Keith A. Ching

Large-scale analysis of the human and mouse transcriptomes

High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (http://expression.gnf.org) that integrates data visualization and curation of current gene annotations.

Ubiquitin-mediated sequestration of normal cellular proteins into polyglutamine aggregates

A hallmark of most neurodegenerative diseases, including those caused by polyglutamine expansion, is the formation of ubiquitin (Ub)-positive protein aggregates in affected neurons. This finding suggests that the Ub system may be involved in common mechanisms underlying these otherwise unrelated diseases. Here we report the finding of ataxin-3 (Atx-3), whose mutation is implicated in the neurodegenerative disease spinocerebellar ataxia type 3, in a bioinformatics search of the human genome for components of the Ub system. We show that wild-type Atx-3 is a Ub-binding protein and that the interaction of Atx-3 with Ub is mediated by motifs homologous to those found in a proteasome subunit. Both wild-type Atx-3 and the otherwise unrelated Ub-binding protein p62/Sequestosome-1 have been shown to be sequestered into aggregates in affected neurons in several neurodegenerative diseases, but the mechanism for this recruitment has remained unclear. In this article, we show that functional Ub-binding motifs in Atx-3 and p62 proteins are required for the localization of both proteins into aggregates in a cell-based assay that recapitulates several features of polyglutamine disease. We propose that the Ub-mediated sequestration of essential Ub-binding protein(s) into aggregates may be a common mechanism contributing to the pathogenesis of neurodegenerative diseases.

A Comparison of the Celera and Ensembl Predicted Gene Sets Reveals Little Overlap in Novel Genes

genes, but the novel genes predicted by both groups are largely nonoverlapping. To validate the existence of the transcript predictions, we used RNA expression profiling and a bank of 13 diverse human tissues. The commercial high-density oligonucleo-The recent description of the human genome and the tide arrays used are based on Expressed Sequence subsequent annotation of putative novel genes has Tags (ESTs) represented in Unigene (release 95). ushered in a new era in biology. One of the revelations of BLASTN was used to assign the transcript predictions the human genome project was the remarkably consistent to a Unigene cluster, and the RNA expression pattern prediction that the genome harbors around 30,000 genes. was determined for the 8,000 known and 5,000 novel This observation was based on independent analyses predicted genes with a corresponding Unigene cluster done by a public genome consortium (29,691 transcripts, on the arrays (see legend to Figure 2 for details). Using Ensembl v0.8) (Lander et al., 2001), by work done at Celera these methods, we found evidence of expression for Genomics (39,114 transcripts) (Venter et al., 2001), and by more than 80% of the known genes in at least one of Green and colleagues using expressed sequence tag the tissue samples analyzed (Figure 2A). Similarly, more (EST) clustering incorporating quality scores (35,000 than 80% of the novel predicted transcripts were genes) (Ewing and Green, 2000). This conclusion was detected as expressed in at least one of the 13 human surprising for two reasons. First, less complex organisms tissues. Hierarchical clustering and visualization of like Arabidopsis (25,000) and C. elegans (19,000) have these expression data revealed a similar fraction of approximately the same number of genes (C. elegans tissue-restricted transcripts for both known and novel Sequencing Consortium, 1998; Arabidopsis Genome genes (Figure 2B). These data support the view that the Initiative, 2000). Second, earlier estimates of gene number novel transcripts predicted by both groups encode bona based on EST clustering and detailed chromosomal fide differentially expressed mRNAs. Since many of analysis were much higher, ranging from 45,000 to 140,000 these verified transcripts were contained in only one of the two predicted transcriptomes, we conclude that the Scott, 1999). While the Celera and Ensembl annotation computational methods used for gene prediction by efforts predicted approximately the same number of either group are inadequate and that the respective genes, a direct comparison of the predicted transcript sets transcriptomes are individually …

