PO-159 Deciphering the function of endogenous E-cadherin glycosylation during ovarian cancer spread

The transmembrane glycoprotein E-cadherin plays a pivotal role in cancer metastasis and is required for cell-cell adhesion. Our recent data on epithelial ovarian cancer demonstrated that E-cadherin-mediated cell-cell adhesion depends on the orchestra of specific glycosphingolipids (globosides) on the cell surface. In order to understand the role of endogenous glycosylation of E-cadherin in more detail, we aimed to C-terminally tag E-cadherin (encoded by CDH1) with haemagglutinin (HA) using the CRISPR-Cas9 system. This allows tracing E-cadherin-specific glycosylation in various cell culture models and tumour xenografts. Material and methods Single-guided RNAs targeting downstream of the CDH1 translation stop site were designed, as well as a single strand oligonucleotide consisting of a BamHI restriction site, HA tag, a translation STOP, and a matching DNA sequence up- and downstream of CDH1 STOP. Our strategy was verified in HEK293T cells by genotyping. Next, we selected ovarian cancer cells BG1 (high E-cadherin expression among various cell lines tested) for knocking in HA characterised by genotyping PCR, Western blot, and immunofluorescence analysis. Immunoprecipitated E-cadherinHA was in-gel digested with Chymotrypsin B following HILIC enrichment. The resulting peptides were separated through a reverse phase column and analysed in an Orbitrap Fusion high-resolution mass spectrometer. Results and discussions Results obtained after clonal selection indicate successful Cas9-activity at the expected genomic locus and knock-in of the HA tag via homologous recombination. Moreover, we immunoprecipitated endogenous as well as exogenous (lentivirally transduced and constitutively expressed) E-cadherin via HA. We could demonstrate that E-cadherin is glycosylated with neuraminic acid using Sambucus nigra agglutinin. Preliminary glycopeptide analysis of E-cadherin using a novel EtHCD fragmentation mass spectrometry workflow revealed cellular growth condition dependent glycosylation. Conclusion Taken together, we have inserted an HA tag in the CDH1 locus following advanced glycopeptide analysis of endogenous E-cadherin. We are currently tracing E-cadherin glycosylation during ovarian cancer cell dissemination in vitro and in vivo in order to further elucidate the role of protein glycosylation during cancer metastasis.