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

 描述（由申请人提供）：使用肽分析和质谱法分析糖基转移酶对乳腺上皮细胞中上皮-间充质转化的活性高通量生物技术已经能够发现识别具有不同表型的癌症亚型的遗传和蛋白质组谱。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类似的profil努力已经滞后。需要这种酶谱分析技术，因为这些酶对细胞表型产生显著影响。例如，前列腺细胞上皮细胞中GalNac-T3和GalNac-T6引起的纤连蛋白糖基化对于TGF-β诱导的上皮-间充质转化（EMT）是必不可少的，EMT是癌细胞劫持以实现转移的发育过程。该实施例与乳腺癌特别相关，因为这种形式的纤连蛋白onfFN在乳腺癌组织中上调，而不存在于正常乳腺组织中。此外，GalNac-T6的过表达在MCF 10A（一种永生化乳腺上皮细胞系）中诱导EMT样表型。尽管有这些证据，但仍不清楚GalNAc-T3和GalNAc-T6是否通过与前列腺细胞中类似的机制调节乳腺组织中的EMT。因此，我假设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作为EMT的新生物标志物。该提案旨在研究GalNAc-T3/-T6对乳腺细胞中TGF-β EMT的作用，并使用创新技术直接从细胞裂解物中分析其活性，称为SAMDI质谱法。该方法使用肽底物阵列来分析样品中的酶活性，并且已经证明了用于研究包括糖基化在内的几种酶化学的有效性。初步数据进一步证明，SAMDI分析是可行的，通过检测内源性半乳糖基转移酶活性的三阴性乳腺癌细胞系，MDA-MB-231。第一个目标将需要合成可以区分GalNAc-T3和GalNAc-T6之间的活性的肽文库，然后通过SAMDI测试GalNAc-T3和GalNac-T6活性是否可以用于分析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是否依赖于GalNAc-T3/-T6的表达以及EMT是否依赖于onfFN的存在。该提案旨在解决两个关键需求：填补与整个糖生物学领域相关的现有技术的空白，以及提供对乳腺癌社区重要的基本信息。

 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.