Non-genomic actions of estrogen in breast cancer

描述(申请人提供)：传统思维是核ER吗？调节乳腺癌发展所必需的基因。虽然这肯定是真的，但雌激素在核外部位的额外作用有助于转录和非转录效应。在我们最初的资助期间，我们建立了重要的新原则，管理ER？的定位、信号和细胞功能。在质膜上。此外，我们还发表了第一个与线粒体ER？在乳腺癌细胞中，有助于细胞在暴露于辐射后存活。我们现在建议进行研究，以阐明乳腺癌中重要的核外ER功能。我们在ER？和ER？以及雄激素和孕激素受体中发现了一个保守的9个氨基酸基序，它决定了棕榈酰化，导致了性激素受体的膜定位。我们现在建议分离和鉴定棕榈酰化类固醇受体的棕榈酰化转移酶(PAT)蛋白(S)和棕榈酰化调节蛋白(例如-HSP27)，每个蛋白都导致膜定位。这些蛋白质可能成为乳腺癌的治疗靶点。在特定的目标2中，我们提出了一种新的涉及ER？在线粒体中。敏感的MCF7和T47D细胞对的反应是线粒体产生强烈的活性氧(ROS)，触发PKC？JNK活化，Bax和Bak向线粒体募集，膜电位降低。这导致了凋亡体的形成，从而触发了效应器caspase的激活和凋亡。在敏感的细胞中，与ER？在线粒体中作为拮抗剂，降低MnSOD活性，这对ROS的产生和随后的死亡信号是重要的。在耐药的MCF7和T47D细胞中，与ER？在线粒体中作为激动剂，刺激MnSOD活性，抑制ROS的形成。用小干扰RNA敲除MnSOD可以将耐药的细胞恢复为敏感细胞，现在可以在那里引起大量的细胞凋亡。这些研究发现了他莫昔芬通过线粒体ER？差异介导的耐药/敏感性的新机制。这些机制是否是芳香酶抑制剂抵抗的基础也将被确定。在人乳腺癌标本中，质膜ER？有助于肿瘤的发展或结果。我们建议在最终目标中使用改进的Allred评分，比较膜和核的ER？在300例乳腺肿瘤中的表达。我们将膜和核ER？Allred表达与许多临床参数有关，包括肿瘤类型、转移、对各种治疗的反应(包括他莫昔芬)、死亡率和15年数据中的BRCA1突变状态。其临床意义在于了解雌激素和三苯氧胺对乳腺癌生物学有重要意义的新作用，可能以膜或线粒体ER为靶点来预防或治疗乳腺癌。 公共卫生相关性：雌激素和雌激素受体对乳腺癌的促进作用来自于核作用，但也可能来自核外效应。然而，这一领域还没有被很好地理解，特别是对于人类的恶性肿瘤，确定雌激素和雌激素受体在质膜和线粒体中的确切作用将鼓励进行特定的治疗干预，从而避免雌激素在核ER？中的一些不良影响。这可能对女性有预防或治疗的好处。

DESCRIPTION (provided by applicant): Traditional thinking is that nuclear ER? modulates the genes that are essential to breast cancer development. Although this is certainly true, additional actions of estrogen at locations outside the nucleus contributes to both transcriptional and non-transcriptional effects. In our initial grant period, we established important new principals governing the localization, signaling, and cell functions of ER? at the plasma membrane. Further, we published the first data implicating mitochondrial ER? in breast cancer cells, contributing to cell survival after exposure to radiation. We now propose studies to clarify important extra-nuclear ER functions in breast cancer. We identified a conserved 9 amino acid motif in ER?, ER?, and both androgen and progesterone receptors that dictates palmitoylation, resulting in membrane localization of the sex steroid receptors. We now propose to isolate and characterize the palmitoylacyltransferase (PAT) protein(s) that palmitoylate the steroid receptors, and palmitoylation-modulating proteins (e.g.-Hsp27), each resulting in membrane localization. These proteins could be therapeutic targets in breast cancer. In specific aim 2, we propose a novel basis for tamoxifen resistance involving ER? in mitochondria. Tam-sensitive MCF7 and T47D cell lines respond to Tam with strong reactive oxygen species (ROS) formation from mitochondria, triggering PKC? and JNK activation, recruitment of Bax and Bak to the mitochondria, and membrane potential decrease. This results in apoptosome formation to trigger effector caspase activation and apoptosis. In Tam-sensitive cells, Tam engages ER? in the mitochondria as an antagonist, decreasing MnSOD activity that is important to ROS generation and subsequent death signaling. In Tam-resistant MCF7 and T47D cells, Tam engages ER? in the mitochondria as an agonist, stimulating MnSOD activity that quenches ROS formation. MnSOD knockdown with siRNA reverts Tam resistant cells to sensitive cells where Tam now causes substantial apoptosis. These studies identify novel mechanisms of Tamoxifen in resistance/sensitivity, mediated differentially through mitochondrial ER?. Whether these mechanisms underlie aromatase inhibitor resistance will also be determined. In human breast cancer specimens, it is unclear whether plasma membrane ER? contributes to tumor development or outcome. We propose in the final aim to use a modified Allred score, comparing membrane and nuclear ER? expression in 300 breast tumors. We will correlate membrane and nuclear ER? Allred expression scores to many clinical parameters, including tumor type, metastasis, response to various therapies (including tamoxifen), mortality, and BRCA1 mutation status from 15 years data. The clinical significance is to understand new actions of estrogen and tamoxifen that are important to breast cancer biology, possibly targeting membrane or mitochondrial ER for preventing or treating breast cancer. PUBLIC HEALTH RELEVANCE: Estrogen and estrogen receptor promotion of breast cancer derives from nuclear actions but probably also from extra-nuclear effects. However, this area is not well understood, particularly for human malignancy and determining the precise actions of estrogen and the estrogen receptor at the plasma membrane and in mitochondria would encourage specific therapeutic interventions that could avoid some undesirable effects of estrogen at nuclear ER?. This could be of preventative or therapeutic benefit to women.