Endothelin Axis Is a Target of the Lung Metastasis Suppressor Gene RhoGDI2

Half of patients treated for locally advanced bladder cancer relapse with often fatal metastatic disease to the lung. We have recently shown that reduced expression of the GDP dissociation inhibitor, RhoGDI2, is associated with decreased survival of patients with advanced bladder cancer. However, the effectors by which RhoGDI2 affects metastasis are unknown. Here we use DNA microarrays to identify genes suppressed by RhoGDI2 reconstitution in lung metastatic bladder cancer cell lines. We identify such RNAs and focus only on those that also increase with tumor stage in human bladder cancer samples to discover only clinically relevant targets of RhoGDI2. Levels of endothelin-1 (ET-1), a potent vasoconstrictor, were affected by both RhoGDI2 reconstitution and tumor stage. To test the hypothesis that the endothelin axis is important in lung metastasis, lung metastatic bladder carcinoma cells were injected in mice treated with the endothelin receptor-specific antagonist, atrasentan, thereby blocking engagement of the up-regulated ET-1 ligand with its cognate receptor. Endothelin antagonism resulted in a dramatic reduction of lung metastases, similar to the effect of reexpressing RhoGDI2 in these metastatic cells. Taken together, these experiments show a novel approach of identifying therapeutic targets downstream of metastasis suppressor genes. The data also suggest that blockade of the ET-1 axis may prevent lung metastasis, a new therapeutic concept that warrants clinical evaluation.

An Integrated Clinical-Genomics Approach Identifies a Candidate Multi-Analyte Blood Test for Serous Ovarian Carcinoma

Purpose: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. Experimental Design: We developed an algorithm to identify secreted proteins encoded among ∼22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. Results: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. Conclusions: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

Data and animal management software for large-scale phenotype screening

The mouse N-ethyl-N-nitrosourea (ENU) mutagenesis program at the Genomics Institute of the Novartis Research Foundation (GNF) uses MouseTRACS to analyze phenotype screens and manage animal husbandry. MouseTRACS is a Web-based laboratory informatics system that electronically records and organizes mouse colony operations, prints cage cards, tracks inventory, manages requests, and reports Institutional Animal Care and Use Committee (IACUC) protocol usage. For efficient phenotype screening, MouseTRACS identifies mutants, visualizes data, and maps mutations. It displays and integrates phenotype and genotype data using likelihood odds ratio (LOD) plots of genetic linkage between genotype and phenotype. More detailed mapping intervals show individual single nucleotide polymorphism (SNP) markers in the context of phenotype. In addition, dynamically generated pedigree diagrams and inventory reports linked to screening results summarize the inheritance pattern and the degree of penetrance. MouseTRACS displays screening data in tables and uses standard charts such as box plots, histograms, scatter plots, and customized charts looking at clustered mice or cross pedigree comparisons. In summary, MouseTRACS enables the efficient screening, analysis, and management of thousands of animals to find mutant mice and identify novel gene functions. MouseTRACS is available under an open source license at http://www.mousetracs.sourceforge.net.

Abstract 940: Identification of Palbociclib response signature across indications

Cellular proliferation is dependent on an orderly movement through the various phases of cell cycle. Progression through the G1 phase in particular requires phosphorylation of the retinoblastoma (Rb) protein which in turn releases E2F transcription factors resulting in transcriptional activation of response gene necessary for progression into S-phase. The cyclin D-CDK4/6 signaling pathway represent a critical regulatory pathway controlling transition from G1 into S-phase and greater than 90% of human tumors have mutations in this pathway. Palbociclib is a potent, selective, and orally bioavailable inhibitor of Cdk4/6. In human tumor xenograft models, Palbociclib has significant antitumor activities. The clinical activities of Palbociclib have also be demonstrated in the phase II PALOMA-1 trial for dramatic efficacy of postmenopausal patients with locally advanced or newly diagnosed estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer in combination with letrozole. To better understand the molecular mechanisms of Palbociclib response, we have identified a common set of gene signatures for ER+ BC as well as melanoma models. The physiological role of these genes regulated by Palbociclib is associated with DNA replication and repair, cell cycle, signal transduction, and Mitosis. Many of these genes have been previously identified as E2F signatures. In this study, we are aiming to expand this analysis to include two additional indications including Head and Neck squamous cell carcinoma (HNSCC) as well as squamous cell lung carcinoma (Sq lung) using Illumina RNASeq technology platform. Results from these studies will be presented and core Palbo-response signature will be discussed. In addition, we will also present the pharmacological effects of Letrozole on the gene expression changes for these core signatures in ER+ BC models. Citation Format: Xianxian Zheng, Mark Ozeck, Zhou Zhu, Keith Ching, David Shields, James Hardwick, Paul Rejto, Todd VanArsdale. Identification of Palbociclib response signature across indications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 940. doi:10.1158/1538-7445.AM2015-940