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

• 批准号: 7799932
• 负责人: JOYCE MARIE SLINGERLAND
• 金额: $ 33.29万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 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/7799932
• 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; 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; 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

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为雌激素受体a （ER）蛋白阴性，预后比雌激素受体a阳性乳腺癌更差。ER是一种配体激活的转录因子。雌激素：内质网结合刺激Src快速激活，并反馈磷酸化内质网，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刺激内质网蛋白水解，这与某些内质网靶基因的激活有关。Src诱导增加内质网活化基因表达和内质网蛋白水解。Src抑制增加内质网稳定性。弱ER +、MDA-MB-361和ER阴性、BT-20乳腺癌细胞系Src均较高，ER蛋白合成容易检测到，但ER t1/2降低。雌激素剥夺和蛋白酶体抑制剂在ER +和ER- line研究中均增加ER。SiRNA修饰Src使BT-20中ER蛋白升高。E6AP是一种泛素连接酶，也可作为ER-共激活剂。我们发现E6AP在体外作为ER的泛素连接酶，并且E6AP介导的ER泛素化通过Src激酶预处理而增加。Src抑制剂在体内和体外破坏了ER的泛素化和降解。与ER阳性乳腺癌相比，原发性ER阴性乳腺癌的Src活性增加。我们进一步研究了当被活化的内质网募集时，Src是否调节转录偶联的内质网蛋白水解。我们的假设是，一部分ER阴性乳腺癌是雌激素反应性的：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水解和内质网靶基因转录。目标1-3:1阐述了这一点。为了进一步测试Src激活如何影响原发性人类乳腺癌和乳腺癌系的内质网水平；2. 在体外和体内测试Src介导的内质网磷酸化或关键共激活因子的磷酸化是否能通过E6AP促进内质网泛素化和内质网降解；和3。分析Src激活是否调节内质网转录活性。致癌Src促进乳腺癌的增殖和存活，也可能加速乳腺癌的内质网蛋白水解。对乳腺癌内质网阴性状态的机制的阐明可能说明为什么它们具有如此强的侵袭性，并为这种治疗抵抗性乳腺癌类型提供新的治疗靶点。