Identifying Novel Breast Cancer Tumor Suppressor Genes

Institution: The Burnham Institute for Medical Research
Investigator(s): Manuel Perucho, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0170 Award: $97,500
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1997)
It is widely accepted that breast cancer arises because of a loss of equilibrium between positive and negative regulators of cell growth. Genes with positive roles in cell growth are called oncogenes. When oncogenes are mutated or overexpressed they can act in dominant fashion to accelerate cell proliferation. In contrast, genes that negatively regulate cell replication are called "tumor suppressor" genes, and when mutated lose their ability to suppress cell growth. Due to the dominant nature of oncogenes, they are easier to identify and isolate. Unfortunately, tumor suppressor genes are more difficult to identify. Thus, at present the number of isolated oncogenes is greater than the number of tumor suppressor genes.

Our goal in this project is to utilize a new experimental approach for cloning tumor suppressor genes from mammary gland cells. To achieve this, we will employ the tools used by evolution. Our approach relies on the generation of diversity followed by selection. In addition, this approach parallels a strategy used by DNA tumor viruses to transform infected normal cells. Previous work has shown that these DNA viruses produce proteins that bind and inactivate the protein products of tumor suppressor genes. This stimulates cell growth to promote viral multiplication.

Following this viral-based strategy, we initially will select from a large collection of artificial bacterial viruses (known as phage libraries) that contain short, random stretches of amino acids (oligopeptides). We propose that these oligopeptides can specifically bind intracellular proteins of mouse mammary gland cells. Next, portions of the enriched, mammary gland-specific phage libraries will be introduced back into breast cells. The idea is to identify phage library-derived peptides that will inhibit undiscovered breast cancer tumor suppressors. We anticipate that the breast cells with inhibited tumor suppressors will have a selective growth advantage. Thus, our unique approach should circumvent the usual difficulties of identification and isolation of tumor suppressor genes. Finally, the isolated viral clones that inhibit tumor suppressors will be used to isolate their genes.

If this approach is successful in the identification and isolation of mouse mammary gland tumor suppressor genes, it will directly impact our understanding of human breast cancer. Any novel breast cancer tumor suppressor genes should contribute to the future development of diagnostic, prognostic and therapeutic approaches.

Final Report (1998)
The main goal of this project was to develop a new approach for the identification and cloning of genes for negative regulators of cell growth or survival (i.e., either tumor suppressors or pro-apoptotic genes) of the mammary gland. Our approach was to use bacterial virus (bacteriophage) 'libraries' that display stretches of amino acids (oligopeptides). This would enable us to detect intracellular proteins from mammary gland cells.

In several pilot experiments we tested our approach by using two known intracellular proteins, the pro-apoptotic protein, called caspase-3, and the product of the Rb tumor suppressor gene. We tested different phage libraries that carry on their surface peptides of different length. Using a bacteriophage library that express oligopeptides of 10 aminoacids, we succeeded in the isolation of phage that bind specifically to caspase-3. The phage that expressed the peptide bind caspase -3 immobilized on the surface of plastic plates as well as in solution. In contrast, these phage don't bind control proteins, such as bovine albumins, gelatins or gluthatione transferase. In a second set of experiments using another phage library that expresses linear peptides of 12 amino acids, we were also able to select phage that bind the product of the Rb tumor suppressor gene and caspase-3. The phage bind these target proteins in solution, which indicates that they recognize the native structure of the proteins. These results support our initial hypothesis, and show that it is possible to identify from phage display libraries certain oligopeptides that bind known intracellular proteins with pro-apoptotic and tumor suppressor function.

The next step will be to express the phage inserts as 'fusion proteins' and to test the binding of the corresponding protein motifs to its targets in vivo. We plan to apply for additional funding to continue with this project. Identification of new tumor suppressor and/or apoptosis related genes would expand our knowledge of breast cancer biology.