Tumor invasion involves the breaching of tissue barriers by cancer cells. Degradation of the extracellular matrix (ECM) is critical for this process, and past studies revealed critical roles for matrix metalloproteases in tumor invasion. However, research in this field has largely ignored the fact that the tumor ECM also contains high levels of hyaluronan (HA). Notably, HA is highly accumulated in tumor stroma, especially that of high-grade adenocarcinomas characterized by desmoplastic changes1,2). The unique biophysical and biochemical properties of HA endow this huge polysaccharide with a large hydrodynamic volume and also enable it to form a matrix that acts as a barrier to invading tumor cells. HA accumulation in tumor stroma also elevates intra-tumoral fluid pressure, which restricts cell movement. Thus, it is reasonable to assume that tumor cells have the ability to degrade HA in the ECM to facilitate their invasion through stromal tissues. The identity of the hyaluronidase responsible for the degradation of HA in this process, however, has been elusive. Although the HYAL family proteins (e.g., HYAL1, HYAL2) have been extensively studied as the major hyaluronidases in mammalian species3), there has been a debate concerning the subcellular localization and the site of action of these hyaluronidases. While HYAL family proteins can be localized on the cell surface, there is also evidence that they are present and function in endosomes and lysosomes, rather than on the cell surface4,5). Against this backdrop, we sought to identify a novel hyaluronidase(s) that physiologically functions on the cell surface. This effort has resulted in the identification of TMEM2 (HGNC gene name: CEMIP2), a transmembrane protein with hitherto unknown function, as a hyaluronidase with the desired properties. Briefly, TMEM2 is localized on the cell surface as a type II transmembrane protein, degrades large HA into fragments as small as ~5 kDa at around near neutral pH, and TMEM2-transfected cells degrade HA at cell-substrate contact sites6). In this project, we investigated the functional significance of TMEM2 in tumor cell adhesion and migration. The results of this study have been published in Irie et al., 20217).TMEM2 degrades substrate-bound HA at focal adhesions (FAs)Using the in situ HA degradation assay6), we first examined endogenous HA degrading activity in a variety of tumor cell lines (U2OS human osteosarcoma, BT474 human breast ductal carcinoma, DU145 human prostate adenocarcinoma, and TRAMP-C2 mouse adenocarcinoma)7). These cells degrade substrate-coated HA in a pattern that resembles the distribution of focal adhesions (FAs) (Figure 1A). Immunostaining for vinculin, a marker for FAs, demonstrates that TMEM2 shows overlapping colocalization with both FAs and sites of HA removal (Figure 1B). Knockdown of TMEM2 almost entirely inhibited in situ HA degradation, whereas knockdown of other hyaluronidases (HYAL1, HYAL2, and KIAA1199) had little effect (Figure 1C). These results indicate that TMEM2 is the predominant, if not the sole, hyaluronidase responsible for contact-dependent degradation of matrix-associated HA.TMEM2 is required for efficient cell adhesion, migration, and FA formation on HA-rich substratesTo test whether TMEM2 plays a functional role in regulating tumor cell behavior, we examined the effect of TMEM2 knockdown on the adhesion and migration of U2OS cells on mixed substrates consisting of HA and type I collagen (Col1)7). Control U2OS cells adhere equally well to a homogeneous Col1 substrate and a mixed Col1/HA substrate. In contrast, adhesion of TMEM2-depleted U2OS cells to the Col1/HA substrate is greatly impaired, even though adhesion of these cells to the homogeneous Col1 substrate is not affected. TMEM2-depleted U2OS cells also exhibit reduced migration in a wound healing-type migration assay on mixed Col1/HA substrates7). Immunostaining for vinculin reveals wild-type U2OS cells at the forefront of migration undergo robust formation of FAs. In TMEM2-depleted U2OS cells, few sites of HA degradation are observed, and the number of FAs is greatly diminished compared with control U2OS cells7). Taken together, these results indicate that TMEM2 plays a critical role in enabling tumor cells to form FAs on HA-containing substrates.91The novel cell surface hyaluronidase TMEM2in tumor cell invasionYu YamaguchiSanford Burnham Prebys Medical Discovery Institute
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