Parametric Analysis Of Gene Set Enrichment Research Articles

Abstract 3433: Identification of a subtype specific molecular field across the mammary gland of breast cancer patients

Abstract Introduction: The role of field cancerization in sporadic breast cancer is not known. Clinical findings of multi-focality and the presence of molecular aberrations in the histologically normal tissue surrounding the primary tumor suggest that there is a field effect. We hypothesized that field cancerization is present in breast cancer and that a subtype specific molecular field can be identified. Methods: RNA exome sequencing was performed using paraffin embedded sections obtained from breast cancer patients undergoing mastectomy; 25 cases were selected from each subtype: hormone receptor (HR) positive, Her2+, triple negative (TN) breast cancer. 4 sites were sampled from each patient: primary tumor (A), adjacent normal parenchyma within 2cm of the tumor (B) 2 separate, histologically normal sites at least 2cm away from the tumor(C &D) or, if available, tissue from the histologically normal contralateral breast (E). Normal breast tissue (N) from cancer free controls was obtained from reduction mammoplasty. Pathway analysis was performed by parametric analysis of gene set enrichment (PAGE). Differential genes and pathways were identified by LIMMA and a FDR adjusted q value of < 0.05 was considered significant. To estimate tumor content across samples, we applied deconvolution analysis to tumor associated genes (TAG) to calculate indices which range from 0 (normal) to 1 (tumor). Genes or pathways that are associated with the field effects were identified by Pearson's correlation coefficient analysis. Results: TAGs within each subtype of breast cancer were derived by identifying genes with at least 2-fold change between tumors (A) as compared to controls (N). Out of a total of 640, 470, and 829 TAGs identified for Her2+, HR, and TN, respectively, 283 TAGs, including 251 downregulated genes and 32 upregulated genes, were shared among all 3 subtypes. Using the tumor content indices calculated by deconvolution analysis, the degree to which the tumor content (A) was shared by histologically normal areas of breast (B,C, D,E) was determined. Overall, across all subtypes, shared tumor contents range from ~50% (E) to 80% (B) in contrast to ~15% of normal controls (N). Pathway scores were calculated for 3,195 gene sets/biological pathways downloaded from Molecular Signatures Database. 38, 18, and 21 deregulated pathways were identified that were shared across the breast parenchyma (samples A-E) in Her2+, HR, and TN, respectively. 3 shared pathways-Immune-, ILS- signaling and CP2_01- were deregulated across the breast parenchyma in all tumor subtypes. Conclusions: Our study suggests the presence of a breast cancer molecular field effect that extends beyond adjacent normal breast tissue and includes the entire mammary gland. This field effect appears to be largely subtype specific. These findings provide new insight about the early changes across the mammary gland that may be contributing to subtype specific breast cancer risk. Citation Format: Isabelle Bedrosian, Zhenlin Ju, Anjana Bhardwaj, Brandon Mistretta, Preethi H. Gunaratne, Jing Wang, Randa El-zein. Identification of a subtype specific molecular field across the mammary gland of breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3433.

Frailty in middle age is associated with frailty status and race-specific changes to the transcriptome.

Frailty is an aging-associated syndrome resulting from diminished capacity to respond to stressors and is a significant risk factor for disability and mortality. Although frailty is usually studied in old age, it is present in mid-life. Given the increases in mortality statistics among middle-aged Americans, understanding molecular drivers of frailty in a younger, diverse cohort may facilitate identifying pathways for early intervention. We analyzed frailty-associated, genome-wide transcriptional changes in middle-aged blacks and whites. Next generation RNA sequencing was completed using total RNA from peripheral blood mononuclear cells (n = 16). We analyzed differential gene expression patterns and completed a parametric analysis of gene set enrichment (PAGE). Differential gene expression was validated using RT-qPCR (n = 52). We identified 5,082 genes differentially expressed with frailty. Frailty altered gene expression patterns and biological pathways differently in blacks and whites, including pathways related to inflammation and immunity. The validation study showed a significant two-way interaction between frailty, race, and expression of the cytokine IL1B and the transcription factor EGR1. The glucose transporter, SLC2A6, the neutrophil receptor, FCGR3B, and the accessory protein, C17orf56, were decreased with frailty. These results suggest that there may be demographic dependent, divergent biological pathways underlying frailty in middle-aged adults.

Abstract 2426: JAK inhibitors as new drugs for treatment of paclitaxel-resistant anaplastic thyroid cancer

