Expression Microarray Studies Research Articles

A Potential Immune-Related miRNAs Regulatory Network and Corresponding Diagnostic Efficacy in Schizophrenia.

Immune-related pathways actively participate in the progression of schizophrenia (SCZ), however, roles of immune-related miRNAs in SCZ are still unclear. A microarray expression study was conducted to explored roles of immune-related genes in SCZ. Functional enrichment analysis by using "clusterProfiler" was used to identify molecular alterations of SCZ. Protein-protein interaction (PPI) network was constructed and helped core molecular factors identification. Based on The Cancer Genome Atlas (TCGA) database, clinical significances of hub immune-related genes in cancers were also been explored. Then, correlation analyses were used to determine immune-related miRNAs. We further validated that hsa-miR-1299 could be an effective diagnostic biomarker for SCZ via analyzing multi-cohorts' data and quantitative real-time PCR (qRT-PCR). A total of 455 mRNAs and 70 miRNAs that were differentially expressed between SCZ and control samples. Functional enrichment analysis based on differentially expressed genes (DEGs) hinted that immune-related pathways were significantly correlated with SCZ. Furthermore, a total of 35 immune-related genes that involved in disease onset and showed significant co-expressed relationships. Hub immune-related gene CCL4 and CCL22 are valuable in tumor diagnosis and survival prediction. Furthermore, we also identified 22 immune-related miRNAs that play important roles in this disease. An immune-related miRNAs-mRNAs regulatory network was constructed to provide miRNAs regulatory roles in SCZ. Core miRNAs expression status of hsa-miR-1299 were also validated in another cohort, which suggested its diagnostic performance for SCZ. Our study reports the downregulation of some miRNAs in the process of SCZ are important. Shared genomics characteristics between SCZ and cancers also provide novel insights for cancers. A significant alteration of hsa-miR-1299 expression is effective as biomarker for the diagnosis of SCZ, suggesting that this miRNA could be a specific biomarker.

Identification of dysregulated canonical pathways associated with pathogenesis and progression of Amyotrophic Lateral Sclerosis-An integrated bioinformatics approach.

The mechanisms responsible for the pathogenesis and progression of Amyotrophic Lateral Sclerosis (ALS) remain poorly understood, making the diagnosis of ALS challenging. We aimed to find the novel gene biomarkers via computationally analyzing microarray expression studies, in three different cell lineages, namely myotube cells, astrocyte cells and oligodendrocyte cells. Microarray gene expression profiles were obtained and analyzed for three cell types: myotube cell lineage (GSE122261), astrocyte, and oligodendrocyte cell lineage (GSE87385). A comprehensive computational pipeline, tailored explicitly for microarray gene expression profiling studies, was devised to analyze the sample groups, wherein the myotube sample group comprised of six control (GSM3462697, GSM3462698, GSM3462699, GSM3462700, GSM3462701, GSM3462702) & six diseased (GSM3462691, GSM3462692, GSM3462693, GSM3462694, GSM3462695, GSM3462696) samples were considered. Similarly, for the astrocyte sample group two samples each for the control (GSM2330040, GSM2330042) and the diseased (GSM2330039, GSM2330041), and for the oligodendrocyte sample group, 2 control (GSM2330043, GSM2330045) samples and two diseased (GSM2330044, GSM2330046) samples were considered for the current study. The in-depth interaction of these DEGs was studied using MCODE and subjected to preliminary functional analysis using ClueGO/CluePedia plug-in. Qiagen's IPA software was employed for enrichment analysis, which generated the key canonical pathways and a list of potential biomarker molecules specific to each sample group. The preliminary analysis yielded 512 DEGs across all 3-sample groups, wherein 139 DEGs belonged to the myotube sample group, 216 DEGs for the astrocyte sample group, and 157 DEGs for the oligodendrocytes sample group. The data suggests growth hormone signaling and its activity, ErbB signaling pathway, and JAK/STAT signaling pathway are some of the pathways that are significantly dysregulated and play a crucial role in the development and progression of ALS. KISS1R and CSHL1 are potential genes that could act as diagnostic biomarkers in myotube cell types. Also, KRAS, TGFB2, JUN, and SMAD6 genes may be used as prognostic biomarkers to differentiate between early and late-stage ALS-diseased patients.

Unraveling the Link between Interferon-α and Systemic Lupus Erythematosus: From the Molecular Mechanisms to Target Therapies.

Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease with a wide range of clinical expressions. The kidney is often affected, usually within 5 years of the onset of SLE, and lupus nephropathy (LN) carries a high risk for increased morbidity. The clinical heterogeneity of the disease is accompanied by complex disturbances affecting the immune system with inflammation and tissue damage due to loss of tolerance to nuclear antigens and the deposition of immune complexes in tissues. Several studies have reported that in human SLE, there is an important role of the Type-I-interferons (INF) system suggested by the upregulation of INF-inducible genes observed in serial gene expression microarray studies. This review aims to describe the transduction pathways of Type-I-interferons, in particular INFα, and its immune-regulatory function in the pathogenesis of SLE and, in particular, in LN. In addition, recent novelties concerning biologic therapy in LN will be discussed.

Gene expression profiling of tumor stroma interactions in retinoblastoma

We aimed to identify the critical molecular pathways altered upon tumor stroma interactions in retinoblastoma (RB). In vitro 2 D cocultures of RB tumor cells (Weri-Rb-1 and NCC-RbC-51) with primary bone marrow stromal cells (BMSC) was established. Global gene expression patterns in coculture samples were assessed using Affymetrix Prime view human gene chip microarray and followed with bioinformatics analyses. Key upregulated genes from Weri-Rb-1 + BMSC and NCC-RbC-51 + BMSC coculture were validated using qRT-PCR to ascertain their role in RB progression. Whole genome microarray experiments identified significant (P ≤ 0.05, 1.1 log 2 FC) transcriptome level changes induced upon coculture of RB cells with BMSC. A total of 1155 genes were downregulated and 1083 upregulated in Weri-Rb-1 + BMSC coculture. Similarly, 1865 genes showed downregulation and 1644 genes were upregulation in NCC-RbC-51 + BMSC coculture. The upregulated genes were significantly associated with pathways of focal adhesion, PI3K-Akt signalling, ECM-receptor interaction, JAK-STAT, TGF-β signalling thus contributing to RB progression. Validation of key genes by qRT-PCR revealed significant overexpression of IL8, IL6, MYC and SMAD3 in the case of Weri-Rb-1 + BMSC coculture and IL6 in the case of NCC-RbC-51 + BMSC coculture. The microarray expression study on in vitro RB coculture models revealed the pathways that could be involved in the progression of RB. The gene signature obtained in a stimulated model when a growing tumor interacts with its microenvironment may provide new horizons for potential targeted therapy in RB.

Microarray expression studies on bone marrow of patients with Shwachman-Diamond syndrome in relation to deletion of the long arm of chromosome 20, other chromosome anomalies or normal karyotype

BackgroundClonal chromosome changes are often found in the bone marrow (BM) of patients with Shwachman-Diamond syndrome (SDS). The most frequent ones include an isochromosome of the long arm of chromosome 7, i (7)(q10), and an interstitial deletion of the long arm of chromosome 20, del (20)(q). These two imbalances are mechanisms of somatic genetic rescue. The literature offers few expression studies on SDS.ResultsWe report the expression analysis of bone marrow (BM) cells of patients with SDS in relation to normal karyotype or to the presence of clonal chromosome anomalies: del (20)(q) (five cases), i (7)(q10) (one case), and other anomalies (two cases). The study was performed using the microarray technique considering the whole transcriptome (WT) and three gene subsets selected as relevant in BM functions. The expression patterns of nine healthy controls and SDS patients with or without chromosome anomalies in the bone marrow showed clear differences.ConclusionsThere is a significant difference between gene expression in the BM of SDS patients and healthy subjects, both at the WT level and in the selected gene sets. The deletion del (20)(q), with the EIF6 gene consistently lost, even in patients with the smallest losses of material, changes the transcription pattern: a low proportion of abnormal cells led to a pattern similar to SDS patients without acquired anomalies, whereas a high proportion yields a pattern similar to healthy subjects. Hence, the benign prognostic value of del (20)(q). The case of i (7)(q10) showed a transcription pattern similar to healthy subjects, paralleling the positive prognostic role of this anomaly as well.

High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.

Background & AimsEsophageal adenocarcinoma (EAC) develops from within Barrett’s esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial–mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells.MethodsSmall interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used.ResultsStatistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin.ConclusionsCollectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.

