterations of the splicing machinery can cause the development of neoplasms, and understanding the splicing machinery is crucial for developing novel therapeutic strategies for malignancies. Colorectal cancer patients need novel strategies not only to enhance the efficacy of the currently available agents but also to utilize newly identified therapeutic targets. This review summarizes the current knowledge about the splice isoforms of VEGFA, UGT1A, PXR, cyclin D1, BIRC5 (survivin), DPD, K-RAS, SOX9, SLC39A14 and other genes, which may be possible therapeutic targets for colorectal cancer. Among them, the VEGFA splice isoforms are classified into VEGFAxxx and VEGFAxxxb, which have proangiogenic and antiangiogenic properties, respectively; UGT1A is alternatively spliced into UGT1A1 and other isoforms, which are regulated by pregnane X receptor isoforms and undergo further splicing modifications. Recently, the splicing machinery has been extensively investigated and novel discoveries in this research field are being reported at a rapid pace. The information contained in this review also provides suggestions for how therapeutic strategies targeting alternative splicing can be further developed.