Transcriptional Regulation of Breast Cancer Metastasis within the Tumor Microenvi

描述(申请人提供)：乳腺癌从原发部位逃逸导致死亡。最近，人们发现肿瘤转移所需的侵袭性可能会在转移的微环境中恢复，从而部分通过逆转E-钙粘蛋白的甲基化来实现播种。最初允许乳腺癌细胞从原发肿瘤扩散的间质转变是通过EGFR家族信号(HER1和HER2)驱动的。然而，在转移性播种过程中，EGFR家族信号下调，E-钙粘蛋白重新表达。我们提供的初步数据显示，KEISO的下调，或从细胞核限制KESO，推动乳腺癌细胞向分化的上皮样表型转变。因此，我们新的转移性种植的基本模型提出了一个关键的分子链接，使乳腺癌细胞能够整合到异位环境中，甚至进入休眠状态，即将Kaiso保留在细胞核外，以便E-cadherin能够重新表达。在这项提案中，我们将证明，即使不能通过调节这种分子级联来减少转移播撒和肿瘤休眠，也可以将其降至最低。要测试的目标是特定的目标1.确定EGF受体信号如何导致Kaiso重新定位到细胞核，以及这是否会改变DNA甲基化和癌细胞的表型。为此，我们将1)确定乳腺癌中自分泌EGFR信号对Kaiso定位/表达和E-cadherin甲基化状态的影响，2)利用药理学和分子构造确定KESO核外滞留时乳腺癌的表型，3)确定EGF受体信号/级联如何协调改变乳腺癌细胞系中Kaiso亚细胞定位和DNA甲基化。具体目标2.1)确定Kaiso重新定位到细胞核的中断是否改变了进入转移的壁龛和/或肿瘤休眠的发展。我们将把癌细胞引入生物反应器，并测定Kaiso在初始转移种植过程中的定位以及E-钙粘附素的表达。2) 确定核外隔离Kaiso定位对器官型肝脏微环境中肿瘤休眠的影响。3)确定核KEISO在动物原发肿瘤逃逸和转移微环境种植两个步骤中的作用。综上所述，通过使用原始人类外植体、已建立的细胞系、创新的人类器官型肝脏生物反应器以及动物模型，我们将确定Kaiso的定位和表达水平，以及与肿瘤微环境的细胞表型和细胞行为相关的KEISO。这些研究的成功完成将对肿瘤生物学的基础科学产生立竿见影的影响，并为乳腺癌转移和进入休眠的分子机制提供范式转换的见解，这是进展中最令人不寒而栗的方面之一。

DESCRIPTION (provided by applicant): Breast carcinoma escape from the primary site leads to mortality. Recently, it has been found that tumor properties of aggressiveness required for dissemination may revert within metastatic microenvironment to enable seeding, in part through reversal of methylation of E-cadherin. The initial mesenchymal transition that allows breast cancer cells to disseminate from the primary tumor is driven via EGFR family signaling (HER1 and HER2). However, during metastatic seeding, EGFR family signaling is downregulated and E-cadherin is re-expressed. We present preliminary data showing that downregulation of Kaiso, or restricting Kaiso from the nucleus, drives breast cancer cells towards a differentiated epithelial-like phenotype. Thus, our novel foundational model of metastatic seeding posits a key molecular link that enables the breast carcinoma cells to incorporate into the ectopic environment and even become dormant, that of retaining Kaiso out of the nucleus so that E-cadherin can be re-expressed. In this proposal we will establish that both metastatic seeding and tumor dormancy can be minimized if not eliminated by modulating this molecular cascade. The aims to be tested are Specific Aim 1. Determine how EGF receptor signaling causes relocalization of Kaiso into the nucleus and whether this alters DNA methylation and the carcinoma cell phenotype. In this aim, we will 1) Determine the influence of autocrine EGFR signaling on Kaiso localization/expression and methylation status of E-cadherin in breast cancer, and 2) Determine breast cancer phenotype upon sequestration of Kaiso outside of the nucleus, using pharmacological and molecular constructs, and 3) Determine how EGF receptor signaling/cascade coordinately alters Kaiso subcellular localization and DNA methylation in breast cancer cell lines. Specific Aim 2. 1) Determine whether disruption of Kaiso relocalization into the nucleus alters entry into the metastatic niche and/or the development of tumor dormancy. We will introduce the carcinoma cells into the bioreactor and determine the localization of Kaiso along with the expression of E-cadherin during initial metastatic seeding. 2) Determine the influence of sequestering Kaiso localization outside of the nucleus on tumor dormancy within the organotypic liver microenvironment. 3) Determine the role of nuclear Kaiso on the two steps of escape from the primary tumor and seeding of the metastatic microenvironment in animals. In summary, through the use of primary human explants, established lines, and an innovative human organotypic liver bioreactor as well as animal models, we will determine Kaiso localization and expression levels and that correlate with cell phenotype and cellular behaviors with the tumor microenvironment. Successful completion of these studies would could have immediate impact be on the basic science of tumor biology, and provide paradigm shifting insights into the molecular mechanisms by which breast carcinomas metastasize and enter dormancy, one of the most chilling aspects of progression.