Highly Deregulated Fibulins in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Introduction The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) develop in a biological continuum from the early cancer stages (ET/PV) to the advanced myelofibrosis stage characterized by huge splenomegaly, bone marrow failure and -fibrosis. Importantly, bone marrow fibrosis also increases from the early stages with reticulin fibrosis only and later deposition of mature collagen as well. Fibulins are glycoproteins that are important constituents of the extracellular matrix (ECM). Thus, fibulins have been shown to modulate cell morphology, growth, adhesion and motility. Dysregulation of fibulins has been reported in several cancers. In addition, upregulation of fibulins and elevated circulating fibulins have been reported in diseases - other than cancers -, in which chronic inflammation is an important pathogenetic factor, such as cardiovascular diseases. Thus, deregulated fibulins have been described in patients with type 2 diabetes mellitus. Herein, using whole blood gene expression profiling, we for the first time report deregulated fibulins in patients with MPNs. Aim To detect if genes associated with pre-atherosclerotic changes in type II diabetes are deregulated in patients with MPNs. Material and methods Gene expression microarrays were applied to generate gene expression profiles of whole blood from control subjects (n=21) and patients with ET (n=19), PV (n=41), and PMF (n=9). Total RNA was purified, amplified to biotin-labeled aRNA and hybridized to microarray chips. The statistical software R was applied to perform initial data processing and statistical analysis of gene expression changes between patients and control subjects. An FDR <0.05 was considered significant. Results We identified 23,657, 25,567, and 17,417 probe sets which were significantly differentially expressed between controls and patients with ET, PV, and PMF, respectively (FDR < 0.05). We focused upon the top 15 upregulated genes from a previous gene expression microarray study performed on arterial tissue from patients with type 2 diabetes compared to non-diabetic patients undergoing artery bypass graft surgery. Several of these genes were significantly deregulated in patients with MPNs (Table 1). In patients with ET, FBLN1, FBLN2,FAM107A, IGF2, MEG3, and ELN were significantly upregulated and ZFP36L2 were significantly downregulated. In patients with PV, FBLN1, FBLN2, ELN, LEPR, FAM107A, IGF2, CRISPLD2, and MEG3 were significantly upregulated and ZFP36L2 and SERPINF1 were significantly downregulated. In patients with PMF, MEG3, LEPR, FBLN1, FAM107A, ELN, IGF2, and VWF were significantly upregulated and ZFP36L2 and SERPINF1 were significantly downregulated. Discussion and conclusions Fibulins regulate several cellular functions including tissue homeostasis and remodeling after injury, angiogenesis, and tumorigenesis. Thus, fibulins have been reported to be upregulated in several cancer types, in which deregulated fibulins have been associated with cancer invasiveness and disease progression. We have for the first time shown that fibulins are also highly deregulated in patients with MPNs. The significance of our findings is presently unknown but since interactions between fibulins and transforming growth factor (TGFbeta) have been demonstrated, upregulated fibulins may enhance the capacities of TGFbeta, which shares several of the regulatory functions excerted by fibulins. In the context of chronic inflammation being a driving force for MPN development during the biological continuum from early cancer stages to the advanced myelofibrosis stage and chronic inflammation likely also accelerates the development of atherosclerosis in MPNs, it is highly intriguing to note that fibulins are also elevated in arteries from patients with type 2 diabetes mellitus. Thus, upregulated fibulins in blood cells may actually indirectly reflect ongoing matrix modelling during atherosclerosis development, including remodeling and turnover of basement membranes in the inflamed endothelium. In conclusion, highly deregulated fibulins have been demonstrated in circulating blood cells by whole blood gene expression profiling. Further studies are needed to assess the significance of our findings in terms of potential associations to modelling of ECM in the bone marrow and during development of atherosclerosis in MPNs. Disclosures Hasselbalch: Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board.

Nutritional status modifies pregnane X receptor regulated transcriptome

Pregnane X receptor (PXR) regulates glucose and lipid metabolism, but little is known of the nutritional regulation of PXR function. We investigated the genome wide effects of the nutritional status on the PXR mediated gene regulation in the liver. Mice were treated with a PXR ligand pregnenolone 16α-carbonitrile (PCN) for 4 days and subsequently either fasted for 5 hours or after 4-hour fast treated with intragastric glucose 1 hour before sample collection. Gene expression microarray study indicated that PCN both induced and repressed much higher number of genes in the glucose fed mice and the induction of multiple well-established PXR target genes was potentiated by glucose. A subset of genes, including bile acid synthesis gene Cyp8b1, responded in an opposite direction during fasting and after glucose feeding. PXR knockout abolished these effects. In agreement with the Cyp8b1 regulation, PCN also modified the bile acid composition in the glucose fed mice. Contribution of glucose, insulin and glucagon on the observed nutritional effects was investigated in primary hepatocytes. However, only mild impact on PXR function was observed. These results show that nutritional status modifies the PXR regulated transcriptome both qualitatively and quantitatively and reveal a complex crosstalk between PXR and energy homeostasis.

