Proteomic and functional characterization of the growth-suppressing action of galectin-4 in colorectal cancer cells

• Applicant: Professor Dr. Magnus von Knebel Doeberitz, since 1/2022
• Subject Area: Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 448311271

Galectin-4 (gal-4) belongs to a family of structurally related carbohydrate-binding proteins (lectins), that translate glycan-encoded information on the cell surface into effects on cell growth and differentiation. Additionally, galectins exhibit distinct intracellular functions including also glycan-independent activities. As a typical representative of tandem-repeat galectins, gal-4 consists of two distinct carbohydrate recognition domains (CRDs) joined by a peptide linker. Its modular structure is expected to carry out crosslinking of two distinct types of ligands on the cell surface as well as to accomplish distinct intracellular interactions. Gal-4 is highly expressed in normal colon-epithelium, but strikingly downregulated in colon cancer tissues. Our previous work showed the growth-suppressing and differentiation-inducing capability of extracellular gal-4 in colorectal cancer (CRC) cells, thereby proving its cell surface activity.This project aims at a comprehensive understanding of extra- and intracellular actions of galectin-4. For that purpose, the cell-surface-associated as well as the intracellular binding partners have to be identified. In order to analyze the galectin intracellular functions a CRC model cell line with inducible gal-4 expression will be generated. In parallel, the effects of exogenously added gal-4 on CRC cells will be investigated in order to decipher signalling pathways and cellular mechanisms induced by its cell surface binding. Initially, mass spectrometry-based proteomics and phosphoproteomics will be applied to identify gal-4 binding partners, gal-4-dependent cell-surface-associated signaling processes as well as intracellular gal-4-induced proteome and protein phosphorylation changes and its effects on cell physiology. At that, different structural variants that enable conclusions on the relationship between structure and function of the tandem-repeat galectin will also be tested. For further characterization and validation of the observed effects with immunological and biochemical methods, in addition to cell cultures also an animal model (gal-4 knockout mice) and human tissue samples will be applied. The KO mice are already available and the gal-4 knockout is proven by molecular, biochemical and immunohistochemical methods. For evaluation in human tissue samples, we will draw on a large cohort of colorectal carcinomas and adenomas with thorough documentation of basic clinical information (age, gender, tumor localization, tumor stage, histology) and molecular data (e.g. microsatellite instability status, BRAF mutation status, KRAS mutation status). These well-characterized samples are already available as paraffin-embedded tissue blocks. Altogether, the planned project will not only increase the knowledge of the pathobiology of colorectal cancer, but also it will contribute to define cellular functions of galectins in general which represents a highly relevant and current issue in glycobiology.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Dr. Malwina Michalak, until 12/2021