Global Gene Expression Data Research Articles

Abstract 3830: Plucked anagen scalp hair: A reproducible surrogate tissue to monitor drug induced transcriptional changes and provide pharmacodynamic biomarker and target engagement information from cancer patients

Abstract Monitoring target engagement is crucial to inform on early drug development decisions, and development of a peripheral tissue-based gene expression signature indicative of pathway inhibition could facilitate the continued clinical development of compounds. Plucked anagen scalp hair is an ideal source of epithelial tissue to monitor drug induced transcriptional changes. High vascularisation of the hair follicle, frequent epithelial origin of tumors, ease of sampling and high degree of congruence of expression in hair of pathways dysregulated in cancers, make the cellular bulb of plucked human scalp hair an excellent surrogate tissue for the non-invasive monitoring of pharmacodynamic (PD) and mechanism of action (MOA) effects in clinical trials. Using our ex vivo plucked hair culture platform, hair bulbs from several healthy donors were exposed to BEZ235, a dual pan-class PI3K and mTOR inhibitor at different concentrations and durations. Total RNA was isolated from the cellular bulb of individual anagen hair post-culture and used for microarray analysis to assess global transcriptional alterations and develop a gene expression signature indicative of BEZ235 exposure. The results of the ex vivo plucked assessment generated a dose dependent appreciable and biologically relevant transcriptional signature following exposure to BEZ235. Transcriptional readouts from patients also revealed strong correlations (0.97-0.99) in the genes expressed in anagen scalp hair between donors and across samples taken from the same donor. In addition, global gene expression data indicated that assessing three plucked scalp hairs or less at each collection point was sufficient to detect significant differential expression (p<0.05 & 1.5 fold change) underpinning the low variance in the majority of target genes in plucked hair as a biomarker platform. Gene signatures for compounds targeting the PI3K/mTOR pathway that were established using the ex vivo plucked scalp hair platform were used to support clinical trials and scalp hair taken from patients receiving treatment showed an excellent post-exposure target signature modulation. In summary, our results substantiate that plucked anagen scalp hair is an ideal non-invasive surrogate tissue to obtain drug-induced pharmacodynamic biomarker and target engagement information from oncology patients. Citation Format: Benjamin J. Reed, Timothy Mefo, Elliott Harrison, Ross Haggart, Emma Grimes, Alan Murdoch, Gino Miele. Plucked anagen scalp hair: A reproducible surrogate tissue to monitor drug induced transcriptional changes and provide pharmacodynamic biomarker and target engagement information from cancer patients [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 3830. doi:10.1158/1538-7445.AM2017-3830

Combining gene expression data from inflamed tissue and machine learning for blood biomarker discovery

Abstract Inflammation is common in virtually all diseases. Since immune cells travel through the blood to inflamed tissues, peripheral blood reflects inflammation at distant sites. However, blood biomarkers are hard to detect in global gene expression data since blood is not the site of perturbation and statistical corrections for multiple testing reduce sensitivity. We hypothesized that combining transcriptional signatures from inflamed tissue and machine learning would be useful to derive blood biomarkers. We first tested this in the mouse model of influenza infection. We identified a set of early genes in the lungs associated with lethality after infection. We then tested their differential abundance early in the blood of mice infected with a range of influenza doses. We applied elastic net to build a biomarker able to predict disease outcome (4 classes: early lethal, late lethal, non-lethal vs. non-infected; accuracy on training and validation sets: 91% and 79%). We next confirmed our hypothesis in lung cancer and breast cancer in humans. We identified sets of genes associated with tumors but not with tumor-free tissues and tested their differential abundance in the blood of patients compared to healthy controls. Using elastic net, we built two blood biomarkers to classify subjects according to disease status (case vs. control; accuracy on training and validation sets in breast cancer: 92% and 67%; in lung cancer: 89% and 78%). In conclusion, combining inflamed tissue signatures and machine learning, we were able to derive blood biomarkers for three different diseases in two different species. We expect that this novel strategy can be applied in other diseases as well. This work was supported by the Intramural Research Program of NIAID, NIH.

Assessment of the short-term toxicity of TiO2 nanofiber in Sprague Dawley rats.

