Gene Cutler

Multiclass cancer classification and biomarker discovery using GA-based algorithms

MOTIVATION The development of microarray-based high-throughput gene profiling has led to the hope that this technology could provide an efficient and accurate means of diagnosing and classifying tumors, as well as predicting prognoses and effective treatments. However, the large amount of data generated by microarrays requires effective reduction of discriminant gene features into reliable sets of tumor biomarkers for such multiclass tumor discrimination. The availability of reliable sets of biomarkers, especially serum biomarkers, should have a major impact on our understanding and treatment of cancer. RESULTS We have combined genetic algorithm (GA) and all paired (AP) support vector machine (SVM) methods for multiclass cancer categorization. Predictive features can be automatically determined through iterative GA/SVM, leading to very compact sets of non-redundant cancer-relevant genes with the best classification performance reported to date. Interestingly, these different classifier sets harbor only modest overlapping gene features but have similar levels of accuracy in leave-one-out cross-validations (LOOCV). Further characterization of these optimal tumor discriminant features, including the use of nearest shrunken centroids (NSC), analysis of annotations and literature text mining, reveals previously unappreciated tumor subclasses and a series of genes that could be used as cancer biomarkers. With this approach, we believe that microarray-based multiclass molecular analysis can be an effective tool for cancer biomarker discovery and subsequent molecular cancer diagnosis.

Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Molecular Signatures of Prostate Stem Cells Reveal Novel Signaling Pathways and Provide Insights into Prostate Cancer

Background The global gene expression profiles of adult and fetal murine prostate stem cells were determined to define common and unique regulators whose misexpression might play a role in the development of prostate cancer. Methodology/Principal Findings A distinctive core of transcriptional regulators common to both fetal and adult primitive prostate cells was identified as well as molecules that are exclusive to each population. Elements common to fetal and adult prostate stem cells include expression profiles of Wnt, Shh and other pathways identified in stem cells of other organs, signatures of the aryl-hydrocarbon receptor, and up-regulation of components of the aldehyde dehydrogenase/retinoic acid receptor axis. There is also a significant lipid metabolism signature, marked by overexpression of lipid metabolizing enzymes and the presence of the binding motif for Srebp1. The fetal stem cell population, characterized by more rapid proliferation and self-renewal, expresses regulators of the cell cycle, such as E2f, Nfy, Tead2 and Ap2, at elevated levels, while adult stem cells show a signature in which TGF-β has a prominent role. Finally, comparison of the signatures of primitive prostate cells with previously described profiles of human prostate tumors identified stem cell molecules and pathways with deregulated expression in prostate tumors including chromatin modifiers and the oncogene, Erg. Conclusions/Significance Our data indicate that adult prostate stem or progenitor cells may acquire characteristics of self-renewing primitive fetal prostate cells during oncogenesis and suggest that aberrant activation of components of prostate stem cell pathways may contribute to the development of prostate tumors.

Identification of transcriptional networks responding to pyrroloquinoline quinone dietary supplementation and their influence on thioredoxin expression, and the JAK/STAT and MAPK pathways.

PQQ (pyrroloquinoline quinone) improves energy utilization and reproductive performance when added to rodent diets devoid of PQQ. In the present paper we describe changes in gene expression patterns and transcriptional networks that respond to dietary PQQ restriction or pharmacological administration. Rats were fed diets either deficient in PQQ (PQQ−) or supplemented with PQQ (approx. 6 nmol of PQQ/g of food; PQQ+). In addition, groups of rats were either repleted by administering PQQ to PQQ− rats (1.5 mg of PQQ intraperitoneal/kg of body weight at 12 h intervals for 36 h; PQQ−/+) or partially depleted by feeding the PQQ− diet to PQQ+ rats for 48 h (PQQ+/−). RNA extracted from liver and a Codelink® UniSet Rat I Bioarray system were used to assess gene transcript expression. Of the approx. 10000 rat sequences and control probes analysed, 238 were altered at the P<0.01 level by feeding on the PQQ− diet for 10 weeks. Short-term PQQ depletion resulted in changes in 438 transcripts (P<0.01). PQQ repletion reversed the changes in transcript expression caused by PQQ deficiency and resulted in an alteration of 847 of the total transcripts examined (P<0.01). Genes important for cellular stress (e.g. thioredoxin), mitochondriogenesis, cell signalling [JAK (Janus kinase)/STAT (signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) pathways] and transport were most affected. qRT-PCR (quantitative real-time PCR) and functional assays aided in validating such processes as principal targets. Collectively, the results provide a mechanistic basis for previous functional observations associated with PQQ deficiency or PQQ administered in pharmacological amounts.

Molecular signatures of the primitive prostate stem cell niche reveal novel mesenchymal-epithelial signaling pathways.

