Chemoprevention Through Activation of Estrogen Receptor Alpha-Induced Senescence

描述(申请人提供)：在正常乳腺中，10-20%的腔上皮细胞表达雌激素受体α(ERA)，这些细胞与ERA阴性的上皮细胞相邻的未分裂细胞相对应。然而，55%的乳腺癌表达ERA，这些癌症的增殖依赖于ERA，激素治疗的反应证明了这一点。对这一难题的一个可能的解释是，检查点介导的失效保护机制，如衰老，阻止了Era及其配体雌激素(E2)诱导的增殖，而这一屏障的丧失是触发Era依赖的增殖的开关之一。不幸的是，与已有文献记载的ERA阴性乳腺癌体外转化模型不同，目前还没有分离ERA阳性乳腺上皮细胞转化途径的体外模型系统，因为只有来自正常乳腺的ERA阴性细胞才能在体外永生化。在一次旨在解决这些困难的调查中，我们提出了以下意见，这些意见构成了这项申请的基础。1)表达Era基因的阴性乳腺上皮细胞在癌前阶段经历了类似于癌基因激活衰老的衰老样生长停滞。2)衰老样生长停滞伴随着P53和Chk2的激活以及衰老相关基因Cst1的诱导。3)ERA阳性细胞最终重新进入细胞周期，这与Chk2和P53的失活有关。但这些细胞不能实现依赖Era-E2的增殖，这暗示了Era介导的增殖的额外信号。我们推测，Era阳性乳腺癌的发生至少有两个主要步骤：1)Chk2/P53介导的衰老样生长停滞通路失活；2)FoxA1等基因的表达，这是Era介导的增殖所必需的。有趣的是，十字花科蔬菜衍生的CHEMO防御剂，如苯基异硫氰酸酯(PEITC)，激活Chk2，这可能导致癌前细胞类似衰老的停滞。利用上述体外检测系统和多瘤中T抗原(PYMT)转基因模型系统，我们将1)研究PEITC对衰老的Era阳性细胞重新进入细胞周期和获取Era：E2依赖的增殖的影响；2)在PYMT转基因乳腺癌模型中，检测PEITC对Era阳性细胞Chk2激活和衰老的影响。我们的长期目标是为ERA阳性乳腺癌的基于衰老的化学预防策略确定分子靶点。

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.