TRANSlational and Functional Onco-Genomics
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Functional genomics to study cancer progression and metastasis
While extensive analysis over the last two decades led to a deep insight into the control of cell proliferation and survival, and their alterations during cancer onset, still much remains to be clarified about the genetic lesions and alterations of cell signalling that lead to aberrant activation of invasive growth, cancer progression and metastasis. To this aim, great advantages may come from the historical changes in perspectives and modality of gaining information that biomedical research is currently facing. Genome-sequencing projects have been completed for many organisms, including Homo sapiens ( ) and Mus musculus ( ). This reversed the conventional approach to biomedical discovery, in which understanding a certain biological function required identification of one or more genes involved in that function. The current situation is that thousands of genes have been sequenced but still wait for any functional information to be assigned to them. The fact that genes of unknown function represent over 70% of all genes suggests that current comprehension of most biological and pathological processes is by far incomplete. This is particularly true in the case of cancer progression, where systematic exploration of gene function is likely to yield a huge amount of information in the next years. There are several ways of obtaining information about gene function, some of which have been evolving at an incredibly high pace. For example, the relatively recent development of the DNA microarray technology currently enables mRNA expression analysis in parallel for thousands of genes. Indeed, being expressed (at least at the RNA level) is an essential prerequisite for a gene to exert its function, and by studying the sites and pathways of regulation of a certain gene expression it is possible to putatively assign it to a broad functional group. In this view, genes with restricted, tissue-specific expression are likely to play key roles in the biochemical and biological processes specifically occurring at the expression sites.
Another powerful approach to gene functional characterization is exploration of the consequences of gene loss-of-function in various model organisms, ranging from unicellular microorganisms to invertebrates, vertebrates and mammals. In particular, generation of mutations in murine ES cells by targeted and random approaches offers a powerful tool for loss-of-function studies in the mouse. ES cells can be grown in vitro as a continuous cell line, genetically modified and subsequently returned to the embryo, where they can generate chimeric mice and eventually contribute to the germ line. Mouse ES cells are now widely used for gene disruption by homologous recombination or chemically induced mutagenesis, to create mutant mice that lack or express an altered form of a specific gene. Recently, an International Mouse Mutagenesis Consortium has been established, with the long-term goal of producing at least one heritable mutation, in either ES cells or mice, in every gene in the mouse genome. In many cases however, functional redundancy or subtle phenotypes may impair functional characterization of the targeted genes. Moreover, this approach is aimed at defining gene function in the context of the organism, but is hard to direct at exploring basic biological and biochemical functions at the cellular level. This latter type of information can be achieved by systematic screenings exploring features of the gene protein product, like subcellular localization, biochemical activity, interactions, and others. Recently, development of small interfering RNA (siRNA)-based approaches rendered loss-of-function studies more easily practicable in cell lines and higher organisms (Science 296:550-553, 2002). Finally, genes can be characterized by gain-of-function approaches, relying on overexpression of cloned genes in cells and organisms (Cancer Res. 61:5861-5868, 2001) or on random activation of gene expression (RAGE; Nat Biotechnol. 19:440-445, 2001).
From this brief outline of the major strategies for gene functional characterization it clearly emerges that a crucial issue in functional genomics is the development of technologies for high-throughput functional analysis. Towards this aim, development of large-scale functional screens focussed on cancer will require a coordinated approach involving complementary competences and establishment of dedicated facilities, for which TRANSFOG intends to provide an optimal organizational and financial framework.
The TRANSFOG project consists of seven research components that will synergistically enable streamlined translation of large-scale genomic screenings into high-impact contributions to cancer diagnosis and therapy. A wide variety of cancer-oriented genomic screenings will be carried out by 14 partners and finally merged with the particular aim of identifying and prioritising novel genes (ESTs or poorly characterised genes) with a clear potential role in cancer metastasis, the candidate genes. Recent works have shown that it is possible to exploit gene expression profiling of tumour samples to define sets of genes (signatures) whose expression correlates, positively or negatively, with metastasis-free survival, e.g. in breast cancer (Nature 415:530-536, 2002; N Engl J Med. 347:1999-2009, 2002.). It has also been found that a general signature associated with metastatic behaviour can be shared between solid tumours of different organs (Nat Genet. 33:49-54, 2003), which indicates that common alterations of basic cellular functions and signalling pathways trigger metastatic progression of cancer. The TRANSFOG screenings will concentrate on breast, lung and colon cancer, which altogether account for the majority of cancer deaths in the general population. Selected tumour samples and cell-based models will be analysed for the identification of molecular signatures for metastasis, through three main approaches:
  • Paired tumour/metastasis samples. In this case, the aim is to identify genes consistently showing differential expression between the primary tumour and its metastasis.
  • Primary tumour samples with no synchronous metastasis, having adequate followup data to assess the clinical history. In this case, the aim is to identify genes whose expression in a primary, non-metastatic tumour is associated with its tendency to give rise to secondary lesions after surgical removal.
  • Organ-specific metastases. The aim is to identify genes whose expression in the primary tumour is associated with metastasis homing to a specific target organ. As a target organ for metastasis, the initial focus will be posed on the liver, the most frequent metastatic target of colon cancer.
Apart from tumours, screenings will also include cancer-oriented experimental models, like serine and tyrosine kinase receptor-driven transcriptional responses, ligand-induced in vitro epithelial morphogenesis and invasive growth, in vitro angiogenesis of endothelial cells. The aim is to obtain a genome-wide exploration of the basic mechanisms of cancer progression. By merging the results of the screenings, we therefore expect to find "common" genes, i.e. genes emerging from more than one screening as associated to invasion and metastasis, and "specific" genes, whose expression is only altered in small subgroups or subtypes of tumours/metastases or cellular models. The relevant genes will have to be ranked for priority towards functional characterization and/or diagnostic validation, with the main priority criterion being their emergence in more than one screening.
Two other activities will produce, respectively, the full-length coding sequences and the siRNA sequences for these genes, enabling their functional, "mirrored" characterization by gain- and loss-of-function in cell lines and model organisms. Up-or down-regulation of these genes in models of tumour aggressiveness will provide proof-of-concept validation of their potential usefulness as new molecular targets for drug screenings. The coding sequences will also be used for systematic protein-protein interaction studies, aimed at defining the signal transduction properties of the identified candidates. The diagnostic potential of the candidate cancer genes will be evaluated and validated by systematic analysis of tumour samples with different techniques, from DNA microarrays to tissue microarrays, to quantitative real-time PCR and finally to diagnostic-grade antibodies.
The huge amount of experimental data generated by the Consortium will require establishment of a shared platform for data handling and gene functional annotation.
OECI week

OECI Week (WG workshops; Scientific Conference and General Assembly) – Budapest, 16 to 18 June 2010.
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