Discovery and Study of Breast Cancer Secreted Proteins

Institution: Cedars-Sinai Medical Center
Investigator(s): Elizabeth Williamson, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6FB-0041 Award: $85,968
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis

Initial Award Abstract (2000)
The identification of serum markers of breast cancer could revolutionize the way we approach this disease. This is most aptly demonstrated in prostate cancer with the development of the measurement of prostate specific antigen (PSA) in menís serum. If I could identify a serum marker in the blood of individuals for breast cancer, it would allow us to: 1. Make early diagnosis of breast cancer and potential use the test for screening breast cancer. 2. Aid in the treatment of breast cancer. I would anticipate that the serum levels of the marker would diminish as the tumor got smaller, and with ineffective therapy, the level of the marker in the serum would remain stable or increase. 3. Use the marker to monitor for recurrence of disease, and therefore allow early retreatment of the disease. This simple blood test could also markedly decrease the cost of detecting and monitoring, and therefore, could become widely available to the underserved members of our society. Because of the simplicity of getting a blood test, this would enhance the number of people who would avail themselves of such a detection system, again, reminiscent of the ease with which prostate cancer is monitored by PSA.

The first goal of my project is to identify potential serum markers by using a method that takes advantage of yeast genetics. Genetically modified yeast will not live unless they contain a gene that allows secretion of an enzyme. The gene is derived from a library of genes from breast cancer cell lines. I have already identified a secreted protein called mammoglobin using this method. Mammoglobin only present in breast tissue and is often present at elevated levels and secreted in breast tumors. The second goal of my project is to develop a detection assay for this protein and determine if it can be used to monitor breast cancer.

In summary, I am investigating breast cancer by identifying serum markers for disease that may allow early diagnosis at a time when breast cancer is potentially curable, for use in monitoring efficacy of therapy and for detecting early recurrence. Mammoglobin may be a potential target because it is secreted and is specific to breast tissue.

Final Report (2003)
The initial goal of this study was to identify serum markers to be used to detect nascent breast cancer and to ultimately use these identified markers to monitor patients in an effort to rapidly discover recurrent breast cancer. However, during the entire project period the investigation did not yield the expected results.

In another attempt to determine other suitable methods of diagnosis and prognosis of breast cancer, I made a novel observation that I felt was worth pursuing. I analyzed a series of breast cancer cell lines for the expression of a variety of proteins and unexpectedly found that the expression of two proteins, BRCA1 and p27Kip1, paralleled each other. BRCA1 is a breast cancer susceptibility gene, which is frequently mutated in hereditary breast cancers. BRCA1 has been suggested to have a number of activities including DNA repair, growth inhibition and transcriptional activation. However very little is understood as to why mutated BRCA1 is associated with the development of breast cancer. The protein p27Kip1is important for slowing cell division by inhibiting most of the cyclin dependent kinases, which drive cellular proliferation. Of particular interest was the observation that cells lines expressing mutant forms of BRCA1 had very low, if any, expression of p27Kip1. This observation suggests that in normal cells BRCA1 activates p27Kip1and may be a mechanism by which BRCA1 can inhibit the cellular growth of breast tissue.

In a series of experiments I determined that BRCA1 did indeed activate the expression of p27Kip1. I was also able to show that BRCA1 responsiveness of the p27Kip1promoter was located to a 35bp region of the promoter and deletion of this sequence abrogated BRCA1 responsiveness of the p27Kip1promoter. In contrast mutated forms of BRCA1 cannot activate p27Kip1expression. I also determined that wild-type BRCA1 could bind to this 35bp DNA sequence, but mutated BRCA1 could not.

Further analysis of the sequence of this 35bp region of the p27Kip1promoter identified a specific sequence of DNA, which could bind another protein called HNF3a. I next determined by Western blot analysis of a series of cell lines that HNF3a was expressed significantly only in estrogen receptor (ER)-positive breast cancer cell lines and had very low expression in ER-negative or BRCA1-containing breast cancer cell lines. Further experiments have demonstrated that HNF3a can indeed activate the expression of p27Kip1alone, and this activity is enhanced when BRCA1 is co-expressed. Mutation of the specific DNA sequence for HNF3a in the p27Kip1promoter reduces the ability of HNF3a to activate the promoter.

Future experiments will include the possible interaction of BRCA1 with HNF3a, the regulation of HNF3a expression in breast tissue and the expression of this protein in breast cancer patients.

I believe that I have identified a defective pathway (BRCA1 induction of p27Kip1) that may contribute to the development of breast cancer, either in patients harboring mutation of BRCA1 or via decreased expression of a necessary co-factor (HNF3a) in patients who develop the more aggressive ER-negative breast tumors. The understanding of this aberrant pathway offers a new downstream target for therapeutic intervention. Identification of the other factors involved may allow for the pharmaceutical stimulation of p27Kip1by a BRCA1-independent route and thus prevent the development of breast cancer.

BRCA1 transactivates the cyclin-dependent kinase inhibitor p27Kip1.
Index Medicus: Oncogene
Authors: Williamson EA, Dadmanesh F, Koeffler HP
Yr: 2002 Vol: 21 Nbr: Abs: Pg:3199-3206