Transcriptional effects and non-transcriptional effects of estrogen in the pathogenesis of Inflammatory Breast Cancer

项目摘要 炎症性乳腺癌（IBC）是最具侵袭性和致命性的乳腺癌之一。的 对IBC的分子机制知之甚少。治疗策略已经从 非IBC乳腺癌，即使HER2阳性和激素治疗的结果有所改善， 尽管三阴性（TN）亚型IBC（ER-/PR-/HER2-）的预后仍然很差。目前，雌激素 非基因组信号传导与TN乳腺癌的进展、运动性和侵袭相关， 最近的IBC。本研究旨在阐明雌激素信号在IBC中的功能作用， 比较雌激素在两种情况下的作用：HER2阳性和三阴性IBC亚型。分析将 进行，以确定雌激素信号传导的影响，由ER α 36和GPR30调节，和交叉- ErbB信号传导的激活在各种致癌表型的获得中是重要的。的靶细胞 分析将是IBC细胞系，SUM149（TNBC）和SUM190（HER2+）。大约40%的IBC是TN乳腺癌 这使得内分泌疗法难以治疗，内分泌疗法在ER +/PR + BC亚型中非常有效。 IBC在约50%的病例中显示ErbB酪氨酸激酶受体，特别是EGFR的过度表达， 但针对该受体的靶向治疗对这种疾病并不有效。一系列的机械研究 评价雌激素在基因敲除后获得致癌表型中的作用 或替代雌激素受体的过表达，以及雌激素信号传导的激活或抑制。 IBC细胞系将用雌激素特异性配体和抑制剂（雌二醇、G1、G15、淫羊藿苷）处理 受体，然后彻底表征EGFR下游激酶的激活和EGFR下游激酶的表达。 与雌激素信号相关的效应蛋白和转录变化。在识别出激酶之后 通过雌激素信号激活，将进行药物反应分析以测试新的治疗靶点。 单药或与EGFR抑制剂联合。雌激素非基因组信号的作用 在原癌基因表型（增殖，运动，和侵袭）将使用一种新的侵袭评估 本发明提供了模拟IBC肿瘤栓塞形成的测定和三维培养系统。我们亦会 确定ER α 36的DNA结合偏好，因为ER α的这种亚型保守配体 结合结构域、DNA结合结构域和核定位信号。通过RNA-seq分析，我们将 在我们的两个细胞系模型中确定与雌激素治疗相关的转录组变化， 受雌激素影响的通路与运动性、侵袭性和/或干性有关。患者的预后较差 与IBC强调需要进一步表征与其侵略性相关的功能变化， 进展以及雌激素非基因组信号传导与EGFR通路的相互作用。从长远来看， 这项研究将帮助我们设计更有效的靶向治疗，可以在动物模型中进行测试， 进一步了解与IBC进展相关的机制。

Project Summary Inflammatory breast cancer (IBC) is one of the most aggressive and lethal form of breast cancer. The molecular mechanisms underlying IBC are poorly understood. Therapeutic strategies have been adopted from non-IBC breast cancers and even though outcomes have been improved for HER2-positive and hormonal receptor IBC, prognosis for triple-negative (TN) subtype IBCs (ER-/PR-/HER2-) is still dire. Currently, estrogen non-genomic signaling has been associated with progression, motility, and invasion of TN breast cancer and most recently of IBC. This proposal seeks to elucidate the functional role of estrogen signaling in IBCs and to compare the effects of estrogen in two contexts: HER2-positive and triple-negative IBC subtypes. Analyses will be performed to define the effects of estrogen signaling, regulated by ERα36 and GPR30, and the cross- activation of ErbB signaling important in the acquisition of various oncogenic phenotypes. The target cells for analysis will be IBC cell lines, SUM149 (TNBC) and SUM190 (HER2+). Around 40% of IBC are TN breast cancers making it hard to treat with endocrine therapy, which is quite effective in ER+/PR+ BC subtypes. IBC shows over-expression of ErbB tyrosine kinase receptors, especially EGFR, in about 50% of the cases, but targeted therapy against this receptor has not been effective for this disease. A series of mechanistic studies will be performed to evaluate the effects of estrogen in the acquisition of oncogenic phenotypes upon knockdown or over-expression of the alternative estrogen receptors, and activation or inhibition of the estrogen signaling. IBC cell lines will be treated with ligands and inhibitors (estradiol,G1,G15, Icaritin) specific to the estrogen receptors to then thoroughly characterized the activation of EGFR downstream kinases and expression of effector proteins and transcriptional changes associated with estrogen signaling. After identifying kinases activated by estrogen signaling, drug response analyses will be done to test novel therapeutic targets with single agents or in combination with EGFR inhibitors. The effects of estrogen non-genomic signaling in pro-oncogenic phenotypes (proliferation, motility, and invasion) will be evaluated using a novel invasion assay and three-dimensional culture system that mimics the formation of IBC tumor emboli. Also, we will determine the DNA binding preferences of ERα36, due to the fact that this isoform of ERα conserved the ligand binding domain, DNA binding domain and nuclear localization signal. In parallel, by RNA-seq analysis we will determine the transcriptome changes associated with estrogen treatment in our two cell line models to identify pathways affected by estrogen relevant in motility, invasion and/or stemness. The poor prognosis for patients with IBC emphasizes the need to further characterize the functional changes associated with its aggressive progression and the interaction of estrogen non-genomic signaling with EGFR pathway. In the long-term, this study will help us design more effective targeted therapies that can be tested in animal models and understand further the mechanisms associated with IBC progression.