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: 8895079
• 负责人: Jason Edward Lee
• 金额: $ 5.6万
• 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/8895079
• 关键词: Apoptotic; Biological Assay; Biological Markers; Breast Cancer Cell; Breast Cancer cell line; Calcium; 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; Health; Heterogeneity; Homeostasis; Integrins; Lead; Life; MCF7 cell; MDA MB 231; 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; reticulum cell; targeted treatment; 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（由申请人提供）：预计2011年将有近300，000名女性被诊断患有乳腺癌，因此乳腺癌是女性中最常见的癌症之一，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，根据生物标志物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深入了解可能会导致新疗法的开发，这些疗法可用于克服疾病的异质性。预后不良与肿瘤转移和耐药有关。因此，有必要建立在目前的理解的机制，细胞迁移和入侵，并阐明广泛的靶向化疗，如紫杉醇的行动和后果。内质网（ER）在乳腺癌和癌症相关细胞过程中的作用尚不清楚。最近的发现确定新的蛋白质参与调节ER结构和ER动力学现在允许研究人员测试有关ER结构和功能的假设。此外，内质网沿着微管（MT）的动力学被表征为尖端附着复合物（TAC）动力学和内质网滑动.此外，最近的研究报道，整合素β 3和蛋白磷酸酶1B（PTP 1B）运输到粘着斑依赖于内质网小管沿着微管延伸。因此，这一建议暗示ER结构/ER动力学和细胞运动性之间的关系，和紫杉醇耐药性，并将阐明乳腺癌细胞中的ER和局灶性粘连之间的关系的机制。该提案的第一个目标包括基础性高分辨率研究，这些研究将使用活细胞和固定细胞荧光显微镜表征非侵袭性（MCF-7）和侵袭性（MDA-MB-231）乳腺癌细胞中的ER结构。此外，ER结构将通过ER结构蛋白的过表达或敲低在细胞中直接调节。ER调节的乳腺癌细胞的性能将在基质胶迁移、跨内皮基质胶侵袭和跟踪测定中测量。最后，ER-细胞器接触的前缘内的分布， 迁移的乳腺癌细胞将通过电子显微镜来阐明。第二个目标将测试紫杉醇介导乳腺癌细胞杀伤的假设，通过破坏ER动力学沿着MT，从而破坏ER依赖性交付的粘着斑蛋白的粘着斑。通过STIM 1表达改变TAC动力学，或通过乳腺癌细胞中HDAC 6表达改变ER滑动，然后在细胞杀伤测定中测试这些细胞对紫杉醇的敏感性，来调节沿着微管的ER动力学。此外，用GFP标记的粘着斑蛋白（整合素-β 3或PTP 1B）和RFP-KDEL（ER标记物）共转染乳腺癌细胞将用于可视化这些蛋白质向粘着斑的ER小管相关递送，并确定该递送过程是否依赖于TAC ER动力学或ER滑动。因此，这些研究的目的是填补目前的理解的亚细胞事件的细胞迁移和紫杉醇治疗的研究ER在这些过程的机制中的作用的空白。