Rehannah Borup

RNA‐binding IMPs promote cell adhesion and invadopodia formation

Oncofetal RNA‐binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss‐of‐function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post‐transcriptional control of the transcripts. In particular, we show that a 5.0 kb CD44 mRNA contained multiple IMP‐binding sites in its 3′UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP‐mediated invadopodia formation. Taken together, our results indicate that RNA‐binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation.

Molecular Composition of IMP1 Ribonucleoprotein Granules

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100–300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study.

Background:MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).Methods:Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.Results:One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.Conclusion:MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

Gene Expression in Skeletal Muscle Biopsies from People with Type 2 Diabetes and Relatives: Differential Regulation of Insulin Signaling Pathways

Background Gene expression alterations have previously been associated with type 2 diabetes, however whether these changes are primary causes or secondary effects of type 2 diabetes is not known. As healthy first degree relatives of people with type 2 diabetes have an increased risk of developing type 2 diabetes, they provide a good model in the search for primary causes of the disease. Methods/Principal Findings We determined gene expression profiles in skeletal muscle biopsies from Caucasian males with type 2 diabetes, healthy first degree relatives, and healthy controls. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 Arrays covering the entire human genome. These arrays have not previously been used for this type of study. We show for the first time that genes involved in insulin signaling are significantly upregulated in first degree relatives and significantly downregulated in people with type 2 diabetes. On the individual gene level, 11 genes showed altered expression levels in first degree relatives compared to controls, among others KIF1B and GDF8 (myostatin). LDHB was found to have a decreased expression in both groups compared to controls. Conclusions/Significance We hypothesize that increased expression of insulin signaling molecules in first degree relatives of people with type 2 diabetes, work in concert with increased levels of insulin as a compensatory mechanism, counter-acting otherwise reduced insulin signaling activity, protecting these individuals from severe insulin resistance. This compensation is lost in people with type 2 diabetes where expression of insulin signaling molecules is reduced.

Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin.

OBJECTIVE To investigate the differences in the gene expression profile of granulosa cells from preovulatory follicles after controlled ovarian hyperstimulation (COH) with recombinant follicle-stimulating hormone (FSH) or urinary human menopausal gonadotropin (hMG) FSH. DESIGN Prospective randomized study. SETTING University-based facilities for clinical services and research. PATIENT(S) Thirty women undergoing treatment with vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). INTERVENTION(S) Patients were randomly allocated to receive recombinant FSH or human (hMG) COH. Granulosa cells were collected from follicular fluid after oocyte retrieval, and mRNA were isolated for gene expression analysis. MAIN OUTCOME MEASURE(S) General gene expression profile. RESULT(S) Ninety-six probe sets (85 genes) showed statistically significant differences in expression level in the two groups of granulosa cells. Expression level of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor gene and genes involved in biosynthesis of cholesterol and steroids were expressed at lower levels in the hMG-treated cells; inositol 1,4,5-triphosphate-3-kinase-A and S100-calcium-binding-protein-P (anti-apoptosis protein) were expressed at higher levels in hMG than in recombinant FSH. CONCLUSION(S) The different hormone compositions of the two drugs used for COH had a statistically significant impact on the gene expression profile of preovulatory granulosa cells. Some of these genes may be important for periovulatory events, which suggests that the preparation used for COH is important for granulosa cell function and may influence the developmental competence of the oocyte and the function of corpus luteum.

