Mechanisms Whereby Src Activates Estrogen Stimulated ER Proteolysis and ER Target

Src 激活雌激素刺激的 ER 蛋白水解和 ER 靶标的机制

基本信息

批准号：
8035886
负责人：
JOYCE MARIE SLINGERLAND
金额：
$ 31.27万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-06 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035886
关键词：
26S proteasome Affect Aggressive Clinical Course Animals Back Binding Biological Assay Breast Cause of Death Chromatin Coupled Data Epidermal Growth Factor Receptor Estrogen Receptors Estrogen receptor positive Estrogens Feedback Feeds Gene Expression Gene Targeting Genetic Genetic Transcription Grant Human In Vitro Label Ligands Link Malignant Neoplasms Mammary gland Mediating Messenger RNA Methionine Microarray Analysis Molecular Target Mus NCOA3 gene Oncogenic Phosphorylation Proteasome Inhibition Proteasome Inhibitor Protein Biosynthesis Proteins Proteolysis Receptor Activation Receptor Protein-Tyrosine Kinases Recruitment Activity Regulation Resistance Series Signal Transduction Testing Time Tissues Transcriptional Activation Transgenic Organisms Tyrosine Phosphorylation Ubiquitin Ubiquitin-Activating Enzymes Ubiquitin-Conjugating Enzymes Woman Work activating transcription factor base cancer type deprivation estrophilin in vivo inhibitor/antagonist malignant breast neoplasm new therapeutic target non-genomic outcome forecast overexpression prevent prognostic public health relevance src-Family Kinases steroid hormone transcription factor tumor ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): Breast cancer is the most frequent womens' cancer. One third of new breast cancers are estrogen receptor a (ER) protein negative and have a worse prognosis than ER positive breast cancers. The ER is a ligand activated transcription factor. Estrogen:ER binding stimulates rapid Src activation that feeds back to phosphorylate ER and increases its transcriptional activity. Estrogen rapidly activates ubiquitin-dependent ER proteolysis which in turn regulates ER activity. Our data suggest that Src-stimulates ER proteolysis and this is linked to activation of certain ER target genes. Src induction increased both ER-activated gene expression and ER proteolysis. Src inhibition increased ER stability. The weakly ER +, MDA-MB-361 and ER negative, BT-20 breast cancer lines both have high Src, and while ER protein synthesis was easily detected, the ER t1/2 was reduced. ER was increased by both estrogen deprivation and proteasome inhibitors in both ER + and ER- line studied. SiRNA to Src increased ER protein in BT-20. E6AP is an ubiquitin ligase that also acts as an ER- coactivator. We show E6AP acts as ubiquitin ligase for ER in vitro and E6AP-mediated ER ubiquitylation was increased by ER pre-treatment with Src kinase. Src inhibitors impaired ubiquitylation and degradation of ER in vivo and in vitro. Src activity was increased in primary ER negative breast cancers compared to ER positive. We further investigate if Src, when recruited by activated ER, regulates transcription-coupled ER proteolysis. Our Hypothesis is that a subset of ER negative breast cancers are estrogen responsive: they express ER mRNA but ER protein is undetectable due to accelerated Src and E6AP-mediated ER proteolysis. Oncogenic Src activation may phosphorylate the ER or key co-regulators to activate both ER proteolysis and ER target gene transcription. This is pursued in Aims 1-3: 1. To further test how Src activation affects ER levels in primary human breast cancers and in breast cancer lines; 2. To test whether Src mediated phosphorylation of the ER or of a key co-activator can promote ER ubiquitylation by E6AP and ER degradation in vitro and in vivo; and 3. To assay if Src activation modulates ER transcriptional activity. Oncogenic Src promotes breast cancer proliferation and survival, and may also accelerate ER proteolysis in breast cancers. Elucidation of mechanisms underlying the ER negative status of breast cancer may indicate why they are so aggressive and yield new therapeutic targets for this treatment-resistant breast cancer type. PUBLIC HEALTH RELEVANCE: One third of new breast cancers are estrogen receptor a (ER) protein negative and have a worse prognosis than ER positive breast cancers. This grant pursues the hypothesis that a subset of ER negative breast cancers are estrogen responsive: they express ER mRNA but ER protein is undetectable due to accelerated Src and E6AP-mediated ER proteolysis. Oncogenic Src activation may phosphorylate the ER or key co- regulators to activate both ER proteolysis and ER target gene transcription. Oncogenic Src promotes breast cancer proliferation and survival. Our work suggests that it may also accelerate ER proteolysis in breast cancers. Elucidation of mechanisms underlying the ER negative status of breast cancer may indicate why they are so aggressive and yield new therapeutic targets for this treatment-resistant breast cancer type.