Abstract Background: Anaplastic thyroid carcinoma (ATC) is a rare but particularly aggressive solid tumor that has a poor prognosis, despite multimodal treatment approaches. New chemotherapy agents are being developed and taxanes have efficacy against ATC. Targeted agents including fosbretabulin, imatinib, erlotinib and gefitinib have also been examined, but were unsuccessful. These results suggest that innovative strategies are required for treatment of ATC. Therefore, our primary aim was to determine the genes and pathways related to sensitivity to paclitaxel as biomarkers in ATC. Our secondary aim was to identify new genes and pathways as therapeutic targets in ATC. Materials and Methods: We analyzed differences in gene expression profiles before and after treatment with paclitaxel in two ATC cell lines: KTA-3 cells, which are sensitive to paclitaxel, and TTA-2 cells, which are resistant to paclitaxel. Parametric Analysis of Gene Set Enrichment (PAGE) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Transcriptional Regulatory Element Database (TRED) were performed to detect significant differences at the level of functional categories such as pathway genes and transcriptionally regulated genes. Results: Genes in the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway (KEGG classification) were downregulated significantly in KTA-3 cells compared with TTA-2 cells after treatment with paclitaxel. PAGE showed that breast cancer susceptibility gene (BRCA)1-related genes including Janus kinase (JAK)1, JAK2 and signal transducer and activator of transcription (STAT)3 were downregulated most significantly in KTA-3 cells compared with TTA-2 cells after paclitaxel treatment. Several JAK inhibitors inhibited cell growth in paclitaxel-resistant ATC cells. Conclusions: JAK-STAT pathway may be a biomarker of paclitaxel sensitivity in ATC and may represent a new target in drug development for ATC therapy. Citation Format: Tomoyuki Fujita, Takayuki Sasaki, Zheng Wang, Kayoko Koshikawa, Hajime Nishimura, Minoru Fujimori. JAK inhibitors as new drugs for treatment of paclitaxel-resistant anaplastic thyroid cancer. [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 2426. doi:10.1158/1538-7445.AM2015-2426

Custom Microarray Analysis for Transcript Profiling of Dormant Vegetative Buds of Japanese Apricot during Prolonged Chilling Exposure

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.

Microarray and gene ontology analyses reveal downregulation of DNA repair and apoptotic pathways in diethylstilbestrol-exposed testicular Leydig cells

This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17β-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories "DNA repair" and "apoptotic program" in DES-exposed cells. PAGE showed that "cell death," which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the "cell death" category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.

Transcriptome analysis of fruit development of a citrus late-ripening mutant by microarray

Mingliutianju ( Citrus reticuiata Blanco cv.Mingliutianju, MP) is a novel late ripening cultivar selected from Chuntianju ( Citrus reticuiata Blanco cv.Chuntianju, CP) through spontaneous bud mutation. Large-scale analysis of gene expression differences between of peels of MP and CP is studied during the course of 12 weeks and 23 weeks after flowering (WAF) using Affymetrix GeneChip ® citrus genome array to provide new insights into the mechanisms behind this bud mutation. Altogether, 395 differentially expressed genes are identified including 132 up-regulated and 263 down-regulated genes. Singular enrichment analysis (SEA) and parametric analysis of gene set enrichment (PAGE) are performed and 7 statistically significant differentially expressed genes are selected finally for further study. Results from semiquantitative RT-PCR performed on these 7 genes show that one gene's expression situation is totally correlated with that observed with the microarray. These seven genes include three UDP-glucosyltransferase genes, two disease resistance-responsive protein-related genes, one chitinase gene and one protein kinase family protein gene, all of which are involved in 18 different biological processes according to the hierarchical tree of annotated gene ontology (GO) terms. The candidate biological process and its relation to bud mutation are discussed.

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction, Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

BackgroundAging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.Methodology/Principal FindingsWe investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.Conclusions/SignificanceThese findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

AgriGO: a GO analysis toolkit for the agricultural community

Gene Ontology (GO), the de facto standard in gene functionality description, is used widely in functional annotation and enrichment analysis. Here, we introduce agriGO, an integrated web-based GO analysis toolkit for the agricultural community, using the advantages of our previous GO enrichment tool (EasyGO), to meet analysis demands from new technologies and research objectives. EasyGO is valuable for its proficiency, and has proved useful in uncovering biological knowledge in massive data sets from high-throughput experiments. For agriGO, the system architecture and website interface were redesigned to improve performance and accessibility. The supported organisms and gene identifiers were substantially expanded (including 38 agricultural species composed of 274 data types). The requirement on user input is more flexible, in that user-defined reference and annotation are accepted. Moreover, a new analysis approach using Gene Set Enrichment Analysis strategy and customizable features is provided. Four tools, SEA (Singular enrichment analysis), PAGE (Parametric Analysis of Gene set Enrichment), BLAST4ID (Transfer IDs by BLAST) and SEACOMPARE (Cross comparison of SEA), are integrated as a toolkit to meet different demands. We also provide a cross-comparison service so that different data sets can be compared and explored in a visualized way. Lastly, agriGO functions as a GO data repository with search and download functions; agriGO is publicly accessible at http://bioinfo.cau.edu.cn/agriGO/.