Cancer-Associated Fibroblasts Enhance Survival and Progression of the Aggressive Pancreatic Tumor Via FGF-2 and CXCL8.

Pancreatic ductal adenocarcinoma remains one of the most challenging human cancers. Desmoplasia is predominant in this disease exhibiting a strong stromal reaction with an abundance of the cancer-associated fibroblasts (CAFs). We aimed in this study to investigate the reciprocal interaction between the tumor cells and the CAFs and its effect on tumor cells survival. We hypothesized that the survival of pancreatic cancer cell with aggressive phenotype is modulated by the Interactions between malignant pancreatic tumor cells and surrounding CAFs. To examine this, we utilized co-culture methods where tumor cells with different malignant potentials, HPAF (low) HPAF-CD11 (moderate/high) co-cultured with CAFs. CAFs-conditioned media increased the growth of HPAF-CD11 but not HPAF cells and increased CXCL8 levels highly in HPAF-CD11 and slightly in HPAF. The growth stimulatory effect and elevated CXCL8 level caused by CAFs-conditioned media were diminished by neutralizing the fibroblast growth factor-2 (FGF-2). In addition, conditioned media of HPAF-CD11 increased CAFs cell number whereas that of HPAF did not, and these effects were suppressed by neutralizing CXCL8. Furthermore, data from gene expression microarray study exhibited different expression profiles between HPAF and HPAF-CD11 when co-culture with CAFs. A significant increase in CXCL8 and FGF-2 expression was observed with HPAF-CD11/CAFs co-culture and to a lower extent with HPAF/CAFs co-culture. Together, these data demonstrate a paracrine bi-directional interaction between pancreatic tumor cells and the CAFs through CXCL8 and FGF-2 that helps the tumor growth. Future in-depth study of these pathways will assist in obtaining diagnostic and therapeutic tools for pancreatic ductal adenocarcinoma.

Genome-Wide Transcriptome Profiling Reveals Genes Associated with Meiotic Drive System of Aedes aegypti.

Aedes aegypti is an important mosquito vector of several arboviruses, including dengue, yellow fever, Zika, and Chikungunya, which cause significant human morbidity and mortality globally. In certain populations of this mosquito, a native meiotic drive system causes abnormal spermatogenesis that results in highly male-biased progenies from some matings. Although the basic genetics and cytogenetics of the drive mechanism were elucidated, very little is known on a transcriptome level about how the meiotic drive phenotype is expressed in individual males. To address this question, we conducted a whole-genome microarray expression study of testes from a meiotic-drive-carrying strain (T37) in comparison with testes from a non-drive-carrying strain (RED). Based on bioinformatics analyses of the microarray data, we identified 209 genes associated with the meiotic drive phenotype that were significantly differentially expressed between the two strains. K-means cluster analysis revealed nine clusters, in which genes upregulated in T37 testes were assigned to five clusters and genes downregulated in T37 testes were assigned to four clusters. Our data further revealed that genes related to protein translation, phosphorylation, and binding, as well as to G-protein-coupled receptor (GPCR) and peptidase activities, are differentially upregulated in testes from males with the meiotic drive genotype. Based on pathway analysis of these differentially expressed genes, it was observed that the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway may play a role in the meiotic drive system. Overall, this investigation enhances our understanding of whole-genome gene expression associated with the meiotic drive system in Ae. aegypti.

RNA isolation from taxonomically diverse photosynthetic protists

AbstractHigh quality RNA is a crucial requirement in a variety of analyses including next generation sequencing, microarray, and gene expression studies. A number of protocols for the extraction of ribonucleic acids from algae are currently in use, but different reagents interfere with buffer capacities and therefore lower the success of the isolations. The diversity of microalgae is reflected in a large variety of biomolecules and so far no standard protocol for the extraction of RNA from these highly divergent groups has been assessed. With this study, 11 conventional protocols are compared and an alternative standardized protocol for the isolation of high quality total RNA is proposed. Furthermore, the extraction method is applied on four different microalgae species of the Chlorophyta, Charophyta, Rhodophyta, and a dinoflagellate. We present an optimized RNA extraction method, yielding total RNA of high purity and integrity from all the investigated protists. The protocol provided in this study is cost‐effective and can assist in future functional genomics, covering an essential step when working with RNA from non‐model microorganisms.

