Dissecting the role of glycan-based tumour promotion in breast cancer

葡聚糖装饰所有活细胞的表面，是最多样化的翻译后修饰之一。在细胞和环境之间的多种相互作用中，糖链在肿瘤发生过程中也会发生很大的变化，而异常的蛋白质糖基化是癌症的一个标志。如果考虑靶向糖基化，目前基于抗体的诊断和治疗试剂的有效性可以得到提高。然而，我们找到正确的靶蛋白糖形式的能力是有限的，并且多糖促进肿瘤的分子细节和相应的糖基转移酶酶是不明确的。这项工作将通过分析糖基转移酶同工酶GalNAc-T4、T6和T14来确定糖基化介导的乳腺癌进展的关键因素。这三种酶在乳腺癌的几种亚型和分期中具有重要的相关性，但它们的蛋白底物和促进疾病的机制尚不清楚。我们将使用一种名为凹凸不平的工程的化学生物学策略来开发所有三种同工酶的功能增益报告系统。该策略使用点击化学来识别蛋白质底物，并通过质谱仪(MS)糖蛋白质组学，以同工酶特异性的方式识别这些底物蛋白质上的糖基化位点。我们将使用这些报告系统首次描述在所有其他糖基转移酶存在的情况下，乳腺癌细胞系和原代人类组织样本中由GalNAc-T4、T6和T14修饰的糖基化位点。我们将通过生物化学、细胞生物学的方法来验证我们的结果，并通过合作，在体内建立播散性肺转移的小鼠模型。因此，我们将开始了解糖基化的复杂性，特别是在恶性过程中。我们的结果不仅将阐明基于葡聚糖的肿瘤促进的细节，还将为未来开发有针对性的诊断和治疗策略提供信息。

Glycans decorate the surface of all living cells and are among the most diverse post-translational modifications. Mediating manifold interactions between cells and their environment, glycans are also heavily altered during tumorigenesis and aberrant protein glycosylation is a hallmark of cancer. The efficacy of current antibody-based reagents for diagnosis and therapy can be enhanced if target glycosylation is considered. However, our capacity to find the right target protein glycoforms is limited, and the molecular details of tumour promotion by glycans and the corresponding glycosyltransferase enzymes are ill-defined. This work will identify key factors of glycosylation-mediated breast cancer progression by profiling the glycosyltransferase isoenzymes GalNAc-T4, T6 and T14. The three enzymes are of significant relevance in several subtypes and stages of breast cancer, but their protein substrates and mechanisms of disease promotion are unclear. We will use a chemical biology strategy termed bump-and-hole engineering to develop gain-of-function reporter systems for all three isoenzymes. The strategy employs click chemistry to identify protein substrates and, by mass spectrometry (MS) glycoproteomics, glycosylation sites on these substrate proteins in an isoenzyme-specific fashion. We will use these reporter systems to profile for the first time the glycosylation sites modified by GalNAc-T4, T6 and T14 in breast cancer cell lines and primary human tissue samples in the presence of all other glycosyltransferases. We will validate our results by methods of biochemistry, cell biology and, by collaboration, in vivo mouse models of disseminated lung metastasis. We will thereby begin to understand the complexity of glycosylation especially in malignant processes. Our results will not only shed light on the details of glycan-based tumour promotion, but also inform future strategies to develop targeted diagnostics and therapeutics.