A Novel Anti-estrogen Resistance Mechanism in Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Kathryn Ely, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8IB-0187 Award: $143,250
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2002)
A widely used treatment of breast cancer patients having estrogen receptor positive (ER+) tumors is to administer anti-estrogens, such as tamoxifen, to block activation of target genes. However, this treatment is effective for only one-half of patients with ER+ tumors, because some tumors display an 'intrinsic' resistance to tamoxifen. In addition, most, if not all, breast tumors that initially respond to anti-estrogens will eventually develop resistance to the treatment. Recently, two breast cancer anti-estrogen resistance genes have been identified that are associated with poor response to tamoxifen and rapid recurrence of disease. The protein products of these genes physically interact and together control activation of cellular signaling pathways. The proteins involved are Bcar1/Cas (a docking protein) and SHEP2 (an exchange factor). Our hypothesis is that if we can understand the physical nature of the interaction between these two proteins, and define the precise region in each protein that is involved, then this knowledge could lead to drug discovery efforts to combat anti-estrogen resistance.

Our research plan is multidisciplinary and combines cell and structural biological approaches. My colleague at the Burnham Institute, Dr. Kristiina Vuori, is responsible for the cell-based studies in this project. First, the suspected interacting domains of Cas and SHEP will be cloned and the proteins produced in both bacterial and other cell culture systems. We will use selective protein degradation and biophysical approaches (e.g., mass spectrometry) to narrow down the interacting elements for each protein. Secondly, we will introduce regions of each protein into MCF7 breast cancer cells and determine the effect on tamoxifen resistance. In some cases we will perform these experiments with mutant forms of the two proteins to confirm the importance of specific amino acid residues. Finally, we propose to crystallize Cas in complex with SHEP to determine the precise three-dimensional structure of this interaction. This interface may represent a novel target for new drugs to block inherent and acquired resistance to the anti-estrogen response.

Bcar1/Cas has just recently been linked to breast cancer anti-estrogen resistance, and the association of Cas and SHEP has been described only within the past year. The results of this research will provide new information on the interactions between these domains and advance our understanding of a key drug resistance clinical problem in breast cancer.


Final Report (2004)
Recent developments have identified Cas protein as a major indicator in breast cancer. Cas is now recognized as a protein encoded by the gene BCAR1 (breast cancer anti-estrogen resistance). The relatively high expression of BCAR1 has been associated with rapid recurrence of the disease and also poorer response to tamoxifen treatment in primary breast cancer cells. The intermolecular contacts that drive Cas signaling in breast cancer can be probed directly by structural biology. With funding from the California Breast Cancer Research Program, we have initiated structural studies on protein interactions of Cas with SHEP proteins, products of another anti-estrogen resistance locus, BCAR3. SHEP proteins contain an SH2 domain and a guanine nucleotide exchange factor (GEF) domain. Through the GEF domain, SHEP proteins bind to GTP-ases such a R-Ras. This same domain associates with Cas, and it has been suggested that Cas inhibits the Ras exchange activity of SHEP2. Since genetic evidence links both Cas and SHEP to antiestrogen resistance, and biochemical evidence indicates that the two protein s physically interact, the goal of this IDEA award was to define the interacting domains of Cas and SHEP as the first step in testing the hypothesis that simultaneous over-expression of the proteins and formation of the Cas/SHEP2 complex affects tamoxifen resistance.

Our main aim in this project was to define the domains of Cas and SHEP2 that are required for their association and to produce them recombinantly for crystallization studies. First, recombinant proteins of different lengths were generated for each partner; until the minimal binding regions were identified. Great progress has been made toward this goal utilizing a strategy to co-express the two proteins in bacteria. The work at this point has focused on SHEP1. For co-expression, one protein was expressed as a GST-fusion protein and the other partner with a hexahistidine fusion tag. Extensive experiments demonstrated that this strategy was feasible and trials with the fusion partners added and then also with the tags reversed revealed that the yield of soluble complex was dramatically improved when the fusion partners were matched in a specific manner; i.e., GST-SHEP and His-Cas. Milligram quantities of the complex have been purified and the material is stable and soluble.

The project now moves on to the biological characterization of these domains and the complex in tumor cell assays. To evaluate the role of this complex functionally, experiments are underway to generate crystal structures of the partners or the complex. Our hypothesis remains that if we can understand the physical nature of the interaction between these two proteins, and define the precise region in each protein that is involved, then this knowledge could lead to drug discovery efforts to combat anti-estrogen resistance in breast cancer treatment.


Symposium Abstract (2003)
Resistance to antiestrogen therapy is a serious obstacle in the treatment of breast cancer. Clearly, a better understanding of the biology of antiestrogen resistance could provide immense help when designing new therapeutic strategies to treat refractory tumors. A new gene BCAR1 has been discovered that is expressed at high levels in breast tumors and is associated with tamoxifen resistance, poor survival and increased metastatic spread. The protein encoded by this gene is Bcar1/Cas (Cas), an intracellular “docking” protein that is a key molecule in cell signaling pathways. It has been proposed that interaction of Cas with another signaling protein, SHEP2, is responsible for tamoxifen resistance. This project will define the domains in Cas and SHEP2 that interact and determine the importance of the association in the response to human breast cancer cells to antiestrogens. First, for the structural studies we are cloning the suspected interacting domains of Cas and SHEP, and the proteins are produced in both bacterial and other cell culture systems. We will use selective protein design and biophysical approaches (e.g., mass spectrometry) to narrow down the interacting elements for each protein. Secondly, for biological testing we plan to introduce the “active” interacting regions of each protein into MCF7 breast cancer cells and test the effect on tamoxifen resistance. In some cases we will perform these experiments with mutant forms of the two proteins to confirm the importance of specific amino acid residues for forming the complex. Finally, we propose to crystallize Cas in complex with SHEP2 to determine the precise three-dimensional structure at the point of contact between the partner molecules. This interface may represent a novel target for new drugs to overcome inherent and acquired resistance to anti-estrogen therapy.

The serine-rich domain from Crk-associated substrate (p130cas) is a four-helix bundle.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Briknarova K, Nasertorabi F, Havert ML, Eggleston E, Hoyt DW, Li C, Olson AJ, Vuori K, Ely
Yr: 2005 Vol: 280 Nbr: 23 Abs: Pg:21908-14

Organization of functional domains in the docking protein p130Cas.
Periodical:Biochemistry and Biophysics Research Communications
Index Medicus: Biochem Biophys Res Commun
Authors: Nasertorabi F, Garcia-Guzman M, Briknarova K, Larsen E, Havert ML, Vuori K, Ely KR.
Yr: 2004 Vol: 324 Nbr: 3 Abs: Pg:993-8