Acid Metabolic Pathways Research Articles

Abstract 2244: Differential DNA methylation analysis of TCGA cholangiocarcinoma data

Abstract Cholangiocarcinoma (CCA), also known as bile duct cancer, is a rare malignancy that develops in bile duct, arising from the epithelial cells (cholangiocytes) lining the bile duct. CCA is the second most common type of primary liver cancer, and its incidence has been rising in the Unites States. Most of the patients with CCA are diagnosed at an advanced stage with a median survival of less than 1 year despite administering chemotherapy. In the current study, we analyze the global patterns of DNA methylation in CCA data from TCGA. Pathway and gene ontology (GO) enrichment analysis of differentially methylated genes (DMGs) enabled us to understand how changes in methylation and gene expression affect the biologic pathways involved in the progression of CCA. TCGA level-3 DNA methylation data were downloaded for 485,578 CpG sites with annotations. Further, we normalized β value by using Beta Mixed Integer-quantile normalization (BMIQ) to adjust type I and type II probes. For a CpG site to be considered differentially methylated, the difference in the median β value in primary tumor and normal samples should be at least 0.2 and the BH adjusted p-value should be less than 0.005 and AUC ≥ 0.7. A total of 12,259 differentially methylated (Δβ >) CpG were observed; out of these 9,534 are hypermethylated and 2,725 are hypomethylated. About 4,714 differentially methylated CpGs (dm-CpGs) were observed within 1.5Kb from the TSS of corresponding genes. Chromosome 1 has maximum number of dm-CpGs, while chromosome 18 has lowest number of dm-CpGs. Differential DNA methylation frequency per Mb was also calculated and it was observed that chromosome 17 has the highest differential methylation frequency (9.61 dm-CpGs/Mb) while chromosome 18 has the lowest (1.47 dm-CpG/Mb). KEGG pathway analysis of genes with differentially methylated CpGs (Δβ ≥ 0.2) observed the enrichment of cancer, cell division and differentiation, amino acid metabolism, xenobiotics degradation, and immune response pathways. Hippo signaling pathway also got affected by DNA methylation (FDR 3.70e-18), which also got affected by differential gene expression. Change in DNA methylation and gene expression level of hippo signaling pathway genes plays a vital role in CCA. Citation Format: Nitish K. Mishra, Niu Meng, Chittibabu Guda. Differential DNA methylation analysis of TCGA cholangiocarcinoma data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2244.

Aerobic Training Remodels Muscle Lipid Composition and Improves Intrinsic Mitochondrial Function in Men with Type 2 Diabetes

The effects of exercise training on the skeletal muscle (SKM) lipidome and mitochondrial function have not been thoroughly explored in individuals with type 2 diabetes (T2D). We investigated the effects of exercise training on molecular signatures within SKM of T2D, employing integrated multi-omics data analyses including lipidomics [MS/MS-shotgun], global DNA methylation and transcriptomics [RNA-Seq]. Muscle tissue and cellular mitochondrial respiration was quantified by high resolution respirometry. Ten weeks of aerobic training increased monolysocardiolipin (MLCL), a marker of mitochondrial remodeling, reduced total and specific species of sphingomyelin (SM), phosphatidylserine (PS) and ceramides lipid content and improved intrinsic mitochondrial respiration in SKM of T2D without alterations in mitochondrial DNA copy number or cardiolipin content. Exercise training differentially modified 50 DNA methylation sites. Among these genes, NOX4, acts as an oxygen sensor and catalyzes the reduction of reactive oxygen species (ROS), and PLCE1, catalyzes the hydrolysis of phosphoinositol 1,4,5-triphosphate (IP3) and DAG, were hypo-methylated and hyper-methylated, respectively, following training. Exercise training differentially regulated 70 transcripts in SKM of T2D that resulted in enrichments of lipid and amino acid metabolism and β-oxidation pathways. In summary, aerobic training improves intrinsic and cell autonomous SKM mitochondrial function and modifies SKM lipid composition in men with T2D, with potentially advantageous augmentations of oxidative stress and insulin signaling pathways. Disclosure M.F. Pino: None. N. Stephens: None. F. Yi: None. A.P. Hodges: None. R.E. Pratley: Other Relationship; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eisai Inc., GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc., Lexicon Pharmaceuticals, Inc., Ligand Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk Inc., Pfizer Inc., Sanofi-Aventis, Takeda Development Center Americas, Inc.. S.R. Smith: None. B.H. Goodpaster: None. L.M. Sparks: None.

Effects of Aerobic and Resistance Training on Circulating Micro-RNA Expression Profile in Subjects with Type 2 Diabetes