Interferon-alfa2 Treatment of Patients with Polycythemia Vera and Related Neoplasms Influences Deregulated Inflammation and Immune Genes in Polycythemia Vera and Allied Neoplasms

Introduction: The Philadelphia-negative myeloproliferative neoplasms are associated with deregulation of inflammation and immune genes implying a gene signature of a chronic inflammatory state and immune deregulation. Several studies have demonstrated interferon-alpha2 (IFN) to be highly efficacious in normalizing elevated blood cell counts and in inducing molecular remissions as well. Using whole blood gene expression, we herein for the first time show that IFN profoundly influences the deregulated inflammation- and immune genes giving rise to a gene signature indicative of decreased inflammation and improved regulation of immune genes during IFN treatment.Material and Methods: Gene expression microarray studies have been performed on eight patients with ET, 21 patients with PV, and 4 patients with PMF. All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole-blood and amplified to biotin-labeled aRNA and hybridized to microarray chips.Results: We identified 6261, 10,008, and 2828 probe sets to be significantly differentially expressed in ET, PV, and PMF, respectively, during treatment with IFN (pvalue <0.05). Previously, we have found 123 inflammation and immune genes to be significantly deregulated in patients with MPN compared to healthy subjects. Here, we extend the gene list with 19 inflammation and immune genes, recently shown to be involved in other cancer. Several of these 142 genes were significantly deregulated during IFN therapy. In response to treatment with IFN, 20 genes were upregulated in ET including CX3CR1, CCL2, CCR1, CCR5, CCR7, HGF, HLA-G, IL1RN, IL10RA, PTX3, TP53, and TGFB1, and 6 genes were downregulated including MAPK1, ORM1, and IL1R1. In patients with PV, ABCF1, CCL2, CCR1, CCR2, CCR5, CCR7, CD3E, CD3G, CD4, CD40LG, CX3CR1, HLA-G, IL1RN, IL8, IL10RA, PTX3, TP53, TGFB1, TNFAIP3 were among the 32 upregulated genes, and BCL6, IL1R1, MAPK1, and ORM1 were among the 10 downregulated genes. During treatment with IFN, 11 genes were upregulated in PMF including CCL2, CCR1, CCR5, CX3CR1, IL1RN, PTX3, and TNFAIP3 and 5 genes were downregulated including BCL6, IL1R1, and ORM1.Discussion and Conclusion: Interferon-alpha2 is increasingly been recognized as a highly efficacious and promising agent in the treatment of MPNs. During recent years several studies have shown that chronic inflammation and immunoderegulation is likely involved in the pathogenesis of MPNs. Thus, elevated blood levels of circulating inflammatory cytokines have been recorded, some even having a prognostic impact and predicting imminent leukemic transformation. Chronic inflammation impairs IFN-signaling and likely therefore the efficacy of IFN in MPNs, although this notion has to be confirmed in MPNs. Immunoderegulation with defective immune surveillance may contribute to the increased risk of second cancers both before and after the MPN-diagnosis. Accordingly, there is an urgent need to explore if IFN might be able to restore deregulated inflammation and immune genes in MPNs. In this study, we have convincingly shown that several genes of significance for inflammation and immune surveillance are positively influenced by IFN treatment implying a significant downregulation of upregulated inflammation genes (e.g. BCL6, IL1R1, MAPK1, ORM1,) and a significant upregulation of downregulated immune genes (e.g. ABCF1, CCR2, CCR5, CCR7, CD3D, CD3E, CD3G, CD4, CD40LG, CXCR1, HLA-G, IL10RA, IL8, TFGB1, TNFAIP3, and TP53). Recently, we have shown significant downregulation of CCR9, CREB1, FAS, LSP1, LTB, NFKB1, SCYE1, SELPG, STAT3, and TNFAIP8L1, and significant upregulation of CCL25, CCL7, CSF3, CXCL9, FOXP3, IL17A, IL17C, IL1F10, IL1F6, IL22, IL4, IL5, ITGB3, in ET, PV, and PMF compared to controls. These genes were no longer significantly deregulated during IFN-treatment. In conclusion, our results have added highly important information on the impact of IFN upon deregulated inflammation and immune genes in MPNs, thereby substantiating the beneficial effects of IFN and its major role as the cornerstone in the future treatment of MPNs. Our study opens the avenue for larger studies exploring the genomic landscape during treatment of patients with MPNs. DisclosuresHasselbalch:Novartis: Research Funding.

