Patricia Soteropoulos

MicroRNA Let-7a Down-regulates MYC and Reverts MYC-Induced Growth in Burkitt Lymphoma Cells

Regulation of the MYC oncogene remains unclear. Using 10058-F4, a compound that inhibits MYC-MAX transcription factor, MYC protein and gene expression were down-regulated in Namalwa cells, a Burkitt lymphoma. Compound 10058-F4 decreased MYC mRNA (45%), MYC protein (50%), and cell growth (32%). MYC-MAX transcription factor was disrupted 24 h after treatment, resulting in transcriptional inhibition of target genes. Because microRNAs (miRNA) disrupt mRNA translation, let-7a, let-7b, and mir-98 were selected using bioinformatics for targeting MYC. Inhibition of MYC-MAX transcription factor with 10058-F4 increased levels of members of the let-7 family. In inhibited cells at 24 h, let-7a, let-7b, and mir-98 were induced 4.9-, 1.3-, and 2.4-fold, respectively, whereas mir-17-5p decreased 0.23-fold. These results were duplicated using microRNA multianalyte suspension array technology. Regulation of MYC mRNA by let-7a was confirmed by transfections with pre-let-7a. Overexpression of let-7a (190%) decreased Myc mRNA (70%) and protein (75%). Down-regulation of Myc protein and mRNA using siRNA MYC also elevated let-7a miRNA and decreased Myc gene expression. Inverse coordinate regulation of let-7a and mir-17-5p versus Myc mRNA by 10058-F4, pre-let-7a, or siRNA MYC suggested that both miRNAs are Myc-regulated. This supports previous results in lung and colon cancer where decreased levels of the let-7 family resulted in increased tumorigenicity. Here, pre-let-7a transfections led to down-regulation of expression of MYC and its target genes and antiproliferation in lymphoma cells. These findings with let-7a add to the complexity of MYC regulation and suggest that dysregulation of these miRNAs participates in the genesis and maintenance of the lymphoma phenotype in Burkitt lymphoma cells and other MYC-dysregulated cancers.

A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas

Human uterine leiomyomas (ULMs) are the most common neoplasms of women. Many genes are dysregulated in ULMs and some of this dysregulation may be due to abnormal expression of micro‐RNAs (miRNAs). In this study, 55 ULMs and matched myometrium were collected from 41 patients for microarray‐based global miRNA expression analysis. Of 206 miRNAs examined, 45 miRNAs were significantly up‐ or down‐regulated in ULMs in comparison to the matched myometrium (P < 0.001). The top five dysregulated miRNAs in ULMs are the let‐7 family, miR‐21, miR‐23b, miR‐29b, and miR‐197. Four polycistronic clusters of miRNAs were either up‐ or down‐regulated, but not in a mixed pattern, indicative of coordinated regulation of these miRNAs. Significance analysis revealed that subsets of miRNAs were strongly associated with tumor sizes and race. By prediction analysis we identified some important tumorigenic genes previously identified in ULMs that may be targeted by the dysregulated miRNAs. HMGA2 was identified as one of target genes of the let‐7 family of miRNAs and has been found to be suppressed by let‐7 in vitro. (This article contains Supplementary material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.)

Global Transcriptional Profile of Mycobacterium tuberculosis during THP-1 Human Macrophage Infection

ABSTRACT During lung infection, Mycobacterium tuberculosis resides in macrophages and subverts the bactericidal mechanisms of these professional phagocytes. Comprehension of this host-pathogen relationship is fundamental for the development of new therapies to cure and prevent tuberculosis. In this work, we analyzed the transcriptional profile of M. tuberculosis infecting human macrophage-like THP-1 cells in order to identify putative bacterial pathogenic factors that can be relevant for the intracellular survival of M. tuberculosis. We compared the gene expression profile of M. tuberculosis H37Rv after 4 h and 24 h of infection of human macrophage-like THP-1 cells with the gene expression profile of the strain growing exponentially in broth cultures. We found 585 genes expressed differentially by intracellular M. tuberculosis. An analysis of the gene expression profile of M. tuberculosis inside THP-1 cells suggests the perturbation of the cell envelope as a major intracellular stress inside THP-1 macrophages.

Human Cytomegalovirus US28 Found in Glioblastoma Promotes an Invasive and Angiogenic Phenotype