Synthetic nanomaterials have many unique chemical and physical properties, mainly due to their high specific surface area and quantum confinement effect. Specifically, titanium dioxide (TiO2 ) nanomaterial has high stability, anticorrosive, and photocatalytic properties. However, there are concerns over adverse biological effects resulting from bioeffects. This study was to investigate adverse effects associated with acute ingestion of TiO2 nanofiber (TDNF). TDNF was fabricated via electrospinning method, followed by dissolution in water. Six- to seven-week-old male Sprague Dawley rats were exposed to a total of 0, 40, and 60 ppm of TDNF for 2weeks via oral gavage. Serum total protein and weight gain during the course of this study displayed marginal concentration-dependent alterations. These findings were followed by a global gene expression analysis to identify which transcripts might be responsive to TNDF toxicity. Differentially expressed mRNA levels were dose-dependently higher in animals exposed to TNDF. The majority of the affected genes were biochemically involved in immune response and inflammation. We believe this is due to the fact that TNDF is unable to penetrate the cell and forms phagocytosis sites that trigger inflammatory and immune response. All results taken together, short-term ingestion of TNDF produced marginal effects indicative of inflammation. Finally, the broad gene expression data were validated through quantification of immunoglobulin heavy chain alpha (Igha). Igha gene was upregulated in treated groups, showing similar expression patterns to the global gene expression data.

Microarray and network-based identification of functional modules and pathways of active tuberculosis.

Diagnose of active tuberculosis (TB) is challenging and treatment response is also difficult to efficiently monitor. The aim of this study was to use an integrated analysis of microarray and network-based method to the samples from publically available datasets to obtain a diagnostic module set and pathways in active TB. Towards this goal, background protein-protein interactions (PPI) network was generated based on global PPI information and gene expression data, following by identification of differential expression network (DEN) from the background PPI network. Then, ego genes were extracted according to the degree features in DEN. Next, module collection was conducted by ego gene expansion based on EgoNetalgorithm. After that, differential expression of modules between active TB and controls was evaluated using random permutation test. Finally, biological significance of differential modules was detected by pathways enrichment analysis based on Reactome database, and Fisher's exact test was implemented to extract differential pathways for active TB. Totally, 47 ego genes and 47 candidate modules were identified from the DEN. By setting the cutoff-criteria of gene size >5 and classification accuracy ≥0.9, 7 ego modules (Module 4, Module 7, Module 9, Module 19, Module 25, Module 38 and Module 43) were extracted, and all of them had the statistical significance between active TB and controls. Then, Fisher's exact test was conducted to capture differential pathways for active TB. Interestingly, genes in Module 4, Module 25, Module 38, and Module 43 were enriched in the same pathway, formation of a pool of free 40S subunits. Significant pathway for Module 7 and Module 9 was eukaryotic translation termination, and for Module 19 was nonsense mediated decay enhanced by the exon junction complex (EJC). Accordingly, differential modules and pathways might be potential biomarkers for treating active TB, and provide valuable clues for better understanding of molecular mechanism of active TB.

DTNI: a novel toxicogenomics data analysis tool for identifying the molecular mechanisms underlying the adverse effects of toxic compounds

Unravelling gene regulatory networks (GRNs) influenced by chemicals is a major challenge in systems toxicology. Because toxicant-induced GRNs evolve over time and dose, the analysis of global gene expression data measured at multiple time points and doses will provide insight in the adverse effects of compounds. Therefore, there is a need for mathematical methods for GRN identification from time-over-dose-dependent data. One of the current approaches for GRN inference is Time Series Network Identification (TSNI). TSNI is based on ordinary differential equations (ODE), describing the time evolution of the expression of each gene, which is assumed to be dependent on the expression of other genes and an external perturbation (i.e. chemical exposure). Here, we present Dose-Time Network Identification (DTNI), a method extending TSNI by including ODE describing how the expression of each gene evolves with dose, which is supposed to depend on the expression of other genes and the exposure time. We also adapted TSNI in order to enable inclusion of time-over-dose-dependent data from multiple compounds. Here, we show that DTNI outperforms TSNI in inferring a toxicant-induced GRN. Moreover, we show that DTNI is a suitable method to infer a GRN dose- and time-dependently induced by a group of compounds influencing a common biological process. Applying DTNI on experimental data from TG-GATEs, we demonstrate that DTNI provides in-depth information on the mode of action of compounds, in particular key events and potential molecular initiating events. Furthermore, DTNI also discloses several unknown interactions which have to be verified experimentally.