The transcription factor Eya2 prevents pressure overload-induced adverse cardiac remodeling

Eyes absent 2 (Eya2) is a transcription factor involved in a number of cellular and developmental processes. We have previously shown that Eya2 is up-regulated during regression of cardiac hypertrophy and blocks phenylephrine-induced development of cardiomyocyte hypertrophy in vitro, suggesting that Eya2 is a negative regulator of cardiac hypertrophy. In this study, we generated transgenic mice with cardiac-specific overexpression of Eya2 to elucidate the in vivo function of Eya2 in cardiac remodeling. Mild cardiac hypertrophy developed in Eya2 transgenic mice under baseline conditions with no obvious structural or functional defects. Eya2 overexpression inhibited development of cardiac hypertrophy as judged by the abrogation of increases in heart weight and cross-sectional cell surface areas and re-activation of fetal genes under pressure overload (4 weeks). Eya2 overexpression also prevented wall thinning, ventricular dilation, and deterioration of cardiac function as well as fibrosis and inflammation in the heart under long-term pressure overload (12 weeks). Gene expression profiling using the parametric analysis of gene set enrichment (PAGE) method revealed that gene sets related to mitochondrial biogenesis and metabolism were elevated in the Eya2 transgenic mice. We also observed that the PI3K/Akt/mTOR signaling cascade was preserved in the Eya2 transgenic mice, while it was significantly reduced in the wild type littermates under pressure overload. These results demonstrate that Eya2 prevents adverse cardiac remodeling under pressure overload partly through altering metabolic gene expression and preserving PI3K/Akt/mTOR signaling pathway.

Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of Bcl-2-over-expressing immortalized breast cell line

Bcl-2 is an anti-apoptotic protein that is frequently over-expressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably over-expressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRT-PCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G1-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 up-regulated and 56 down-regulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Down-regulation of the phosphatase CDC25A could solely be responsible for the slow growth phenotype in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results.

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Fat But Healthy

Resveratrol is a polyphenolic compound produced by plants and found in red wine. It has been reported to extend life span in various model organisms, presumably through its ability to stimulate the deacetylase activity of sirtuins (there are seven isoforms in mammals, SIRT1 through 7). Baur et al . report that in mice, the addition of resveratrol to a high-calorie, high-fat diet (HCR) prevented much of the aging and disease associated with obesity without affecting body weight. Mice fed the HCR diet showed increased life span compared with high-calorie, high-fat diet (HC)-fed mice, and longevity was similar to that of mice on a standard diet. HCR mice exhibited greater insulin sensitivity and decreased circulating concentrations of glucose, insulin, and insulin-like growth factor 1 (IGF-1). The activity of adenosine monophosphate-activated protein kinase (AMPK), which is a crucial regulator of metabolism, was increased to a greater extent in the livers of the HCR-fed mice than in the livers of the HC-fed mice. Histology and assessment of various markers for the function of liver, pancreas, and heart indicated that the HCR diet prevented the damage associated with obesity. Acetylation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) was decreased in the livers of the HCR-fed mice. PGC-1α is a substrate for SIRT1, and its acetylation status is used as a measure of SIRT activity. Parametric analysis of gene set enrichment (PAGE), a computational method for analyzing microarray data, suggested that resveratrol affected 127 biochemical and signaling pathways. Notable were the down-regulation of genes associated with insulin signaling and metabolism and the up-regulation of genes associated with STAT3 (signal transducer and activator of transcription 3) signaling. STAT3 signaling declines with age and may contribute to liver regeneration. Although it would obviously be best to limit caloric intake and exercise, this study suggests that there may be pharmacological interventions that can prevent some of the diseases associated with obesity (see Kaeberlein and Rabinovitch). M. Kaeberlein, P. S. Rabinovitch, Grapes versus gluttony. Nature 444 , 280-281 (2006). [PubMed] J. A. Baur, K. J. Pearson, N. L. Price, H. A. Jamieson, C. Lerin, A. Kalra, V. V. Prabhu, J. S. Allard, G. Lopez-Lluch, K. Lewis, P. J. Pistell, S. Poosala, K. G. Becker, O. Boss, D. Gwinn, M. Wang, S. Ramaswamy, K. W. Fishbein, R. G. Spencer, E. G. Lakatta, D. Le Couteur, R. J. Shaw, P. Navas, P. Puigserver, D. K. Ingram, R. de Cabo, D. A. Sinclair, Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444 , 337-342 (2006). [PubMed]

PAGE: Parametric Analysis of Gene Set Enrichment

BackgroundGene set enrichment analysis (GSEA) is a microarray data analysis method that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. GSEA is especially useful when gene expression changes in a given microarray data set is minimal or moderate.ResultsWe developed a modified gene set enrichment analysis method based on a parametric statistical analysis model. Compared with GSEA, the parametric analysis of gene set enrichment (PAGE) detected a larger number of significantly altered gene sets and their p-values were lower than the corresponding p-values calculated by GSEA. Because PAGE uses normal distribution for statistical inference, it requires less computation than GSEA, which needs repeated computation of the permutated data set. PAGE was able to detect significantly changed gene sets from microarray data irrespective of different Affymetrix probe level analysis methods or different microarray platforms. Comparison of two aged muscle microarray data sets at gene set level using PAGE revealed common biological themes better than comparison at individual gene level.ConclusionPAGE was statistically more sensitive and required much less computational effort than GSEA, it could identify significantly changed biological themes from microarray data irrespective of analysis methods or microarray platforms, and it was useful in comparison of multiple microarray data sets. We offer PAGE as a useful microarray analysis method.