Transformierender Wachstumsfaktor-β3: ein potenzieller neuer Regulator der Zusammensetzung und Funktionalität von Heparansulfat in Krebs
Zusammenfassung der Projektergebnisse
Within the project, genome-wide loss-of-function CRISPR-Cas9 screens in human A375 melanoma cells identified genes, other than those encoding heparan sulfate (HS) biosynthetic enzymes, which are potentially involved in the regulation of cell surface HS. TGFB3 was among the top screening hits. To evaluate how TGFB3 gene expression regulates the transcription/translation of glycosaminoglycan (GAG) biosynthetic enzymes or orchestrates their action to achieve the final GAG composition, TGFB3- and TGFB1-deficient mutant cell lines were generated by CRISPR-Cas9 gene targeting. HS and chondroitin sulfate (CS) analysis by liquid chromatography-mass spectrometry demonstrated reduced amounts of both GAGs on the cell surface of TGFB3-deficient cells, while TGFB1 inactivation only decreased cell surface CS levels. Likewise, the binding of HS- and CS-dependent antibodies to the cell surface was lower after TGFB3 inactivation proving that ligand binding structures present in the GAG chains of HS and CS proteoglycans (PGs) are affected. Supplementation with soluble TGF-β3 reversed these effects. The influence of a dysregulated TGFB3 expression was further studied by evaluating the growth of melanoma cells in vitro and in vivo. Here, TGFB1 depletion fostered the formation of large tumor cell colonies in collagen gels, while in contrast, TGFB3 inactivation reduced the colony number and size. In addition, the tumor growth of TGFB3-deficient melanoma cells in nude mice was significantly decreased compared to wildtype cells. Moreover, small, drug-like agents, which were previously identified in a drug screen, where evaluated regarding their potential to increase the expression of HS in cells with diminished capacity to produce HS. Here, a N-aryl-2-aminothiazole derivative was shown to enhance the binding of HS-dependent ligands to the cell surface as well as to increase the total amount of HS. Overall, this project provided insights into the mechanisms that cells use to regulate their GAG composition. This is of particular relevance since numerous human diseases are associated with alterations in HS formation, such as melanoma and multiple hereditary exostoses (MHE).
Projektbezogene Publikationen (Auswahl)
- Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate. Nat Chem Biol. 2021;17(6):684-692
R.J. Weiss, P.N. Spahn, A.W.T. Chiang, Q. Liu, J. Li, K.M. Hamill, S. Rother, T.M. Clausen, M.A. Hoeksema, B.M. Timm, K. Godula, C.K. Glass, Y. Tor, P.L.S.M. Gordts, N.E. Lewis, J.D. Esko
(Siehe online unter https://doi.org/10.1038/s41589-021-00776-9)
