Functional analysis of BRCA1 in breast cancer development

Purpose

Mutations in BRCA1 strongly predispose to breast and ovarian cancer. Breast tumors in mutation carriers invariably show loss of the wildtype allele, indicating that BRCA1 acts as a tumor suppressor gene. However, somatically acquired BRCA1 mutations in non-inherited breast cancer have very rarely been reported, shedding doubt on the role of BRCA1 in sporadic breast cancer. In this project we wish to investigate this singular feature of BRCA1, by building on the hypothesis that functional inactivation of the normal BRCA1 cellular activity is particularly critical in the very early stages of normal breast glandular development. In addition, we want to address the possibility that somatic mutations haven’t been detected more frequently because they occur only in a small proportion of tumors with a certain phenotype and genotype, as predicted by current knowledge on BRCA1 cellular function.

Plan of investigation

Our approach will combine research on human material derived from selected breast cancer patients, and analysis of conditional Brca1 knock-out mouse models.

1. Human studies. Our working model predicts the presence of somatic mutations in at least a small proportion of early-onset sporadic breast tumors with similar genotypic and phenotypic features as typical BRCA1-linked breast tumors (morphology, immuno-phenotype, LOH- and gene mutation-patterns). Conversely, breast tumors which developed in carriers of a BRCA1 mutation, but at extraordinary late ages (e.g., over 60), are predicted to have in fact followed a genetic pathway independent of BRCA1-inactivation, and hence would share features characteristic of non-inherited breast cancer. Both predictions can be tested by genetic analyses of sufficient numbers of the defined breast tumors.
2. Mouse studies. We have developed a Brca1 knock-out model in our laboratory, carrying an allele in which exon 20 is disturbed (Brca1^1700T). We will make this mutation conditional by crossing it to a mouse transgenic for a BAC-clone containing the complete human BRCA1-gene bracketed by loxP sites. A recently published conditional knock-out mouse (Xu et al., Nat Genet, 22: 37, 1999), in which the Cre-mediated switch to a homozygous Brca1^del11 mutation in breast epithelial ducts occurs during pregancy, shows defects in normal mammary gland development, hyperplasia, and tumor formation with long latency. The phenotype of our homozygous Brca1^1700T mice predicts that a conditional variant will confer an even more severe phenotype than the Brca1^del11 mutation. Furthermore, the Brca1^1700T mutation will also be present in the normally occurring alternatively spliced variant lacking exon 11, so that interpretation of the phenotype will be more straightforward. We will manipulate Cre expression by employing the recently published tet-on switch (Utomo et al., Nat Biotechnol 17: 1091, 1999). This will allow us to study the phenotypic effect of Brca1 inactivation in all relevant stages of mammary gland development, instead of only during pregnancy. These studies will be supported by detailed expression analyses to examine the spatial and temporal expression of Brca1 protein in all tissues and all major stages of mouse development. For these studies we will generate a mouse which has the complete genomic mouse Brca1 sequence, tagged with GFP at the 3' end, in a BAC clone as the transgene.

Possible results

This project will shed more light on the role of BRCA1 in non-inherited, sporadic breast cancer. It will further our understanding of the physiological role of the Brca1 protein in breast cancer development, as well as in the very early stages of normal mammary gland development. This knowledge may provide clues for the development of (hormonal) intervention strategies in BRCA1 carriers.

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