Upregulation of the Oct3/4 Network in Basal Breast Cancer Is Associated with Its Metastatic Potential and Shows Tissue Dependent Variability.

Adaptive plasticity of Breast Cancer stem cells (BCSCs) is strongly correlated with cancer progression and resistance, leading to a poor prognosis. In this study, we report the expression profile of several pioneer transcription factors of the Oct3/4 network associated with tumor initiation and metastasis. In the triple negative breast cancer cell line (MDA-MB-231) stably transfected with human Oct3/4-GFP, differentially expressed genes (DEGs) were identified using qPCR and microarray, and the resistance to paclitaxel was assessed using an MTS assay. The tumor-seeding potential in immunocompromised (NOD-SCID) mice and DEGs in the tumors were also assessed along with the intra-tumor (CD44+/CD24-) expression using flow cytometry. Unlike 2-D cultures, the Oct3/4-GFP expression was homogenous and stable in 3-D mammospheres developed from BCSCs. A total of 25 DEGs including Gata6, FoxA2, Sall4, Zic2, H2afJ, Stc1 and Bmi1 were identified in Oct3/4 activated cells coupled with a significantly increased resistance to paclitaxel. In mice, the higher Oct3/4 expression in tumors correlated with enhanced tumorigenic potential and aggressive growth, with metastatic lesions showing a >5-fold upregulation of DEGs compared to orthotopic tumors and variability in different tissues with the highest modulation in the brain. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted the sustained upregulation of Sall4, c-Myc, Mmp1, Mmp9 and Dkk1 genes in metastatic lesions with a 2-fold higher expression of stem cell markers (CD44+/CD24-). Thus, Oct3/4 transcriptome may drive the differentiation and maintenance of BCSCs, promoting their tumorigenic potential, metastasis and resistance to drugs such as paclitaxel with tissue-specific heterogeneity.

Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza A Virus Infection in C57BL/6 Mice.

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

Multiple domains in the 50 kDa form of E4F1 regulate promoter-specific repression and E1A trans-activation.

The 50 kDa N-terminal product of the cellular transcription factor E4F1 (p50E4F1) mediates E1A289R trans-activation of the adenovirus E4 gene, and suppresses E1A-mediated transformation by sensitizing cells to cell death. This report shows that while both E1A289R and E1A243R stimulate p50E4F1 DNA binding activity, E1A289R trans-activation, as measured using GAL-p50E4F1 fusion proteins, involves a p50E4F1 transcription regulatory (TR) region that must be promoter-bound and is dependent upon E1A CR3, CR1 and N-terminal domains. Trans-activation is promoter-specific, as GAL-p50E4F1 did not stimulate commonly used artificial promoters and was strongly repressive when competing against GAL-VP16. p50E4F1 and E1A289R stably associate in vivo using the p50E4F1 TR region and E1A CR3, although their association in vitro is indirect and paradoxically disrupted by MAP kinase phosphorylation of E1A289R, which stimulates E4 trans-activation in vivo. Multiple cellular proteins, including TBP, bind the p50E4F1 TR region in vitro. The mechanistic implications for p50E4F1 function are discussed.

In-vivo fusion of human cancer and hamster stromal cells permanently transduces and transcribes human DNA.

After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection P-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other gene functions of the donor DNA. Using heterospecific in-vivo cell fusion, genes encoding oncogenic and organogenic traits may be identified.

Molecular profiles of pyramidal neurons in the superior temporal cortex in schizophrenia.

Disrupted synchronized oscillatory firing of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30-100 Hz) mediates many of the cognitive deficits and symptoms of schizophrenia. In fact, the density of dendritic spines and the average somal area of pyramidal neurons in layer 3 of the cerebral cortex, which mediate both long-range (associational) and local (intrinsic) corticocortical connections, are decreased in subjects with this illness. To explore the molecular pathophysiology of pyramidal neuronal dysfunction, we extracted ribonucleic acid (RNA) from laser-captured pyramidal neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem brains from schizophrenia and normal control subjects. We then profiled the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identified 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong to the transforming growth factor beta (TGF-ß) and the bone morphogenetic proteins (BMPs) signaling pathways. Disturbances of these signaling mechanisms may in part contribute to the altered expression of other genes found to be differentially expressed in this study, such as those that regulate extracellular matrix (ECM), apoptosis, and cytoskeletal and synaptic plasticity. In addition, we identified 10 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of their predicted gene targets revealed signaling pathways and gene networks that were found by microarray to be dysregulated, raising an interesting possibility that dysfunction of pyramidal neurons in schizophrenia may in part be mediated by a concerted dysregulation of gene network functions as a result of the altered expression of a relatively small number of miRNAs. Taken together, findings of this study provide a neurobiological framework within which specific hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.

