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LABORATORY OF: Functional Genomics
CONTACT PERSON: Dr.Ulrich Pfeffer (IST)
Phone +39 010 5737462 E-mail: ulrich.pfeffer@istge.it

Description of Laboratory and Expertise:

The Laboratory of Functional Genomics is dedicated to high throughput genomic analyses: Gene expression profiling, exon arrays, micro-RNA expression analysis, array CGH and SNP-arrays.

Abstract of Activities:

At present, the Laboratory of Functional Genomics is involved in two main research projects:
Analysis of micro-RNA expression by human tumors (mammary carcinoma, uveal melanoma, laryngeal carcinoma) aimed at the identification of miRNAs that correlate with clinical and biological features and outcome of the tumors.
Analysis of the progressive loss of normal splicing control during the evolution of human cancers.
In addition, the laboratory works as a service provider for the research community of Genova in the field of high-throughput genomics on the Affymetrix platform.

Detailed Research Activities:

Analysis of micro-RNA expression by human tumors (mammary carcinoma, uveal melanoma, laryngeal carcinoma) aimed at the identification of miRNAs that correlate with clinical and biological features and outcome of the tumors.
Micro-RNAs (miRNAs) are small RNAs that are encoded by the nuclear genome. The primary transcripts of miRNAs are processed by the cell to yield short (21 to 23-mer) single stranded RNAs that can bind in a not completely understood manner to complementary mRNA molecules, thereby inhibiting their translation or inducing de-capping and de-adenylation and, as a consequence, their degradation. Hence, they are heavily involved in the regulation of gene expression. Evidence has accumulated for important control functions in normal and neoplastic cell physiology of these tiny RNAs. As a consequence, miRNAs are of high interest in cancer research.
We are contributing to this field through microarray-based screenings of human cancers for expression of miRNAs. MiRNA expression profiles will be generated and correlated to mRNA expression profiles in the attempt to understand whether miRNA expression simply reflects molecular classes of human cancers identified by mRNA expression profiles or whether miRNA expression allows for the identification of subtypes of cancers with specific biologic and clinical behaviors.
The choice of cancers to be examined is essentially based on the high advancement of molecular classification for mammary cancers. Breast cancer types and subtypes can therefore be matched to miRNA expression classes.
We also analyze laringeal carcinomas, since we believe that they represent a particular class of tumors that probably are distinct from tumors of other localizations in the oral cavity. Finally, we have started to analyze uveal melanomas, a rare melanoma of the eye, that is characterized by its propensity to metastatise to the liver.

The expected outcome of this project is the generation of combined mRNA-miRNA signatures with an elevated prognostic power. In addition, we hope to identify new functional aspects of miRNAS in the process of tumor progression and metastasis.
Analysis of the progressive loss of normal splicing control during the evolution of human cancers.

During the maturation of eukaryotic messenger RNAs, non coding intronic regions are removed and exons are spliced. Splicing is carried out by the spliceosome, a multi protein-RNA complex containing at least 100 protein species. It is still not completely clear how the spliceosome recognizes the correct splice sites in the precursor RNA. Differential usage of splice sites normally occurs during development and in different tissues. It is estimated that abnormal splicing accounts for 15% of all heritable diseases. In cancer, aberrant splicing has been described for many genes.

Alternative splicing appears to depend on the quantitative composition, the spliceosome. The many proteins that compose the spliceosome are encoded by genes spread over the whole genome. Genomic alterations such as chromosome copy number changes, deletions or amplifications, which are frequent events in advanced stages of cancer, are therefore highly likely to affect the expression levels of one or several splicing factors and are expected to lead to abnormal splicing of many genes. This in turn may contribute to cancer progression to a more aggressive behavior.

To address these aspects of splicing we have performed bioinformatic analyses in collaboration with Marco Muselli, Genoa (CNR-IEIIT), aimed at classifying splice sites based on the surrounding sequences. Through the application of Shadow Clustering, a rule generating algorithm, to the problem of splicing, we were able to identify classes of splice sites that obey to specific rules. These rules are characterized by discrete sequence requirements. In this way, we have identified new sequence elements that might contribute to splice site selection.

We plan to correlate the splice site classes with alternative splicing events through whole genome screenings of exon expression. In parallel, we are seeking in re-sequencing databases for mutations that affect nucleotides outside the splice consensus predicted to be informative for splice site selection.

Applications and Developments:

Production of miRNA microarrays
Development of splice site classification

Managed core facilities:

1. Affymetrix gene expression microarrays
2. Affymetrix copy number and SNP arrays

Ongoing collaborations:

Sarah Coupland, University of Liverpool, UK, uveal melanoma
Carlo Mosci, National. Cancer Research Institute, Genova, Italy, uveal melanoma
Marco Muselli, CNR-IEIIT, Genova, Italy, splice site detection
Roberto Gherzi, ABC, Genova, Italy, patterns of miRNA-specific maturation processes

Most recent and significant publications:

Ferrari N., Pfeffer U., Dell’Eva R., Ambrosini C., Noonan D.M. and Albini A. The TGF-beta family members BMP-2 and MIC-1 as mediators of the anti-angiogenic activity of 4-Hydroxyphenylretinamide. Clinical Cancer Research 11: 4610-4619, 2005.

Anfosso L., Efferth T., Albini A. and Pfeffer U.. Microarray Expression Profiles of Angiogenesis-Related Genes Predict Tumor Cell Response to Artemisinins. The Pharmacogenomics J., 2006, 6, 269-278.

Benelli R., Larghero P., Minghelli S., Travaini G., Venè R., Ferrari N., Pfeffer U., Noonan D. and Albini A. Lipoic acid activates anti oxidative stress responses in endothelial cells up-regulating Thioredoxin reductase 1 and modulating angiogenesis. Carcinogenesis, 28, 1008-20, 2007.

Indraccolo S, Minuzzo S., Pfeffer U., Gola E., Esposito G., Roni V, Ferrari N., Anfosso L., Albini A., Noonan D., Chieco-Bianchi L., Amadori A. Identification of genes selectively regulated by interferons in endothelial cells. J. Immunol., 178, 1122-35, 2007.

Bachmeier B. E., Nerlich A.G., Iancu C.A., Cilli M., Jochum M., Albini A. and Pfeffer U. The Chemopreventive Polyphenol Curcumin Prevents Hematogenous Breast Cancer Metastases in Immunodeficient Mice Through Induction of Apoptosis and Inhibition of NFkB/AP-1/CRE Dependent Transcription of Matrix Metalloproteases. Cell. Physiol. Biochem., 19, 137-52, 2007.

Pedemonte N, Caci E., Sondo E., Caputo A., Rhoden K., Pfeffer U., Di Candia M., Ravazzolo R., Zegarra-Moran O., Galietta L.G.V. Thiocyanate Transport Mechanisms in Resting and IL-4 Stimulated Human Bronchial Epithelial Cells. J. Immunol. 2007, in press.

Bachmeier BE, Mohrenz IV, Mirisola V, Schleicher E, Romeo F, Höhneke C, Jochum M, Nerlich AG, Pfeffer U. Curcumin Down-Regulates the Inflammatory Cytokines CXCL1 and -2 in Breast Cancer Cells via NF{kappaCarcinogenesis. 2007 Nov 13

Albini, A., Mirisola, V., and Pfeffer, U. Metastasis signatures: genes regulating tumor–microenvironment interactions predict metastatic behavior. Cancer Met. Rev., 2007, [Nov 29 e-pub ahead of print].

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