MSI-target gene-dependent alterations of cell surface glycosylation signatures in MSI colorectal cancer cell lines and primary tumors

微卫星不稳定（MSI）肿瘤表现出多种组织临床病理学特征，包括粘液性组织学和改善预后相比，他们的微卫星稳定（MSS）的同行。一些MSI靶基因的双等位基因突变失活经常被观察到，并且通常被认为是驱动MSI肿瘤发生的原因。近年来，针对这些MSI肿瘤的发病机制，我们发现了特定MSI靶基因的功能失活与细胞表面糖基化模式之间的潜在相关性。目前的提议旨在探索MSI结直肠癌细胞系中细胞表面糖基化特征的MSI靶基因依赖性改变，所述MSI结直肠癌细胞系被工程化以允许两种最频繁突变的MSI靶基因（ACVR 2、TGFBR 2）的诱导型表达。在第一种通用筛选方法中，将通过凝集素-FACS分析在靶基因熟练（ACVR 2+，TGFBR 2+）和缺陷（ACVR 2-，TGFBR 2-）MSI结直肠癌细胞系中以及通过凝集素组织化学在原发性MSI肿瘤中使用广泛的一组不同凝集素来确定差异糖基化谱。基于这些结果，将通过凝集素亲和层析、2D凝胶电泳和质谱鉴定引起特异性变化的糖蛋白。从所得的差异糖基化蛋白质组中，将详细表征两种候选物（一种ACVR 2依赖性和一种TGFBR 2依赖性）关于改变的糖表位结构。最后，我们试图通过比较MSI和MSS肿瘤细胞中鉴定的糖肽的丰度来证明MSI肿瘤特异性。从本研究中获得的结果具有重要的临床意义，因为它们将定义并提供与已知MSI肿瘤特异性移码肽抗原互补的MSI肿瘤特异性糖蛋白表位的新来源，从而能够开发MSI肿瘤的新诊断和治疗策略。

Microsatellite unstable (MSI) tumors exhibit a variety of histoclinicopathological features including mucinous histology and improved prognosis when compared to their microsatellite stable (MSS) counterparts. Biallelic mutational inactivation of some MSI target genes is frequently observed and is generally believed to drive MSI tumorigenesis. Focussing on the pathogenetic mechanisms of these MSI tumors we recently uncovered a potential correlation between functional inactivation of specific MSI target genes and cell surface glycosylation pattern. The current proposal aims to explore MSI target gene-dependent alterations of cell surface glycosylation signatures in MSI colorectal cancer cell lines engineered to allow inducible expression of two of the most frequently mutated MSI target genes (ACVR2, TGFBR2). In a first general screening approach differential glycosylation profiles will be determined by Lectin-FACS analysis in target gene proficient (ACVR2+, TGFBR2+) and deficient (ACVR2-, TGFBR2-) MSI colorectal cancer cell lines and by lectin-histochemistry in primary MSI tumors, using a broad panel of different lectins. Based on these results the glycoproteins underlying the specific changes will be identified by lectin affinity chromatography, 2D-gel electrophoresis and mass spectrometry. From the resulting set of differentially glycosylated proteins two candidates (one ACVR2-dependent and one TGFBR2-dependent) will be characterized in detail with regard to the altered glycoepitope structures. Finally, we seek to demonstrate the MSI-tumor-specificity by comparing the abundance of the identified glycopeptides in MSI and MSS tumor cells. The results obtained from this study are of major clinical relevance because they will define and provide a novel source of MSI tumor-specific glycoprotein epitopes complementary to the known MSI-tumor specific frameshift peptide antigens enabling the development of new diagnostic and therapeutic strategies for MSI tumors.