Structured exercise programs are of great benefit in order to obtain a proper glycemic control in subjects with type diabetes (T2D). However, whether the metabolic effects of aerobic (AER) and resistance (RES) exercise exert specific changes in the expression profile of circulating miRNAs (c-miRNAs) is still largely unknown. We studied 24 individuals with T2D from the RAED2 Trial (NCT01182948), randomly assigned to AER or RES training protocols (NAER=12; NRES=12). Firstly, the baseline and post-training levels of 179 c-miRNAs were measured by RT-PCR in 6 individuals (NAER=3; NRES=3). Nineteen c-miRNAs exhibited at least 20% fold-change variation from baseline and were thereafter measured in the whole group. Compared to baseline, the expression levels of miR-423-3p, miR-451a and miR-766-3p resulted significantly upregulated after exercise, irrespective of exercise type (type I error rate threshold: 0.0026; 0.05/19 miRNAs). Bioinformatic analysis by miRSystem and Diana Tools revealed that these c-miRNAs targeted genes involved in pathways relevant to fatty acids biosynthesis and metabolic regulation, including LKB1, PI3K, mTOR and IRS signaling pathways. Interestingly, miR-451a and miR-423-3p changes were positively correlated with fat loss after training (rho=0.45 and 0.43, respectively; p<0.05). Both miRNAs, alone or in combination, resulted as independent predictors of fat loss, when entered in generalized linear models including exercise type as covariate. However, only miR-451a eventually survived as predictor of fat loss after further correction for age, sex, BMI and hemoglobin A1c. In conclusion, physical exercise in individuals with T2D is associated with specific changes of c-miRNAs profile, predominantly clustered along pathways with regulatory significance for fatty acid metabolism. Further investigations are warranted to confirm our findings and to unravel their mechanistic significance. Disclosure D. Olioso: None. M. Dauriz: None. E. Bacchi: None. C. Negri: None. E. Bonora: None. P. Moghetti: None.

DNA Methylation at Birth Is Associated with Adiposity and Infant Growth during the First Six Months of Life in the Healthy Start Study

In utero exposures have been shown to impart increased risk for obesity in the offspring and are associated with epigenetic changes. DNA methylation was measured in 351 umbilical cord blood (UCB) samples within the Healthy Start Study, a well-characterized prebirth cohort that is following mother-infant pairs from pregnancy-early childhood. The primary objective was to test if DNA methylation at birth was associated with fat mass (FM) gains and infant growth (weight-for-age Z-score, WAZ) during the first 6 months (mos) of life to identify potential biomarkers of future obesity risk. DNA methylation was assessed in UCB via the Illumina 450K array. Adiposity was measured at birth and 4-6 mos of age via PeaPod. Infant weight was obtained from medical records. After adjusting for cellular heterogeneity, sex, and race we found 742 methylation sites (CpGs) through a univariable analysis that were associated with the rate of FM (g/day) in the first 6 mos of life (p < 0.001). Then, partial least squares (PLS) fitting using a multivariable analysis selected a subset of 235 predictor CpGs in this list for KEGG pathway analysis. Similarly, 1314 CpGs were associated with WAZ (p < 0.001); PLS selected a subset of 198 CpGs for KEGG analysis. Using a FDR < 0.001 for KEGG analysis, 3 pathways were associated with FM gain (Metabolic pathways, Glyoxylate and dicarboxylate metabolism, Neurotrophin signaling) and 8 pathways for WAZ gain (Gastric acid secretion, Oocyte meiosis, Metabolic pathways, Endocytosis, Tight Junction, Oxytocin signaling, GnRH signaling, and Inflammatory mediator regulation of TRP channels). The KEGG: Metabolic pathways was identified for both FM and WAZ gain, and 3 gene targets enriched in this pathway, ATP5C1, CYP26A1, and IDH3A are known to be involved in fat metabolism. Our studies have identified potential candidate methylation biomarkers, present at birth, which can be used for early risk detection in order to mitigate development of childhood obesity. Disclosure S.J. Borengasser: None. A.P. Starling: None. W. Zhang: None. J. Friedman: Advisory Panel; Self; Janssen Pharmaceuticals, Inc.. R.F. Hamman: None. I. Yang: None. K. Kechris: None. D. Dabelea: None.

Fructose Consumption Induces Strain-Specific Transcriptomic Response Perturbing Different Pathways in Genetically Diverse Mouse Strains

Metabolic syndrome (MetS) predisposes individuals to type 2 diabetes mellitus and cardiovascular diseases. Epidemiological and clinical studies support fructose intake as an environmental risk for MetS and the associated metabolic diseases. To explore the role of genomic variability in determining metabolic responses to fructose, we fed three strains of mice, namely C57/B6 (B6), DBA and FVB, with 8% fructose for 12 weeks. Fructose-fed DBA mice gained a significantly higher amount of body weight and glucose intolerance from the 4th to the 12th week, while B6 and FVB showed no differences in these phenotypes over 12 weeks. In addition, elevated insulin levels were found in fructose-fed DBA and FVB mice, and cholesterol levels were uniquely elevated in B6 mice. To explore the molecular underpinnings, we applied RNA sequencing to investigate the effect of fructose on the transcriptional profiles of liver, adipose and hypothalamus tissues. Different strains showed distinct patterns of transcriptional and pathway perturbations in a tissue-specific manner. Among pathways altered in the liver tissue, fatty acid and cholesterol metabolic pathways were prominent in B6 mice, while DBA mice showed unique over-representation of pathways related to PPAR signaling pathway. In adipose tissue, pathways are more related to fatty acid metabolism and oxidation in B6 mice, whereas no metabolic pathways were found to be enriched for differential genes in DBA and FVB mice. In hypothalamus tissue, only B6 showed significant enrichment for pathways involved in protein folding, pancreatic secretion and fatty acid beta-oxidation. Using network modeling, we predicted potential key regulators of fructose response such as FGF-21 and Sqle in the liver, Cav1 and DPP-4 in adipose tissue and Fmod in the hypothalamus. Our findings provide molecular insights into the mechanisms by which fructose contributes to the development of MetS and metabolic diseases. Disclosure G. Zhang: None. H. Byun: None. Z. Ying: None. Y. Zhao: None. F. Gomez-Pinilla: None. X. Yang: None.