Molecular profiles of parvalbumin-immunoreactive neurons in the superior temporal cortex in schizophrenia.

Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder.

Downregulation of GABAA receptor protein subunits α6, ß2, δ, ε, γ2, θ, and ρ2 in superior frontal cortex of subjects with autism.

We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABAA) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABAA and GABAB subunits and overall reduced protein expression for GABAA receptor alpha 6 (GABRα6), GABAA receptor beta 2 (GABRß2), GABAA receptor delta (GABRδ), GABAA receptor epsilon (GABRε), GABAA receptor gamma 2 (GABRγ2), GABAA receptor theta (GABRθ), and GABAA receptor rho 2 (GABRρ2) in superior frontal cortex from subjects with autism. Our data demonstrate systematic changes in GABAA&B subunit expression in brains of subjects with autism, which may help explain the presence of cognitive abnormalities in subjects with autism.

miR-126 contributes to Parkinson's disease by dysregulating the insulin-like growth factor/phosphoinositide 3-kinase signaling.

Dopamine (DA) neurons in sporadic Parkinson's disease (PD) display dysregulated gene expression networks and signaling pathways that are implicated in PD pathogenesis. Micro (mi)RNAs are regulators of gene expression, which could be involved in neurodegenerative diseases. We determined the miRNA profiles in laser microdissected DA neurons from postmortem sporadic PD patients' brains and age-matched controls. DA neurons had a distinctive miRNA signature and a set of miRNAs was dysregulated in PD. Bioinformatics analysis provided evidence for correlations of miRNAs with signaling pathways relevant to PD, including an association of miR-126 with insulin/IGF-1/PI3K signaling. In DA neuronal cell systems, enhanced expression of miR-126 impaired IGF-1 signaling and increased vulnerability to the neurotoxin 6-OHDA by downregulating factors in IGF-1/PI3K signaling, including its targets p85ß, IRS-1, and SPRED1. Blocking of miR-126 function increased IGF-1 trophism and neuroprotection to 6-OHDA. Our data imply that elevated levels of miR-126 may play a functional role in DA neurons and in PD pathogenesis by downregulating IGF-1/PI3K/AKT signaling and that its inhibition could be a mechanism of neuroprotection.

The viral theory of schizophrenia revisited: abnormal placental gene expression and structural changes with lack of evidence for H1N1 viral presence in placentae of infected mice or brains of exposed offspring.

Researchers have long noted an excess of patients with schizophrenia were born during the months of January and March. This winter birth effect has been hypothesized to result either from various causes such as vitamin D deficiency (McGrath, 1999; McGrath et al., 2010), or from maternal infection during pregnancy. Infection with a number of viruses during pregnancy including influenza, and rubella are known to increase the risk of schizophrenia in the offspring (Brown, 2006). Animal models using influenza virus or Poly I:C, a viral mimic, have been able to replicate many of the brain morphological, genetic, and behavioral deficits of schizophrenia (Meyer et al., 2006, 2008a, 2009; Bitanihirwe et al., 2010; Meyer and Feldon, 2010; Short et al., 2010). Using a murine model of prenatal viral infection, our laboratory has shown that viral infection on embryonic days 9, 16, and 18 leads to abnormal expression of brain genes and brain structural abnormalities in the exposed offspring (Fatemi et al., 2005, 2008a,b, 2009a,b). The purpose of the current study was to examine gene expression and morphological changes in the placenta, hippocampus, and prefrontal cortex as a result of viral infection on embryonic day 7 of pregnancy. Pregnant mice were either infected with influenza virus [A/WSN/33 strain (H1N1)] or sham-infected with vehicle solution. At E16, placentas were harvested and prepared for either microarray analysis or for light microscopy. We observed significant, upregulation of 77 genes and significant downregulation of 93 genes in placentas. In brains of exposed offspring following E7 infection, there were changes in gene expression in prefrontal cortex (6 upregulated and 24 downregulated at P0; 5 upregulated and 14 downregulated at P56) and hippocampus (4 upregulated and 6 downregulated at P0; 6 upregulated and 13 downregulated at P56). QRT-PCR verified the direction and magnitude of change for a number of genes associated with hypoxia, inflammation, schizophrenia, and autism. Placentas from infected mice showed a number of morphological abnormalities including presence of thrombi and increased presence of immune cells. Additionally, we searched for presence of H1N1 viral-specific genes for M1/M2, NA, and NS1 in placentas of infected mice and brains of exposed offspring and found none. Our results demonstrate that prenatal viral infection disrupts structure and gene expression of the placenta, hippocampus, and prefrontal cortex potentially explaining deleterious effects in the exposed offspring without evidence for presence of viral RNAs in the target tissues.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy.

