EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins

EWI-2 and EWI-F, two members of a novel subfamily of Ig proteins, are direct partners of tetraspanins CD9 (Tspan29) and CD81 (Tspan28). These EWI proteins contain a stretch of basic charged amino acids in their cytoplasmic domains that may act as binding sites for actin-linking ezrin-radixin-moesin (ERM) proteins. Confocal microscopy analysis revealed that EWI-2 and EWI-F colocalized with ERM proteins at microspikes and microvilli of adherent cells and at the cellular uropod in polarized migrating leukocytes. Immunoprecipitation studies showed the association of EWI-2 and EWI-F with ERM proteins in vivo. Moreover, pulldown experiments and protein-protein binding assays with glutathione S-transferase fusion proteins containing the cytoplasmic domains of EWI proteins corroborated the strong and direct interaction between ERMs and these proteins. The active role of ERMs was further confirmed by double transfections with the N-terminal domain of moesin, which acts as a dominant negative form of ERMs, and was able to delocalize EWIs from the uropod of polarized leukocytes. In addition, direct association of EWI partner CD81 C-terminal domain with ERMs was also demonstrated. Functionally, silencing of endogenous EWI-2 expression by short interfering RNA in lymphoid CEM cells augmented cell migration, cellular polarity, and increased phosphorylation of ERMs. Hence, EWI proteins, through their direct interaction with ERM proteins, act as linkers to connect tetraspanin-associated microdomains to actin cytoskeleton regulating cell motility and polarity.     

Antimetastatic activity of pinosylvin, a natural stilbenoid, is associated with the suppression of matrix metalloproteinases

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.     

Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

Contrasting effects of EWI proteins, integrins, and protein palmitoylation on cell surface CD9 organization

CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of alpha3beta1 and alpha6beta4 integrins and by palmitoylation of the CD9 and beta4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression.     

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

Background

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Experimental

Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.

Conclusion

This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.     

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.     

Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis

The traditional treatments for fibrosarcoma have limited efficacy. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored. Accumulating evidence indicates that programmed cell death (PCD) is closely related to anticancer therapy. Many studies have shown that tumor cells treated with anticancer drugs experience the induction of type I PCD, apoptosis, and type II PCD, autophagy. In the present study, we investigated the anticancer effects of ionizing radiation (IR) combined with arsenic trioxide (ATO) in human fibrosarcoma cells in vitro and in xenograft tumors in SCID mice in vivo. We found that IR increased the population of HT1080 cells in the G₂/M phase in a time-dependent manner within 9 h. IR treatment combined with ATO at this time point induced a significantly prolonged G₂/M arrest and consequently enhanced cell death. Furthermore, damage of mitochondria membrane potential could be involved in the underlying mechanisms. The enhanced cytotoxic effect of combined treatment occurred due to the increased induction of more autophagy and apoptosis through the inhibition of Akt and the activation of ERK1/2 signaling pathways in HT1080 cells. The combined treatment of HT1080 cells pretreated with Z-VAD or 3-MA resulted in a significant reduction in AO-positive cells, apoptotic cells and cytotoxicity. In in vivo studies, the combination of IR and ATO significantly reduced the tumor volume in SCID mice that had received a subcutaneous injection of HT1080 cells. The data suggest that a combination of IR and ATO could be a new potential therapeutic strategy for the treatment of fibrosarcoma.     

Interplay between steroid receptors and neoplastic progression in sarcoma tumors

Steroid hormones are expressed at low levels in mesenchymal cells and are highly expressed in soft tissue sarcoma. In human soft tissue fibrosarcoma cell line (HT-1080), the epidermal growth factor (EGF) stimulates the express of matrix metal (MMPs) expression through a Src-dependent mechanism. In human fibrosarcomas, increased expression of MMPs correlates with the metastatic progression. Our recent data in human breast cancer cell line MCF-7, demonstrates that EGF stimulates estradiol receptor (ER) phosphorylation on tyrosine at position 537 thereby promoting the association of a complex among EGF receptor (EGFR), androgen receptor (AR), ER, and Src that activates EGF-dependent signaling pathway. In the present study, we demonstrate that, in HT-1080 cells, the Src kinase activity is involved in EGFR phosphorylation and this activity is regulated by an interplay between Src, steroid receptors, and EGFR. In these cells, estradiol (E(2) )/ER and synthetic androgen (R1881)/AR trans-activate EGFR leading to the downstream signaling and to ERK activation. Indeed, the association between ER/AR and EGFR enhances metastatic progression of fibrosarcoma tumors. A population pilot study performed on 16 patients with soft tissue neoplasias highlights that MMPs expression correlates with progression of anaplastic sarcoma as well as overexpression of EGFR. These findings suggest that there is a crosstalk among AR, ER, and EGFR that lead to src activation also in fibrosarcoma cells.     

