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

聚糖修饰所有活细胞的表面，是最多样化的翻译后修饰之一。介导细胞与其环境之间的多种相互作用，聚糖在肿瘤发生过程中也发生严重改变，异常的蛋白质糖基化是癌症的一个标志。如果考虑到靶糖基化，目前基于抗体的诊断和治疗试剂的疗效可以提高。然而，我们找到正确靶蛋白糖型的能力是有限的，而且聚糖和相应的糖基转移酶促进肿瘤的分子细节还不清楚。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个亚型和分期中具有重要的相关性，但它们的蛋白底物和疾病促进机制尚不清楚。我们将使用一种化学生物学策略，称为凹凸孔工程，以开发所有三种同工酶的功能获得报告系统。该策略采用点击化学来鉴定蛋白质底物，并通过质谱（MS）糖蛋白组学，以同工酶特异性的方式鉴定这些底物蛋白上的糖基化位点。我们将使用这些报告系统首次在所有其他糖基转移酶存在的情况下，分析乳腺癌细胞系和原代人组织样本中GalNAc-T4、T6和T14修饰的糖基化位点。我们将通过生物化学、细胞生物学的方法验证我们的结果，并通过合作，建立弥散性肺转移的小鼠体内模型。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。我们的研究结果不仅将揭示基于聚糖的肿瘤促进的细节，而且还将为未来开发靶向诊断和治疗的策略提供信息。

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.