Nathan Elliott

Stress Induction and Mitochondrial Localization of Oxr1 Proteins in Yeast and Humans

ABSTRACT Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.

BackgroundThe NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1.ResultsWe find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7s oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei.ConclusionNCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.

A Multiplex Assay to Measure RNA Transcripts of Prostate Cancer in Urine

The serum prostate-specific antigen (PSA) test has a high false positive rate. As a single marker, PSA provides limited diagnostic information. A multi-marker test capable of detecting not only tumors but also the potentially lethal ones provides an unmet clinical need. Using the nanoString nCounter gene expression system, a 20-gene multiplex test was developed based on digital gene counting of RNA transcripts in urine as a means to detect prostate cancer. In this test, voided urine is centrifuged to pellet cells and the purified RNA is amplified for hybridization to preselected probesets. Amplification of test cell line RNA appeared not to introduce significant bias, and the counts matched well with gene abundance levels as measured by DNA microarrays. For data analysis, the individual counts were compared to that of β2 microglobulin, a housekeeping gene. Urine samples of 5 pre-operative cases and 2 non-cancer were analyzed. Pathology information was then retrieved. Signals for a majority of the genes were low for non-cancer and low Gleason scores, and 6/6 known prostate cancer markers were positive in the cases. One case of Gleason 4+5 showed, in contrast, strong signals for all cancer-associated markers, including CD24. One non-cancer also showed signals for all 6 cancer markers, and this man might harbor an undiagnosed cancer. This multiplex test assaying a natural waste product can potentially be used for screening, early cancer detection and patient stratification. Diagnostic information is gained from the RNA signatures that are associated with cell types of prostate tumors.

A Functional Genomics Approach to Identify and Characterize Oxidation Resistance Genes

In order to develop a more complete understanding of the genes required for resistance to oxidative DNA damage, we devised methods to identify genes that can prevent or repair oxidative DNA damage. These methods use the oxidative mutator phenotype of a repair deficient E. coli strain to measure the antimutator effect resulting from the expression of human cDNAs. The method can be adapted to characterize the function, and to determine the active site domains, of putative antimutator genes. Since bacteria do not contain subcellular compartments, genes that function in mitochondria, the cytoplasm, or the nucleus can be identified. Methods to determine the localization of genes in their normal host organism are also described.

Abstract 2441: NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein

Introduction: Development of improved cancer diagnostics and therapeutics requires detailed understanding of the genomic, transcriptomic, and proteomic profiles in the tumor microenvironment. Current technologies can excel at measuring a single analyte, but it remains challenging to simultaneously collect high-throughput DNA, RNA, and protein data from small samples. We have developed an approach that uses optical barcodes to simultaneously profile DNA, RNA, and protein from as little as 5ng DNA, 25ng RNA, and 250ng protein or just 2 5µm FFPE slides, and simplifies data analysis by generating digital counts for each analyte. Methods: The approach uses paired capture and reporter oligonucleotide probes and optical barcodes to enumerate up to 800 targets. The platform was initially developed to measure RNA, and we have adapted it to measure DNA single nucleotide variants (SNVs), proteins, and phospho-proteins. SNVs are detected by direct hybridization of sequence discriminating probes to the wild-type and mutant sequence of interest. Proteins are detected via binding of oligonucleotide-conjugated antibodies. Results: Combinations of DNA, RNA, and protein in biological and experimental contexts. SNV probes are able to detect variant alleles down to 5% abundance within a wild type population and can discriminate variants within mutation hotspots. It was >96% accurate at identifying variants from samples displaying a range of allele frequencies and DNA integrity when benchmarked against next-generation sequencing. Protein detection has been developed for cell surface, cytosolic, and nuclear proteins, as well as phospho-proteins. It was validated against flow cytometry, western blot, and mass spectrometry using cell lines with ectopic target expression and primary cells. To demonstrate concurrent measurement of DNA, RNA, and protein from a single system, BRAFWT or BRAFV600E cell lines were treated with the BRAFV600E inhibitor vemurafenib and the MEK inhibitor trametinib. We measured the allele usage at the BRAFV600 locus, as well as BRAFV600E dependent changes in mRNA expression, protein expression and protein phosphorylation in a single experiment. Conclusions: 3D Biology has several advantages over other analytical approaches. Direct, single-molecule digital counting allows detection over a broad dynamic range with high reproducibility, often over 98% concordance between technical replicates. The simultaneous interrogation of DNA, RNA, and protein maximizes the amount of data obtained from precious samples and minimizes instrumentation demands by leveraging a single detection platform. The 3D Biology approach allows holistic, digital analysis of biological samples with high specificity and precision. This technology is currently available for research use, but may also have clinical application in the future. Citation Format: Chris Lausted, Yong Zhou, Jinho Lee, Christopher Vellano, Karina A. Eterovic, Ping Song, Lin-ya Tang, Gloria Fawcett, Tae-Beom Kim, Ken Chen, Gary Geiss, Gavin Meredith, Qian Mei, Gokhan Demirkan, Dwayne Dunaway, Dae Kim, P. Martin Ross, Elizabeth Manrao, Nathan Elliott, Sarah Warren, Christina Bailey, Chung-Ying Huang, Joseph Beechem, Gordon Mills, Leroy Hood. NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2017-2441

Preliminary validation of nCounter PanCancer immune profiling of FFPE slides and pbmc in CITN-05, a CITN study of the immunological effects of an IDO1 inhibitor in patients with ovarian carcinoma.

Meeting abstracts The Cancer Immunotherapy Trials Network (CITN) is conducting a pilot study in patients with advanced epithelial ovarian, fallopian tube or primary peritoneal carcinoma to determine the extent to which a regimen of an IDO1 inhibitor that normalizes Kyn/Trp ratios might be