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

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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.