Definition of Microenvironment in Breast Cancer

描述（由申请人提供）：恶性进展的早期表现之一是组织组织的丧失。该建议继续检验结构完整性对于维持正常乳房功能以及抑制肿瘤形成至关重要的假设。我们假设，阐明在乳腺组织的腺泡和导管内建立和维持极性和结构的“信号整合计划”将揭示恶性肿瘤中间步骤的预后和治疗相关标记。因此，我们假设并提供了证据表明肌上皮细胞（MEPs）部分通过层粘连蛋白-111 （Ln-1）的产生和信号传导为管腔上皮细胞（LEPs）提供了关键的结构和功能线索。我们进一步假设，现在提供了额外的证据，在传统的细胞培养条件下，纯化的LEP几乎立即发展出LEP/MEP杂交表型，获得MEP基因表达的方面，特别是发展出通常由MEP在体内赋予的肿瘤抑制功能和制造腺泡蛋白的能力，这通常需要MEP的存在。因此，为了建立研究人类LEP-MEP相互作用的相关模型，我们必须确定指导这些细胞类型保持其原始功能的条件。因此，我们开发了新的多功能微环境阵列（MEArrays）来探测mep和lep是如何形成和保持的。我们现在建议将我们的研究结果扩展为三个具体目标。我们将具体地：1-使用设计MEArrays和设计介质确定允许在培养中保留MEP和LEP特定功能的途径。然后，我们可以探索除MEPs产生的Ln-1外，桥粒蛋白和其他调节分子在MEPs和LEPs如何相互作用以保持极性、结构和功能中的重要性。2-使用细胞组学和其他设备以及设计的3D微环境，在高通量分析中使用抑制肽、阻断抗体、突变细胞和shRNA消融完成对乳腺特异性功能的Ln-1信号级联成分的鉴定。3-利用基因表达阵列的生物信息学分析、全基因组甲基化谱和染色质结构的其他变化、影响细胞结构的mirna鉴定，以及利用独特的3D屏幕在我们的整合图上定位我们鉴定的新基因，识别和表征acini结构完整性的“信号整合计划”中的中心参与者和连接。总的来说，这些实验表明了MEP/LEP相互作用在维持乳腺组织极性腺泡结构中的重要性，也可以为其他组织提供原理证明。由于这些信号失去适当的平衡和/或整合会导致恶性肿瘤，我们的研究结果将推进基础知识，并为早期诊断和治疗策略提供新的标记物，以限制和/或逆转乳腺肿瘤的进展。公共卫生相关性：本提案旨在确定细胞微环境对人类乳腺细胞的影响，特别关注肌上皮细胞和腔上皮细胞相互作用。基于我们对微环境在正常人类乳房形态发生中的作用的研究，我们反复表明，微环境的变化可以导致恶性人类乳房肿瘤细胞重新获得正常细胞的关键属性，并且我们特别证明了肌上皮细胞在防止人类乳房细胞变成恶性细胞中起着核心作用。这一建议继续我们对这一重要领域的研究；我们的研究结果将进一步加深对复杂的相互作用的理解，这些相互作用有助于控制甚至逆转人类乳腺肿瘤。

DESCRIPTION (provided by applicant): One of the earliest manifestations of malignant progression is loss of tissue organization. This proposal continues to examine the hypothesis that architectural integrity is crucial for maintenance of normal breast function as well as for suppression of neoplasia. We have postulated that elucidation of a "signaling integration plan" that establishes and maintains polarity and structure within the acini and ducts of breast tissue will uncover prognostic and therapeutically-relevant markers of intermediary steps in malignancy. As such, we postulated and provided evidence that myoepithelial cells (MEPs) provide crucial structural and functional cues to luminal epithelial cells (LEPs) partly via production of, and signaling through, laminin-111 (Ln-1). We further postulated, and now provide additional evidence, that in traditional cell culture conditions, purified LEPs almost immediately develop a hybrid LEP/MEP phenotype acquiring aspects of MEP gene expression and in particular developing the tumor suppressor functions normally conferred in vivo by MEPs and an ability to make acini, which normally requires the presence of MEPs. Consequently, in order to make relevant models for the study of human LEP-MEP interactions we must identify the conditions that instruct these cell types to retain their original functions. Accordingly, we have developed new and versatile microenvironmental arrays (MEArrays) to probe how MEPs and LEPs become, and remain, determined. We now propose to expand our findings in 3 specific aims. We will specifically: 1- identify pathways that allow retention of MEP- and LEP- specific functions in culture using designer MEArrays and designer media. We then could probe the importance of desmosomal proteins and other regulatory molecules in addition to Ln-1 produced by MEPs, in how MEPs and LEPs interact to retain polarity, architecture and function. 2- complete the identification of Ln-1 signaling cascade components for mammary specific functions using inhibitory peptides, blocking antibodies, mutant cells and shRNA ablation in high throughput assays using Cellomics and other devices and designer 3D microenvironments. 3- identify and characterize central players and connections in the 'signaling integration plan' for structural integrity of acini using bioinformatics analysis of gene expression arrays, genome-wide methylation profiles and other changes in chromatin structure, identification of miRNAs affecting cellular architecture, and by positioning the new genes we identified using a unique 3D screen, on our integration map. Collectively these experiments address the importance of MEP/LEP interactions in maintenance of polar acinar structures in breast tissue, and could also provide a proof of principle for other tissues. Since loss of appropriate balance and/or integration of these signals leads to malignancy, our results will both advance fundamental knowledge and yield novel markers for early diagnosis and therapeutic strategies to limit and/or reverse breast tumor progression. PUBLIC HEALTH RELEVANCE: This proposal seeks to define the influences of cellular microenvironments on human breast cells, with particular attention to the effects of myoepithelial and luminal epithelial cells on one another. Building on our studies characterizing the role of the microenvironment in normal human breast morphogenesis, we have repeatedly shown that changes in the microenvironment can cause malignant human breast tumor cells to re-acquire critical attributes of normal cells, and we have specifically demonstrated that myoepithelial cells play a central role in preventing human breast cells from becoming malignant. This proposal continues our research into this important field of inquiry; our results will further common understanding of the complex interactions which serve to contain and even reverse human breast tumors.