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.     

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly to promote cell motility

Fibronectin (FN) matrix assembly is an essential process in normal vertebrate development, which is frequently lost in tumor cells. Here we show that membrane-type 1 matrix metalloproteinase (MT1-MMP) regulates FN matrix assembly. MT1-MMP knockdown induced FN assembly in breast carcinoma cells. Ectopic expression of MT1-MMP reduced specifically the assembled FN matrix level without affecting whole FN production in fibroblasts. Treatment of fibrosarcoma HT1080 cells with dexamethasone (DEX) enhanced FN synthesis, resulting in short fibrils but not dense matrix formation. Combined treatment of DEX and MT1-MMP inhibitor accelerated FN matrix assembly, which mediated cellular adhesion and reduced cell migration and invasion. These results indicate that MT1-MMP stimulates cell migration and invasion by negatively regulating FN assembly.     

Superoxide plays critical roles in electrotaxis of fibrosarcoma cells via activation of ERK and reorganization of the cytoskeleton

Direct-current electrical field (DCEF) induces directional migration in many cell types by activating intracellular signaling pathways. However, the mechanisms coupling the extracellular electric stimulation to the intracellular signals remain largely unknown. In this study, we show that DCEF directs migration of HT-1080 fibrosarcoma cells to the cathode, stimulates generation of hydrogen peroxide and superoxide through the activation of NADPH oxidase, induces anode-facing cytoskeleton polarization, and activates ERK signaling. Subsequent studies demonstrate that the electrotaxis of HT-1080 fibrosarcoma cells is abolished by NADPH oxidase inhibitor and overexpression of manganese superoxide dismutase (MnSOD), an enzyme that hydrolyzes superoxide. In contrast, overexpression of catalases, which hydrolyze hydrogen peroxide, does not affect electrotaxis. MnSOD overexpression also eliminates cytoskeleton polarization as well as the activation of AKT, ERKs, and p38. In contrast, under catalase overexpression, the cytoskeleton still polarizes and p38 activation is affected. Finally, we show that inhibition of ERK activation also abolishes DCEF-induced directional migration and cytoskeleton polarization. Collectively, our results indicate that superoxide plays critical roles in DCEF-induced directional migration of fibrosarcoma cells, possibly by regulating the activation of ERKs. This study provides novel insights into the current understanding of DCEF-mediated cancer cell directional migration and metastasis.     

Glucocorticoid receptor-induced down-regulation of MMP-9 by ginseng components, PD and PT contributes to inhibition of the invasive capacity of HT1080 human fibrosarcoma cells

We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HT1080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP-9 by PD and PT affects the invasive capacity of HT1080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HT1080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT. These results suggest that GR-induced down-regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HT1080 cells.     

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.     

Continuous requirement for pp60-Src and phospho-paxillin during fibronectin matrix assembly by transformed cells

Fibronectin (FN) matrix assembly is an integrin-mediated process that is regulated by both the extracellular environment and intracellular signaling pathways. The activity of Src-family kinases is important for initiation of FN assembly by normal fibroblasts. Here we report that in HT1080 fibrosarcoma cells, Src kinase activity is required not only for the assembly of FN matrix but also for the maintenance of FN matrix fibrils at the cell surface. Dexamethasone-induced FN fibril formation by these cells was completely blocked for at least 24 h when Src-family kinase activity was inhibited by either PP1 or SU6656. Inhibition of Src after significant matrix had already been assembled, resulted in an increased rate of loss of detergent-insoluble FN. Binding of activation-dependent integrin antibodies reveals a role for Src in maintaining integrin activity. The requirement for Src kinase activity appears to depend, in part, on phosphorylation of paxillin at tyrosine 118 (Y118). Phospho-paxillin co-localized with FN fibrils, and overexpression of GFP-paxillin but not of GFP-paxillinY118F enhanced cell-mediated assembly of FN. Our results indicate that Src maintains FN matrix at the cell surface through its effect on integrin activity and paxillin phosphorylation.     

Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.