Endoplasmic reticulum structure and dynamics in breast cancer cell metastasis and

描述(申请人提供)：2011年预计将有近300,000名女性被诊断出患有乳腺癌，从而使乳腺癌成为女性最常见的癌症之一，也是一种高度优先的疾病。乳腺癌被认为是一种异质性疾病，根据生物标记物的表达和药物敏感性进行分类。然而，对乳腺癌细胞所利用的基本细胞过程的更多了解可能会导致新疗法的发展，这种疗法可以用来克服疾病的异质性。预后不良与肿瘤转移和耐药有关。因此，有必要建立在目前对细胞迁移和侵袭机制的理解基础上，并阐明广泛靶向化疗的作用和后果，如紫杉醇。内质网(ER)在乳腺癌和与癌症相关的细胞过程中的作用尚不清楚。最近发现了参与调节内质网结构和内质网动态的新蛋白质，这使得研究人员可以测试与内质网结构和功能相关的假说。此外，电流变沿微管(MTS)的动力学特征是尖端附着复合体(TAC)动力学和电流变滑动。此外，最近的研究报道，整合素-�3和蛋白磷酸酶1B(PTP1B)转运到局灶性粘连依赖于内质网小管沿微管的延伸。因此，这一建议暗示了ER结构/ER动力学与细胞运动和紫杉醇耐药性之间的关系；并将阐明ER与乳腺癌细胞局部粘连之间关系的潜在机制。这项建议的第一个目标是基础的高分辨率研究，这些研究将使用活细胞和固定细胞荧光显微镜来表征非侵袭性(MCF-7)和侵袭性(MDA-MB-231)乳腺癌细胞的ER结构。此外，ER结构蛋白的过度表达或敲除将直接调节细胞内的ER结构。ER调控的乳腺癌细胞的性能将在基质迁移、跨内皮基质侵袭和跟踪试验中进行测量。最后，ER-细胞器接触在前缘的分布。 迁移的乳腺癌细胞将通过电子显微镜来阐明。第二个目标将检验这样的假设，即紫杉醇通过扰乱MTS沿线的ER动力学，从而扰乱依赖ER的焦点黏附蛋白向焦点粘连的递送，从而介导乳腺癌细胞的杀伤。在乳腺癌细胞中，通过STIM1表达改变TAC动力学，或通过HDAC6表达ER滑动，然后在细胞杀伤实验中测试这些细胞对紫杉醇的敏感性，将调节微管上的ER动力学。此外，将绿色荧光蛋白标记的焦点黏附蛋白(整合素-�3或PTP1B)和RFP-KDEL(ER标记物)共转染乳腺癌细胞，将被用于可视化这些蛋白质通过内质网小管相关的递送到焦点粘连，并确定这一递送过程是否依赖于TACER动力学或ER滑动。因此，这些研究旨在通过研究内质网在这些过程中的作用来填补目前对细胞迁移和紫杉醇治疗的亚细胞事件的了解的空白。

DESCRIPTION (provided by applicant): Nearly 300,000 women were predicted to be diagnosed with breast cancer in 2011, thus making breast cancer one of the most common cancers in women and a disease of high priority. Breast cancer is considered to be a heterogeneous disease that is categorized based upon biomarker expression and drug sensitivity. However, a greater understanding of the basic cellular processes that are exploited by breast cancer cells may lead to the development of novel therapeutics, which can be used to overcome disease heterogeneity. Poor prognosis has been correlated to tumor metastasis and drug resistance. Therefore, there is a need to build on the present understanding of the mechanisms underlying cell migration and invasion, and to elucidate the actions and consequences of broad-targeting chemotherapies, such as taxol. The role of the endoplasmic reticulum (ER) in breast cancer and in cancer-relevant cellular processes is unclear. Recent discoveries identifying novel proteins involved in regulating ER structure and ER dynamics now permit investigators to test hypotheses relating ER structure and function. Additionally, ER dynamics along microtubules (MTs) have been characterized as tip- attachment complex (TAC) dynamics and ER sliding. Moreover, recent studies have reported that integrin-�3 and protein phosphatase 1B (PTP1B) trafficking to focal adhesions was dependent on ER tubule extension along microtubules. Thus, this proposal implicates a relationship between ER structure/ER dynamics and cell motility, and taxol resistance; and will elucidate the mechanisms underlying the relationship between the ER and focal adhesions in breast cancer cells. The first aim of this proposal consists of foundational high-resolution studies that will characterize ER structure in non-invasive (MCF-7) and invasive (MDA-MB-231) breast cancer cells using live- cell and fixed-cell fluorescence microscopy. Furthermore, ER structure will be directly modulated in cells by overexpression or knockdown of ER structural proteins. The performance of ER-modulated breast cancer cells will be measured in matrigel migration, transendothelial-matrigel invasion and tracking assays. Finally, the distribution of ER-organellar contacts within the leading edge of migrating breast cancer cells will be elucidated by electron microscopy. The second aim will test the hypothesis that taxols mediate breast cancer cell killing by disrupting ER dynamics along MTs, thus disrupting ER-dependent delivery of focal adhesion proteins to focal adhesions. ER dynamics along microtubules will be modulated by altering TAC dynamics via STIM1 expression, or ER sliding via HDAC6 expression in breast cancer cells, then testing the sensitivity of these cells to taxol in cell killing assays. Furthermore, co-transfection of breast cancer cells with GFP-tagged focal adhesion proteins (integrin-�3 or PTP1B) and RFP-KDEL (ER marker) will be used to visualize ER tubule-associated delivery of these proteins to focal adhesions, and to determine whether this delivery process is dependent on TAC ER dynamics or ER sliding. Thus, these studies aim to fill a void within the current understanding of the subcellular events underlying cell migration, and taxol treatment by investigating the role of the ER in the mechanisms of these processes.