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.

mRNA and protein levels for GABAAalpha4, alpha5, beta1 and GABABR1 receptors are altered in brains from subjects with autism.

We have shown altered expression of gamma-aminobutyric acid A (GABA(A)) and gamma-aminobutyric acid B (GABA(B)) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3 GABA(A) subunits previously associated with autism (GABRalpha4; GABRalpha5; GABRbeta1). Three GABA receptor subunits demonstrated mRNA and protein level concordance in superior frontal cortex (GABRalpha4, GABRalpha5, GABRbeta1) and one demonstrated concordance in cerebellum (GABBetaR1). These results provide further evidence of impairment of GABAergic signaling in autism.

Hepatic gene expression in morbidly obese women: implications for disease susceptibility.

The objective of this study was to determine the molecular bases of disordered hepatic function and disease susceptibility in obesity. We compared global gene expression in liver biopsies from morbidly obese (MO) women undergoing gastric bypass (GBP) surgery with that of women undergoing ventral hernia repair who had experienced massive weight loss (MWL) following prior GBP. Metabolic and hormonal profiles were examined in MO vs. MWL groups. Additionally, we analyzed individual profiles of hepatic gene expression in liver biopsy specimens obtained from MO and MWL subjects. All patients underwent preoperative metabolic profiling. RNAs were extracted from wedge biopsies of livers from MO and MWL subjects, and analysis of mRNA expression was carried out using Affymetrix HG-U133A microarray gene chips. Genes exhibiting greater than twofold differential expression between MO and MWL subjects were organized according to gene ontology and hierarchical clustering, and expression of key genes exhibiting differential regulation was quantified by real-time-polymerase chain reaction (RT-PCR). We discovered 154 genes to be differentially expressed in livers of MWL and MO subjects. A total of 28 candidate disease susceptibility genes were identified that encoded proteins regulating lipid and energy homeostasis (PLIN, ENO3, ELOVL2, APOF, LEPR, IGFBP1, DDIT4), signal transduction (MAP2K6, SOCS-2), postinflammatory tissue repair (HLA-DQB1, SPP1, P4HA1, LUM), bile acid transport (SULT2A, ABCB11), and metabolism of xenobiotics (GSTT2, CYP1A1). Using gene expression profiling, we have identified novel candidate disease susceptibility genes whose expression is altered in livers of MO subjects. The significance of altered expression of these genes to obesity-related disease is discussed.

The role of LANP and ataxin 1 in E4F-mediated transcriptional repression.

The leucine-rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1--a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)--we tested whether ataxin 1 can alter the E4F-LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP-E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1.

Interaction of the hepatitis B virus protein HBx with the human transcription regulatory protein p120E4F in vitro.

Infection with the hepatitis B virus has been identified as one of the major causes of liver cancer. A large body of experimental work points to a central role for the virally encoded protein HBx in this form of carcinogenesis. HBx is expressed in HBV-infected liver cells and interacts with a wide range of cellular proteins, thereby interfering in cellular processes including cell signaling, cycle regulation and apoptosis. In order to identify possible new targets of the HBx protein, we performed a yeast two-hybrid screen using a truncated protein mini-HBx(18-142) as the bait. In addition to known interacting partners, such as RXR and UVDDB1, we identified several new candidates including the human transcriptional regulatory protein p120E4F, which has been implicated in the regulation of mitosis and the cell cycle. In vitro pull down experiments confirmed the interaction and transcription activation assays in the yeast demonstrated that HBx protein was able to repress GAL4AD-p120E4F-dependent activation of a reporter gene under the control of E4F binding sites found in the adenovirus E4 promoter and the HBV enhancer II region. We also showed that the cysteine residues in HBx are necessary for its interaction with UVDDB1 but not for the interaction with RXR or p120E4F. The possible functional relevance of the interaction between HBx and E4F proteins is discussed in the contexts of cellular transformation and host-virus co-evolution.

Association of p14ARF with the p120E4F transcriptional repressor enhances cell cycle inhibition.

The p14(ARF) tumor suppressor is a key regulator of cellular proliferation and is frequently inactivated in human cancer. This tumor suppressor functions in the p53 and pRb cell cycle regulatory pathways and can effectively activate both pathways to induce growth arrest or cell death. We now report that p14(ARF) forms a complex with the E1A-regulated transcriptional repressor, p120(E4F). p120(E4F) contacts p14(ARF) and p53 to form a ternary complex in vivo and enhances p14(ARF)-induced G(2) cell cycle arrest in a p53-dependent manner. We suggest that the interaction of p14(ARF) and p120(E4F) forms an important link between the p14(ARF) and p53 tumor suppressor proteins, both of which exhibit enhanced cell cycle inhibitory activity in the presence of this transcriptional repressor.