Uncontrolled cell proliferation is the basis of malignant transformation of breast epithelium. Ca2+/calmodulin participates in normal cell replication and motility, but the intermediate proteins through which it transduces these effects have not been identified. The levels of calmodulin and its binding proteins (e.g., IQGAP1) are altered in malignancy, suggesting that modification of these interactions could be an effective target for chemotherapy. The hypothesis to be evaluated in this proposal is that defective coupling between calmodulin and selected calmodulin-binding proteins contributes to neoplastic transformation of breast tissue. Our objective is to use a novel, state-of-the-art strategy to identify calmodulin-binding proteins that are directly involved in the regulation of cell transformation and to dissect out their function with biochemical and molecular biology techniques. To determine whether IQGAP1 - the first target identified by this approach - and calmodulin are components of aberrant cell growth, wild-type and mutant IQGAP1 will be overexpressed in breast carcinoma cells; the effects on growth and transformation will be examined in the transfected cells. Because in vitro evidence reveals that Ca2+/calmodulin regulates Cdc42 via IQGAP1, modification of Cdc42 activity by Ca2+/calmodulin through IQGAP1 in intact cells will be analyzed with fusion proteins, cell fractionation, and immunocytochemistry. To establish whether IQGAP1 and calmodulin modulate E-cadherin and beta-catenin function, adhesion assays ion manipulation, transfection and immunocytochemistry will be performed. To identify other calmodulin-binding proteins that may contribute to neoplasia, affinity chromatography or immunoprecipitation with a highly-specific anti-calmodulin antibody will be performed; proteins that are expressed in malignant breast cells at levels different from those in untransformed breast cells will be unambiguously identified by the novel technique of nanoelectrospray tandem mass spectrometry. These studies should provide insight into the molecular mechanisms that underlie the malignant transformation of breast tissue. This knowledge could potential lead to the development of novel markers and specifically targeted pharmacologic agents for breast carcinoma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA075205-02
Application #
6173032
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Ault, Grace S
Project Start
1999-09-01
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
2
Fiscal Year
2000
Total Cost
$310,926
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
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White, Colin D; Li, Zhigang; Sacks, David B (2011) Calmodulin binds HER2 and modulates HER2 signaling. Biochim Biophys Acta 1813:1074-82
Brown, Matthew D; Sacks, David B (2009) Protein scaffolds in MAP kinase signalling. Cell Signal 21:462-9
White, Colin D; Brown, Matthew D; Sacks, David B (2009) IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett 583:1817-24
Mataraza, Jennifer M; Li, Zhigang; Jeong, Ha-Won et al. (2007) Multiple proteins mediate IQGAP1-stimulated cell migration. Cell Signal 19:1857-65