Johan A. den Boon

MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins.

Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3′ UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3′ UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3′ UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin γ1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.

Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus–Positive and Human Papillomavirus–Negative Head/Neck and Cervical Cancers

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.

Organelle-Like Membrane Compartmentalization of Positive-Strand RNA Virus Replication Factories

Positive-strand RNA virus genome replication is invariably associated with extensively rearranged intracellular membranes. Recent biochemical and electron microscopy analyses, including three-dimensional electron microscope tomographic imaging, have fundamentally advanced our understanding of the ultrastructure and function of organelle-like RNA replication factories. Notably, for a range of positive-strand RNA viruses embodying many major differences, independent studies have revealed multiple common principles. These principles include that RNA replication often occurs inside numerous virus-induced vesicles invaginated or otherwise elaborated from a continuous, often endoplasmic reticulum-derived membrane network. Where analyzed, each such vesicle typically contains only one or a few genome replication intermediates in conjunction with many copies of viral nonstructural proteins. In addition, these genome replication compartments often are closely associated with sites of virion assembly and budding. Our understanding of these complexes is growing, providing substantial new insights into the organization, coordination, and potential control of crucial processes in virus replication.

Cytoplasmic viral replication complexes.

Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bound RNA replication complexes and how these complexes function in close coordination with virion assembly and budding. The structure, function, and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions and more general similarities with the replication factories of cytoplasmic DNA viruses.

Random-set methods identify distinct aspects of the enrichment signal in gene-set analysis

A prespecified set of genes may be enriched, to varying degrees, for genes that have altered expression levels relative to two or more states of a cell. Knowing the enrichment of gene sets defined by functional categories, such as gene ontology (GO) annotations, is valuable for analyzing the biological signals in microarray expression data. A common approach to measuring enrichment is by cross-classifying genes according to membership in a functional category and membership on a selected list of significantly altered genes. A small Fishers exact test

Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

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Identification of Sequences in Brome Mosaic Virus Replicase Protein 1a That Mediate Association with Endoplasmic Reticulum Membranes

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Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping

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An Amphipathic α-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function

table is indicative of enrichment. Other category analysis methods retain the quantitative gene-level scores and measure significance by referring a category-level statistic to a permutation distribution associated with the original differential expression problem. We describe a class of random-set scoring methods that measure distinct components of the enrichment signal. The class includes Fishers test based on selected genes and also tests that average gene-level evidence across the category. Averaging and selection methods are compared empirically using Affymetrix data on expression in nasopharyngeal cancer tissue, and theoretically using a location model of differential expression. We find that each method has a domain of superiority in the state space of enrichment problems, and that both methods have benefits in practice. Our analysis also addresses two problems related to multiple-category inference, namely, that equally enriched categories are not detected with equal probability if they are of different sizes, and also that there is dependence among category statistics owing to shared genes. Random-set enrichment calculations do not require Monte Carlo for implementation. They are made available in the R package allez.

Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling

To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.