Identification of Novel Secreted Proteins of Breast Cancer

Institution: Cedars-Sinai Medical Center
Investigator(s): Phillip Koeffler, M.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5IB-0069 Award: $75,750
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis

Initial Award Abstract (1999)
Successful treatment of breast cancer still depends on the early detection of the disease. Mammography and MRI depend on physical detection and have limitations as to cost, availability of experts to interpret the images, and access to women in rural and underserved areas. What is needed is a simpler, informative, and quantitative method with acceptable risks. Our interest is the development of serum markers for breast cancer, which could greatly facilitate discovery of disease recurrence and progress in treatment regimens. Currently, the CA15-3 serum marker is used for monitoring therapy of breast cancer, but it is fraught with lack of specificity and sensitivity, rendering it of little use as a screening tool.

Our aim is to develop novel serum biomarkers for breast cancer. For this project we will use a molecular technique that is designed specifically to detect proteins secreted from cells and possibly present in the blood of women having breast cancer. We are using a new yeast-based molecular screen that detects the genes for proteins that are secreted from cells. This employs a ‘signal sequence trap’ technique, because secretory breast cancer proteins are expected to contain a molecular ‘signal’ for cellular export. Initially, we have constructed a cDNA library that has been enriched for breast cancer-related genes. This library has been placed in our yeast-based signal sequence trap vector in order to identify genes encoding secreted proteins. The first part of the project will be to identify breast cancer cDNA clones of interest. Then, we will confirm these clones, express them as protein fragments, and prepare specific antibodies. We hope to develop this information in future work to utilize these antibodies against breast cancer secreted proteins as potential cancer detection agents.

We hope that this technology will evolve to the point where both known and novel breast cancer secreted proteins can be measured in a manner similar to the PSA test for prostate cancer diagnosis. Moreover, a by-product of this project may be the discovery of novel secreted or membrane-associated proteins specific to breast cancer, such as growth factors or their receptors, which may contribute to clarifying the pathogenesis of these cancers

Final Report (2001)
To identify genes involved in breast cancer, PCR-selected cDNA subtraction was utilized to construct a breast cancer subtracted library. Differential screening of the library isolated the growth factor inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDAMB-231, T47D and MDA-MB-157; very low levels were found in less tumorgenic MCF7 and BT20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF 12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 2- fold in MCF12A and 3- to 5- fold in MCF7 cells after 24 and 48 hrs of exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI 182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MC12A normal breast cells induced their tumor formation in nude mice. These results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.

Breast cancer: Cyr61 is over-expressed, estrogen inducible and associated with more advanced disease
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Xie D, Miller CW, O'Kelley J, Nakachi K, Sakashita A, Said JW, Gornbein J, Koeffler HP
Yr: 2001 Vol: 276 Nbr: 17 Abs: Pg:14187-94