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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：
8228083
负责人：
JOYCE MARIE SLINGERLAND
金额：
$ 30.28万
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/8228083
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 Health 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 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(申请人提供)：乳腺癌是女性最常见的癌症。三分之一的新发乳腺癌是雌激素受体a(ER)蛋白阴性，预后比ER阳性乳腺癌更差。ER是一种配体激活的转录因子。雌激素：ER结合刺激快速的Src激活，反馈到磷酸化的ER并增加其转录活性。雌激素迅速激活泛素依赖的ER蛋白分解，进而调节ER的活性。我们的数据表明，Src刺激ER蛋白分解，这与某些ER靶基因的激活有关。SRC诱导可增加ER激活基因的表达和ER蛋白的降解。SRC抑制增加了内质网的稳定性。ER+、MDA-MB-361和ER阴性的BT-20乳腺癌细胞株都具有较高的Src，虽然ER蛋白的合成容易被检测到，但ER的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可增加ER。SiRNA对Src的作用增加了BT-20中的ER蛋白。E6AP是一种泛素连接酶，也是一种内质网共激活因子。我们发现，E6AP在体外可以作为ER的泛素连接酶，并且E6AP介导的ER泛素化可被ER用Src激酶处理而增加。SRC抑制剂在体内和体外均可抑制ER的泛素化和降解。与ER阳性乳腺癌相比，ER阴性乳腺癌中SRC活性增强。我们进一步研究当Src被激活的内质网招募时，是否调节转录偶联的ER蛋白分解。我们的假设是，ER阴性乳腺癌的一部分是雌激素反应的：它们表达ER mRNA，但由于加速了Src和E6AP介导的ER蛋白分解，因此检测不到ER蛋白。致癌的Src激活可能使内质网或关键的协同调节因子磷酸化，从而激活内质网蛋白分解和内质网靶基因转录。这是在AIMS 1-3：1.进一步测试Src激活如何影响原发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或关键共激活子的磷酸化是否可以通过E6AP促进ER泛素化和ER在体外和体内的降解；以及3.检测Src激活是否调节ER转录活性。致癌基因Src促进乳腺癌的增殖和生存，也可能加速乳腺癌中ER蛋白的分解。阐明乳腺癌ER阴性状态的潜在机制可能表明它们为什么如此具有侵袭性，并为这种耐药乳腺癌类型产生新的治疗靶点。公共卫生相关性：三分之一的新发乳腺癌是雌激素受体a(ER)蛋白阴性，预后比ER阳性乳腺癌更差。这项资助追求的假设是，ER阴性乳腺癌的一部分是雌激素反应的：它们表达ER mRNA，但由于加速了Src和E6AP介导的ER蛋白分解，无法检测到ER蛋白。致癌的Src激活可能使内质网或关键的协同调节因子磷酸化，从而激活内质网蛋白分解和内质网靶基因转录。致癌基因Src可促进乳腺癌的增殖和生存。我们的工作表明，它还可能加速乳腺癌中ER蛋白的分解。阐明乳腺癌ER阴性状态的潜在机制可能表明它们为什么如此具有侵袭性，并为这种耐药乳腺癌类型产生新的治疗靶点。