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样乳腺癌的三阴性亚组。 目前还没有已知的针对这种疾病的靶向疗法。 这些癌症的特征在于由于促进细胞迁移、侵袭和存活的因子的失调而导致的高转移倾向。 此外，三阴性乳腺癌通常含有p53突变。 最近研究表明突变型p53通过多种机制促进癌细胞的转移，所有这些都需要在三阴性环境中进一步研究。 三阴性乳腺癌细胞系显示p53抑制剂HDM2和HDMX的特征性低表达。 由于p53通过其反式激活结构域而不是通常突变的DNA结合结构域与这些蛋白质结合，因此我们目前正在使用针对反式激活结构域的钉合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中的调节。 表达野生型p53和高水平抑制性蛋白的MCF7细胞系用作肽活性的阳性对照。初步实验表明，在存在或不存在用作化学引诱物的表皮生长因子（EGF）的情况下，用5 - 10 μ M SAH-p53肽抑制MDA-MB-231细胞通过基质胶的迁移和侵袭。 此外，MTT和台盼蓝测定分别显示线粒体代谢和活力的剂量依赖性降低，表明SAH-p53在三阴性细胞中的作用不依赖于p53的转录活性。 有趣的是，MTT和台盼蓝数据也适用于p53无效的三阴性乳腺癌细胞系MDA-MB-157。 SAH-p53对p53-空细胞系的迁移和侵袭特性的影响目前正在进行中，并且有可能证实肽对三阴性细胞的转移潜力的p53非依赖性作用。 所观察到的效果保证调查的信号通路受SAH-p53在三阴性线。 我们已经确定，在MCF7细胞中诱导的经典p53转录途径在MDA-MB-231细胞中不被SAH-p53诱导。 我们目前正试图查明突变细胞中改变的途径。 我们正在研究SAH-p53对p63/p63活性改变和甾醇生物合成调节所引起的事件的影响，这两者都已知在线粒体和细胞迁移中起作用，尽管这还没有得到很好的表征。 SAH-p53在过度表达HDM2或HDMX的细胞中的作用与在具有非常低水平的细胞中的作用一样强的事实表明SAH-p53可能具有第二靶点，或者这些蛋白质具有引起这种反应的其他结合伴侣。 为此，我们设计了生物素化的可光活化的SAH-p53肽，其用于在UV光存在下将肽交联至其靶标。 这些肽可用于与链霉亲和素珠缀合以抑制靶蛋白。 我们已经确定，这些肽成功地结合到HDM2上，并可以与HDM2交联，HDMX的工作正在进行中。 我们目前正在将来自MDA-MB-231和MCF7细胞的全蛋白裂解物暴露于这些肽，并有初步结果表明它们确实与多种蛋白质交联。 在不久的将来，实验将确定这些蛋白质的身份。 此外，正在建立稳定的HDMX shRNA MDA-MB-231等基因细胞系，以确定HDMX在对SAH-p53肽的应答中的必要性。

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. The MCF7 cell line that expresses wild-type p53 and high levels of the inhibitory proteins is used as a positive control for peptide activity. Preliminary experiments have showed an inhibition of MDA-MB-231 cell migration and invasion through Matrigel with 5-10 uM of the SAH-p53 peptide in the presence or absence of epidermal growth factor (EGF) used as a chemoattractant. In addition, MTT and trypan blue assays have shown a dose-dependent decrease in mitochondrial metabolism and viability, respectively, suggesting that the effects of SAH-p53 in triple-negative cells are independent of the transcriptional activity of p53. Interestingly, the MTT and trypan blue data also held true for a p53-null triple-negative breast cancer cell line MDA-MB-157. The effects of SAH-p53 on the migration and invasion properties on the p53-null line are currently underway and have the potential to confirm a p53-independent effect of the peptide on the metastatic potential of triple-negative cells. The observed effects warrant investigations into the signaling pathways affected by SAH-p53 in triple-negative lines. We have established that the canonical p53 transcriptional pathway that is induced in MCF7 cells is not induced by SAH-p53 in MDA-MB-231 cells. We are currently attempting to pinpoint the pathways that are altered in the mutant cells. We are investigating the effect of SAH-p53 on events resulting from alterations in p63/p63 activity and modulation of sterol biosynthesis, both of which are known to have roles at the mitochondria and in cell migration, though this has not been well characterized. The fact that effects of SAH-p53 are as strong in cells that overexpress HDM2 or HDMX as in cells with very low levels suggests that SAH-p53 may have a secondary target or these proteins have other binding partners that elicit this response. To this end, we have designed biotinylated photoactivatable SAH-p53 peptides that are used to cross-link the peptide to its target in the presence of UV light. These peptides can be used in conjugation with strepavidin beads to elute target proteins. We have established that these peptides successfully bind to and can be cross-linked to HDM2, and the work on HDMX is underway. We are currently exposing whole protein lysates from MDA-MB-231 and MCF7 cells to these peptides and have preliminary results suggesting that they do cross-link to multiple proteins. Experiments in the near future will determine the identity of these proteins. In addition, a stable HDMX shRNA MDA-MB-231 isogenic cell line is being created to determine the necessity of HDMX in the response to the SAH-p53 peptides.