Endoplasmic reticulum structure and dynamics in breast cancer cell metastasis and

乳腺癌细胞转移和转移中的内质网结构和动力学

• 批准号: 8455005
• 负责人: Jason Edward Lee
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455005
• 关键词: Apoptotic; Biological Assay; Biological Markers; Breast Cancer Cell; Calcium; Cancer cell line; Cell physiology; Cells; Cellular biology; Complex; Development; Diagnosis; Disease; Drug Metabolic Detoxication; Drug Targeting; Drug resistance; Electron Microscopy; Endoplasmic Reticulum; Equilibrium; Event; Fluorescence Microscopy; Focal Adhesions; HDAC6 gene; Heterogeneity; Homeostasis; Integrins; Lead; Life; MCF7 cell; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Microtubules; Names; Neoplasm Metastasis; Nocodazole; Organelles; Paclitaxel; Patients; Performance; Pharmaceutical Preparations; Plastics; Process; Protein Biosynthesis; Protein phosphatase; Proteins; Reporting; Research Personnel; Research Proposals; Resistance; Resolution; Role; STIM1 gene; Signal Transduction; Slide; Structural Protein; Structure; Testing; Transfection; Woman; base; cell fixing; cell killing; cell motility; chemokine receptor; chemotherapy; drug sensitivity; malignant breast neoplasm; matrigel; migration; novel; novel therapeutics; outcome forecast; overexpression; prevent; public health relevance; reticulum cell; trafficking; tumor

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.

描述（由适用提供）：预计2011年被诊断出近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，并且是高度优先级的疾病。乳腺癌被认为是一种基于生物标志物表达和药物敏感性分类的异质疾病。但是，对乳腺癌细胞所利用的基本细胞过程有更深入的了解可能导致新型治疗的发展，这些疗法可用于克服疾病异质性。预后不良与肿瘤转移和耐药性有关。因此，有必要建立对细胞迁移和侵袭机制的当前理解，并阐明诸如紫杉醇等广泛靶向化学疗法的作用和后果。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。最近发现涉及调节ER结构和ER动力学的新蛋白质的发现现在允许研究人员测试与ER结构和功能有关的假设。另外，沿微管（MT）的ER动力学已被描述为尖端连接复合物（TAC）动力学和ER滑动。此外，最近的研究报道说，整联蛋白 - 3和蛋白质光培养酶1b（PTP1B）运输到焦点粘合剂上取决于沿微管沿着ER管扩展。这就是该提案实现了ER结构/ER动力学与细胞运动性与紫杉醇耐药性之间的关系。并将阐明乳腺癌细胞中ER与局灶性粘合剂之间关系的机制。该提案的第一个目的是由基础高分辨率研究组成，这些研究将表征非侵入性（MCF-7）和侵入性（MDA-MB-231）乳腺癌细胞中的ER结构，使用活细胞和固定细胞荧光显微镜。此外，ER结构将通过过表达或敲低ER结构蛋白直接调节。 ER调节的乳腺癌细胞的性能将在基质迁移，跨内皮式摩trigel侵袭和跟踪测定中进行测量。最后，在领先的前沿ER-Organellar接触的分布 通过电子显微镜阐明迁移的乳腺癌细胞。第二个目的将检验以下假设：紫杉醇通过破坏MT沿MT的ER动力学而杀死乳腺癌细胞的中位数，从而破坏了ER依赖性局灶性粘合剂蛋白向局灶性粘合剂的递送。沿着微管的ER动力学将通过通过STIM1表达改变TAC动力学，或通过HDAC6在乳腺癌细胞中滑动来调节ER动力学，然后测试这些细胞对细胞杀死阿萨斯的紫杉醇的敏感性。此外，乳腺癌细胞与GFP标记的局灶性粘附蛋白（整联蛋白 - 或PTP1B）和RFP-KDEL（ER标记）的共转染将用于可视化与ER管相关的ER管相关的蛋白质递送到焦点粘合剂中，以确定此递送过程是否依赖于Tac ERIDENT DYSTIDERTIND DYSTIDENT DYSTIDENT DYSTIDENT DYSTIDENT DYSTIDERTIDERTIDERTIDENT。这是这些研究旨在通过研究ER在这些过程机制中的作用来填补当前对细胞迁移基础事件的空隙和紫杉醇治疗。