Mechanisms Whereby Src Activates Estrogen Stimulated ER Proteolysis and ER Target

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是雌激素受体a（ER）蛋白阴性的，其预后比ER阳性乳腺癌更差。ER是配体激活的转录因子。雌激素：ER结合刺激快速Src激活，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的ER蛋白水解，进而调节ER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乳腺癌细胞系均具有高Src，并且虽然ER蛋白合成容易检测，但ER t1/2降低。雌激素剥夺和蛋白酶体抑制剂均可增加ER +和ER-细胞系的ER。Src的SiRNA增加BT-20中的ER蛋白。E6 AP是一种泛素连接酶，也可作为ER-共激活剂。我们发现E6 AP在体外作为ER的泛素连接酶，并且E6 AP介导的ER泛素化通过用Src激酶预处理ER而增加。Src抑制剂在体内和体外损害ER的泛素化和降解。与ER阳性相比，原发性ER阴性乳腺癌中Src活性增加。我们进一步调查，如果Src，激活ER招募时，调节转录偶联ER蛋白水解。我们的假设是，ER阴性乳腺癌的一个子集是雌激素反应：他们表达ER mRNA，但ER蛋白是检测不到的，由于加速Src和E6 AP介导的ER蛋白水解。致癌Src激活可使ER或关键辅助调节因子磷酸化以激活ER蛋白水解和ER靶基因转录。这是在目标1-3：1中追求的。进一步测试Src活化如何影响原发性人乳腺癌和乳腺癌细胞系中的ER水平; 2.测试Src介导的ER或关键共激活剂的磷酸化是否可以通过E6 AP促进ER泛素化和体外和体内ER降解;和3.分析Src激活是否调节ER转录活性。致癌Src促进乳腺癌的增殖和存活，也可能加速乳腺癌中的ER蛋白水解。阐明乳腺癌ER阴性状态的机制可能表明为什么它们如此具有侵袭性，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）蛋白阴性的，其预后比ER阳性乳腺癌更差。这项资助追求的假设是，ER阴性乳腺癌的一个子集是雌激素反应：他们表达ER mRNA，但ER蛋白是无法检测到的，由于加速Src和E6 AP介导的ER蛋白水解。致癌Src激活可能会磷酸化ER或关键辅调节因子，以激活ER蛋白水解和ER靶基因转录。致癌Src促进乳腺癌增殖和存活。我们的研究表明，它也可能加速乳腺癌中的ER蛋白水解。阐明乳腺癌ER阴性状态的机制可能表明为什么它们如此具有侵袭性，并为这种耐药乳腺癌类型产生新的治疗靶点。

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.