Transcript profiling of genes expressed during fibre development in diploid cotton (Gossypium arboreum L.)

BackgroundCotton fibre is a single cell and it is one of the best platforms for unraveling the genes express during various stages of fibre development. There are reports devoted to comparative transcriptome study on fiber cell initiation and elongation in tetraploid cultivated cotton. However, in the present investigation, comparative transcriptome study was made in diploid cultivated cotton using isogenic fuzzy-lintless (Fl) and normal fuzzy linted (FL) lines belong to Gossypium arboreum, diploid species at two stages, 0 and 10 dpa (days post anthesis), using Affymetrix cotton GeneChip genome array.ResultScanning electron microscopy (SEM) analysis uncovered the occurrence of few fibre cell initials in the Fl line as compared to many in Normal FL at −2 and 0 dpa. However, at 10 dpa there were no fibre cells found elongated in Fl but many elongated cells were found in FL line. Up-regulation of transcription factors, AP2-EREBP, C2H2, C3H, HB and WRKY was observed at 0 dpa whereas in 10 dpa transcription factors, AP2-EREBP, AUX/IAA, bHLH, C2H2, C3H, HB, MYB, NAC, Orphans, PLATZ and WRKY were found down regulated in Fl line. These transcription factors were mainly involved in metabolic pathways such as phytohormone signaling, energy metabolism of cell, fatty acid metabolism, secondary metabolism and other signaling pathways and are related directly or indirectly in fiber development. Quantitative real-time PCR was performed to check fold up or down-regulation of these genes and transcription factors (TFs) down regulated in mutants as compared to normal at 0 and 10 dpa.ConclusionThis study elucidates that the up-regulation of transcription factors like AP2-EREBP, C2H2, C3H, HB, WRKY and phytohormone signaling genes at 0 dpa and their down-regulation at the 10 dpa might have constrain the fibre elongation in fuzzy-lintless line. Along with this the down-regulation of genes involved in synthesis of VLCFA chain, transcripts necessary for energy and cell wall metabolism, EXPANSINs, arabinogalactan proteins (AGPs), tubulin might also be the probable reason for reduced growth of fibres in the Fl. Plant receptor-like kinases (RLKs), Leucine Rich Repeats) LRR- family protein and signal transduction coding for mitogen-activated protein kinase (MAPK) cascade, have been engaged in coordination of cell elongation and SCW biosynthesis, down-regulation of these might loss the function leads to reduced fibre growth.

Abstract 2507: Using 1H NMR metabolomics to study breast cancer and endothelial cell metabolic interactions

Abstract Breast cancer is the second most commonly diagnosed cancer among women worldwide. Patient death is typically caused by metastasis development rather than the primary tumor. Metastasis in breast cancer has been shown to occur via blood and lymphatic vessels. Research shows that breast cancer cells ‘educate’ lymphatic and blood endothelial cells to support tumor growth by stimulating growth factor secretion; specifically VEGF, PDGF-BB and EGF secretion. In addition, cell metabolism is altered during malignant transformation. Cancer cells have increased energy and macromolecule biosynthesis requirements to sustain rapid proliferation. Increased angiogenesis observed in tumors points to a need for an increased nutrient supply. However, it remains unclear how tumor endothelial cell metabolism is altered and how metabolism regulates tumor angiogenesis. Therefore, this study aims to use 1H NMR metabolomics to identify breast cancer-endothelial cell metabolic interactions in a high-throughput manner. Firstly, we studied if in vitro co-culture with breast cancer altered endothelial cell metabolism. We identified distinct metabolic profiles for HUVECs grown in monoculture or co-culture with three different breast cancer cell lines. Metabolism of HUVECs co-cultured with MCF7 and SKBR3 breast cancer cell lines was significantly altered compared to mono-culture control. The most impacted metabolic pathways by co-culture were amino acid metabolism, energy metabolism and lipid biosynthesis pathways. Culturing HUVECs with MDA-MB-231 triple negative breast cancer cells did not have a significant impact on HUVEC metabolism, however. Together, these results indicate that culturing HUVECs with certain breast cancer lines causes significant changes in endothelial metabolism. Alterations in endothelial cell metabolism in response to breast cancer co-culture can further our understanding of tumor-vascular interactions and may lead to identification of metabolic biomarkers or therapeutic targets that can disrupt tumor angiogenesis. Citation Format: Suehelay Acevedo-Acevedo, Sean P. Palecek. Using 1H NMR metabolomics to study breast cancer and endothelial cell metabolic interactions [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 2507. doi:10.1158/1538-7445.AM2017-2507

Abstract LB-163: Biological differences underlying racial disparity in breast cancer

