Retroviral insertional mutagenesis screens have been used for many years as a tool for cancer gene discovery. In recent years, completion of the mouse genome sequence as well as improved technologies for cloning and sequencing of retroviral insertions have greatly facilitated the retrieval of more complete data sets from these screens. The concomitant increase of the size of the screens allows researchers to address new questions about the genes and signalling networks involved in tumour development. In addition, the development of new insertional mutagenesis tools such as DNA transposons enables screens for cancer genes in tissues that previously could not be analysed by retroviral insertional mutagenesis.
The integration of slow transforming retroviruses into the genome of host cells can lead to deregulation of nearby genes. Cells can be infected repeatedly, acquiring multiple mutations that can lead to the development of a tumour.
Genes implicated in tumour formation are found through the identification of proviral or transposon insertions in independent tumours at frequencies higher than would be expected by chance. Genes in loci that recurrently contain proviral or transposon insertions in independent tumours show a significant overlap with known human oncogenes and tumour suppressor genes.
The arrival of next-generation sequencing technologies allows identification of most if not all insertions in a tumour. This will enable a more comprehensive identification of cancer genes and cooperating pairs of cancer genes and facilitate the establishment of the functional role of these cancer genes in tumour development.
Examining the genes that collaborate with common mutations in the Myc, p53, Rb and Ras pathways may identify genes that are essential for activating or abrogating the tumorigenic effects of these mutations. Using retrovirus- or transposon-mediated insertional mutagenesis to identify genes that collaborate with genes of the Myc, p53, Rb and Ras pathways may expand the range of targets for developing novel cancer therapeutics.
Retrovirus- or transposon-mediated insertional mutagenesis screens can be used to identify genes that confer resistance to drugs both in vitro and in vivo and may thus provide pivotal information to improve existing therapeutic strategies.
Although most proviral insertions are activating rather than inactivating, a substantial fraction of the insertions are found to abrogate gene function, thus enabling the identification of tumour suppressor genes.
Cross-comparison of insertional mutagenesis data from mice with mutation data from human tumour panels may help identify the 'driver' mutations in human cancer. With the development of insertional mutagenesis strategies for solid tissues these cross-comparisons will become even more powerful, as they will allow direct comparison of human tumour data with insertions derived from their cognate mouse tumours.
Scrutinizing concurrent and mutually exclusive insertions in clonal cell populations of tumours or in single cells of a tumour in combination with high-quality expression profiling and proteomics analysis may teach us about underlying mechanisms of tumour heterogeneity and their role in tumour initiation and maintenance.
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We thank A. Uren, A. Sparmann, J. Jonkers and M. van Lohuizen for discussions and critically reading the manuscript. J.K. was supported by the De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) Genomics Programme and the Cancer Genomics Centre, which is supported by the Netherlands Genomics Initiative (NGI).
The authors declare no competing financial interests.
The continuous presence of infectious viruses in an organism.
- Viraemic phase
The stage of viral infection of an organism in which infectious viruses are present in the body.
The integration of a retrovirus or transposon in the genomic DNA of a host cell through an RNA intermediate.
- Massive parallel sequencing
Next-generation sequencing technologies that are able to sequence millions of DNA fragments in parallel.
- DNA transposon system
DNA transposons are genetic elements that can be excised from a genome or episomal element and integrate (transpose) into another DNA site. The transposase protein is required for this process.
- Gene trap
A vector element consisting of a splice acceptor site and polyadenylation signal that is intended to create a fusion of the cellular transcript and vector sequences, resulting in termination of transcription of the cellular gene.
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