Abstract 246: An integrative genetic epidemiologic approach to identify susceptibility genes for post-menopausal breast cancer

Abstract Background: Up to 30% of common (peri- and post-menopausal) breast cancer cases in the general population are believed to be due to segregation of mutations and variations with low to medium risk [i.e., 1.5&amp;lt; relative risk &amp;lt;20] for breast cancer. The majority of these genes remain to be identified. We used a hypothesis-driven integrative genetic epidemiologic approach to test the association of post-menopausal breast cancer with Nucleotide Excision Repair (NER) genes using several gene expression microarray and genome-wide association studies (GWAS) datasets. Methods: As per inclusion criteria, all datasets contained Caucasian cases with invasive ductal breast carcinoma diagnosed post-menopause (≥50 years of age) and healthy controls (with no personal history of breast cancer). Raw data from four gene expression microarray datasets (GSE5764, GSE10797, GSE10780, E-TABM-276) were used for pathway and meta-analysis. Raw data from three GWAS datasets, the Cancer Genetic Markers of Susceptibility (CGEMS) (1089 cases, 1093 controls), a nested case-control (351 cases, 884 controls) within National Surgical Adjuvant Breast and Bowel Project (NSABP), and a nested case-control (465 cases and 1394 controls) within the Women's Health Initiative (WHI) Hormone Therapy Trials, were analyzed using logistic regression models adjusting for covariates. Linkage disequilibrium patterns and deviations from Hardy-Weinberg Equilibrium (HWE) were assessed for single nucleotide polymorphisms (SNPs) within all candidate genes. Individual haplotypes and diplotypes were determined for all subjects. Odd ratios (OR) and 95% confidence intervals (CI) were calculated. Results: Individual analysis of gene expression microarray datasets and meta-analysis of GSE10780 and E-TABM-276 identified significant up-regulation of several NER genes including ERCC6 (P=7.95*10-6) and ERCC8 (P=4.67*10-6). Increased risk of breast cancer was found in association with variations in ERCC6 in all GWAS datasets. ERCC6 SNPs were in strong LD in all datasets with no deviations from HWE. In CGEMS, increased risk of breast cancer was found with ERCC6 rs4253082 (OR=1.32, 95%CI: 1.09-1.61, P=0.005), rs3750749 (OR=1.40, 95%CI: 1.07-1.8, P=0.01), and rs3750751 (OR=2.91, 95%CI: 1.05-8.06, P=0.04). In NSABP, increased risk of breast cancer was found with ERCC6 rs2229760 (OR=1.37, 95%CI: 1.02-1.83, P=0.03). In WHI, increased risk of breast cancer was found with ERCC6 rs1012553 (OR=1.35, 95%CI: 1.07-1.71, P=0.01) and rs2228528 (OR=1.29, 95%CI: 1.01-1.66, P=0.04). Gene-gene interaction analysis was significant for interactions between ERCC6 and several NER genes, including between specific diplotypes in ERCC6 and ERCC8 (Pinteraction=0.0009 in CGEMS). Conclusion: Genes involved in the NER pathway, including ERCC6 and ERCC8, may be associated with low to medium risk for susceptibility to post-menopausal breast cancer. Citation Format: Roxana Moslehi, Nur Zeinomar, Cristy Stagnar, Hui-Shien Tsao, Amiran Dzutsev. An integrative genetic epidemiologic approach to identify susceptibility genes for post-menopausal breast cancer [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 246.