Abstract Compared to European American (EA) women, African American (AA) women have higher rates of estrogen receptor negative (ER-) breast cancer, more specifically triple-negative (TN) subtype, and show poorer prognosis and survival. Increasing evidence suggests biological differences may contribute to the observed racial disparity. Studies evaluating racial differences in gene expression in breast tumor tissue have reported inconsistent results. Study of normal breast tissue in healthy women may provide new insight into biological mechanisms for cancer development prospectively before tumor occurrence. However, racial influence on gene expression in normal breast tissue has not been extensively studied. In this study, we evaluated racial differences in gene expression in normal breast tissue obtained from 23 healthy women as well as tumor tissue obtained from 108 breast cancer patients. We analyzed RNA-sequencing data and performed differential expression analysis between AA and EA women. We found that differentially expressed genes in normal breast tissue between AA and EA women were enriched in biological pathways related to amino acid metabolism, immune response and signaling, lipid and protein biosynthesis, and cell proliferation and survival. Many of these biological differences persist in tumor tissue between EA and AA women in a subtype-specific manner. For example, in ER- breast cancer, racial differences in gene expression were enriched in amino acid metabolism, cell proliferation and survival pathways, with particular enrichment in immune response and signaling and lipid metabolism pathways in TN breast cancer. For ER+ breast cancer, we did not find any major biological pathway enrichment. In addition, we identified several microRNAs differentially expressed in normal breast tissue between EA and AA women. They may be involved in racial differences in breast carcinogenesis but their potential downstream effects are yet to be determined. Future studies are needed to validate our findings and understand racial-specific biological mechanisms in breast cancer development. Citation Format: Aditi Shendre, Yunlong Liu, Anna Maria Storniolo, Bryan P. Schneider, Jiali Han, Chunyan He. Biological differences underlying racial disparity in breast cancer [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 LB-163. doi:10.1158/1538-7445.AM2017-LB-163

Transcriptome Analysis of Differentially Expressed Genes Induced by Low and High Potassium Levels Provides Insight into Fruit Sugar Metabolism of Pear.

Potassium (K) deficiency is a common abiotic stress that can inhibit the growth of fruit and thus reduce crop yields. Little research has been conducted on pear transcriptional changes under low and high K conditions. Here, we performed an experiment with 7-year-old pot-grown “Huangguan” pear trees treated with low, Control or high K levels (0, 0.4, or 0.8 g·K2O/kg soil, respectively) during fruit enlargement and mature stages. We identified 36,444 transcripts from leaves and fruit using transcriptome sequencing technology. From 105 days after full blooming (DAB) to 129 DAB, the number of differentially expressed genes (DEGs) in leaves and fruit in response to low K increased, while in response to high K, the number of DEGs in leaves and fruit decreased. We selected 17 of these DEGs for qRT-PCR analysis to confirm the RNA sequencing results. Based on GO enrichment and KEGG pathway analysis, we found that low-K treatment significantly reduced K nutrient and carbohydrate metabolism of the leaves and fruit compared with the Control treatment. During the fruit development stages, AKT1 (gene39320) played an important role on K+ transport of the leaves and fruit response to K stress. At maturity, sucrose and acid metabolic pathways were inhibited by low K. The up-regulation of the expression of three SDH and two S6PDH genes involved in sorbitol metabolism was induced by low K, promoting the fructose accumulation. Simultaneously, higher expression was found for genes encoding amylase under low K, promoting the decomposition of the starch and leading the glucose accumulation. High K could enhance leaf photosynthesis, and improve the distribution of the nutrient and carbohydrate from leaf to fruit. Sugar components of the leaves and fruit under low K were regulated by the expression of genes encoding 8 types of hormone signals and reactive oxygen species (ROS). Our data revealed the gene expression patterns of leaves and fruit in response to different K levels during the middle and late stages of fruit development as well as the molecular mechanism of improvement of fruit sugar levels by K and provided a scientific basis for improving fruit quality with supplemental K fertilizers.

RNA-seq for comparative transcript profiling of kenaf under salinity stress

Kenaf (Hibiscus cannabinus L.) is an economically important global natural fiber crop. As a consequence of the increased demand for food crops and the reduction of available arable land, kenaf cultivation has increasingly shifted to saline and alkaline land. To investigate the molecular mechanism of salinity tolerance in kenaf, we performed Illumina high-throughput RNA sequencing on shoot tips of kenaf and identified 71,318 unigenes, which were annotated using four different protein databases. In total, 2,384 differentially expressed genes (DEGs) were identified between the salt-stressed and the control plants, 1,702 of these transcripts were up-regulated and 683 transcripts were down-regulated. Thirty-seven transcripts belonging to 15 transcription-factor families that respond to salt stress were identified. Gene ontology function enrichment analysis revealed that the genes encoding antioxidant enzymes were up-regulated. The amino acid metabolism and carbohydrate metabolism pathways were highly enriched among these DEGs under salt stress conditions. In order to confirm the RNA-seq data, we randomly selected 20 unigenes for analysis using a quntitative real-time polymerase chain reaction. Our study not only provided the large-scale assessment of transcriptome resources of kenaf but also guidelines for understanding the mechanism underlying salt stress responses in kenaf.

Transcriptomic profiling of soybean in response to UV-B and Xanthomonas axonopodis treatment reveals shared gene components in stress defense pathways

Soybean (Glycine max [L.]) has evolved to survive under abiotic and biotic stress conditions by utilizing multiple signaling pathways. Although several studies have revealed shared defense signaling pathways in plants, the majority of components at the convergence points of signaling pathways triggered by both abiotic and biotic stress remain poorly understood. Here, we profiled the overall transcriptional responses of soybean to two different types of stress using the UV-B-resistant cultivar, Buseok, and the UV-B-sensitive cultivar, Cheongja 3, as well as two near isogenic lines carrying bacterial leaf pustule (BLP) disease-resistant and -susceptible alleles. We compared transcript abundance and identified genes that commonly respond to UV-B stress and BLP disease. In addition, we surveyed the co-localization of differentially expressed genes (DEGs) and their paralogs with abiotic and biotic stress-related quantitative trait loci (QTLs) on the soybean genome. Among 14 DEGs that respond to both stresses, five DEGs are involved in the jasmonic acid (JA) metabolic pathway, encoding Jasmonate ZIM (Zinc-finger protein expressed in Inflorescence Meristem) domain-containing protein 1 (JAZ 1), a negative regulator of JA signaling. Two DEGs for JAZ 1 were co-localized with biotic stress-related QTLs. One DEG encoding the stress-induced protein starvation-associated message 22 and its two paralogs were co-localized with both abiotic and biotic stress-related QTLs. The results of this study help elucidate general responses to abiotic and biotic stress in soybean, thereby helping breeders improve stress-resistant soybean cultivars.

