Therapeutic implication of estrogen receptor-p53 interaction in mitochondria

描述（由申请人提供）：雌激素受体-1（ER）和肿瘤抑制因子p53在乳腺癌的发生和进展中起重要但相反的作用。与其他癌症相比，乳腺癌中p53突变的总频率约为20%;然而，野生型p53在功能上是衰弱的。ER和p53都位于细胞核和线粒体中，在两个区室中都具有重要的功能。已经报道ER结合并抑制细胞核中的野生型p53功能。在理解线粒体p53如何被ER拮抗方面存在根本性的差距。这个缺口代表了一个重要的问题，因为破译ER在抑制线粒体p53中的作用对于理解野生型p53在乳腺癌中失活的机制至关重要。长期目标是了解p53的核和线粒体功能在乳腺癌中受损的机制。本申请的目的是分析线粒体ER-p53相互作用及其功能后果。核心假设是ER和p53在线粒体内相互作用，导致重要的功能后果。提出的研究的基本原理是，鉴于线粒体p53在响应致癌信号和调节细胞代谢中的重要性，了解线粒体ER和p53之间的相互作用及其功能后果将提供新的治疗靶点，除了可以用于更好的干预策略的机制见解。这一假设将通过追求三个具体目标来检验：1）分析乳腺癌细胞中线粒体中ER-p53相互作用; 2）研究ER-p53相互作用对线粒体中氧化磷酸化（OXPHOS）的影响;和3）确定ER-p53相互作用对线粒体基因转录和凋亡的影响。在第一个目标下，已经在申请人的实验室中建立的已经证实的免疫沉淀（IP）测定、RNA干扰（RNAi）方法和基因转染方法将用于表征线粒体内的ER-p53相互作用。在第二个具体目标下，将使用诸如呼吸复合物测定和定量实时PCR（qPCR）测定的方法沿着申请人已经开发和验证的OXPHOS-芯片表达阵列来分析ER-p53相互作用对OXPHOS的影响，所述方法在申请人的实验室中建立。第三个目的将使用申请人已经优化的多种技术方法来解决ER-p53相互作用如何影响线粒体基因转录和细胞凋亡。这项研究是创新的，因为它代表了一种新的和实质性的偏离现状，即以综合的方式分析乳腺癌细胞线粒体中的ER和p53功能，而不是将它们作为单独途径的独立调节剂。该提案意义重大，因为它是连续研究的第一步，预计将导致更好地了解线粒体ER和p53在乳腺癌中的作用，可用于开发新的治疗策略。

DESCRIPTION (provided by applicant): Estrogen receptor-1 (ER) and tumor suppressor p53 play important, but opposite, roles in the onset and progression of breast cancer. Compared to other cancers, overall frequency of p53 mutation in breast cancer is about 20%; however, wild type p53 is functionally debilitated. Both ER and p53 are localized in the nuclei as well as mitochondria and are functionally important in both the compartments. ER has been reported to bind and inhibit wild type p53 function in the nucleus. There is a fundamental gap in understanding how mitochondrial p53 is antagonized by ER. This gap represents an important problem because deciphering the role of ER in suppressing mitochondrial p53 is essential in understanding the mechanisms by which wild type p53 is inactivated in breast cancer. The long-term goal is to understand the mechanisms by which nuclear and mitochondrial functions of p53 are compromised in breast cancer. The objective in this particular application is to analyze mitochondrial ER-p53 interaction and its functional consequences. The central hypothesis is that ER and p53 interact within mitochondria leading to important functional consequences. The rationale that underlies the proposed research is that given the importance of mitochondrial p53 in responding to oncogenic signaling and regulation of cellular metabolism, understanding the interaction between mitochondrial ER and p53 and its functional consequences would provide new therapeutic targets in addition to mechanistic insights that could be exploited for better intervention strategies. This hypothesis wil be tested by pursuing three specific aims: 1) Analyze ER-p53 interaction in mitochondria in breast cancer cells; 2) Investigate effect of ER- p53 interaction on oxidative phosphorylation (OXPHOS) in mitochondria; and 3) Determine the effect of ER- p53 interaction on mitochondrial gene transcription and apoptosis. Under the first aim, already proven immunoprecipitation (IP) assay, RNA interference (RNAi) approach, and gene transfection approaches, which have been established in the applicants' laboratories, will be used to characterize ER-p53 interaction within mitochondria. Under the second specific aim, effect of ER-p53 interaction on OXPHOS will be analyzed using approaches such as respiratory complex assays and quantitative real-time PCR (qPCR) assay that are established in the applicants' laboratories along with a OXPHOS-Chip expression array already developed and validated by the applicants. The third aim will address, using multiple technical approaches already optimized by the applicants, how ER-p53 interaction affect mitochondrial gene transcription and apoptosis. The research is innovative because it represents a new and substantive departure from the status quo, namely analyzing ER and p53 function in breast cancer cell mitochondria in an integrated manner instead of pursuing them as independent regulators of separate pathways. The proposal is significant because it is the first step in a continuum of research that is expected to lead to a better understanding of the roles of mitochondrial ER and p53 in breast cancer that could be exploited for developing new therapeutic strategies.