Principal Investigator: Yu Yamaguchi
Grant Title: The Novel Cell Surface Hyaluronidase TMEM2 in Tumor Cell Invasion
Abstract
Objectives: Tumor invasion involves the breaching of tissue
barriers by cancer cells. Degradation of extracellular matrix(ECM) is critical for this process, and past studies revealed criticalroles for matrix metalloproteases in tumor invasion. However,research in this field have largely ignored the fact that the tumor
ECM also contains high levels of hyaluronan (HA). The uniquebiophysical and biochemical properties of HA endow this hugepolysaccharide with a large hydrodynamic volume and also enable it to form a matrix that acts as abarrier to invading tumor cells. HA accumulation in tumor stroma also elevates intra-tumoral fluidpressure, which further restricts cell movement. Thus, it is thought that tumor cells have the ability
to degrade HA to facilitate their invasion through HA-rich stromal tissues. The identity of thehyaluronidase responsible for this process, however, has been elusive. We have recently
demonstrated that TMEM2, a transmembrane protein of previously unknown function, is such acell surface hyaluronidase. In this project, we investigate the functional significance of TMEM2 intumor cell adhesion and migration.
Methods: We used in situ HA degradation assays to characterize
the ability of a variety of tumor cells to degrade substrate-bound
HA and a battery of in vitro assays to determine the function ofTMEM2 in tumor cell adhesion and migration on HA-richsubstrate. Expression of TMEM2 in tumor cells was manipulatedby transfection of siRNA and truncated TMEM2 constructs.
Results: We found that a variety of tumor cells exhibit the ability
to eliminate substrate-bound HA in a tightly localized patterncorresponding to the distribution of focal adhesions (FAs) andstress fibers. Immunostaining of these cells for vinculin reveals
that the spots of HA degradation coincide with FAs. Knockdownof TMEM2 almost entirely inhibited in situ HA degradation. On
the other hand, knockdown of other hyaluronidases (HYAL1,HYAL2, and KIAA1199) had little effect on in situ HAdegradation, indicating that TMEM2 is the hyaluronidase
primarily responsible for contact-dependent HA degradation.
mCherry-tagged TMEM2 exhibits overlapping colocalization withboth vinculin-positive puncta and sites of HA removal. TMEM2
depletion attenuates the ability of U2OS cells to attach,
Figure 1. A model for the role of TMEM2 inmigrate, and form FAs on HA-containing substrates.
integrin-mediated cell adhesion and FA
Importantly, TMEM2 directly binds at least two integrins,formation. 1, High levels of HA in the ECM areinhibitory to the direct engagement of integrins
namely a5b1 and aLb2 (LFA-1). This interaction isto their ECM ligands. 2, In the presence ofdemonstrated by cell surface crossTMEM2, HA in the ECM is locally removed,
which generates a microenvironment that islinking/coimmunoprecipitation assays and by direct binding
permissible to the direct integrin–ECM
assays with recombinant integrins and TMEM2. The integrin- engagement. 3, The association between
TMEM2 and integrins promotes the FA
TMEM2 interaction is mediated by interaction between
formation and maturation via further removal of
extracellular domains of respective proteins. Our findings
HA in the vicinity of the integrin–ECMengagement. 4, This in turn facilitates integrin
demonstrate a critical role for TMEM2-mediated HAclustering, integrin-mediated downstream
degradation in the adhesion and migration of cells on HA-rich signaling, and cellular responses.
ECM substrates and provide novel insight into the early phaseof FA formation.