CAV-1, Stat5a Signaling, and Estrogen-Dependent Breast Cancer

描述(申请人提供)：人小窝蛋白-1(Cav-1)基因作为乳腺肿瘤抑制因子。我们之前已经在高达35%的雌激素受体(ER)阳性乳腺癌患者中发现了Cav-1失活(显性-阴性(DN))突变。我们的假设是，ER水平和活性的上调是由Cav-1失活突变引起的。由于Cav-1是JAK-2激酶的抑制因子，我们认为Stat5a激活是Cav-1功能丧失导致ER-α水平升高的机制。为了支持这一假设，我们提出了新的证据，即Stat5a的激活足以上调ER阴性的人乳腺癌细胞中ER-α的水平。因此，我们的初步研究现在已经确定了导致乳腺癌的一种新的信号通路：Cav-1基因失活(DN突变)--&gt；Stat5a激活--&gt；ER-α上调--&gt；Cyclin D1过表达。该项目的三个具体目标是： 1)确定Stat5a激活和ER-α在Cav-1相关的乳腺增生、增殖和3D管腔形成中的作用。我们将分析Cav-1/Stat5a双基因敲除小鼠的乳腺，并研究这些小鼠乳腺上皮细胞原代培养的体外行为。 2)探讨Stat5a活化和ER-α在Cav-1相关乳腺肿瘤发生和转移中的作用。为此，我们将对在人类乳腺癌中发现的表达Cav-1显性阴性(DN)突变(如P132L)的Met-1细胞进行原位移植。将使用Stat5a和Jak-2的DN突变体来评估Stat5a信号的作用。雌激素的作用将通过卵巢切除和补充雌激素颗粒来评估。对他莫昔芬的耐药性也将进行调查。 3)确定ER(+)人乳腺癌标本中Cav-1突变是否与Stat5a激活共分离。在这里，我们建议使用Cav-1突变、ER-α表达水平和Stat5a激活作为新的预后标记物，来检验这一新定义的信号通路在人类乳腺癌发病机制中的相关性。由于超过40%的ER-α阳性患者表现出他莫昔芬耐药，我们还将检查Cav-1突变和Stat5a激活是否是他莫昔芬耐药的关键预测因素。

DESCRIPTION (provided by applicant): The human Caveolin-1 (Cav-1) gene acts as a mammary gland tumor suppressor. We have previously identified Cav-1 inactivating (dominant-negative (DN)) mutations in up to 35 % of estrogen receptor (ER) positive breast cancer patients. Our hypothesis is that up-regulation of ER levels and activity are caused by Cav-1 inactivating mutations. As Cav-1 functions as an inhibitor of the Jak-2 kinase, we propose that Stat5a activation is the mechanism by which loss of Cav-1 function results in increased ER-alpha levels. In support of this hypothesis, we present novel evidence that Stat5a activation is sufficient to upregulate ER-alpha levels in ER-negative human breast cancer cells. As such, our preliminary studies have now defined a novel signaling pathway leading to breast cancer: Cav-1 gene inactivation (DN-mutations) --> Stat5a activation --> ER-alpha upregulation --> Cyclin D1 over-expression. The three Specific Aims of the project are: 1) Determine the role of Stat5a activation and ER-alpha in Cav-1-related mammary hyperplasia, proliferation, and 3D lumen formation. We will analyze the mammary glands of Cav-1/Stat5a double- knockout mice and study the ex vivo behavior of primary cultures of mammary epithelia from these mice. 2) Determine the role of Stat5a activation and ER-alpha in Cav-1-related mammary tumorigenesis and metastasis. For this purpose, we will perform orthotopic transplantation of Met-1 cells expressing Cav-1 dominant-negative (DN) mutants (such as P132L) that are found in human breast cancer. The role of Stat5a signaling will be assessed using DN mutants of Stat5a and Jak-2. The role of estrogen will be assessed by ovariectomy and supplementation with estrogen pellets. Tamoxifen-resistance will also be investigated. 3) Determine if Cav-1 mutations co-segregate with Stat5a activation in ER(+) human breast cancer samples. Here, we propose to examine the relevance of this newly defined signaling pathway in human breast cancer pathogenesis, using Cav-1 mutations, ER-alpha expression levels, and Stat5a activation as novel prognostic markers. Since greater than 40% of ER-alpha positive patients show tamoxifen-resistance, we will also examine if Cav-1 mutations and Stat5a activation are critical predictors of tamoxifen-resistance.