Estrogen-Estrogen Receptor axis in non-transformed breast epithelial cells: studies beyond MCF-7

 说明(申请人提供)：雌激素(E2)通过核受体雌激素受体α(ER-α)在乳腺癌中起主要作用。大约70%的乳腺癌表达ERα，ERα阳性乳腺癌患者接受抗雌激素治疗。然而，抗雌激素治疗的耐药性是一个主要的临床问题。这一领域至少有两个关键问题尚未解决：1)在正常乳腺中，ERα在10-20%的未分裂细胞中表达。这些ERα阳性细胞通过旁分泌作用支持ERα阴性细胞对E2的反应。与正常细胞相比，ERα阳性的癌细胞本身对雌二醇的反应是增殖的。调控这种ERα功能的开关的机制在很大程度上是未知的，这是本提案的重点：2)抗雌激素抵抗的机制。我们目前对ERα生物学的大部分知识，包括全基因组结合模式(ERα序列)、先驱因子/染色质修饰在引导ERα序列中的作用以及E2调节的基因表达模式(E2-转录组)都来自于使用癌细胞株MCF-7进行的研究，而不是正常/未转化细胞，因为在技术上还不可能培养表达ERα的未转化细胞。这一局限性阻碍了我们理解ERα在正常乳腺中的功能，以及控制癌症相关ERα功能转变的机制。为了解决这些关键问题，我们通过使用最近描述的上皮细胞重编程试验从正常乳腺繁殖细胞，然后使用端粒酶逆转录酶将其永生化，从而产生了几个ERα阳性的乳腺上皮细胞系。与预测的ERα在原代细胞中的表达模式一致，ERα在这些细胞系的腔前体细胞和成熟细胞中表达，而在这些细胞系的干细胞部分中不表达，这证明了该系统的相关性。其中一株细胞的RNAseq分析表明，ER、α：E2在抑制基因表达方面具有优势作用。相反，ERα信号涉及癌细胞的激活和抑制，并与PI3K-Cyclin D1-cMyc增殖网络以及正常细胞中未见的非典型泛素化系统很好地整合。利用这些新资源，我们将解决这样的假设，即ERα周期和E2转录组从抑制模式切换到激活模式是ERα阳性乳腺癌的关键早期步骤。其具体目标是：1)确定未转化的乳腺上皮细胞所特有的ERα周期；2)通过整合ERα周期与转录组，研究未转化和转化的乳腺上皮细胞中调控ERα功能的信号节点。影响：据我们所知，这是第一次确定未转化的乳腺上皮细胞中ERα功能的研究。开发的资源将对研究界重新评估与ERα阳性乳腺癌和抗雌激素耐药相关的信号网络具有价值。特别是，与ERα阳性乳腺癌中特有的基因组异常有关的信号缺陷可以被调查，并且可以使用这个资源来识别针对特定异常的药物。

 DESCRIPTION (provided by applicant): Estrogen (E2) acting through the nuclear receptor estrogen receptor alpha (ERα) plays a major role in breast cancer. Approximately 70% of breast cancers express ERα and patients with ERα-positive breast cancers receive anti-estrogens. However, resistance to anti-estrogen therapy is a major clinical problem. There are at least two critical unresolved issues in this field: 1) in the normal breast, ERα is expressed in 10-20% of non- dividing cells. These ERα-positive cells support proliferation of ERα-negative cells in response to E2 through paracrine action. In contrast to normal cells, ERα-positive cancer cells themselves proliferate in response to E2. Mechanisms governing switch in this ERα function are largely unknown and is the focus of this proposal; 2) Mechanisms of anti-estrogen resistance. Most of our current knowledge of ERα biology including genome- wide binding pattern (ERα cistrome), the role of pioneer factors/chromatin modifications in guiding ERα cistrome, and E2-regulated gene expression pattern (E2-transcriptome) are from studies using the cancer cell line MCF-7 but not normal/non-transformed cells because it has not been technically possible to culture ERα- expressing non-transformed cells. This limitation has prevented us from understanding the function of ERα in the normal breast and mechanisms that govern cancer-associated shift in ERα function. To address these critical issues, we generated several ERα-positive breast epithelial cell lines by propagating cells from normal breast using recently described epithelial cell-reprogramming assay followed by immortalization using the hTERT. Consistent with the predicted expression pattern of ERα in primary cells, ERα is expressed in the luminal progenitor and mature cell but not in stem cell fraction of these cell lines, which authenticates the relevance of the system. RNA-seq analysis of one of these cell lines showed dominant action of ERα:E2 in repressing gene expression. In contrast, ERα-signaling involved both activation and repression in cancer cells and was well integrated with PI3K-Cyclin D1-cMyc proliferation network as well as an atypical ubiquitination system not seen in normal cells. Using these new resources, we will address the hypothesis that switch in ERα cistrome and E2-transciptome from a repressor mode to a activator mode is a critical early step in ERα- positive breast cancer. The specific aims are to: 1) Define ERα cistrome that is unique to non-transformed breast epithelial cells; 2) Investigate signaling nodes that govern ERα function in non-transformed and transformed breast epithelial cells by integrating ERα cistrome with transcriptome. Impact: To our knowledge, this is the first study to determine ERα function in non-transformed breast epithelial cells. The resources developed will be valuable to the research community to reassess signaling networks associated with ERα-positive breast cancer and anti-estrogen resistance. In particular, signaling defects linked to genomic aberrations uniquely enriched in ERα-positive breast cancers can be investigated and drugs targeting specific aberration can be identified using this resource.