Abstract LB-182: Notch signaling pathway is transcriptionally elevated in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC), contrary to well-differentiated papillary thyroid cancer (PTC), is one of the most aggressive human malignancies. Anaplastic thyroid cancer is responsible for more than half of all thyroid cancer deaths, with an overall mortality rate about 70% at 6 months and 81% at 12 months, despite only accounting for &amp;lt;2% of thyroid cancer incidence [1]. Both PTC and ATC are MAPK-driven cancers that have two mutually exclusive drivers with distinct signaling consequences: BRAF V600E mutation and RAS mutation [2-3]. In PTC, BRAF (V600E)-like tumors showed higher MAPK transcriptional output and lower expression of genes related with iodine metabolism, and RAS-like tumors showed lower MAPK signaling and comparatively higher expression of iodine metabolism genes. However, in ATC both BRAF mutant and RAS mutant tumors showed BRAF-like tumor transcriptional output. Down-regulation of Notch signaling by gamma-secretase inhibitor (GSI) or Notch1 RNAi reduced PTC cell proliferation [3]. Here by combining multiple gene expression microarray studies (TCGA, GSE27155, GSE29265 and GSE33630), we showed that compared with normal thyroid tissue the Notch signaling pathway was up-regulated in both PTC and ATC. However, ATC and PTC showed different activation patterns in terms of expression of Notch receptors, ligands and downstream targets. Compared with normal thyroid tissue, Notch1, Notch3 and JAG1 were up-regulated in both ATC and PTC, but were much higher in ATC. Compared with normal thyroid tissue, HEY1, HES4 and NRARP were up-regulated in both ATC and PTC. Comparing with PTC, HEY1 was also higher in ATC. However, HES1 was down-regulated in ATC but up-regulated in PTC. It was also reported that both Notch1 and Hes1 were up-regulated by activation of MAPK signaling in thyroid follicular cells with RET/PTC3 or BRAF (T1799A) [4]. Notch1 protein level was also higher in PTC [3]. All of the evidence above supports the hypothesis that Notch pathway may be a key signaling component along with aberrant activation of MAPK pathway in ATC. These data suggest inhibition of Notch signaling alone and/or combination with MAPK signaling inhibitors, such as BRAF inhibitor or ERK inhibitor, may be of benefit in ATC, and support investigation in a clinical trial. 1. Anaplastic Thyroid Carcinoma: Treatment Outcome and Prognostic Factors. Cancer 103 1330-1335,2015. 2. Integrated Genomic Characterization of Papillary Thyroid Carcinoma. Cell 159, 676-690, 2014. 3. Notch Pathway is activated by MAPK signaling and influences papillary thyroid cancer proliferation. Translational oncology, 6,197-205, 2013. 4. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. JCI, 126, 1052-1066, 2016. Citation Format: Yan Ding, Hui-Rong Qian, Bharvin K R Patel, Gerald Joseph Oakley, Karim A. Benhadji, Emma Bowden, Amit Aggarwal. Notch signaling pathway is transcriptionally elevated in anaplastic thyroid cancer [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 LB-182.

System-wide coordinates of higher order functions in host-pathogen environment upon Mycobacterium tuberculosis infection

Molecular signatures and their interactions behind the successful establishment of infection of Mycobacterium tuberculosis (Mtb) inside macrophage are largely unknown. In this work, we present an inter-system scale atlas of the gene expression signatures, their interactions and higher order gene functions of macrophage-Mtb environment at the time of infection. We have carried out large-scale meta-analysis of previously published gene expression microarray studies andhave identified a ranked list of differentially expressed genes and their higher order functions in intracellular Mtb as well as the infected macrophage. Comparative analysis of gene expression signatures of intracellular Mtb with the in vitro dormant Mtb at different hypoxic and oxidative stress conditions led to the identification of the large number of Mtb functional groups, namely operons, regulons and pathways that were common and unique to the intracellular environment and dormancy state. Some of the functions that are specific to intracellular Mtb are cholesterol degradation and biosynthesis of immunomodulatory phenolic compounds. The molecular signatures we have identified to be involved in adaptation to different stress conditions in macrophage environment may be critical for designing therapeutic interventions against tuberculosis. And, our approach may be broadly applicable for investigating other host-pathogen interactions.

RT-qPCR for Fecal Mature MicroRNA Quantification and Validation.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase (RT) followed by TaqMan® minor-groove binding (MGB) probe, real-time quantitative PCR analysis on exfoliated enriched colonocytes in stool, using human (Homo sapiens, hsa) micro(mi)RNAs to monitor changes of their expression at various stages of colorectal (CRC) progression, this method allows for the reliable and quantitative diagnostic screening of colon cancer (CC). Although the expression of some miRNA genes tested in tissue shows less variability in normal or cancerous patients than in stool, the noninvasive stool by itself is well suited for CC screening. An miRNA approach using stool promises to offer more sensitivity and specificity than currently used genomic, methylomic, or proteomic methods for CC screening.To present an application of employing miRNAs as diagnostic markers for CC screening, we carried out global microarray expression studies on stool colonocytes isolated by paramagnetic beads, using Affymetrix GeneChip miRNA 3.0 Array, to select a panel of miRNAs for subsequent focused semiquantitative PCR analysis studies. We then conducted a stem-loop RT-TaqMan® MGB probes, followed by a modified real-time qPCR expression study on 20 selected miRNAs for subsequent validation of the extracted immunocaptured total small RNA isolated from stool colonocytes. Results showed 12 miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p, and miR214) to have an increased expression in stool of CC patients, and that later TNM stages exhibited more increased expressions than adenomas, while 8 miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222, and miR-938) showed decreased expressions in stool of CC patients, which becomes more pronounced as the cancer progresses from early to late TNM stages (0-IV).

