The Role of ESE-1 in HER2-Positive Breast Cancer

描述（由申请人提供）：ETS转录因子家族是高度冗余的，其中许多成员用于促进肿瘤发生。 几种ETS因子与乳腺癌有关，但ESE-1是最相关的。 ESE-1 mRNA和蛋白在人乳腺癌中过表达，其与HER 2的共表达与高度恶性表型相关。 事实上，ESE-1和HER 2之间存在正反馈回路。 尽管有这些进展，任何ETS因子转化人乳腺上皮细胞（MEC）的能力仍然未知。 我们发现ESE-1通过一种新的细胞质机制启动良性、ESE-1阴性MCF-12 A和MCF-10 A人乳腺上皮细胞系的转化，其中其独特的富含40个氨基酸、丝氨酸和天冬氨酸的结构域是转化所必需和足够的。 随着我们对致瘤性乳腺癌细胞系的研究进展，我们发现ESE-1在维持乳腺肿瘤发生中的额外的经典核转录作用。 使用我们产生的高特异性单克隆抗ESE-1抗体（mAb 405和mAb 1534），我们发现ESE-1在已建立的管腔和HER 2+乳腺癌细胞系中是核，而乳腺癌组织微阵列的初步IHC研究显示ESE-1蛋白在细胞核和细胞质中表达。 此外，将稳定表达HA-ESE-1的T47 D细胞注射到裸鼠中导致原位肿瘤比载体对照大2倍。 此外，我们已经表明，通过显性负性，反义或shRNA方法干扰ESE-1功能或表达，足以逆转MCF-7，T47 D和HER 2 + ZR-75-1乳腺癌细胞中的转化表型，揭示了ESE-1在维持乳腺肿瘤发生中的关键作用，通过经典的核转录机制。 我们的目标是确定ESE-1顺式组，驱动HER 2+乳腺癌细胞的转化，从而阐明ESE-1的机制，并确定新的治疗靶点。 该提议的假设是ETS转录因子ESE-1通过控制ESE-1特异性促生长靶基因（ESE-1顺式组）的转录来维持HER 2+乳腺癌细胞中的转化表型。 为了验证这一假设，我们提出了四个具体目标：（1）确定ESE-1在维持乳腺癌细胞系中转化表型的HER 2依赖性和机制作用;（2）应用无偏染色质免疫沉淀（ChIP）和DNA酶I-深度DNA测序，结合全基因组RNA表达研究，鉴定ESE-1顺式组;（3）通过异位表达符合严格选择标准的候选物并测试其拯救shESE-1的转化抑制作用的能力来鉴定关键下游ESE-1靶标;和（4）使用高度特异性的抗ESE-1单克隆抗体来探测乳腺癌标本，以确定细胞核和/或细胞质ESE-1表达是否与肿瘤细胞类型、分级或结果相关。 我们已经聚集了专家合作者，并生成了支持项目可行性的新数据。 获得的见解将提供新的药物靶点和标记物，用于我们与这种致命疾病的斗争。 公共卫生相关性：乳腺癌仍然是女性中最常见和最致命的恶性肿瘤之一，HER 2+亚型显示出不良的总体预后和对HER 2特异性治疗的频繁最终耐药性。 到目前为止，我们仍然不了解导致HER 2+乳腺肿瘤的所有生物学机制，以及它们如何获得对HER 2+靶向治疗的耐药性。 在这项提案中，我们计划使用我们产生的高度特异性抗ESE-1单克隆抗体和对曲妥珠单抗敏感和耐药的HER 2+细胞系（赫赛汀）治疗，以确定ESE-1在维持转化状态中的作用、HER 2/ESE-1相互依赖性、关键的促肿瘤发生ESE-1调节基因，并探测人肿瘤样品中ESE-1的表达，以便与肿瘤亚型和临床行为相关联。

DESCRIPTION (provided by applicant): The ETS transcription factor family is highly redundant, with many members serving to promote tumorigenesis. Several ETS factors have been implicated in breast cancer, but ESE-1 is the most relevant. ESE-1 mRNA and protein are over-expressed in human breast cancer and its co-expression with HER2 correlates with a highly malignant phenotype. Indeed, a positive-feedback loop exists between ESE-1 and HER2. Despite these advances, the ability of any ETS factor to transform human mammary epithelial cells (MECs) remained unknown. We discovered that ESE-1 initiates transformation of benign, ESE-1-negative MCF-12A and MCF-10A human mammary epithelial cell lines via a novel cytoplasmic mechanism in which its unique 40-amino acid, serine and aspartic rich domain is necessary and sufficient for transformation. As our studies progressed with tumorigenic breast cancer cell lines, we discovered an additional, classical nuclear transcriptional role for ESE-1 in maintaining mammary tumorigenesis. Using highly-specific monoclonal anti-ESE-1 antibodies that we generated (mAb405 and mAb1534), we found ESE-1 to be nuclear in established luminal and HER2+ breast cancer cell lines, while preliminary IHC studies of breast cancer tissue microarrays revealed ESE-1 protein to be expressed both in the nucleus and cytoplasm. Furthermore, T47D cells stably expressing HA-ESE-1 injected into nude mice resulted in orthotopic tumors that were 2-fold larger than vector controls. Also, we've shown that interfering with ESE-1 function or expression, via dominant-negative, antisense or shRNA methods, is sufficient to reverse the transformed phenotype in MCF-7, T47D and HER2+ ZR-75-1 breast cancer cells, revealing a crucial role for ESE-1 in maintaining mammary tumorigenesis, via a classic nuclear transcriptional mechanism. Our goal is to define the ESE-1 cistrome that drives transformation in HER2+ breast cancer cells, thus elucidating ESE-1 mechanism and identifying novel therapeutic targets. The hypothesis of this proposal is that the ETS transcription factor, ESE-1, maintains the transformed phenotype in HER2+ breast cancer cells by controlling the transcription of ESE-1-specific, growth-promoting target genes (ESE-1 cistrome). To test this hypothesis, we propose four specific aims: (1) to determine the HER2-dependence and mechanistic role of ESE-1 in maintaining the transformed phenotype in breast cancer cell lines; (2) to apply unbiased chromatin immunoprecipitation (ChIP)- and DNAse I-deep DNA sequencing, coupled with genome-wide RNA expression studies, to identify the ESE-1 cistrome; (3) to identify key downstream ESE-1 targets by ectopically expressing candidates that meet rigorous selection criteria and testing their ability to rescue the transformation-inhibitory effect of shESE-1; and (4) to use highly-specific anti-ESE-1 mAbs to probe breast cancer specimens, to determine whether nuclear and/or cytoplasmic ESE-1 expression correlates with tumor cell type, grade or outcome. We have gathered expert collaborators and generated new data supporting the project feasibility. Insights gained will provide new drug targets and markers to use in our battle against this deadly disease. PUBLIC HEALTH RELEVANCE: Breast cancer remains the most common and one of the deadliest malignancies in women, with the HER2+ subtype displaying a poor overall prognosis and frequent eventual resistance to HER2-specific therapies. To date, we still do not understand all of the biological mechanisms causing HER2+ breast tumors, and how they acquire resistance to HER2+-targeted therapies. In this proposal, we plan to use highly-specific anti-ESE-1 monoclonal antibodies that we generated and HER2+ cell lines that are sensitive and resistant to trastuzumab (Herceptin) treatment, to define the role of ESE-1 in maintaining the transformed state, the HER2/ESE-1 interdependence, the key pro-tumorigenic ESE-1-regulated genes, and to probe for ESE-1 expression in human tumor samples, in order to correlate with tumor subtype and clinical behavior.