Background Signals between stem cells and stroma are important in establishing the stem cell niche. However, very little is known about the regulation of any mammalian stem cell niche as pure isolates of stem cells and their adjacent mesenchyme are not readily available. The prostate offers a unique model to study signals between stem cells and their adjacent stroma as in the embryonic prostate stem cell niche, the urogenital sinus mesenchyme is easily separated from the epithelial stem cells. Here we investigate the distinctive molecular signals of these two stem cell compartments in a mammalian system. Methodology/Principal Findings We isolated fetal murine urogenital sinus epithelium and urogenital sinus mesenchyme and determined their differentially expressed genes. To distinguish transcripts that are shared by other developing epithelial/mesenchymal compartments from those that pertain to the prostate stem cell niche, we also determined the global gene expression of epidermis and dermis of the same embryos. Our analysis indicates that several of the key transcriptional components that are predicted to be active in the embryonic prostate stem cell niche regulate processes such as self-renewal (e.g., E2f and Ap2), lipid metabolism (e.g., Srebp1) and cell migration (e.g., Areb6 and Rreb1). Several of the enriched promoter binding motifs are shared between the prostate epithelial/mesenchymal compartments and their epidermis/dermis counterparts, indicating their likely relevance in epithelial/mesenchymal signaling in primitive cellular compartments. Based on differential gene expression we also defined ligand-receptor interactions that may be part of the molecular interplay of the embryonic prostate stem cell niche. Conclusions/Significance We provide a comprehensive description of the transcriptional program of the major regulators that are likely to control the cellular interactions in the embryonic prostatic stem cell niche, many of which may be common to mammalian niches in general. This study provides a comprehensive source for further studies of mesenchymal/epithelial interactions in the prostate stem cell niche. The elucidation of pathways in the normal primitive niche may provide greater insight into mechanisms subverted during abnormal proliferative and oncogenic processes. Understanding these events may result in the development of specific targeted therapies for prostatic diseases such as benign prostatic hypertrophy and carcinomas.

A comparative analysis of HGSC and Celera human genome assemblies and gene sets

MOTIVATION Since the simultaneous publication of the human genome assembly by the International Human Genome Sequencing Consortium (HGSC) and Celera Genomics, several comparisons have been made of various aspects of these two assemblies. In this work, we set out to provide a more comprehensive comparative analysis of the two assemblies and their associated gene sets. RESULTS The local sequence content for both draft genome assemblies has been similar since the early releases, however it took a year for the quality of the Celera assembly to approach that of HGSC, suggesting an advantage of HGSCs hierarchical shotgun (HS) sequencing strategy over Celeras whole genome shotgun (WGS) approach. While similar numbers of ab initio predicted genes can be derived from both assemblies, Celeras Otto approach consistently generated larger, more varied gene sets than the Ensembl gene build system. The presence of a non-overlapping gene set has persisted with successive data releases from both groups. Since most of the unique genes from either genome assembly could be mapped back to the other assembly, we conclude that the gene set discrepancies do not reflect differences in local sequence content but rather in the assemblies and especially the different gene-prediction methodologies.

Advancing therapeutic discovery through phenotypic screening of the extracellular proteome using hydrodynamic intravascular injection.

Objective: Although the human genome encodes ∼ 20,000 protein-coding genes, only a very small fraction of these have been explored as potential targets for therapeutic development. The challenge of identifying and validating new protein targets has contributed to the significant reduction in the productivity of the pharmaceutical industry in the recent decade, highlighting the continued need to find new therapeutic targets. Research design and methods: The traditional methods to discover new targets are expensive, low throughput and time consuming, usually taking years to validate or invalidate a target. To address these limitations, as a proof of concept, we explored the hydrodynamic tail vein (HTV) injection as a gene delivery method for direct in vivo phenotypic screening of novel secreted factor targets for Type II diabetes therapeutics. Results: High levels and sustained expression of target proteins were observed in diabetic mouse models tested, allowing us to identify multiple novel hormones that may regulate glucose metabolism. Conclusions: These results suggest that HTV is a low-cost, high-throughput method for direct in vivo phenotypic drug screening in metabolic disorders and could be applicable to many other disease areas as well. This method if combined with other approaches such as human genetic studies could provide a significant value to future drug discovery.

Genomic Resources for Cancer Biologists

Cancer is fundamentally a genetic disease caused by a combination of several mutations that make a tumor cell distinct from a normal cell. The new generation of cancer therapies is aimed at targeting this difference, for example, by inhibiting the activity of the product of a hyperactive or overexpressed oncogene. The utilization of genomic approaches to identify cancer-causing mutations and understand the underlying biological mechanisms has made significant advances in recent years. Notably, profiling of gene expression in tumor samples by microarrays has provided new insights into the molecular characterization of many types of cancer. Other genomic approaches including genomewide profiling of amplifications and deletions and analysis of single-nucleotide polymorphisms (SNPs), hold the promise of enabling the systematic description of all mutations in a given tumor and, thus, the possibility of personalizing cancer diagnosis and treatment. Because of the explosion in the use of genomic approaches in the field, knowledge and expertise in the efficient utilization of bioinformatics resources and tools has become essential for cancer researchers. This chapter is intended to help cancer biologists navigate through the complexity of current genomic databases to find the information they need.