描述（由申请人提供）：乳腺癌是最常见的女性癌症。三分之一的新发乳腺癌为雌激素受体 a (ER) 蛋白阴性，且预后比 ER 阳性乳腺癌更差。 ER 是配体激活的转录因子。雌激素：ER 结合刺激 Src 快速激活，从而反馈磷酸化 ER 并增加其转录活性。雌激素迅速激活泛素依赖性内质网蛋白水解，进而调节内质网活性。我们的数据表明 Src 刺激 ER 蛋白水解，这与某些 ER 靶基因的激活有关。 Src 诱导增加了 ER 激活基因表达和 ER 蛋白水解。 Src 抑制增加 ER 稳定性。弱 ER+、MDA-MB-361 和 ER 阴性、BT-20 乳腺癌系均具有高 Src，虽然很容易检测到 ER 蛋白质合成，但 ER t1/2 降低。在所研究的 ER+ 和 ER- 系中，雌激素剥夺和蛋白酶体抑制剂均增加了 ER。 Src 的 siRNA 增加了 BT-20 中的 ER 蛋白。 E6AP 是一种泛素连接酶，也可作为 ER 辅激活剂。我们发现 E6AP 在体外充当 ER 的泛素连接酶，并且用 Src 激酶预处理 ER 可以增加 E6AP 介导的 ER 泛素化。 Src 抑制剂在体内和体外损害 ER 的泛素化和降解。与 ER 阳性原发性 ER 阴性乳腺癌相比，Src 活性增加。我们进一步研究 Src 在被激活的 ER 招募时是否调节转录偶联的 ER 蛋白水解。我们的假设是，一部分 ER 阴性乳腺癌对雌激素有反应：它们表达 ER mRNA，但由于 Src 和 E6AP 介导的 ER 蛋白水解加速，无法检测到 ER 蛋白。致癌 Src 激活可能使 ER 或关键协同调节因子磷酸化，从而激活 ER 蛋白水解和 ER 靶基因转录。这是目标 1-3 中所追求的： 1. 进一步测试 Src 激活如何影响原发性人类乳腺癌和乳腺癌细胞系中的 ER 水平； 2. 测试Src介导的ER或关键共激活因子的磷酸化是否可以通过E6AP促进ER泛素化以及体外和体内ER降解； 3. 测定 Src 激活是否调节 ER 转录活性。 Oncogenic Src 促进乳腺癌增殖和存活，还可能加速乳腺癌中的 ER 蛋白水解。阐明乳腺癌 ER 阴性状态的潜在机制可能会表明它们为何如此具有侵袭性，并为这种难治性乳腺癌类型产生新的治疗靶点。公共健康相关性：三分之一的新乳腺癌是雌激素受体 a (ER) 蛋白阴性，且预后比 ER 阳性乳腺癌更差。这项资助的假设是，一部分 ER 阴性乳腺癌具有雌激素反应性：它们表达 ER mRNA，但由于 Src 和 E6AP 介导的 ER 蛋白水解加速，无法检测到 ER 蛋白。致癌 Src 激活可能使 ER 或关键共调节因子磷酸化，从而激活 ER 蛋白水解和 ER 靶基因转录。 Oncogenic Src 促进乳腺癌增殖和存活。我们的工作表明它还可能加速乳腺癌中的内质网蛋白水解。阐明乳腺癌 ER 阴性状态的潜在机制可能会表明它们为何如此具有侵袭性，并为这种难治性乳腺癌类型产生新的治疗靶点。