Abstract 4489: Using functional data from Roadmap Epigenomics to inform analysis of rare variants linked to gene expression in a large colorectal cancer study

Abstract To investigate the role of low frequency and rare genetic variation in colorectal cancer (CRC) susceptibility, the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colorectal Cancer Family Registry (CCFR) conducted whole genome sequencing and imputed into genome-wide association studies (GWAS) of 14,718 CRC cases and 12,186 controls. These data provide a unique opportunity to investigate rare variants, which contribute to the majority of the variation in the genome. To improve power for discovering rare CRC susceptibility variants (<1% MAF), Roadmap Epigenomics data were used to construct biologically relevant testing sets of enhancers, promoters and exons for gene-based association testing across the genome. Since enhancers exert their effects by impacting expression of target genes, we defined enhancer-gene networks by linking enhancer(s) to target gene expression using Roadmap chromatin state maps and gene expression. Variants in linked enhancers from digestive and immune tissues were aggregated together with variants in the promoter and non-synonymous coding variants in the target gene. We tested 9,884 variant sets for association with CRC risk using the Mixed effects Score Test (MiST). Our most significant findings are for acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor-ACADS (p = 1×10−4), AlkB homologs, including AlkB homolog 1-ALKBH1 (p = 2×10−4), and SRA stem-loop interacting RNA binding protein-SLIRP (p = 2×10−4). We will replicate these findings within the Colorectal Cancer Transdisciplinary Study (CORECT), as well as additional samples currently genotyped in CCFR and GECCO (over 25,000 CRC cases and controls). Although the top findings are statistically non-significant in this initial dataset, each of these genes linked to molecular pathways implicated in CRC carcinogenesis (fatty acid metabolism, DNA/RNA repair, and Nuclear Receptor signaling pathway, which interacts with the Wnt, beta-catenin pathways to result in a diverse array of cellular effects including altered cellular adhesion, tissue morphogenesis, and oncogenesis). Our current findings suggest that although functional insight can improve power for novel discovery, even larger sample sizes and/or pathway-based analyses are necessary to understand the role of rare variants in CRC carcinogenesis. Citation Format: Stephanie A. Bien, Tabitha A. Harrison, Paul L. Auer, Flora Qu, Jeroen Huyghe, Barbara Banbury, Peyton Greenside, Goncalo R. Abecasis, Sonja I. Berndt, Stephane Bézieau, Hermann Brenner, Graham Casey, Andrew T. Chan, Jenny Chang-Claude, Sai Chen, Joshua D. Smith, Loic Le Marchand, Christopher Carlson, Polly A. Newcomb, Christian Fuchsberger, Marty L. Slattery, Hyun M. Kang, Emily White, John Potter, Steven J. Gallinger, Michael Hoffmeister, Stephen B. Gruber, Deborah A. Nickerson, Ulrike Peters, Anshul Kundaje, Li Hsu. Using functional data from Roadmap Epigenomics to inform analysis of rare variants linked to gene expression in a large colorectal cancer study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4489.

Gene expression profile analysis of pig muscle in response to cold stress

ABSTRACTLow-temperature injury is one of the most significant causes of livestock damage worldwide. To identify genes that respond to cold stress, the transcriptome profile of the skeletal muscle of the Min pig (Sus scrofa) after cold stress was analysed using the deep sequencing technique. One group was raised in the normal environment (18 ± 2°C, n = 3) as the thermos-neutral group; another group was raised in the low-temperature environment (4 ± 2°C, n = 3) as the cold stress group. After 24 h, the gene expression profile of skeletal muscle was analysed. The results showed that the expression levels of 662 genes were significantly up-regulated, and those of 1058 genes were significantly down-regulated in the thermos-neutral and the cold stress groups (P < 0.01). It was found through Gene Ontology analysis that the significantly differently expressed genes were mainly distributed in the cytoplasm, mitochondrion, organelle envelope and ribosomal subunit. They enriched ribosome, fatty acid metabolism, RNA transport and metabolic pathways. It means that cold stress could significantly change the gene expression profile of pig muscle.

