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Characterization of the Tumor Suppresssor RASSF1A in Breast Cancer Initiation and Progression
Background: The RASSF1A locus at 3p21.3 is epigenetically inactivated at a high frequency in breast cancer indicating that its loss may be important for breast cancer progression. Clinical data suggests that RASSF1A is lost in very early stages of breast cancer, including epithelial hyperplasia. RASSF1A can inhibit proliferation and revert tumorgenicity in human cancer cells lines. Preliminary data suggests that RASSF1A may translationally repress cyclin D1 levels thereby inhibiting cell cycle progression.
Objective/Hypothesis: We hypothesize that RASSF1A gene inactivation promotes tumorgenicity by generating a permissive environment for uncontrolled cell cycle progression. Previous data suggests that RASSF1A negatively regulates the cell cycle by inhibiting cyclin D1 protein expression. The objectives of these studies are to determine if RASSF1A gene inactivation can directly promote oncogenic transformation of human mammary epithelia and to identify the mechanistic relationship between RASSF1A and cell cycle regulation.
Specific Aims: 1) Establish if loss of RASSF1A can directly contribute to development of tumorigenic phenotypes in an organotypic mammary epithelial cell tumor-progression model system. 2) Define the mechanistic relationship between RASSF1A and cell-cycle regulation through analysis of RASSF1A-specific molecular partners and the contribution of RASSF1A to the regulation of cyclin D1 protein levels.
Study Design: We have developed an in vitro tumor progression model starting from normal diploid human mammary epithelial cells that recapitulates mammary ducts. We have developed protocols to specifically silence gene expression in this model. We will combine shRNA-mediated loss-of-function analysis with this organotypic tumor progression model system to directly evaluate the contribution of RASSF1A inactivation to the generation of tumorigenic phenotypes. We will probe the mechanistic relationship between RASSF1A and cell cycle regulation through a combination of gain-of-function and loss-of-function analysis of RASSF1A and RASSF1A-specific interacting proteins to the regulation of cyclin D1 translation in mammary epithelial cells.
Potential Outcomes and Benefits of the Research: Numerous genes are silenced by promoter methylation and careful investigation of these candidates, including loss of function analysis, is required to establish a protein as a tumor suppressor. By identifying RASSF1A as a tumor suppressor and its loss as causative for tumor formation, we will potentially unveil a new molecular marker for early disease progression. By determining the mechanism of RASSF1A function, we will also potentially reveal new molecular targets for therapy.
The cells that constitute breast tissue carry out elaborate mechanisms for growth, repair and death. Normal breast cells have many checkpoints that regulate how fast they divide, and these mechanisms are in place to prevent uncontrolled growth, which results in breast cancer. Cancer cells have lost their ability to divide at a normal rate and instead multiply quickly, resulting in a mass of cells that has no physiological function. We know that the ability of cancer cells to grow uncontrollably is due, in part, to the loss of proteins that regulate cell division. This loss is a very early event in breast cancer progression and even precedes mammographic detection. By determining which proteins are changed early in breast cancer, we will be able to improve detection and therapy. This proposal will determine if the protein RASSF1A is important for breast tumor formation, and could therefore be used as a prognostic indicator for breast cancer at its earliest stages. RASSF1A is present in normal cells and appears to regulate cell division, but is often not present in pre-malignant lesions such as epithelial hyperplasia or breast tumors. When we add RASSF1A back to cancer cells, they cease to divide and lose properties that classify them as cancerous. We believe the loss of RASSF1A may be an important early event in breast cancer progression, and it is important to determine if this loss predisposes one to develop breast cancer. The studies proposed here will determine if loss of RASSF1A contributes to breast cancer initiation, laying important groundwork for future clinical studies assessing the prognosis of pre-cancerous lesions lacking RASSF1A. Furthermore, this study will characterize the mechanism by which RASSF1A exerts its anti-tumorigenic function and potentially reveal new molecular targets for therapy.