The role of the mTOR signaling pathway in ER-positive breast cancer

描述(申请人提供)：背景：临床上，高达60%的乳腺癌ER阳性，表明癌细胞生长依赖雌激素。ER阳性乳腺癌可以通过抗雌激素(如他莫昔芬)或芳香酶抑制剂(AIs)进行治疗。然而，只有大约一半的ER阳性乳腺癌对内分泌治疗有反应，并且经常产生耐药性。一个重要的临床策略是与其他途径的抑制剂联合治疗，以提高内分泌治疗的疗效，抑制或诱导耐药的发生或逆转。调控细胞生长和增殖的最重要的途径之一是雷帕霉素复合体的哺乳动物靶点1(MTORC1)。抑制mTORC1途径与内分泌治疗相结合，在早期临床试验中提供了令人鼓舞的结果。虽然在了解内分泌治疗敏感性和出现耐药的机制方面已经取得了很大进展，但细节还不完全清楚。目的/假设：我们的初步研究表明，ER？和mTOR途径，促进乳腺癌细胞的增殖，特别是通过雌激素上调关键的mTOR效应因子S6Kinase1(S6K1，由RPS6KB1基因编码)，以及mTORC1介导的ER？和14-3-3？，以及一个新的蛋白SPATS2。因此，我们建议研究乳腺癌中mTORC1/S6K1和ER通路之间关系的机制、治疗和预后方面的潜在疾病进展和治疗抵抗。具体目标：我们建议研究mTORC1和ER通路之间的三个不同的联系。我们试图(1)研究mTORC1介导的14-3-3？(2)阐明RPS6KB1的雌激素表达机制；(3)明确mTORC1信号在ER？磷酸化和激活。研究设计：我们将使用染色质免疫沉淀(CHIP)对直接ER？与S6K1启动子区域结合，以及与其他转录因子，如GATA-3和Err？的功能相互作用。为了探讨mTOR介导的ER？的磷酸化方式和S6K1靶蛋白的作用，我们将采用生化和基于细胞的激酶活性测量，以及报告分子分析来测量ER？转录活性。最后，我们将使用药理学和RNAi介导的抑制这些途径来评估这些蛋白在内分泌耐药中的表型效应。我们将研究增殖率、生长、存活和凋亡的变化。癌症相关性：我们的建议侧重于研究ER与mTORC1在调节癌症的发展和进展中合作的分子机制。具体地说，我们将确定这种关系如何影响内分泌治疗的敏感性和耐药性的发展。长期目标是通过瞄准最有可能从特定药物组合中受益的患者来改进乳腺癌患者的治疗方案。

DESCRIPTION (provided by applicant): Background: Clinically, up to 60% of breast cancers are ER-positive, indicative of estrogen dependence for cancer cell growth. ER-positive breast cancers can be targeted therapeutically by antiestrogens (such as tamoxifen) or aromatase inhibitors (AIs). However, only about half of ER-positive breast cancers respond to endocrine treatments, and resistance frequently develops. An important clinical strategy is to use a combination therapy approach with inhibitors of other pathways in order to enhance the efficacy of endocrine therapy, and suppress the development or induce reversal of resistance. One of the most important pathways in regulating cellular growth and proliferation is the mammalian Target of Rapamycin Complex 1 (mTORC1). Inhibition of the mTORC1 pathway in combination with endocrine therapy has provided encouraging results in early clinical trials. Although great strides have been made in understanding the mechanisms of endocrine therapy sensitivity and emergence of resistance, the details are not fully understood. Objective/hypothesis: Our preliminary studies suggest that a co-regulatory relationship exists between the ER? and mTOR pathways, promoting breast cancer cell proliferation, specifically through estrogenic upregulation of a key mTOR effector, S6 Kinase 1 (S6K1, encoded by the RPS6KB1 gene), and mTORC1- mediated phosphorylation of ER? and 14-3-3?, and a novel protein SPATS2. Therefore, we propose to study the mechanistic, therapeutic and prognostic aspects of the relationship between mTORC1/S6K1 and ER pathways underlying disease progression and therapeutic resistance in breast cancer. Specific aims: We propose to investigate three distinct connections between the mTORC1 and ER pathways. We seek to (1) study mTORC1-mediated phosphorylation of 14-3-3? and SPATS2; (2) elucidate the mechanism of estrogenic expression of RPS6KB1; (3) define the role of mTORC1 signaling in ER? phosphorylation and activation. Study design: We will use Chromatin Immunoprecipitation (ChIP) to perform a comprehensive analysis of direct ER? binding to S6K1 promoter regions, and functional interactions with other transcription factors, such as GATA-3 and ERR?. To address the mode of mTOR-mediated phosphorylation of ER?, and the role of S6K1 target proteins, we will employ biochemical and cell-based measurements of kinase activity, and reporter assays for measurements of ER? transcriptional activity. Finally, we will use pharmacological and RNAi- mediated inhibition of these pathways to assess the phenotypic effects of these proteins on endocrine resistance. We will investigate changes in proliferative rates, growth, survival and apoptosis. Cancer relevance: Our proposal focuses on investigating the molecular mechanism by which ER cooperates with mTORC1 in regulating development and progression of cancer. Specifically, we would determine how this relationship affects sensitivity to endocrine therapy and development of resistance. The long-term goal is to improve therapeutic regiments for breast cancer patients by targeting patients most likely to benefit from a particular drug combination.