Abstract 4232: Small molecule phenotypic targeting of aggressive B-cell lymphomas

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Combination of gene expression and metabolic profiling of DLBCLs uncovered two biologically relevant entities: BCR-DLBCL (∼60% of DLBCL pts) characterized by a glycolytic metabolism and B-cell receptor (BCR) signaling activation; and OXPHOS-DLBCL characterized by enhanced mitochondrial energy transduction and increased glutathione levels. To identify small molecules that can serve as biological probes and potential therapeutics for these subgroups, we conducted a phenotypic screening in BCR-DLBCLs vs. OXPHOS-DLBCLs. We first tested cell growth inhibition capacity of 84,000 drug-like small molecules in one BCR (OCI-Ly7) vs. OXPHOS (Toledo) cell lines for 48 h at 20 µM. We identified 3,684 compounds that inhibited &amp;gt;90% the growth of either or both cell lines. In a secondary screening we selected compounds that showed a dose-response effect lower than 10 µM. We identified 31 BCR-selective, 8 OXPHOS-selective and 5 non-selective compounds. We then determined potency and phenotypic-selectivity of active compounds in an expanded panel of BCR-glycolytic (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, SU-DHL6) vs. OXPHOS (OCI-Ly4, Karpas422, Toledo, WSU-DLCL2) cell lines. Five compounds were validated as BCR-glycolytic specific and 3 as bi-specific and their GI50 values (50% growth inhibition) were determined. We further characterized the most selective (&amp;gt;5-fold) and potent (low µM) BCR-glycolytic specific small molecules, termed LI-2 and LI-5. These compounds showed GI50 between 500 nM - 3 µM in BCR-glycolytic vs 9-20 µM in OXPHOS DLBCLs. LI-2 and LI-5 selectively induced 60-80% caspase-dependent apoptosis (p≤0.05) after 24 h at 2.5 µM in BCR-glycolytic but not in OXPHOS. To determine phenotypic changes upon treatment with LI-2 and LI-5, we performed RNA-sequencing on BCR-glycolytic DLBCL OCI-Ly7 and SUDHL-6 cells. LI-2 signature included 91 up- and 76 down-regulated transcripts (FC&amp;gt;1.2, FDR adjusted p&amp;lt;0.05), whereas LI-5 comprised 493 up- and 248 down-regulated genes. Computational analysis revealed that both compounds affected survival and cell cycle pathways (p=5.2x10-7) as well as amino acid and glucose metabolism pathways (p=2.4x10-8). LI-2 specifically affected DNA mismatch repair and replication pathways (p=3.9x10-5), and LI-5 specifically affected cytokine and growth factors signaling pathways (p=2.4x10-24). When compared to other known drug effects, LI-2 showed an expression signature similar to valinomycin (an OXPHOS uncoupler) and to pyrvinium (that kills cancer cells upon glucose starvation), suggesting that activity of TCA and glycolytic pathways are both critical for BCR-glycolytic cells survival. LI-5 relates to the protein synthesis inhibitor anisomycin and the cell cycle inhibitor podophyllotoxin. In sum, phenotypic screening provides a basis for discovery of distinct susceptibilities of DLBCL subtypes that could be capitalized towards individualized therapies. Citation Format: Mariano G. Cardenas, Matt R. Teater, Nieves Calvo-Vidal, Shao Ning Yang, J F. Glickman, Ari Melnick, Leandro Cerchietti. Small molecule phenotypic targeting of aggressive B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2014-4232

Abstract 219: Tissue-Specific RNAseq Identifies Novel and Cardiometabolic Disease--Associated Long Intergenic Noncoding RNAs in Human Adipose

Objective: The purpose is to identify novel long intergenic noncoding RNAs (lincRNAs) in human adipose, examine the association of their genetic variants with cardiometabolic traits, and explore their function in adipocytes. Methods and results: We performed deep high-throughput RNAseq (&gt;200 million reads/sample) in adipose tissues of 13 volunteers. Of 54,944 human lincRNAs, 6,558 were detected in adipose. We utilized multiple GWAS meta-analysis of adipose-related cardiometabolic traits (e.g. BMI, triglyceride and HDL cholesterol) and interrogated 6,558 adipose LincRNAs for their overlap with GWAS SNPs. After performing Bonferroni correction for the number of adipose lincRNA SNPs tested, we identified 46 SNPs in 9 lincRNAs, 28 SNPs in 13 lincRNAs and 13 SNPs in 6 lincRNAs that were significantly associated with waist-hip ratio adjusted BMI, HDL and triglyceride respectively. Notably, RP11-392O17.1, more abundantly expressed in adipose, and linc-NFE2L3-1, predominantly detected in adipose and skeleton muscle, contain 22 and 4 SNPs reaching genome wide significance for BMI (lead SNPs rs12031603, P=2.15х10 -9 and rs10267498, P=2.73х10 -8 respectively). Linkage disequilibrium analysis confirmed RP11-392O17.1 and linc-NFE2L3-1 harbor the strongest GWAS signals in the loci, suggesting causality for GWAS association with BMI. Several adipose lincRNAs, including RP11-392O17.1, linc-NFE2L3-1 and linc-AQPEP, were induced during in vitro human adipocyte differentiation. As an example of adipose lincRNA functional impacts, Linc-AQPEP, an abundant adipose-specific lincRNA, was knocked down by 70% with lentiviral-based short hairpin RNAs, resulting in ~35% decrease of triglyceride content in human adipocytes. RNAseq analysis demonstrated differential expression (DE) in 3,291 of 13,653 adipocyte genes with Linc-AQPEP knockdown (FDR&lt;0.01) and significant enrichments of DE genes in lipid biosynthesis, fatty acid metabolism and mitochondrion pathways. Additional studies are ongoing to further investigate biological roles of human lincRNAs in adipocytes. Conclusion: Our data suggest human LincRNAs may play specific roles in adipocyte biology and that their genetic variations may contribute to cardiometabolic traits.

Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis

Activated fibroblasts, also known as myofibroblasts, are mediators of several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. We previously identified Niemann-Pick type C2 (NPC2) protein as a negative regulator of fibroblast activation (Csepeggi, C., Jiang, M., Kojima, F., Crofford, L. J., and Frolov, A. (2011) J. Biol. Chem. 286, 2078-2087). Here we report that NPC2-deficiency leads to a dramatic up-regulation of the arachidonic acid (AA) metabolic pathway in human fibroblasts. The major enzymes in this pathway, cPLA2 type IVA, COX-2, and mPGES-1, were dramatically up-regulated at both the transcriptional and translational levels. The specific phenotypic changes resulted in a >10-fold increase in the production and secretion of a key modulator of inflammation and immunity, prostaglandin E2. More importantly, AA metabolome profiling by liquid chromatography/tandem mass-spectrometry revealed the very specific nature of prostaglandin E2 up-regulation as the other analyzed AA metabolites derived from the COX-2, cytochrome P450, 5/15-lipoxygenase, and non-enzymatic oxidative pathways were mostly down-regulated. Blocking activity of cPLA2 efficiently suppressed expression of inflammatory cytokines, IL-1β and IL-6, thereby identifying cPLA2 as an important regulator of the inflammatory program in NPC2-null cells. Altogether, these studies highlight NPC2 as a specific regulator of AA metabolism and inflammation that suggests potential for NPC2 protein or its related signaling in the treatment of inflammatory diseases characterized by the presence of activated fibroblasts.

Abstract 5418: Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells .

Abstract Many cancer cells generate energy by rapidly converting glucose to lactate in the cytosol, a process termed aerobic glycolysis. This metabolic phenotype is recognized as one of the hallmarks of cancer and is enabled by lactate dehydrogenase (LDH), which catalyzes pyruvate to lactate inter-conversion. We find that hepatocellular carcinoma cells express micromolar quantities of LDH5 and that LDH5 protein down-regulation takes about 5 days allowing time for the cells to adapt their metabolism. Since metabolic processes happen in minutes, addressing consequences of LDH5 inhibition by protein down-regulation is inadequate. We screened the GSK compound library and identified a series of quinoline acids as NADH-competitive LDH5 inhibitors. Subsequent lead optimization yielded molecules with LDH5 inhibitory potencies as low as 2-3 nM and selectivity over LDH1 on the order of 10-100-fold. These molecules were cell-permeable and did not have any appreciable activity against a panel of approximately fifty common enzymes, receptors and ion channels, making them the most potent and selective LDH5 inhibitors identified to date. Using these tool inhibitors, we find that rapid chemical inhibition of LDH5 in Snu398 hepatocellular carcinoma cells results in profound inhibition of lactate production and increase in pyruvate as measured by mass spectrometric analysis. Real-time analysis by NMR spectroscopy of live Snu398 cells fed with 13C-labeled glucose demonstrated that chemical LDH5 inhibition led to a rapid decrease in glucose uptake and concomitant slow-down of lactate production. Comprehensive analysis of more than 500 metabolites upon LDH5 inhibition in Snu398 cells revealed that the cytosolic glycolysis pathway was significantly impeded with some up-stream intermediates increasing as much as 40-fold. As the cell lost its ability for cytosolic glucose processing, the TCA cycle activity increased indicating that pyruvate entered the mitochondria and restored their activity resulting in increased oxygen consumption upon LDH5 inhibition. Several pathways that rely on glycolytic and TCA intermediates were also upregulated, including fatty acid metabolism and pentose phosphate pathway. LDH5 inhibition also strongly potentiated PKM2 activity. These profound metabolic alterations greatly impaired cell survival and induced cell death in Snu398 cells. In summary, we have shown that rapid chemical inhibition of LDH5 leads to profound metabolic alterations and impairs cell survival in hepatocellular carcinoma cells making it a compelling strategy for treating solid tumors relying on aerobic glycolysis. Citation Format: Julia Billiard, Roland Annan, Jennifer Ariazi, Jacques Briand, Kristin Brown, Nino Campobasso, Subhas Chakravorty, Deping Chai, Mariela Colón, Elizabeth Davenport, Christopher Dodson, Nathan Gaul, Seth Gilbert, Anthony Jurewicz, Hong Lu, Dean McNulty, Jeanelle McSurdy-Freed, Lisa Miller, Kelvin Nurse, Paru Rao Nuthulaganti, Chad Quinn, Jessica Schneck, Gilbert Scott, Tony Shaw, Christian Sherk, Angela Smallwood, Sharon Sweitzer, James Villa, Gregory Waitt, Richard Wooster, Kevin Duffy. Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2013-5418

Differential gene expression in liposarcoma: Insight into pathways for dedifferentiation?

