Project 1: Roles and Regulation of Mutant p53 and ANp63 in Breast Cancer Cells Carol Prives, Ph.D. Increasing evidence supports the likelihood that, in stark contrast to wild-type p53, tumor-derived mutant forms of p53 play roles in fostering tumor development, invasion and metastasis. To gain insight into the mechanism by which mutant p53 may play a role in breast cancer we adopted the "3D" protocol in which placing normal mammary epithelial cells (such as MCF10A cells) into an extracellular matrix-rich environment leads to their assembling into acinar structures with hollow lumens that resemble the terminal ducts of the mammary gland. We discovered that expression of mutant p53 in MCF10A cells leads to acini with filled rather than hollow lumens, while shRNA mediated down-regulation of mutant p53 in 2 breast cancer cell lines leads to reduced invasive behavior in one case (MDA-231 cells) and conversion of disordered structures into proper hollow acini in the other case (MDA-468 cells). To understand how mutant p53 prevents mammary cells from forming proper acini we will pursue the observation that mutant p53 up-regulates integrin ?4 in 3D culture conditions and integrin ?4 regulates invasive behavior in MD-231 cells. In collaboration with Scott Lowe and Arnold Levine we will pursue another discovery that mutant p53 increases levels of several enzymes involved in sterol biosynthesis when cell are plated in 3D culture conditions, and that treatment of such cells with statins leads to reduced invasive behavior and cell death. In each case we will seek the mechanism by which mutant p53 regulates these pathways. Another potential regulator of cell proliferation is the ?Np63a isoform of p63. We previously discovered that levels of ?Np63a are maintained by a novel protein (Stxbp4) and reduced by another protein (Rack 1) whose ability to induce degradation of ?Np63a is repressed by Stxbp4. We have now identified the APC/C E3 ubiquitin ligase complex as another regulator of ?Np63a turnover and have shown that it's degradation of ?Np63a is blocked by Stxbp4. Since our data and that of our co-program member Carlos Cordon-Cardo indicate that ?Np63 likely plays pro-proliferative and tumorigenic roles (including prevention of acinar luminal clearing in 3D cultures) we will elucidate the role of Stxbp4 in stabilizing ?Np63 and seek ways to prevent their interaction. Finally we will investigate the roles of p63 and p73 in regulation of formation of acinar like structures in the 3D culture setting and will determine whether the abilities of ?Np63a and mutant p53 to prevent luminal hollowing work by similar or different mechanisms.
Both mutant forms of p53 and the AN forms of p63 have been implicated in breast and bladder cancer; two of the major forms of human cancer. The goal of this program is to determine the mechanisms by which mutant p53 and DeltaNp63 regulate novel pathways discovered under the auspices of this program that are involved in these and other forms of cancer. The experiments we propose in many cases are derived from and dependent on inter-program collaborations.
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