Genome‐wide Screening Uncovers Transforming Growth Factor‐β3 as Regulator of Glycosaminoglycan Assembly

Abstract

Heparan sulfate (HS) and chondroitin sulfate (CS) are glycosaminoglycans covalently linked to proteoglycans (PG) present on all cell surfaces and in the extracellular matrix. They play a complex role in carcinogenesis as they are important for extracellular matrix remodeling as well as cell‐cell and cell‐matrix interactions. These processes are altered during malignancy, when tumor cells invade and systemically spread in different tissues. We have developed a genome wide screening method using CRISPR‐Cas9 gene targeting to identify novel genes involved in glycosaminoglycan metabolism. The approach utilizes agents that bind specifically to cell surface HS. One of the top hits was transforming growth factor‐β3 (TGF‐β3), a growth factor that controls multiple genes with tumor‐suppressing and tumor‐promoting effects. Cell surface levels of PG were evaluated in A375 melanoma cells after treatment with soluble TGF‐β1 or TGF‐β3 in the presence or absence of a TGF‐β type I receptor inhibitor (SB‐431542) using specific agents that bind to glycosaminoglycans, including mAbs 10E4 or CS56, guanidinylated neomycin and fibroblast growth factor‐2 (FGF2). Both TGF‐β isoforms stimulated cell surface HSPG and CSPG expression, that was blocked by SB‐431542. Inactivation of TGF‐β3 expression by CRISPR/Cas9 had the opposite effect. These findings show that TGF‐β3 is a newly identified regulatory factor of PG assembly in melanoma cells and that tonic expression of TGF‐β3 regulates PG production in a cell autonomous manner. Further exploration how TGF‐β affects disorders associated with altered PG formation in cancer might lead to innovative solutions to alter tumor cell growth and metastasis.Support or Funding InformationWe acknowledge financial support by the DFG (research fellowship, project number 420160411) and the NIH (fellowship award, K12HL141956).