Definition of the Microenvironment in Breast Cancer

 描述(申请人提供)：核细胞和组织结构丧失是恶性进展的最早表现之一。为了使组织正常运作，必须维持组织的结构。30年前，我们假设在细胞外基质(ECM)蛋白和细胞核之间存在相互作用的“信号整合计划”。如果这种交流保持平衡，组织内环境就会保持稳定。然而，互惠信号的任何一步的中断最终都会打开恶性进展的机会。重大发现包括发现了层粘连蛋白-111(LN1)不可或缺的作用， 基底膜(BM)的一个成分。LN1的非程序性蛋白水解酶活性或LN1表面受体(如特异性整合素或营养不良多糖)及其下游介体的功能障碍破坏了LN1的整合计划，导致乳腺上皮细胞无法正确极化和静止，从而导致恶性进展。在之前的提交中，我们定义了一些参与LN1/乳腺细胞相互作用的通路，这些通路介导整合，当被破坏时，会导致组织紊乱和恶性，如果在3D中纠正，则会逆转恶性表型。在上一个资金周期中，我们获得了意想不到的数据，即LN1通过加速肌动蛋白(N-肌动蛋白)从细胞核输出而发出停顿信号，如果这一点被阻止，细胞将不会进入停顿。我们 发现LN1激活P53并产生不同的P53亚型。N-肌动蛋白的输出和P53状态的改变都提供了新的和未知的途径，将细胞外基质与细胞核中的变化联系起来，导致基因表达的变化，从而决定细胞的动态平衡和恶性进展。我们已经组建了一支优秀的专家团队，在这些研究上进行合作。我们将使用同基因乳腺癌进展系列、乳房缩小整形术的原代乳腺细胞和转基因小鼠的细胞来研究BM中的LN1如何调节N-肌动蛋白输出和P53状态。我们将使用RNAseq、MS Spec、ChIPseq、带有光漂白方法的超分辨率显微镜等技术，以及更常规的细胞、分子和生化技术，如Western blotts、IP，如果使用来自我们合作者的更多具有良好特征的抗体，来研究基因表达的变化、表观遗传修饰以及导致LN1信号导致表型显著变化的p53亚型的变化。我们提议的工作将提供有关组织如何保持健康的基础知识，并提供潜在的新分子和途径，针对微环境和由此产生的细胞内信号级联，在乳腺癌进展过程中可能出错。

 DESCRIPTION (provided by applicant): Loss of nuclear- cellular- and tissue- architecture is one of the earliest manifestations of malignant progression. In order for a tissue to function properly, tissue architecture must be maintained. We postulated 3 decades ago that there is a reciprocal `signaling integration plan' between the extracellular matrix (ECM) proteins and the nucleus. If this communication remains balanced, tissue homeostasis is maintained. However disruption of any step in reciprocal signaling eventually opens an opportunity for malignant progression. Significant findings include discovery of the indispensible role of laminin-111 (Ln1), a component of the basement membrane (BM). Destruction of Ln1 by unscheduled proteinase activity or dysfunction of surface receptors for Ln1 (such as specific integrins or dystroglycan) and their downstream mediators, disrupts integration plan and leads to inability of breast epithelial cells to correctly polarize and become quiescent thus leading to malignant progression. In previous submission we have defined a number of pathways involved in Ln1/breast cell interactions that mediate integration and, when disrupted, lead to disorganization and malignancy and, if corrected in 3D, revert the malignant phenotype. During last funding cycle we obtained unexpected data that Ln1 signals for quiescence by accelerating the export of actin from the nucleus (N-actin), and if this is prevented, the cells will not enter quiescence. We found Ln1 activates p53 and production of different p53 isoforms. Both the export of N-actin and changes in p53 status provide novel and uncharted pathways linking ECM to changes in the nucleus which lead to altered gene expression that dictate cellular homeostasis vs malignant progression. We have assembled a stellar team of experts to collaborate on these studies. We will use an isogenic breast cancer progression series, primary breast cells from reduction mammoplasty, and cells from genetically engineered mice to investigate how Ln1 in the BM modulates N-actin export and p53 status. We will use techniques such as RNAseq, Mass Spec, ChIPseq, super-resolution microscopy with photobleaching approaches, along with more routine cell, molecular and biochemical techniques such as western blots, IP, IF using additional well-characterized antibodies from our collaborators to investigate changes in gene expression, epigenetic modifications, and changes in p53 isoforms responsible for dramatic alterations in phenotypes as a result of Ln1 signaling. Our proposed work will provide fundamental knowledge about how tissues can remain healthy, as well as providing potential new molecules and pathways that target the microenvironment and the resulting intracellular signaling cascades that can go awry during breast cancer progression.