Programme: The Genetics of Breast Cancer
Molecular-genetic as well cytogenetic studies have revealed that loss of heterozygosity (LOH) at the long arm of chromosome 16 (16q) belongs to the most frequent genetic alterations occurring in breast cancer. In a previous study we identified two separate smallest regions of overlap (SRO) involved in LOH at 16q22.1 and 16q24.3. In lobular breast cancer we unmasked E-cadherin at 16q22.1 as a major target for LOH showing inactivating mutations in about 60% of the tumours. In an effort to identify the tumour suppressor genes (TSGs) involved in ductal breast we constructed a complete physical and transcription map of the SRO at 16q24.3 in collaboration with in the international Fanconi Anaemia A/Breast Cancer Consortium. Although this map was instrumental for the identification of the Fanconi anaemia gene this gene and most other transcripts identified in this region have been exluded and thus the TSG(s) at 16q24.3 remain(s) elusive. Because of the biological complexity of LOH data, probably targeting more than one gene at 16q, as well as technical problems, the precise SRO boundaries remain uncertain. For this reason the relialability of SROs as sole starting poin for positional cloning can be questioned. Here we propose a novel "brute force" positional cloning approach to identify the breast cancer TSG(s) at 16q. This approach accounts for inaccuracies in the SRO boundaries and entails: 1) identification of genes located at 16q, 2)identification of the subset of genes expressed in breast epithelium, 3) selection of best candidates, 4)high-throughput mutation screening and 5) validation of remaining candidate TSGs.
From the 1000-2000 genes on 16q it is estimated that + 100 will be located at 16q24.3 and + 150 at 16q22.1. With current technology, it will be feasible to analyze a set of about 40 of the most likely candidate genes for inactivating mutations in tumours during the course of this project. The priority of candidate genes to be investigated will be determined by the following ranking: 1) located in the extended LOH region at 16q24.3 2) expression in normal mammary epithelial cells 3) decreased expression in some breast cancer cell lines 4) function or homology compatible with tumour suppression. To identify genes and their sequence we will use data available from electronic databases, large-scale sequencing efforts and from the data of our collaborators in Adelaide who are sequencing band 16q24.3 and characterizing genes in that region. Expression will be determined by microarray analysis with genes and ESTs located on 16q, as probes. Cy3- and Cy5- labeled RNAs from normal breast epithelial cells and breast cancer cell lines, will be used as targets. A facility for microarray analysis recently has been installed at our institute. We will start with using genes and ESTs selected from. the I.M.A.G.E. 40,000 cDNA set as probes. This set will be complemented by genes localized on 16q during the course of the project. Information on (putative) gene function will be retrieved from databases or inferred from sequence homology. Prior to mutation exon-intron boundaries will be sequenced to design primers for mutation analysis and sequencing. To maximize the probability on detection of a mutation, candidate genes will be screened for mutations by fluorescent CSGE (conformation sensitive gel electrophoresis) in a panel of 50 tumours representing different histological subtypes and showing LOH at the gene locus, preferably restricted to the region that harbours this gene and in a breast cancer cell line panel with 16q homozygosity. We have a panel of 500 well-characterized breast tumours available to determine mutation frequencies in genes with validated mutations. Functional analysis entails transfection of full length cDNA to breast tumour cell lines and investigation of effects on proliferation, apoptosis and cell cycle progression.
The two smallest regions of overlapping LOH on 16q22.1 and 16q24.3 have been delineated to 1.3 cM and 650 kb, respectively (figure). Within the scope of a newly established consortium, the Fanconi anaemia/Breast Cancer Consortium, a complete cosmid contig has been constructed from the 650 kb critical region on 16q24.3 and a dense transcription map is being assembled. Screening for mutations in candidate genes and newly identified transcripts in tumors showing LOH is in progress (figure). The E-cadherin gene on 16q22.1 has been identified as a classical tumor suppressor gene which is inactivated by truncating mutations and LOH. Intriguingly, this occurs only in a histological subset of breast tumors, i.e. the infiltrating lobular type. In two-thirds of the lobular cases the E-cadherin gene is mutated. In infiltrating ductal carcinomas no E-cadherin mutations could be identified indicating that another tumor suppressor involved in these tumors gene must exist on 16q22.1.
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