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

描述(申请人提供)：ETS转录因子家族是高度冗余的，许多成员服务于促进肿瘤发生。有几个ETS因素与乳腺癌有关，但ESE-1是最相关的。ESE-1mRNA和蛋白在人乳腺癌中过表达，其与HER2的共表达与高度恶性表型相关。事实上，在ESE-1和HER2之间存在一个正反馈循环。尽管取得了这些进展，但任何ETS因子转化人乳腺上皮细胞(MECs)的能力仍不清楚。我们发现，ESE-1通过一种新的细胞质机制启动良性的、ESE-1阴性的MCF-12A和MCF-10A人乳腺上皮细胞系的转化，在这种机制中，其独特的40个氨基酸、丝氨酸和天冬氨酸富含结构域是转化所必需的和充分的。随着我们对致癌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还有一个额外的、经典的核转录作用。使用我们产生的高度特异的抗ESE-1的单抗(mAb405和mAb1534)，我们发现ESE-1在已建立的腔和HER2+乳腺癌细胞系中是有核的，而对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都有表达。此外，将稳定表达HA-ESE-1的T47D细胞注射到裸鼠体内，可产生比载体对照组大2倍的原位肿瘤。此外，我们还表明，通过显性-阴性、反义或shRNA方法干扰ESE-1的功能或表达足以逆转MCF-7、T47D和HER2+ZR-75-1乳腺癌细胞的转化表型，揭示了ESE-1通过经典的核转录机制在维持乳腺肿瘤发生中的关键作用。我们的目标是确定驱动HER2+乳腺癌细胞转化的ESE-1周期，从而阐明ESE-1的机制并确定新的治疗靶点。该方案的假设是，ETS转录因子ESE-1通过控制ESE-1特异的、促进生长的靶基因(ESE-1周期)的转录来维持HER2+乳腺癌细胞的转化表型。为了验证这一假设，我们提出了四个特定的目标：(1)确定ESE-1在维持乳腺癌细胞系转化表型中的HER2依赖性和机制作用；(2)应用无偏染色质免疫沉淀(ChIP)和DNase I深度DNA测序，结合全基因组RNA表达研究，识别ESE-1环路；(3)通过异位表达符合严格选择标准的候选基因并测试它们拯救shESE-1转化抑制效应的能力，识别关键的下游ESE-1靶点；(4)用高度特异的抗ESE-1单抗检测乳腺癌标本，以确定ESE-1的胞核和/或胞浆表达是否与肿瘤细胞类型、分级或预后有关。我们收集了专家合作者，并生成了支持该项目可行性的新数据。所获得的见解将为我们抗击这种致命疾病提供新的药物靶点和标记物。

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.