Molecular Analysis of BRCA1 Function

Institution: Salk Institute for Biological Studies
Investigator(s): Quan Zhu, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9FB-0172 Award: $85,557
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2003)
Mutations in the hereditary breast cancer suppressor gene BRCA1 account for almost half of the familial breast cancers and the majority of the combined familial mammary and ovarian malignancies. Inherited mutations in BRCA1 predispose the carrier at high risk about 60 to 80%, whereas most sporadic breast cancers show lower expression levels of BRCA1. The normal biological function of the protein is essential for understanding the mechanism of disease formation. Presently, BRCA1 is associated with numerous cell functions, including cell cycle control, controlling protein degradation, DNA damage repair, and transcriptional (gene activity) regulation.

Although mounting evidence has shown that BRCA1 is involved in multiple cellular pathways, its precise biochemical functions in suppressing tumor formation remain to be elucidated. Furthermore, most of the data were obtained from experiments in tissue culture cells or mice lacking functional BRCA1 genes. However, BRCA1 deficient mice die at an early developmental stage, hampering further analysis of the role of the gene in later developmental stages. Therefore, it is imperative to develop better BRCA1animal models, so that the biological functions of human BRCA genes can be dissected.

To establish a better breast cancer mouse model, we attempt to use a highly advanced gene transfer technique, the lentiviral vectors, to create transgenic mice with tightly-controlled, inducible expression of human BRCA1. Lentiviral vectors have been proven to be a powerful tool in introducing transgenes in a variety of tissue culture or primary cells, as well as in experimental animals with remarkably high efficiency and stable expression. To avoid toxicity of BRCA1 overexpression and to mimic the physiological expression levels of BRCA1, the expression of the transgene will be controlled by administration of an inducing drug in the drinking water of the animals. We hypothesize that such BRCA1 transgene with modulated expression will be functionally equivalent to the mouse BRCA1 gene so that the function of BRCA1 in normal development can be studied. In other proposed experiments we plan to use this transgenic system to study the distinct BRCA1 function domains. We hope to identify those domains that can reconstitute the function of full length protein.

This project will shed light on both basic breast cancer pathogenesis research and gene therapy, because BRCA1 mutants found in human breast cancers can be readily studied using this system. Successful selection of protein domains of BRCA1 that can recapitulate the full-length gene will deepen our understanding of the function and structure of the tumor suppressors. This could lead to the use of smaller BRCA1 fragments as therapy targets to prevent cancer progression.


Final Report (2005)
Women with inherited mutations in BRCA1 are predisposed at high risk at a level of about 80%, whereas most sporadic breast cancers show lower expression levels of BRCA1. Presently, BRCA1 is associated with numerous cell functions, including cell cycle control, controlling protein degradation, DNA damage repair, and transcriptional (gene activity) regulation. Although mounting evidence has shown that BRCA1 is involved in multiple cellular pathways, its precise biochemical functions in suppressing tumor formation remain to be elucidated. Furthermore, most of the data were obtained from experiments in tissue culture cells or mice lacking functional BRCA1 genes. However, BRCA1 deficient mice die at an early developmental stage, hampering further analysis of the role of the gene in later developmental stages. Therefore, it is imperative to develop better BRCA1 animal models, so that the biological functions of human BRCA genes can be dissected.

To establish a better breast cancer mouse model, we attempted to create transgenic mice harboring human BRCA1 using a highly advanced gene transfer technique, the lentiviral vectors. Through Southern analysis, we found that six founders were generated containing human BRCA1 directed by PGK promoter and one founder was identified to carry a human BRCA1 gene with tightly-controlled and inducible expression. To avoid toxicity of BRCA1 overexpression and to mimic the physiological expression levels of BRCA1, the expression of the transgene are being controlled by administration of an inducing drug in the drinking water of the animals. Unfortunately, we were not able to detect any transgene expression in any of the lines by Northern blotting and Western blotting. Furthermore, no mice were rescued by mating with mice are defective for mouse BRCA1. Therefore, although lentiviral vectors have been proven to be a powerful tool in introducing transgenes in experimental animals with remarkably high efficiency, no human BRCA1 transgenic animals were successfully generated in this project.