Impact of Tumor-Associated Glycosylation on the Multi-Functionality of TIMP-1 (Tissue Inhibitor of Metalloproteinases-1)

• Applicant: Professor Dr. Achim Krüger
• Subject Area: Hematology, Oncology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 454309898

It is necessary to understand the exact role of individual proteins in specific processes of tumor progression and metastasis to define better targets for diagnosis and cancer therapy. Beyond the era of genomics, it is now increasingly appreciated that protein variants resulting from expression from one gene exhibit even further differential functionalities as a consequence of post-translational modifications. Protein N-glycosylation is the most versatile post-translational modification in eukaryotic cells and crucially contributes to the regulation of tumor-promoting cellular processes, such as signal transduction, immuno-modulation or cell-matrix interactions. Tumor-associated changes of the ‘glycome’ of proteins was recently appreciated as crucial ‘Enabling Characteristic’ for the establishment of all hallmarks of cancer. However, the impact of glycosylation on protein function is rather rarely considered in experimental set-ups in mice or in vitro, when recombinant proteins with undefined glycosylation patterns are employed in functional assays. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a secreted multi-functional glycoprotein with two N-glycosylation sites at N30 and N78 involved in tissue homeostasis through its anti-proteolytic activity. In cancer, it is systemically elevated in a biological sex-(male-)specific manner, and creates a hepatic pre-metastatic niche by binding to the tetraspanin CD63 on hepatic stellate cells and their subsequent activation. This cytokine function modulates the function of various immune cells including bone marrow-derived accomplices of pre-metastatic niche formation (neutrophils). In the 1st granting period, we established that three possible glycosylation variants (double-(dgTIMP-1), mono-, and non-glycosylated) can be detected in the blood of healthy donors. The proportion of dgTIMP-1 significantly increases in pancreatic cancer patients. Incremental occupation of glycosylation sites correlated with the shift from the anti-proteolytic to the tumor-promoting cytokine-like activity of TIMP-1 in vitro, resolving the molecular basis for the long-debated paradoxical correlation of TIMP-1 with poor prognosis of virtually all inflammation-associated diseases including cancer. In the 2nd period we aim to investigate the in vivo-impact of circulating recombinant murine TIMP-1 glycosylation variants by sn/scRNA-seq and subsequent construction of gene regulatory networks (GRN) in liver and bone marrow cells from female and male mice. Experimental metastasis assays will show a correlation between the respective GRNs and metastasis efficacy. The broader significance and conceptual advance of this study is the biological sex-specific appreciation of the impact of glycosylation patterns of TIMP-1 on other disease challenges involving the liver and the bone marrow, of which TIMP-1 emerges as biomarker. Furthermore, the insight may be translated to other clinically relevant glycoproteins including interleukins.

• DFG Programme: Research Grants