芳香化酶与PI3K/AKT通路的串话对芳香化酶抑制剂敏感性的调节作用的研究

Breast cancer is the most frequently diagnosed women malignant disease world widely, and has recently become the "TOP Killer" for Chinese women. About 60-75% human breast tumors express estrogen receptor (ER)α which is an estrogen-dependent transcriptional factor facilitating breast cancer tumorigenesis and progression. Aromatase (Arom) is the rate limiting enzyme during estrogen synthesis and provided the most promising target for the majority of postmenopausal breast cancer patients. However, breast cancer resistance to Arom inhibitors (AI) is inevitable. While increasing studies have focused on crosstalk between growth factor receptor pathways and ERα, to uncover the mechanisms underlying AI resistance, none have addressed whether Arom itself could play an active role in the regulation of AI sensitivity. Our preliminary studies have detected a c-src dependent interaction between Arom and PI3K p85 subunit. This interaction might be mediated via a known c-src phosphorylation of Arom at Tyr 361 which resides at a potential p85 binding motif (YXXM), suggesting that Arom might be involved in the regulation of PI3K pathway. We then demonstrated that overexpression of dominant active AKT could potentiate the endogenous Arom activity without affecting its RNA expression. Eventually we determined that an Arom fragment harboring a potential AKT phosphorylation site (Thr 268) could be phosphorylated by immunoprecipitated AKT. Taken together, our data strongly suggest that there might be a cross-regulation between Arom and PI3K/AKT pathway. In this proposal, we will extend our preliminary studies to define the mechanisms underlying this crosstalk and evaluate its impact on breast cancer sensitivity to AI. ..The Specific Aims are:..I: to determine whether Arom could affect AI sensitivity through modulating PI3K/AKT pathway both in vitro and in vivo.We will first confirm whether c-src dependent interaction between p85 and Arom is indeed mediated via Arom phosphorylation at Y361 using breast cancer cells overexpressing wild type or Y361 mutated Arom, in combination with pY361 blocking peptide and different inhibitors. Then we will perform in vivo and in vitro studies to evaluate the impact of Arom Y361 phosphorlation on PI3K/AKT activity and AI sensitivity using different approaches including the pY361 Arom antibody we developed. ..II: to examine whether PI3K/AKT could modulate the activity and AI responsiveness of Arom through direct phosphorylation of Arom both in vivo and in vitro..We will first define the AKT phosphorylation site on Arom through both in vivo and in viro approaches, and then we will develop an antibody against AKT phosphorylated Arom and examine whether AKT phosphorylation of Arom could affect Arom activity and AI sensitivity both in vivo and in vitro.

芳香化酶（Arom）是乳腺癌治疗的重要靶点，即使第三代芳香化酶抑制剂（AI）也不可避免地产生耐药。目前对AI耐药机制的研究主要集中在某些信号途径和雌激素受体（ER）的串话机制上，而对Arom本身在AI耐药发生中的主动调节作用则缺少认识。我们前期研究发现Arom和PI3K/AKT途径之间存在相互作用。 Arom和p85 PI3K 之间存在可被c-src激酶抑制剂抑制的结合，这种结合可能与最近发现的在Arom的第361位酪氨酸的磷酸化有关，该酪氨酸位于一个潜在的p85结合位点上；我们还发现AKT 可以增强乳腺癌细胞中的Arom的活性且在体外可以磷酸化Arom。 这些发现提示在芳香化酶和PI3K/AKT途径之间存在着相互调节作用，而这种相互调节作用可能导致了AI 耐药的发生。本项目将研究Arom 和PI3K/AKT之间的相互作用机制，将为克服AI耐药提供基础。具有极大地理论意义和潜在实用价值。

芳香化酶抑制剂（aromatase inhibitors, AIs）是绝经后雌激素受体（ER）阳性的乳腺癌患者内分泌治疗的一线方案。然而，AIs的耐药成为临床应用中的最大问题，其耐药机制不明确。 对耐药机制的研究主要集中于雌激素受体（ER）与生长因子受体信号途径的串话或ER本身的突变，而对于芳香化酶本身在AI耐药发生中的作用则没有研究。本项目的前期研究表明芳香化酶可以与PI3K调节亚基p-85结合而c-Src激酶的抑制剂AZD0530（PP2）可以阻断这种结合。而据报道c-src可以磷酸化芳香化酶（arom）第361位的酪氨酸。因此我们推测：第361位的酪氨酸磷酸化的芳香化酶（pY361Arom）可以介导与p-85的结合继而活化PI3K/AKT/ mTOR途径而引起AI耐药的发生。因此本项目的主要研究内容即为1，验证arom对PI3K/AKT/ mTOR的活化机制，2，验证pY361Arom的表达与AI耐药的关系。重要结果为： 1， Arom的表达对乳腺癌细胞PI3K/AKT/ mTOR的活化是必须的，2，Arom可以上调体内氧自由基（ROS）水平， 进而活化c-src激酶从而导致Arom磷酸化为pY361Arom，伴随着PI3K/AKT/ mTOR的活化，3， 利用我们研制的pY361Arom的抗体进行的多中心的临床研究（共485例标本，根据手术前/后辅助治疗分为2个队列）， 多因素分析发现pY361Arom的表达只与经AI治疗的患者的生存显著相关（p=0.02）， 与患者年龄、组织学分级、分期、ER、PR、Her2表达无相关性。 表明pY361Arom是预测AI耐药的一个独立指标。科学意义：发现Arom对PI3K/AKT/ mTOR的活化机制并为乳腺癌的个体化治疗提供了一个独立的预后标志。