Chemoprevention Through Activation of Estrogen Receptor Alpha-Induced Senescence

描述（由申请人提供）：在正常乳腺中，10-20%的管腔上皮细胞表达雌激素受体α（ER α），这些细胞对应于位于ER α阴性上皮细胞附近的非分裂细胞。然而，55%的乳腺癌表达ER α，这些癌症的增殖取决于ER α，如对激素治疗的反应所证明的。对于这个难题的一个可能的解释是，检查点介导的故障安全机制（例如衰老）阻止ER a及其配体雌激素（E2）诱导增殖，并且该屏障的丧失是触发ER a依赖性增殖的开关之一。不幸的是，与文献充分记载的ER α阴性乳腺癌的体外转化模型不同，不存在剖析参与ER α阳性乳腺上皮细胞转化的途径的体外模型系统，因为只有来自正常乳腺的ER α阴性细胞在体外永生化。在为解决这些困难而进行的调查中，我们提出了以下意见，这些意见构成了本申请的基础。1)ER α阴性乳腺上皮细胞工程表达ER α经历衰老样生长停滞类似癌基因激活的衰老在癌前阶段。2)衰老样生长停滞伴随着p53和检查点激酶Chk 2的激活以及衰老相关基因Cst 1的诱导。3)ER α阳性细胞最终重新进入细胞周期，这与Chk 2和p53活化的丧失相关。但是这些细胞不能实现ER α-E2依赖性增殖，这表明ER α介导的增殖有额外的信号。我们假设在ER α阳性乳腺癌的起始中至少有两个主要步骤：1）Chk 2/p53介导的衰老样生长停滞途径的失活，2）ER α介导的增殖所需的基因如FoxA 1的表达。有趣的是，芸苔属植物来源的化学预防剂如异硫氰酸苯乙酯（PEITC）激活Chk 2，这可能导致癌前细胞的衰老样停滞。使用上述体外测定系统以及多瘤中T抗原（PyMT）转基因模型系统，我们将1）研究PEITC对衰老ER α阳性细胞重新进入细胞周期和获得ER α：E2依赖性增殖的影响，和2）确定PEITC对乳腺癌的PyMT转基因模型中增生、腺瘤、早期和晚期腺癌阶段中ER α阳性细胞的Chk 2活化和衰老的影响。我们的长期目标是确定用于ER α阳性乳腺癌的基于衰老的化学预防策略的分子靶标。

DESCRIPTION (provided by applicant): In normal breast, 10-20% of luminal epithelial cells express estrogen receptor alpha (ERa) and these cells correspond to non-dividing cells located adjacent to ERa-negative epithelial cells. Yet 55% of breast cancers express ERa and proliferation of these cancers depends on ERa as evidenced by response to hormonal therapy. One possible explanation for this conundrum is that checkpoint-mediated failsafe mechanisms such as senescence prevent ERa and its ligand, estrogen (E2), from inducing proliferation and loss of this barrier is one of the switches that triggers ERa-dependent proliferation. Unfortunately, unlike the well-documented in vitro transformation model for ERa-negative breast cancers, no in vitro model system to dissect pathways involved in transformation of ERa-positive mammary epithelial cells exists because only ERa-negative cells from the normal breast immortalize in vitro. During an investigation aimed to tackle these difficulties, we made the following observations, which form the basis of this application. 1) ERa-negative mammary epithelial cells engineered to express ERa undergo senescence-like growth arrest similar to oncogene-activated senescence during premalignant stages. 2) Senescence-like growth arrest was accompanied with activation of p53 and the checkpoint kinase Chk2 and induction of the senescence-associated gene Cst1. 3) ERa- positive cells eventually re-entered cell cycle, which correlated with loss of Chk2 and p53 activation. But these cells fail to achieve ERa-E2-dependent proliferation suggesting additional signals for ERa-mediated proliferation. We hypothesize that there are at least two major steps in the initiation of ERa-positive breast cancers: 1) inactivation of the Chk2/p53 mediated senescence-like growth arrest pathway, 2) expression of genes such as FoxA1, which is required for ERa-mediated proliferation. Interestingly, brassica vegetable- derived che mo preventive agents such as phenethyl isothiocyanate (PEITC) activate Chk2, which may lead to senescence-like arrest of premalignant cells. Using the above in vitro assay system as well as a polyoma middle T antigen (PyMT) transgenic model system, we will 1) investigate the effect of PEITC on re-entry of senescent ERa-positive cells to cell cycle and acquisition of ERa:E2-dependent proliferation and 2) determine the effect of PEITC on Chk2 activation and senescence of ERa-positive cells in hyperplastic, adenoma, early and late adenocarcinoma stages in the PyMT transgenic model of breast cancer. Our long- term goal is to identify molecular targets for senescence-based chemoprevention strategies for ERa-positive breast cancers.