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

 描述（由申请人提供）：雌激素（E2）通过核受体雌激素受体α（ERα）发挥作用，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，对抗雌激素治疗的抗性是一个主要的临床问题。在这一领域至少有两个关键的未解决的问题：1）在正常乳腺中，ERα在10-20%的非分裂细胞中表达。这些ERα阳性细胞通过旁分泌作用支持ERα阴性细胞对E2的增殖反应。与正常细胞相反，ERα阳性癌细胞本身对E2有反应而增殖。控制ERα功能转换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生物学的大多数了解，包括全基因组结合模式（ERα顺式组）、先锋因子/染色质修饰在引导ERα顺式组中的作用以及E2调节的基因表达模式（E2-转录组），都来自使用癌细胞系MCF-7而非正常/非转化细胞的研究，因为在技术上不可能培养表达ERα的非转化细胞。这种局限性使我们无法理解ERα在正常乳腺中的功能以及控制癌症相关ERα功能变化的机制。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测定法从正常乳腺增殖细胞，然后使用hTERT永生化，产生了几种ERα阳性乳腺上皮细胞系。与原代细胞中ERα的预测表达模式一致，ERα在管腔祖细胞和成熟细胞中表达，但在这些细胞系的干细胞部分中不表达，这验证了该系统的相关性。其中一个细胞系的RNA-seq分析显示ERα：E2在抑制基因表达中起主导作用。相反，ERα-信号通路在癌细胞中涉及激活和抑制，并与PI 3 K-Cyclin D1-cMyc增殖网络以及正常细胞中未观察到的非典型泛素化系统良好整合。利用这些新的资源，我们将解决这样的假设，即ERα顺式组和E2-transciptome从阻遏模式到激活模式的转换是ERα阳性乳腺癌的关键早期步骤。具体目标是：1）定义非转化乳腺上皮细胞所特有的ERα顺反子组; 2）通过整合ERα顺反子组与转录组来研究在非转化和转化乳腺上皮细胞中控制ERα功能的信号传导节点。影响：据我们所知，这是第一项确定ERα在非转化乳腺上皮细胞中功能的研究。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抵抗相关的信号网络具有价值。特别是，可以研究与ERα阳性乳腺癌中独特富集的基因组畸变相关的信号传导缺陷，并且可以使用该资源鉴定靶向特定畸变的药物。

 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.