Human cytomegalovirus (HCMV) infections are seen often in glioblastoma multiforme (GBM) tumors, but whether the virus contributes to GBM pathogenesis is unclear. In this study, we explored an oncogenic role for the G-protein-coupled receptor-like protein US28 encoded by HCMV that we found to be expressed widely in human GBMs. Immunohistochemical and reverse transcriptase PCR approaches established that US28 was expressed in approximately 60% of human GBM tissues and primary cultures examined. In either uninfected GBM cells or neural progenitor cells, thought to be the GBM precursor cells, HCMV infection or US28 overexpression was sufficient to promote secretion of biologically active VEGF and to activate multiple cellular kinases that promote glioma growth and invasion, including phosphorylated STAT3 (p-STAT3) and endothelial nitric oxide synthase (e-NOS). Consistent with these findings, US28 overexpression increased primary GBM cell invasion in Matrigel. Notably, this invasive phenotype was further enhanced by exposure to CCL5/RANTES, a US28 ligand, associated with poor patient outcome in GBM. Conversely, RNA interference-mediated knockdown of US28 in human glioma cells persistently infected with HCMV led to an inhibition in VEGF expression and glioma cell invasion in response to CCL5 stimulation. Analysis of clinical GBM specimens further revealed that US28 colocalized in situ with several markers of angiogenesis and inflammation, including VEGF, p-STAT3, COX2, and e-NOS. Taken together, our results indicate that US28 expression from HCMV contributes to GBM pathogenesis by inducing an invasive, angiogenic phenotype. In addition, these findings argue that US28-CCL5 paracrine signaling may contribute to glioma progression and suggest that targeting US28 may provide therapeutic benefits in GBM treatment.

Global Analysis of Pub1p Targets Reveals a Coordinate Control of Gene Expression through Modulation of Binding and Stability

ABSTRACT Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1Δ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1Δ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1Δ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3′ untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.

Energy metabolism pathways in rat muscle under conditions of simulated microgravity

Evidence from rats flown in space suggests that there is a decrease in the ability of the soleus muscle to oxidize long chain fatty acids during space flight. The observation suggests that a shift in the pathways involved in muscle fuel utilization in the absence of load on the muscle has occurred. It is also possible that the reduction is part of a general down-sizing of metabolic capacity since energy needs of inactive muscle are necessarily less. The rodent hind limb suspension model has proved to be a useful ground based model for studying the musculo-skeletal systems changes that occur with space flight. Microarray technology permits the screening of a large number of the enzymes of the relevant pathways thereby permitting a distinction to be made between a shift fuel utilization pattern or a general decrease in metabolic activity. The soleus muscle was isolated from 5 control and 5 hindlimb suspended rats (21 days) and the Affymetrix system for assessing gene expression used to determine the impact of hindlimb unloading on fuel pathways within the muscle of each animal. RESULTS: Suspended rats failed to gain weight at the same rate as the controls (337 +/- 5 g vs 318 +/- 6 g, p < 0.05) and muscle mass from the soleus was reduced (135 +/- 3 mg vs 48 +/- 4 mg, p < 0.05). There was a consistent decrease (p < 0.05) in gene expression of proteins involved in fatty acid oxidation in the suspended group whereas glycolytic activity was increased (p < 0.05). Gene expressions of individual key regulatory enzymes reflected these changes. Carnitine palmitoyltransferase I and II were decreased (p < 0.05) whereas expression of hexokinase, phosphofructokinase and pyruvate kinase were increased (p < 0.05). CONCLUSION: Disuse atrophy is associated with a change in mRNA levels of enzymes involved in fuel metabolism indicative of a shift in substrate utilization away from fat towards glucose.

Profiling and Functional Analyses of MicroRNAs and Their Target Gene Products in Human Uterine Leiomyomas

Background Human uterine leiomyomas (ULM) are characterized by dysregulation of a large number of genes and non-coding regulatory microRNAs. In order to identify microRNA::mRNA associations relevant to ULM pathogenesis, we examined global correlation patterns between the altered microRNA expression and the predicted target genes in ULMs and matched myometria. Methodology/Principal Findings Patterns of inverse association of microRNA with mRNA expression in ULMs revealed an involvement of multiple candidate pathways, including extensive transcriptional reprogramming, cell proliferation control, MAP kinase, TGF-β, WNT, JAK/STAT signaling, remodeling of cell adhesion, and cell-cell and cell-matrix contacts. We further examined the correlation between the expression of the selected target gene protein products and microRNAs in thirty-six paired sets of leiomyomas and matched myometria. We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level. The comparative genomic hybridization (CGH) in eight ULM patients revealed that partially shared deletions of two distinct chromosomal regions might be responsible for loss of cancer–associated microRNA expression and could thus contribute to the ULM pathogenesis via deregulation of target mRNAs. Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro. Conclusions/Significance We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro. Our findings provide a broad overview of molecular events underlying the tumorigenesis of uterine ULMs and identify select genetic and regulatory events that alter microRNA expression and may play important roles in ULM pathobiology by positively regulating tumor growth while maintaining the non-invasive character of ULMs.

Prostaglandin J2 alters pro-survival and pro-death gene expression patterns and 26 S proteasome assembly in human neuroblastoma cells.

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a “repair” response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.

Identification of Direct Transcriptional Targets of the Kaposi's Sarcoma-Associated Herpesvirus Rta Lytic Switch Protein by Conditional Nuclear Localization

ABSTRACT Lytic reactivation from latency is critical for the pathogenesis of Kaposis sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rtas direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.

Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication

ABSTRACT Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.