Cell Cycle M-Phase Genes Are Highly Upregulated in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) accounts for only 3% of thyroid cancers, yet strikingly, it accounts for almost 40% of thyroid cancer deaths. Currently, no effective therapies exist. In an effort to identify ATC-specific therapeutic targets, we analyzed global gene expression data from multiple studies to identify ATC-specific dysregulated genes. The National Center for Biotechnology Information Gene Expression Omnibus database was searched for high-throughput gene expression microarray studies from human ATC tissue along with normal thyroid and/or papillary thyroid cancer (PTC) tissue. Gene expression levels in ATC were compared with normal thyroid or PTC using seven separate comparisons, and an ATC-specific gene set common in all seven comparisons was identified. We investigated these genes for their biological functions and pathways. There were three studies meeting inclusion criteria, (including 32 ATC patients, 69 PTC, and 75 normal). There were 259 upregulated genes and 286 downregulated genes in ATC with at least two-fold change in all seven comparisons. Using a five-fold filter, 36 genes were upregulated in ATC, while 40 genes were downregulated. Of the 10 top globally upregulated genes in ATC, 4/10 (MMP1, ANLN, CEP55, and TFPI2) are known to play a role in ATC progression; however, 6/10 genes (TMEM158, CXCL5, E2F7, DLGAP5, MME, and ASPM) had not been specifically implicated in ATC. Similarly, 3/10 (SFTA3, LMO3, and C2orf40) of the most globally downregulated genes were novel in this context, while 7/10 genes (SLC26A7, TG, TSHR, DUOX2, CDH1, PDE8B, and FOXE1) have been previously identified in ATC. We experimentally validated a significant correlation for seven transcription factors (KLF16, SP3, ETV6, FOXC1, SP1, EGFR1, and MAFK) with the ATC-specific genes using microarray analysis of ATC cell lines. Ontology clustering of globally altered genes revealed that "mitotic cell cycle" is highly enriched in the globally upregulated gene set (44% of top upregulated genes, p-value <10-30). By focusing on globally altered genes, we have identified a set of consistently altered biological processes and pathways in ATC. Our data are consistent with an important role for M-phase cell cycle genes in ATC, and may provide direction for future studies to identify novel therapeutic targets for this disease.

In silico clustering of Salmonella global gene expression data reveals novel genes co-regulated with the SPI-1 virulence genes through HilD.

A wide variety of Salmonella enterica serovars cause intestinal and systemic infections to humans and animals. Salmonella Patogenicity Island 1 (SPI-1) is a chromosomal region containing 39 genes that have crucial virulence roles. The AraC-like transcriptional regulator HilD, encoded in SPI-1, positively controls the expression of the SPI-1 genes, as well as of several other virulence genes located outside SPI-1. In this study, we applied a clustering method to the global gene expression data of S. enterica serovar Typhimurium from the COLOMBOS database; thus genes that show an expression pattern similar to that of SPI-1 genes were selected. This analysis revealed nine novel genes that are co-expressed with SPI-1, which are located in different chromosomal regions. Expression analyses and protein-DNA interaction assays showed regulation by HilD for six of these genes: gtgE, phoH, sinR, SL1263 (lpxR) and SL4247 were regulated directly, whereas SL1896 was regulated indirectly. Interestingly, phoH is an ancestral gene conserved in most of bacteria, whereas the other genes show characteristics of genes acquired by Salmonella. A role in virulence has been previously demonstrated for gtgE, lpxR and sinR. Our results further expand the regulon of HilD and thus identify novel possible Salmonella virulence genes.

APOL1 Renal-Risk Variants Induce Mitochondrial Dysfunction.

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions.

Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level. Systems biology-Microgravity-Pathways and networks-Bacteria. Astrobiology 16, 677-689.