Abstract 5164: A pyrrole-Imidazole polyamide conjugate targeting KRAS oncogenic mutations is a promising approach against KRAS mutated colorectal and pancreatic cancers

Abstract Although a tremendous amount of studies has been made to directly target oncogenic drivers, such as RAS and MYC, no drug is clinically available because of difficulties to develop RAS- or Myc-targeted anti-cancer therapeutics due to the smooth 3D surface topology. One of major limitations of targeting the RAS pathway may be intrinsic or acquired resistance as seen in the other molecular target therapy. A new approach that directly target driver genes may provide a more direct route to help address unmet medical needs for refractory cancer conquest. We therefore synthesized the Pyrrole-Imidazole polyamide conjugate, KR12, specifically alkylated KRAS codon 12 mutant DNA (G12D or G12V). KR12 reduced expression of the mutated oncogenic-protein by RNA transcription inhibition and induced cancer cell death at low dose (1 to 50 nM). We also applied a novel enrichment-based sequencing and computational pipeline, which first identifies a PIP’s genomic binding sites through next-generation sequencing followed by gene expression microarray studies. The computational analysis suggested non-KRAS binding overall had little contribution to changes at the phenotypic level in colorectal cancer cell lines. We also the anti-cancer event is not only for colorectal but also pancreatic ductal adenocarcinoma (PDAC). Significant tumor reduction was observed in a mouse model of spontaneous PDAC (LSL-KrasG12D/+,Ptf1aCre/+,Tgfbr2flox/flox). In animal studies we also observed KR12 intakes and retentions in tumor but not any other organs 24 hours after I.V. administration. We will summarize the sequence-dependent alkylating approach using antibiotic mimics of alkylating PI-polyamide conjugates, may open a new strategy targeting the driver oncogene mutated DNA sequence. This approach should be used for medicine against variety of unfavorable KRAS mutated cancers. Note: This abstract was not presented at the meeting. Citation Format: Hiroki Nagase, Atsushi Takatori, Takayoshi Watanabe, Nobuko Koshikawa, Jason Lin. A pyrrole-Imidazole polyamide conjugate targeting KRAS oncogenic mutations is a promising approach against KRAS mutated colorectal and pancreatic cancers [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 5164. doi:10.1158/1538-7445.AM2017-5164

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Gestational Diabetes Alters Functions in Offspring's Umbilical Cord Cells With Implications for Cardiovascular Health.

Because noncommunicable diseases such as type 2 diabetes mellitus have their roots in prenatal development and conditions such as maternal gestational diabetes mellitus (GDM), we aimed to test this hypothesis in primary cells derived from the offspring of mothers with GDM compared with control subjects. We have assessed primary umbilical cord-derived cells such as human umbilical vein endothelial cells (HUVECs) and Wharton's jelly-derived mesenchymal stem cells from the offspring of mothers with and without GDM. We have compared the primary isolates in cell-based assays measuring proliferation, mitochondrial oxygen consumption, and the ability to support blood vessel growth. We conducted gene expression microarray studies with subsequent pathway analysis and candidate gene validation. We observed striking differences between the two groups, such as lower metabolic rates and impairment of endothelial tube formation in cells with GDM background. HUVECs from subjects with maternal GDM have lower expression of the antiapoptotic protein BCL-xL, suggesting compromised angiogenic capabilities. Comparative gene expression analysis revealed blood vessel formation as a major pathway enriched in the GDM-derived HUVECs with the surface marker CD44 as a gene underexpressed in the GDM group. Functional validation of CD44 revealed that it regulates tube formation in HUVECs, thereby providing insights into a pathway imprinted in primary umbilical cord-derived cells from GDM offspring. Our data demonstrate that primary cells isolated from the umbilical cord of offspring born to mothers with GDM maintain metabolic and molecular imprints of maternal hyperglycemia, reflecting an increased risk for cardiovascular disease later in life.

γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury.

Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the γδ T cell receptor (TCR) rather than the αβ TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in γδ T cells; deficiency in γδ T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and γδ T cells are associated with human hypertension. Male C57BL/6 wild-type and Tcrδ-/- mice, which are devoid of γδ T cells, or wild-type mice injected IP with control isotype IgG or γδ T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR γ constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between γδ T cells and SBP. Seven- and 14-day Ang II infusion increased γδ T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcrδ-/- mice (P<0.01). Anti-TCRγδ antibody-induced γδ T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcrδ-/- mice (P<0.01). In humans, the association between SBP and γδ T cells was demonstrated by a multiple linear regression model integrating whole blood TCR γ constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). γδ T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. γδ T cells might contribute to the development of hypertension in humans.