Structural and Biochemical Studies of p53 Family Function

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Breast cancer that lacks expression of the estrogen receptor (ER), progesterone receptor(PR), and the epidermal growth factor receptor 2 (Her2/Neu/ErbB2) is known as the triple negative subset of basal-like breast cancers. There are currently no known targeted therapies for this disease. These cancers are characterized by a high propensity to metastasize due to the dysregulation of factors contributing to cell migration, invasion and survival. In addition, triple-negative breast cancers commonly harbor mutations of p53. It has been shown recently that mutant p53 contributes to the metastasis of cancer cells by multiple mechanisms, all of which require further study in the triple negative setting. Triple-negative breast cancer cell lines show characteristically low expression of the p53 inhibitors HDM2 and HDMX. Because p53 binds to these proteins via its transactivation domain and not the commonly mutated DNA binding domain, we are currently using a stapled alpha helical peptide against the transactivation domain (SAH-p53) to study the regulation of mutant p53 in the triple-negative breast cancer cell line MDA-MB-231. Our results show that the SAH-p53 peptide is able to disrupt the mutant p53/HDMX and mutant p53/HDM2 complexes in the triple-negative background. However, we have also determined that SAH-p53 can inhibit cell migration independently of HDMX as well as independently of mutant p53 and HDM2, indicating the possibility of an additional target of SAH-p53. SAH-p53 not only inhibits migration of cells that exhibit motility in the absence of a stimulus, but also that which is induced by EGF, TGF-beta, HGF, and PDGF, suggesting a convergence of pathways that can be inhibited by the peptide. Furthermore, focal adhesion complexes are disrupted by SAH-p53. As determined by microscopic visualization of vinculin, paxillin, and p-FAK, all primary focal complex proteins, the complexes appear much smaller and less intense in cells treated with SAH-p53. A decrease in the activation of Akt with peptide alone or even upon growth factor stimulation confirms the lack of a stable signaling pathway necessary for motility or invasion. SAH-p53 also decreases the stability of actin stress fibers and the formation of lamellipodia, both of which are structures necessary for cell migration. Together these results confirm that SAH-p53 inhibits cell migration by an alternative mechanism not yet established for this peptide. We have done peptide localization studies in the lab in order to investigate an alternate mechanism. We have found that SAH-p53 localizes to vesicles within an hour of treating cells. We have yet to determine the exact type of vesicle, but the vesicles also contain EGFR, another protein commonly overexpressed in triple-negative breast cancer. Further studies must be done in order to determine the identity of these vesicles, the specificity of the peptide for these vesicles, and the role of the EGFR in peptide localization. Ideally the answers to these questions will help to understand how the internalization of the peptide affects actin polymerization and focal adhesion disruption to ultimately prevent cell migration. Upon completion of this part of the project, we hope to explore the possibility of using this peptide to inhibit metastases in a mouse xenograft model of triple-negative breast cancer. Ultimately these studies with SAH-p53 will aid in the understanding of the mechanisms that drive the metastasis of triple-negative breast cancer cells. This knowledge has the potential to aid in the design of more effective therapeutic strategies for the prevention of metastatic disease.