Molecular Mechanisms Contributing to the Growth and Physiology of an Extremophile Cultured with Dielectric Heating.

The residual heating of living organisms in the microwave region of the electromagnetic spectrum has complicated the identification of radiation-only effects using microorganisms for 50 years. A majority of the previous experiments used either mature cells or short exposure times with low-energy high-frequency radiation. Using global differential gene expression data, we identified molecular processes unique to dielectric heating using Thermus scotoductus SA-01 cultured over 30 h in a commercial microwave digestor. Genes associated with amino acid metabolism, carbohydrate metabolism, and defense mechanisms were upregulated; the number of repressed genes with unknown function increased; and at all time points, transposases were upregulated. These findings serve as a platform for future studies with mesophiles in order to better understand the response of microorganisms to microwave radiation.

Abstract 1232: A-type proanthocyanidins selectively target acute myeloid leukemia cells in vitro and in vivo

Abstract Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. A-type proanthocyanidins (A-PACs) are a unique class of compounds found in cranberries (Vaccinium macrocarpon Ait.) that we have found to be effective against several leukemia cell lines and primary AML samples in vitro. Moreover, A-PACs possess a unique ether bond and have ortho-hydroxyl phenolic groups that have the potential to bind to iron, alter redox status, and other biological effects. We found that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p&amp;lt;0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 μg/ml A-PACs was decreased (90.6%, n = 3, p&amp;lt;0.001), whereas normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction consisting of A-PACs that ranged from 2 to 7 degrees of polymerization were more effective than individual A-PACs. This information led us to investigate the anti-leukemia effects of A-PACs in AML patient-derived xenografts (AML-PDX) and to further investigate the mechanisms associated with these compounds. AML-PDX mice (n = 15), were treated for 2.5 weeks via intraperitoneal injections of A-PACs (25 or 50 mg/kg dose every 3 days) or a vehicle control (PBS every 3 days). Mice were sacrificed and leukemia engraftment was evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, oxidative stress, cytokine response, and survival pathways by gene expression analysis or flow cytometry. Administration of A-PACs to AML-PDX tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 12.51% (95% CI: 4.9, 20.11; n = 5), whereas the mice treated with the 50 mg/kg and 25 mg/kg A-PACs showed a level of engraftment of 5.2% (95% CI: 1.5, 8.9; n = 5) and 5.4% (95% CI: 2.3, 8.5; n = 5), respectively. These results indicated more than a 50% reduction in engraftment, which was better or equal to the effects we observed in mice treated with high-dose cytarabine, a standard care drug. Moreover, no toxic effects were observed in the mice. It was also found that both cells and mice treated with A-PACs lead to the production of specific subset of cytokines. Global gene expression data showed consistent upregulation of some of these cytokines, and also upregulation of NF-κB and enzymes indicative of oxidative stress. The results indicate that A-PACs not only target primary AML cells in vitro, but are also effective in vivo by a potentially novel mechanism. Further elucidation of this mechanism may uncover new vulnerabilities of this disease. Citation Format: Laura M. Bystrom, Luis Andres Lara-Martinez, Bernardo Gomel, Burak Isal, Hongliang Zong, Sabrina Martinez, Catherine Neto, Stefano Rivella, Monica L. Guzman. A-type proanthocyanidins selectively target acute myeloid leukemia cells in vitro and in vivo. [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 1232.

The effects of lactate and acid on articular chondrocytes function: Implications for polymeric cartilage scaffold design

Poly (lactic-co-glycolic acid) (PLGA) and poly-l-lactate acid (PLLA) are biodegradable polymers widely utilized as scaffold materials for cartilage tissue engineering. Their acid degradation products have been widely recognized as being detrimental to cell function. However, the biological effects of lactate, rather than lactic acid, on chondrocytes have never been investigated. This is the major focus of this study. The amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100mM and exposure duration of 8h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds. Statement of SignificanceLactic acid is a widely used substrate for polymers synthesis, PLGA and PLLA in particular. Although physical and biological modifications have been made on these polymers to make them be better cartilage scaffolds, little concern has been given on the biological effect of lactic acid, the main degradation product of these polymers, on chondrocytes. Our finding illustrates the differential biological function of lactate and acid on chondrocytes matrix synthesis. These results can facilitate future design of lactate polymers-based cartilage scaffolds

WNT5A: a motility-promoting factor in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

HOXA5 determines cell fate transition and impedes tumor initiation and progression in breast cancer through regulation of E-cadherin and CD24.