Down-regulation of microRNAs controlling tumourigenic factors in follicular thyroid carcinoma

The molecular determinants of thyroid follicular nodules are incompletely understood and assessment of malignancy is a diagnostic challenge. Since microRNA (miRNA) analyses could provide new leads to malignant progression, we characterised the global miRNA expression in follicular adenoma (FA) and follicular carcinoma (FC). Comparison of carcinoma and adenoma with normal thyroid revealed 150 and 107 differentially expressed miRNAs respectively. Most miRNAs were down-regulated and especially miR-199b-5p and miR-144 which were essentially lost in the carcinomas. Integration of the changed miRNAs with differentially expressed mRNAs demonstrated an enrichment of seed sites among up-regulated transcripts encoding proteins implicated in thyroid tumourigenesis. This was substantiated by the demonstration that pre-miR-199b reduced proliferation when added to cultured follicular thyroid carcinoma cells. The down-regulated miRNAs in FC exhibited a substantial similarity with down-regulated miRNAs in anaplastic carcinoma (AC) and by gene set enrichment analysis, we observed a significant identity between target mRNAs in FC and transcripts up-regulated in AC. To examine the diagnostic potential of miRNA expression pattern in distinguishing malignant from benign nodules we employed a supervised learning algorithm and leave-one-out-cross-validation. By this procedure, FA and FC were identified with a negative predicted value of 83% (data generated by microarray platform) and of 92% (data generated by qRT-PCR platform). We conclude that follicular neoplasia is associated with major changes in miRNA expression that may promote malignant transformation by increasing the expression of transcripts encoding tumourigenic factors. Moreover, miRNA profiling may facilitate the diagnosis of carcinoma vs adenoma.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia‐inducible factor 1 (HIF‐1). HIF‐1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF‐1 and mediates the hypoxic repression of a set of nuclear‐encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear‐encoded mitochondrial genes where it directly antagonizes c‐Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short‐hairpin RNA (shRNA)‐expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)‐6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays in wild‐type and IL‐6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post‐lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with ∼12 400 probe sets corresponding to ∼10 000 different murine genes (MG_U74Av2). A robust, conventional statistical method (two‐way anova) was employed to select the genes significantly affected. An orderly pattern of gene responses was clearly detected, with genes being up‐ or down‐regulated at specific timings consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma. IL‐6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post‐lesion timing, which presumably underlies the poor capacity of IL‐6 knockout mice to cope with brain damage. The results highlight the importance of IL‐6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study.

Highly glycosylated α1‐acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils

α‐1‐Acid glycoprotein (AGP) is an acute‐phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil‐derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma‐derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer‐binding protein (C/EBP)‐ɛ in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

Microarray-based classification of diffuse large B-cell lymphoma

Abstract:  Objective: Hierarchical clusterings of diffuse large B‐cell lymphoma (DLBCL) based on gene expression signatures have previously been used to classify DLBCL into Germinal Center B‐cell (GCB) and Activated B‐cell (ABC) types. To examine if it was feasible to perform a cross‐platform validation on the Affymetrix HG‐U133A oligonucleotide arrays and improve the classification, we determined the expression profiles of pretreatment, diagnostic samples from 52 primary nodal DLBCL. Methods and results: First, three previously published gene lists were converted to the HG‐U133A probe sets and used for hierarchical clustering. In this way, three subtypes, including the GCB type (n = 20), the ABC type (n = 25) and an intermediate group, Type‐3 (n = 5), were distinguished. The CD10 and Bcl‐6 expression as well as t(14;18) translocation were prevalent, but not exclusive to the GCB type. By contrast, MUM1 was only expressed in the ABC and in Type‐3 samples. The 5‐year survival was similar between the groups, but GCB patients showed a better initial response to CHOP or CHOP‐like regimens than the remaining patients and tended to have less advanced disease and lower IPI scores. As a next step, an improved set of classifier genes was generated by analysis of 34 patients that were consistently classified as GCB or ABC in the above analyses. Seventy‐eight genes were selected and demonstrated on two previously published data sets (Shipp et al. Nat Med 2002;8:68–74 and Houldsworth et al. Blood 2004;103:1862–1868) to exhibit a higher specificity than the original gene lists. Conclusion: We conclude that gene expression profiling with Affymetrix Genechips is efficient to distinguish between GCB and ABC types of DLBCL and that these are likely to represent separate biological entities. The Genechip platform is highly standardised and therefore useful for future prospective investigations to establish the value of gene expression profiling in the clinical management of DLBCL.