Non-genomic actions of estrogen in breast cancer

描述（由申请人提供）：传统的想法是，核ER？调节对乳腺癌发展至关重要的基因。虽然这是肯定的，雌激素在细胞核外的其他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的原则，管理的本地化，信号和细胞功能的ER？在质膜上。此外，我们发表了第一个数据牵连线粒体ER？在乳腺癌细胞中，有助于暴露于辐射后的细胞存活。我们现在提出的研究，以澄清重要的核外ER功能在乳腺癌。我们在ER？中鉴定了一个保守的9个氨基酸基序，急诊室？雄激素和孕激素受体决定棕榈酰化，导致性类固醇受体的膜定位。我们现在提出分离和表征棕榈酰化类固醇受体的棕榈酰酰基转移酶（PAT）蛋白，以及棕榈酰化调节蛋白（例如，Hsp 27），每一个导致膜定位。这些蛋白质可能是乳腺癌的治疗靶点。在具体的目标2，我们提出了一个新的基础，他莫昔芬耐药涉及ER？在线粒体中。Tam敏感的MCF 7和T47 D细胞系响应于Tam，线粒体形成强活性氧（ROS），触发PKC？JNK激活、Bax和巴克向线粒体的募集以及膜电位降低。这导致溶酶体形成以触发效应物半胱天冬酶活化和凋亡。在塔姆敏感细胞，塔姆从事ER？在线粒体中作为拮抗剂，降低MnSOD活性，这对ROS产生和随后的死亡信号传导很重要。在抗TAM的MCF 7和T47 D细胞中，Tam参与ER？在线粒体中作为激动剂，刺激MnSOD活性，猝灭ROS的形成。用siRNA敲低MnSOD使Tam抗性细胞恢复为敏感细胞，其中Tam现在引起大量细胞凋亡。这些研究确定了他莫昔芬耐药/敏感性的新机制，通过线粒体ER？差异介导。这些机制是否是芳香酶抑制剂耐药性的基础也将被确定。在人乳腺癌标本中，尚不清楚是否质膜ER？有助于肿瘤的发展或结果。我们建议在最后的目标，使用修改后的Allred评分，比较膜和核ER？在300例乳腺肿瘤中表达。我们将膜和核ER联系起来？Allred表达评分与许多临床参数相关，包括肿瘤类型、转移、对各种治疗（包括他莫昔芬）的反应、死亡率和来自15年数据的BRCA 1突变状态。临床意义在于了解雌激素和他莫昔芬对乳腺癌生物学的重要作用，可能靶向膜或线粒体ER以预防或治疗乳腺癌。 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核作用，但也可能来自核外效应。然而，这一领域还没有得到很好的理解，特别是对于人类恶性肿瘤，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特定的治疗干预，可以避免雌激素在核ER？中的一些不良影响。这可能对女性有预防或治疗作用。

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.