Structural and Biochemical Studies of p53 Family Function

缺乏雌激素受体(ER)、孕激素受体(PR)和表皮生长因子受体2(Her2/Neu/ErbB2)表达的乳腺癌被称为基底细胞样乳腺癌的三重阴性亚群。目前还没有已知的针对这种疾病的靶向治疗方法。这些癌症的特点是由于促进细胞迁移、侵袭和生存的因素的失调而导致高转移倾向。此外，三阴性乳腺癌通常含有p53突变。近来研究表明，突变型P53通过多种机制参与癌细胞的转移，这些机制都需要在三重负的背景下进一步研究。三阴性乳腺癌细胞株表现出典型的低表达P53抑制物hdm2和hdmx。由于p53通过其反式激活结构域而不是通常突变的DNA结合域与这些蛋白结合，因此我们目前正在使用针对反式激活结构域的装订的α螺旋肽(SAH-P53)来研究突变型P53在三阴性乳腺癌细胞系MDA-MB-231中的调控。我们的结果表明，SAH-P53多肽能够在三阴性背景下破坏突变的P53/HDMX和突变的P53/Hdm2复合体。然而，我们也确定SAH-P53可以独立于HDMX以及突变型P53和Hdm2抑制细胞迁移，这表明SAH-P53可能是一个额外的靶点。SAH-P53不仅抑制在没有刺激的情况下表现出运动能力的细胞的迁移，而且还抑制由EGF、转化生长因子-β、HGF和PDGF诱导的迁移，这表明可以被多肽抑制的通路的汇聚。此外，焦点黏附复合体被SAH-P53破坏。通过对纽蛋白、巴西林和p-FAK这三种主要的焦点复合体蛋白的显微镜观察，在SAH-p53处理的细胞中，复合体看起来要小得多，强度也较低。仅用多肽或甚至在生长因子刺激下Akt的激活减少，证实了缺乏运动或侵袭所需的稳定信号通路。SAH-P53还降低了肌动蛋白应激纤维的稳定性和片状脂膜的形成，这两者都是细胞迁移所必需的结构。综上所述，这些结果证实了SAH-P53通过一种尚未为该肽建立的替代机制来抑制细胞迁移。我们在实验室中进行了多肽定位研究，以探索另一种机制。我们发现SAH-P53在处理细胞后一小时内定位于囊泡。我们尚未确定囊泡的确切类型，但这些囊泡也含有EGFR，这是另一种在三阴性乳腺癌中通常过度表达的蛋白质。必须进行进一步的研究，以确定这些小泡的身份，这些小泡的特异性，以及EGFR在多肽定位中的作用。理想情况下，这些问题的答案将有助于理解多肽的内化如何影响肌动蛋白聚合和局部黏附破坏，最终阻止细胞迁移。在这部分项目完成后，我们希望探索使用这种多肽来抑制三阴性乳腺癌小鼠异种移植模型转移的可能性。最终，这些对SAH-P53的研究将有助于理解驱动三阴性乳腺癌细胞转移的机制。这些知识有可能有助于设计更有效的治疗策略来预防转移性疾病。

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.