10048 Background: Liposarcoma (LPS) dedifferentiation signifies conversion to a clinically aggressive phenotype, but the biologic processes required for this change have not been determined. We describe differential gene expression patterns between well-differentiated (WD) and dedifferentiated (DD) tumors to determine pathways involved in LPS dedifferentiation. Methods: From 1999 to 2006, 121 fatty tumors were resected at a single institution. Twenty tumors, consisting of atypical lipomatous tumors (ALT), WD LPS or DD LPS, were randomly selected and clinicopathologic characteristics were retrospectively reviewed. Gene expression profiling was performed on extracted RNA using the Affymetrix GeneChip platform. Differentially expressed genes were obtained and gene network analysis was done using GeneGO by MetaCore. Results: Median age was 59 years and 70% of cases were male. WD tumors, consisting of 3 ALT and 6 WD LPS, were compared with 11 DD LPS. After a median follow-up of 64 months, 7 patients had died of whom 6 had DD LPS. DD histology was associated with lower overall survival (p&lt;0.05). Significance Analysis of Microarrays for WD tumors vs. DD LPS using a 0% false discovery rate showed differential expression of 188 genes. Network analysis of genes from WD tumors vs. DD LPS showed significant (p&lt;0.001) differential regulation of glucose-activated transcription factor ChREBP (carbohydrate response element binding protein), a key element involved in lipogenesis, gluconeogenesis and glycolysis. There was also significant differential regulation of insulin signaling, PI3K-dependent and PKA signal transduction pathways and of amino acid, fatty acid and glucose metabolism pathways (p&lt;0.05). These pathways, based on Gene Ontology cellular processes, mapped to gene networks primarily involved in lipid metabolism (p&lt;0.05). Conclusions: Differential expression of genes involved in lipid metabolism networks is seen in DD LPS and changes in lipid metabolism may be associated with dedifferentiation. These differential gene expression patterns may help identify fatty tumors potentially at risk for progressing to a malignant or DD state and provide prognostic factors and therapeutic targets for patients with LPS.

001 NOVEL BIOMARKERS OF KNEE OSTEOARTHRITIS IDENTIFIED USING METABOLIC PROFILING

Purpose: There is a pressing need to develop reliable molecular biomarkers that can inform on the process of joint destruction in osteoarthritis (OA). Such biomarkers could aid in drug development by identifying fast progressors and early response to therapy. Recent advance in metabolomics (the quantitative analysis of all metabolites present within a biological sample) has opened new avenues for biomarker identification. The aim of the study, therefore, was to identify serum metabolic biomarkers for OA using a metabolomics approach. Methods: 123 knee OA cases and 299 controls were selected from the TwinsUK cohort. Knee OA was defined as either radiographic, self-reported OA, or total knee joint replacement due to primary OA. All the subjects were unrelated Caucasian females. Their frozen serum samples were retrieved and assessed for targeted metabolite profiling using API4000 Q TRAP LC/MS/MS System (Applied Biosystems, Darmstadt, Germany) equipped with Schimadzu Prominence LC20AD pump and SIL-20AC auto sampler with AbsoluteIDQTM Kit (Biocrates life sciences AG, Austria). Results: A total of 163 serum metabolites were assessed and their concentrations were obtained. The mean of the coefficient of variation (CV) for the 163 metabolites was 0.07±0.05 and 90% of the metabolites had a CV of less than 0.10. The ratios of all pair metabolite concentrations were calculated and tested for the association with knee OA. Overall 14 ratios were significantly associated with knee OA with p ≤ 1.9×10-6 which was the significance level after conservative Bonferroni correction. These ratios were mainly involved in valine and acylcarnitine, namely the ratios of valine to arginine, glutamine, glycine, histidine, tyrosine, and dodecanedioyl-L-carnitine, and the ratios of fumaryl-L-carnitine to propenoyl-L-carnitine, dodecanedioyl-L-carnitine, and lysophosphatidylcholine acyl C26:1. The association between these ratios and knee OA remained strong even after adjustment for age and BMI. Conclusions: This is the first study reporting the identification of novel OA biomarkers using a metabonomics approach. The results suggest that amino acid and acylcarnitine metabolic pathways involving valine and fumaryl-L-carnitine are implicated in the development of OA and have potential clinical use as biomarkers.

Changes of gene expression profiling and pathways related fat metabolisim in femoral head of steroid-induced osteonecrotic rats

Objective To understand the changes of gene expression profiling and pathways related to fat metabolism in femoral head of steroid-induced osteonecrotic rats in contribution to pathogenesis of steroid-induced osteonecrosis. Methods We applied lipopolysaccharide (LPS) and methylprednisolone (MPSL) to prepare a rat model of steroid-induced osteonecrosis (SIO). Rats were divided into two groups: normal (N) group and model (M) group. All rats were sacrificed after 6 weeks. Then we applied a genome wide cDNA microarray technology to analyze genes expressed in femoral head of two groups and also serum levels of cholesterol (TC) were measured in the two groups. Results (1) There were a total of 111 expression genes which were changed by a minimum of two-fold and also it was found that 18 pathways had significant changes in these differential genes by molecule annotation system (MAS) analysis in femoral head of model group compared with normal rats. (2) There were 6 upregulated genes related to fatty acid metabolism (FAM) pathway ( Acadl, Cpt2, Acaa2, Acat1, Acsl1, Hadhb ), 2 upregulated genes related to fatty acid elongation in mitochondria (FAEM) ( Acaa2, Hadhb ), 3 upregulated genes related to adipocytokine signaling pathway (ACK) ( Cpt2, Prkaa2, Acsl1 ) et al in femoral head of model group compared with normal rats. (3) Serum levels of cholesterol significantly increased 21.2% in the model group compared with the normal group (1.754 ± 0.264 mol/L vs 1.447 ± 0.142 mol/L, p Conclusions The data suggest that the upregulated genes related to fat metabolism in the pathways of femoral head of steroid-induced osteonecrotic rats might play an important role in promoting the synthesis of fatty acid and cholesterol and fat metabolism could be mediated by FAM, FAEM and ACK pathways. The changes of multiple gene expression and numerous pathways could play major roles in steroid-induced osteonecrosis pathogenesis. (This study was supported by National Natural Science Foundation of China, NSFC 30472173).