Programme: The Genetics of Breast Cancer
E-cadherin mutational inactivation occurs in the majority of lobular breast cancers and is an early event that may arise in the pre-invasive stage. E-cadherin inactivating mutations have also been found in diffuse gastric cancer, both sporadic and hereditary cases. E-cadherin protein is involved in cellular adhesion and signal transduction. The role of E-cadherin in adhesion, its downregulation in various cancers, and results from in vitro experiments have suggested an invasion suppressor role for E-cadherin. However, our observation of E-cadherin inactivation in pre-invasive breast cancer, as well as data from the literature, suggest an additional role for E-cadherin as a 'gatekeeper' in control of cell growth and early carcinogenesis of lobular breast tumours. The work proposed here is aimed at the identification of genes and possible molecular pathways involved in this 'gatekeeper' role of E-cadherin.
We want to identify those molecules in mammary epithelial cells that are directly or indirectly affected by loss of E-cadherin function at the level of transcription. This will supply us with a better insight into the molecular mechanisms that are the result of E-cadherin inactivation, especially the early pre-invasive events, which is important for understanding lobular breast carcinogenesis. A few molecules have been identified to be involved in E-cadherin mediated growth inhibition and tumour suppression such as p27KIP1, however the majority is as yet unknown. In order to identify the genes whose expression is directly or indirectly dependent on the presence of functional E-cadherin we propose an approach for the investigation of the expression from a large number of genes simultaneously using the novel technique of cDNA microarraying.
The relative expression patterns of E-cadherin negative breast tumour cell lines will be compared with those of the same cell lines transfected with an E-cadherin encoding cDNA. For this purpose cDNA microarrays will be constructed on glass slides containing cDNAs that represent genes involved in signalling, growth, cell cycle control, apoptosis, cellular structure and morphology as well as all the possible homologues of these genes, the latter representing novel candidate genes involved in these processes. The cDNA probes that are used for this analysis will be selected from a well documented 40,000 clone library containing partial or total cDNAs of known and novel genes. The data resulting from these expression analyses will be studied using clustering algorithms that have been applied successfully to several microarray studies. Possible cascades of transcriptional alterations will be studied by comparing different time-points after induction of E-cadherin expression from an inducible promotor. In a second stage of the project this analysis will be applied to primary lobular breast tumours with and without inactivating E-cadherin mutations.
In the first year, with extension to the second year, the microarrays will be constructed and hybridised with RNA from transfectants, and later with RNA from primary loular breast tumours with and without E-cadherin mutations. The second and third year will be dedicated to verification and analysis of the results and characterisation of possible novel genes. This analysis will result in the ascertainment of the role of known genes and identification novel genes in mammary epithelial cells whose RNA expression is modified by the presence or absence of functional E-cadherin. Eradication of this molecule plays a key role in early lobular breast carcinogenesis and the unravelling of molecular pathways that are involved in this event are important for understanding the carcinogenic processes in this tumour type and possible other tumours in which E-cadherin inactivation plays a role. Furthermore we expect to identify new genes that are implicated in these pathways.
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