Loss of HOXA5 expression occurs frequently in breast cancer and correlates with higher pathological grade and poorer disease outcome. However, how HOX proteins drive differentiation in mammalian cells is poorly understood. In this paper, we investigated cellular and molecular consequences of loss of HOXA5 in breast cancer, and the role played by retinoic acid in HOXA5 function. Analysis of global gene expression data from HOXA5-depleted MCF10A breast epithelial cells, followed by validation, pointed to a role for HOXA5 in maintaining several molecular traits typical of the epithelial lineage such as cell-cell adhesion, tight junctions and markers of differentiation. Depleting HOXA5 in immortalized MCF10A or transformed MCF10A-Kras cells reduced their CD24+/CD44lo population, enhanced self-renewal capacity, and reduced expression of E-cadherin (CDH1) and CD24. In the case of MCF10A-Kras, HOXA5 loss increased branching and protrusive morphology in Matrigel, all features suggestive of epithelial to basal transition. Further, orthotopically implanted xenografts of MCF10A-Kras-scr grew as well-differentiated pseudo-luminal carcinomas, while MCF10A-Kras-shHOXA5 cells formed aggressive, poorly differentiated carcinomas. Conversely, ectopic expression of HOXA5 in aggressive SUM149 or SUM159 breast cancer cells reversed the cellular and molecular alterations observed in the HOXA5-depleted cells. Retinoic acid is a known upstream regulator of HOXA5 expression. HOXA5 depletion in MCF10A cells engineered to express doxycycline-induced shHOXA5 slowed transition of cells from a less differentiated CD24−/CD44+ to the more differentiated CD24+/CD44+ state. This transition was promoted by retinal treatment which upregulated endogenous HOXA5 expression, and caused re-expression of, Occludin, and claudin-7 (CLDN7). Expression of CDH1 and CD24 was transcriptionally upregulated by direct binding of HOXA5 to their promoter sequences as demonstrated by luciferase and ChIP analyses. Thus, loss of HOXA5 in mammary cells leads to loss of epithelial traits, an increase in stemness and cell plasticity, and the acquisition of more aggressive phenotypes.

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data.

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery.

Suppression of Antitumor Immune Responses by Human Papillomavirus through Epigenetic Downregulation of CXCL14.

ABSTRACTHigh-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK), CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression.

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation

SummaryMetazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

NCOA2 is a candidate target gene of 8q gain associated with clinically aggressive prostate cancer.

Prostate carcinomas harboring 8q gains are associated with poor clinical outcome, but the target genes of this genomic alteration remain to be unveiled. In this study, we aimed to identify potential 8q target genes associated with clinically aggressive prostate cancer (PCa) using fluorescence in situ hybridization (FISH), genome-wide mRNA expression, and protein expression analyses. Using FISH, we first characterized the relative copy number of 8q (assessed with MYC flanking probes) of a series of 50 radical prostatectomy specimens, with available global gene expression data and typed for E26 transformation specific (ETS) rearrangements, and then compared the gene expression profile of PCa subsets with and without 8q24 gain using Significance Analysis of Microarrays. In the subset of tumors with ERG fusion genes (ERG+), five genes were identified as significantly overexpressed (false discovery rate [FDR], ≤ 5%) in tumors with relative 8q24 gain, namely VN1R1, ZNF417, CDON, IKZF2, and NCOA2. Of these, only NCOA2 is located in 8q (8q13.3), showing a statistically higher mRNA expression in the subgroup with relative 8q gain, both in the ERG+ subgroup and in the whole series (P = 0.000152 and P = 0.008, respectively). Combining all the cases with NCOA2 overexpression, either at the mRNA or at the protein level, we identified a group of tumors with NCOA2 copy-number increase, independently of ETS status and relative 8q24 gain. Furthermore, for the first time, we detected a structural rearrangement involving NCOA2 in PCa. These findings warrant further studies with larger series to evaluate if NCOA2 relative copy-number gain presents prognostic value independently of the well-established poor prognosis associated with MYC relative copy-number gain.

