Vorschau

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Abstract

DNA mismatch repair-deficient colorectal tumors exhibit a high-frequency of microsatellite instability (MSI-H) and accumulate somatic frameshift mutations in genes harboring repetitive DNA sequences. Biallelic frameshift mutations in the A8 coding repeat of the activin receptor type 2 A (ACVR2A or ACVR2) gene occur at high frequency in these tumors thereby abrogating normal receptor and signaling function. Nevertheless, it is not clear if protein deficiency is modifying the glycosylation pattern as well as the whole proteomic constellation of cells, since it has been shown that proteomic and glycomic alterations have emerging significance in cancer cells. Here, we compensated the loss of function by reconstitution of ACVR2 into a MSI-H colon cancer cell line and analyzed its impact on the protein pattern of these cells. As a model system we used the MSI colorectal cancer cell line HCT116-AWE that enables doxycycline-inducible expression of target genes. Applying retroviral genomic targeting and recombination-mediated cassette exchange (RMCE) technology we have generated stable clones that allow dox-regulated expression of a single copy ACVR2 transgene. Fragment analysis, determination of the transgene transcript level, dox-inducible expression of wildtype ACVR2 protein and functional analysis by ligand-stimulated activation of signal transduction and expression of specific target genes confirmed successful gene reconstitution. Upon induction of receptor expression, glyco-gene chip expression analysis of ACVR2-induced versus -uninduced cells revealed ACVR2-dependent upregulation of the glycosyltransferase LNFG, an important regulator of Notch signaling. Moreover, metabolic labeling experiments showed a significant decrease in fucose incorporation and a modest increase in mannosamine uptake, indicating significant glycan alterations of newly synthesized proteins due to ACVR2 re-expression. By applying a Click-it chemistry approach and subsequent mass spectrometry analysis a list of proteins, differentially expressed between ACVR2-deficient and -proficient cells, was identified. These results suggest that ACVR2 signaling can affect glycomic and proteomic modifications that might cause alterations in many cellular processes like growth suppression, cell death, cell adhesion and communication properties or invasion and metastasis of these MSI tumor cells. This study is of major relevance because it may define and provide a novel source of MSI tumor-specific carbohydrate epitope changes in cell surface levels, thereby initiating analysis of glycan function related to MSI. At the same time new tumor markers could be established, which are not only monitors for diagnosis or therapy, but also represent the biological characteristics of cancer cells.