TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation

SIMPLE SUMMARY: Tumor cells receive signals from the surrounding extracellular matrix that affect their growth and survival. An important component of the extracellular matrix is the large polysaccharide hyaluronan, which binds and activates certain receptors at the cell surface, including CD44. Activation of CD44 initiates several signaling pathways; one of them involves the cleavage of CD44 by proteases, leading to the release of the intracellular domain of CD44, which after translocation to the nucleus affects the transcription of certain genes. In the present report, we elucidate the mechanism by which CD44 is cleaved, and show that this occurs at an increased rate in stem-like tumor cells grown in spheres. We also show that CD44 cleavage promotes the migration of tumor cells. Since the mechanism we have elucidated promotes tumorigenesis, it is possible that inhibition of this pathway may be beneficial in the treatment of tumor patients. ABSTRACT: The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.