Clinical outcome and global gene expression data support the existence of the estrogen receptor-negative/progesterone receptor-positive invasive breast cancer phenotype.

The presence or absence of estrogen and progesterone steroid hormone receptor expression (ER, PR) is an essential feature of invasive breast cancer and determines prognosis and endocrine treatment decisions. Among the four ER/PR receptor phenotypes, the ER-/PR+ is infrequent, and its clinical relevance has been controversially discussed. Thus, we investigated its clinical significance and gene expression pattern in large datasets. In a retrospective clinical study of 15,747 breast cancer patients, we determined the ER/PR subtype survival probabilities using Kaplan-Meier and Cox regression analyses. From The Cancer Genome Atlas (TCGA) breast cancer dataset, PAM50 expression signature and pathway analyses were performed to test for distinct molecular features. In our cohort, the ER-/PR+ phenotype has been observed at a frequency of 4.1% and was associated with an improved 10-year survival for stage I cancers compared to the ER+/PR+ reference subtype (median; 95% CI 88.1%; 83-93 vs. 84.3%; 82-86%, P=0.024) as was confirmed by multivariate analysis over the entire follow-up (HR 0.59, 95% CI 0.38-0.92, P=0.021). This association lacked significance when including all stages. ER-/PR+ patients treated with antihormonal agents (34.5%) had shorter survival compared to their non-treated counterparts (Log-rank P=0.0001). PAM50 signatures suggest a distinct configuration for the ER-/PR+ phenotype. This specific phenotype has been further separated by a set of 59 uniquely expressed genes. Our study supports the notion of the existence of an ER-/PR+ phenotype with clinical and molecular features distinct from the large group of ER+/PR+ patients.

Tumor Vs Normal Gene Expression Profiles Identify Deregulated Pathways in ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia: A Microarray-Based Meta-Analysis

Acute lymphoblastic leukemia (ALL), a clinically and biologically heterogeneous disease, is the most common type of childhood cancer. Approximately 25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most frequent genetic aberration in pediatric ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, generating the fusion oncogene ETV6/RUNX1 (synonym TEL/AML1). Recent studies in various animal models have strengthened the view that ETV6/RUNX1 positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy. However, the effects triggered by the expression of ETV6/RUNX1 and the associated additional mutated genes are not well understood.We compared global gene expression data from ETV6/RUNX1 positive ALL and normal hematopoietic progenitor cells to identify expression signatures underlying the malignant phenotype. Our meta-analysis comprised Affymetrix HG-U133A und HG-U133 Plus 2.0 data from a total of 321 patients with ETV6/RUNX1 positive ALL, different ETV6/RUNX1 positive cell models, leukemias with 11q23 rearrangements and hyperdiploid ALL, as well as 251 samples from various normal controls including normal bone marrow, hematopoietic stem cells (HSCs) and normal B-(progenitor) cells (multipotent progenitor, pro-, pre-, immature-, naive-, centrocyte-, centroblast-, memory-, plasmablast-B cells). To eliminate potential lab batch effects, we performed RMA normalization of all microarray CEL files and next applied the batch effect removal algorithm ComBat. For determination of differentially expressed genes and pathway analyses, the R/Bioconductor packages limma and SPIA were used, respectively.We generated a gene expression landscape of the normal B-cell development and were able to show that pre-B cells are the closest normal counterpart of ETV6/RUNX1 positive ALL. Furthermore, in comparisons with normal HSCs the p53-, mTOR-, PI3K-AKT-, Apoptosis-, and JAK-STAT signaling pathways were found to be deregulated in those ALL. The combination of multiple global gene expression data sets from ETV6/RUNX1 positive ALL offers the possibility of an integrated large-scale meta-analysis and reveals novel insights into deregulated gene networks in this type of leukemia. DisclosuresNo relevant conflicts of interest to declare.