Tetraspanin protein contributions to cancer

Among the 33 human tetraspanin proteins, CD151, CD9 and Tspan12 play particularly important roles in cancer. Tetraspanin CD151, in partnership with integrins α6β1 and α6β4, modulates tumour cell growth, invasion, migration, metastasis, signalling and drug sensitivity. Tetraspanin CD9 has suppressor functions in multiple tumour cell types. Major CD9 partner proteins, such as EWI-2 and EWI-F, may modulate these tumour-suppressor functions. Tetraspanin Tspan12 mutations are linked to a human disease called familial exudative vitreoretinopathy. In addition, as a regulator of the metalloprotease ADAM10 (a disintegrin and metalloprotease 10) maturation and function, Tspan12 probably contributes to the pro-tumorigenic functions of ADAM10.     

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3β1 versus α6β4 integrin

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.     

Activation-induced internalization differs for the tetraspanins CD9 and Tspan8: Impact on tumor cell motility

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8-CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8-CD9-CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8-INS2-CD49d-clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.     

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.     

Distorted leukocyte migration,angiogenesis, wound repair and metastasis in Tspan8 and Tspan8/CD151 double knockout mice indicate complementary activities of Tspan8 and CD51

The tetraspanin Tspan8 supports via associated integrins and proteases tumor progression and angiogenesis. To shed light on its activities in non-transformed cells, we generated a Tspan8 knockout (ko) mouse, comparing leukocyte migration, angiogenesis, wound healing and tumor growth with wild type, CD151ko and Tspan8/CD151ko (dbko) mice. CD151ko mice were included as CD151 activities resemble that of Tspan8, and dbko mice to exclude mutual substitution. Tspan8ko and dbko mice show no pathological phenotype. However, delayed type hypersensitivity reactions are mitigated in Tspan8ko mice, angiogenesis is severely impaired in Tspan8ko, CD151ko and dbko mice, with Tspan8 mostly affecting lymphangiogenesis. Distinct contributions of CD151 and Tspan8 to skin wound healing rely on preferentially CD151 anchoring basal keratinocytes and Tspan8 promoting motility. Proliferation of wounded skin keratinocytes is not affected. Metastasis formation of a melanoma and a Tspan8-expressing pancreatic cancer line was impaired in Tspan8ko and dbko mice, pointing towards a contribution of host Tspan8 to tumor progression. In line with the importance of tetraspanins in exosome-mediated intercellular communication, defects became mitigated by Tspan8/CD151-competent serum exosomes, which offers a most promising therapeutic option for chronic wounds and arteriosclerosis.     

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.     

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane “partner” proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.     

Tetraspanins in the humoral immune response

The tetraspanins represent a large superfamily of four-transmembrane proteins that are expressed on all nucleated cells. Tetraspanins play a prominent role in the organization of the plasma membrane by co-ordinating the spatial localization of transmembrane proteins and signalling molecules into 'tetraspanin microdomains'. In immune cells, tetraspanins interact with key leucocyte receptors [including MHC molecules, integrins, CD4/CD8 and the BCR (B-cell receptor) complex] and as such can modulate leucocyte receptor activation and downstream signalling pathways. There is now ample evidence that tetraspanins on B-lymphocytes are important in controlling antibody production. The tetraspanin CD81 interacts with the BCR complex and is critical for CD19 expression and IgG production, whereas the tetraspanin CD37 inhibits IgA production and is important for IgG production. By contrast, the tetraspanins CD9, Tssc6 and CD151 appear dispensable for humoral immune responses. Thus individual tetraspanin family members have specific functions in B-cell biology, which is evidenced by recent studies in tetraspanin-deficient mice and humans. The present review focuses on tetraspanins expressed by B-lymphocytes and discusses novel insights into the function of tetraspanins in the humoral immune response.     

Tetraspanins interweave EV secretion,endosomal network dynamics and cellular metabolism

Tetraspanin proteins organize membrane nanodomains related to cell adhesion and migration. An essential feature conserved along the superfamily is their cone-shaped tertiary structure, which allows tetraspanins to be enriched in highly curved membrane structures. Their conical shape, together with their ability to associate to transmembrane receptors and to bind to cystoskeletal and signaling scaffolds, are key in their ability to regulate endosomal network dynamics and Extracellular Vesicle biogenesis and cargo selection. Recent evidence suggests that tetraspanins have a relevant impact in mitochondria turnover and regulation of cellular metabolism. In this review we highlight those reports that point to tetraspanins as key regulators in the communication between the endosomal network, EVs and the cellular metabolism.     

Multiple levels of interactions within the tetraspanin web

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31.

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP-1 and SHP-2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP-1 and SHP-2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC-gamma1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP-1, SHP-2, SHIP and PLC-gamma1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate-stimulated THP-1 cells. These results suggest that overlapping immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based activation motif-like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP-1, SHP-2, SHIP and PLC-gamma1.     

The CD9/CD81 Tetraspanin Complex and Tetraspanin CD151 Regulate α3β1 Integrin-Dependent Tumor Cell Behaviors by Overlapping but Distinct Mechanisms

Integrin α3β1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3β1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3β1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3β1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3β1 association with other tetraspanins and impairs α3β1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3β1 functions is unclear. To begin to clarify which aspects of α3β1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3β1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3β1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3β1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3β1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3β1 integrin function. Importantly, some of CD9/CD81''s α3β1 regulatory functions may not require CD9/CD81 to be physically linked to α3β1 by CD151.     

Role of tetraspanin CD151-α3/α6 integrin complex: Implication in angiogenesis CD151-integrin complex in angiogenesis

Tetraspanin CD151 mainly associates with laminin-binding integrins and forms CD151-integrin complex. We previously reported that CD151 could be a potential target for angiogenesis, but the mechanisms involved are still unclear. This study investigated the role of CD151-integrin complex in angiogenesis and the signaling mechanisms involved. Here we showed that CD151 and CD151-AAA mutant were both well expressed at the protein level. CD151 gene transfer promoted angiogenesis and improved skin temperature of the lateral ischemic hindlimb, whereas CD151-AAA mutant abrogated the increase in capillary density and skin temperature. Further, CD151-AAA mutant failed to activate the FAK, ERK, PI3K/Akt/eNOS, and Rac1/Cdc42 signaling pathways. Moreover, CD151-AAA mutant was unavailable to promote bovine aortic endothelial cells (BAECs) proliferation and migration, in contrast to the effects of CD151. The results suggested that formation of CD151-integrin complex was likely to be a prerequisite for CD151-induced angiogenesis and signaling pathways.     

Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site

Integrin α9β1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9β1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9β1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9β1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9β1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.     

Dances with leukocytes: how tetraspanin-enriched microdomains assemble to form endothelial adhesive platforms

Rather than just providing an unstructured adhesive surface for leukocytes, cytokine-activated endothelial cells assemble preexisting tetraspanin-enriched microdomains to form endothelial adhesive platforms (EAPs) and endothelial docking structures. In this issue of the Journal of Cell Biology, Barreiro et al. (Barreiro, O., M. Zamai, M. Yáñez-Mó, E. Tejera, P. López-Romero, P.N. Monk, E. Gratton, V.R. Caiolfa, and F. Sánchez-Madrid. 2008. J. Cell Biol. 183:527–542) show how the immunoglobulin superfamily adhesion molecules intercellular adhesion molecule (ICAM)–1 and vascular cell adhesion molecule (VCAM)–1 form nanoclusters with the tetraspanins CD9 and CD151 in a physiologically relevant system. Furthermore, convincing biochemical data suggest that these structures are distinct from lipid rafts.