Profiling Glycosyltransferase Activities on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using peptide arrays and mass spectrometry

使用肽阵列和质谱分析乳腺上皮细胞上皮-间质转化中的糖基转移酶活性

• 批准号: 9441727
• 负责人: Jose-Marc Techner
• 金额: $ 4.95万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9441727
• 关键词: Address; Biological Assay; Biological Markers; Biotechnology; Breast; Breast Cancer cell line; Breast Epithelial Cells; Cell Line; Cell model; Cells; Chemistry; Clinical; Communities; Complex; Data; Detection; Development; Developmental Process; Effectiveness; Enzymes; Epithelial; Epithelial Cells; Fetal Tissues; Fibronectins; GALNT3 gene; Galactosyltransferases; Generations; Genetic; Glycobiology; Glycoproteins; Goals; Lead; Lung; MALDI-TOF Mass Spectrometry; MCF10A cells; MDA MB 231; Malignant - descriptor; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary gland; Maps; Mass Spectrum Analysis; Measures; Mediating; Mesenchymal; Metastatic breast cancer; Methods; Negativism; Neoplasm Metastasis; Outcome; Peptide Library; Peptides; Phenotype; Post-Translational Protein Processing; Process; Prostate; Protein Glycosylation; Protein Isoforms; Proteomics; Research; Role; Sampling; Small Interfering RNA; Spectrometry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Techniques; Technology; Testing; Time; Tissues; Transforming Growth Factor beta; Transitional Cell Carcinoma; Work; base; cancer cell; cancer subtypes; enzyme activity; enzyme substrate; epithelial to mesenchymal transition; experimental study; genome sequencing; glycosylation; glycosyltransferase; high throughput screening; improved; innovative technologies; knock-down; loss of function; malignant breast neoplasm; monolayer; novel; novel marker; overexpression; public health relevance; sugar; therapeutic target; treatment response; triple-negative invasive breast carcinoma; tumor DNA; whole genome

 DESCRIPTION (provided by applicant): Profiling Glycosyltransferase Activity on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using Peptide Asrrays and Mass Spectrometry High-throughput biotechnology has enabled discovery of genetic and proteomic profiles that identify cancer subtypes with distinct phenotypes. However, similar efforts to profil based on the posttranslational modifications mediated by glycosyltransferases have lagged due to lack of efficient high-throughput assays. Such enzymatic profiling technology is needed because these enzymes exert dramatic effects on cell phenotype. For example, fibronectin glycosylation by GalNac-T3 and GalNac-T6 in prostate cells epithelial cells is essential for the TGF-beta induced epithelial-mesenchymal transition (EMT), a developmental process that cancer cells hijack to enable metastasis. This example is particularly relevant to breast cancer because this form of fibronectin, onfFN, is upregulated in breast cancer tissue and not present in normal mammary tissue. Moreover, overexpression of GalNac-T6 induce an EMT-like phenotype in MCF10A, an immortalized breast epithelial cell line. Despite these evidence it remains unclear if GalNAc-T3 and GalNAc-T6 regulate EMT in breast tissue by a similar mechanism as that in prostate cells. Therefore my hypothesis that GalNAc-T3 and GalNAc-T6 promote EMT in breast cells via glycosylation of fibronectin and that their activity can serve as novel biomarkers of EMT. This proposal seeks to investigate the role of GalNAc-T3/-T6 on TGF-beta EMT in breast cells and use an innovative technology to profile their activity directly from cell lysate, known as SAMDI mass spectrometry. This method uses peptide substrate arrays to profile enzyme activities in a sample and has demonstrated effectiveness for studying several enzymatic chemistries, including glycosylation. Preliminary data further demonstrate that SAMDI profiling is feasible via detection endogenous galactosyltransferase activity in the triple-negativ breast cancer cell line, MDA-MB-231. The first aim will entail synthesis of a peptide library that can distinguish activities between GalNAc-T3 and GalNAc-T6 and then test by SAMDI whether GalNAc-T3 and GalNac-T6 activities can be used to profile EMT. In the second aim, I will investigate the extent to which GalNAc-T3 and -T6 regulate EMT in breast epithelial cells. Using overexpression and knockdown studies, I will test whether EMT is dependent on the expression of GalNAc-T3/-T6 and whether EMT is dependent on the presence of onfFN. This proposal aims to address two critical needs: fill a gap in available technology relevant to the field of glycobiology as a whole as well as provide fundamental information important to the breast cancer community.

 描述（由适用提供）：使用肽Asrrays和质谱高通量生物技术在乳腺上皮细胞中上皮间质转化上的糖基转移酶活性的分析已使遗传和蛋白质组学特征发现鉴定出具有不同势型的癌症的遗传和蛋白质组学特征。然而，由于缺乏有效的高通量测定，基于糖基转移酶介导的翻译后修饰的努力也滞后。需要这种酶促分析技术，因为这些酶对细胞表型产生了巨大的影响。例如，前列腺细胞中GalNAC-T3和GALNAC-T6上皮细胞中通过纤连蛋白糖基化对于TGF-BetA诱导的上皮 - 间质转变（EMT）是必不可少的，这是一种癌细胞的发育过程，癌细胞抢劫以促进转移。这个例子与乳腺癌尤其重要，因为这种形式的纤连蛋白ONFFN在乳腺癌组织中进行了更新，而在正常的乳腺组织中不存在。此外，GalNAC-T6的过表达诱导MCF10A（一种永生的乳腺上皮细胞系）中的EMT样表型。尽管有这些证据，但尚不清楚Galnac-T3和GalNAC-T6是否通过与前列腺细胞中的类似机制调节乳腺组织中的EMT。因此，我的假设是GalNAC-T3和GalNAC-T6通过纤连蛋白的糖基化促进乳腺细胞中的EMT，并且它们的活性可以用作EMT的新型生物标志物。该提案旨在研究GalNAC-T3/-T6在TGF-βEMT中的作用，并使用创新技术直接从细胞裂解物（称为SAMDI质谱法）中介绍其活性。该方法使用肽底物阵列来介绍样品中的酶活性，并证明了研究多个酶促化学家（包括糖基化）的有效性。初步数据进一步表明，通过检测三型乳腺癌细胞系MDA-MB-231中的内源性半乳糖基转移酶活性，SAMDI分析是可行的。第一个目的将需要综合肽库，该肽库可以区分GalNAC-T3和GalNAC-T6之间的活动，然后通过SAMDI测试GalNAC-T3和GalNAC-T6活动是否可以用于介绍EMT。在第二个目标中，我将研究GalNAC -T3和-T6在乳腺上皮细胞中调节EMT的程度。使用过表达和敲低研究，我将测试EMT是否取决于GalNAC-T3/-T6的表达以及EMT是否取决于ONFFN的存在。该建议旨在满足两个关键需求：填补与整个糖生物学领域有关的可用技术的空白，并提供对乳腺癌社区重要的基本信息。