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

Constantinos Kolliopoulos,Paraskevi Heldin,Athanasios Chatzopoulos,Spyros S Skandalis,Carl-Henrik Heldin

doi:10.3390/cancers13051021

Constantinos Kolliopoulos, Paraskevi Heldin + Show 3 more

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https://doi.org/10.3390/cancers13051021

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Journal: Cancers	Publication Date: Mar 1, 2021
Citations: 7	License type: CC BY 4.0

Affiliation: Uppsala University, University of Patras

Abstract

Simple SummaryTumor 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.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.

Highlights

Transforming growth factor β (TGFβ) promoted the cleavage of CD44 and formation of the CD44 TM-intracellular domain (ICD) domain in a time-dependent manner in the lung adenocarcinoma A549 cell line (Figure 1a), as did several other growth factors (Figure S1a)
We investigated the effect of proteasomal (MG132) and lysosomal (chloroquine (CQ)) inhibitors on the accumulation of CD44 cleavage products in untreated and TGFβ-treated cells
We found that TRAF6 is important for the cleavage of CD44 and release of its intracellular domain in mouse embryo fibroblasts (MEFs), as well as in

Summary

Introduction

Hyaluronan, a prominent constituent of the extracellular matrix, is synthesized by three hyaluronan synthase isoforms, i.e., HAS1, HAS2, and HAS3, and is catabolized by hyaluronidases, including HYAL1, HYAL2, and TMEM2. In addition to its structural role in the extracellular matrix, hyaluronan exerts signaling effects via binding to specific cell surface receptors, such as CD44 [1,2,3], which is a transmembrane receptor that mediates signals affecting cell proliferation and migration [4,5]. The human CD44 gene is composed of 19 exons, i.e., 10 “constant exons” encoding the standard CD44 molecule (CD44s) and nine “variable exons”, which in different combinations can be inserted in the membraneproximal stem region through alternative splicing to generate CD44v isoforms [6].

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