Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion.

BACKGROUND: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. RESULTS AND METHODS: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal-epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT-PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. CONCLUSION: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.

Severely reduced female fertility in CD9-deficient mice.

CD9 is a widely expressed cell surface molecule that belongs to the tetraspanin superfamily of proteins. The tetraspanins CD9, KAI-1/CD82, and CD63 are involved in metastasis suppression, an effect that may be related to their association with beta1 integrins. Knockout mice lacking CD9 were created to evaluate the physiological importance of CD9. CD9-/- females displayed a severe reduction of fertility. Oocytes were ovulated but were not successfully fertilized because sperm did not fuse with the oocytes from CD9-/- females. Thus, CD9 appears to be essential for sperm-egg fusion, a process involving the CD9-associated integrin alpha6beta1.

Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.

We used a proteomic approach to identify proteins that commonly induce an antibody response in lung cancer. Sera from 64 newly diagnosed patients with lung cancer, 99 patients with other types of cancer, and 71 noncancer controls were analyzed for antibody-based reactivity against lung adenocarcinoma proteins resolved by two-dimensional PAGE. Unlike controls, autoantibodies against a protein identified by mass spectrometry as protein gene product 9.5 (PGP 9.5) were detected in sera from 9 of 64 patients with lung cancer. Circulating PGP 9.5 antigen was detected in sera from two additional patients with lung cancer, without detectable PGP 9.5 autoantibodies. PGP 9.5 is a neurospecific polypeptide previously proposed as a marker for non-small cell lung cancer, based on its expression in tumor tissue. Using A549 lung adenocarcinoma cell line, we have demonstrated that PGP 9.5 was present at the cell surface, as well as secreted. Thus, the findings of PGP 9.5 antigen and/or antibodies in serum of patients with lung cancer suggest that PGP 9.5 may have utility in lung cancer screening and diagnosis.

Transcriptional regulation of the human CD9 gene: characterization of the 5'-flanking region.

The CD9 antigen, initially discovered on B lineage leukemic cells, belongs to the tetraspan superfamily of surface molecules. If no precise function has been assigned to any of these molecules, there are some indications that they could be involved in cell adhesion and cell migration, as well as malignant progression. The CD9 antigen is associated with surface proteins such as VLA integrins or HB-EGF precursor. Transfection of CD9 in melanoma cells reduces tumor growth and metastasis. The heterogenous distribution of the CD9 antigen suggests a complex regulation of its expression. We have previously characterized the CD9 gene and shown that transcription could be initiated at several sites in the TATA-less 5'-flanking region. We show here, using as a model two human leukemic cell lines with erythromegakaryocytic potential, HEL and K562, that the [-205, -154] region supports a promoter activity when cloned ahead of a CAT reporter gene. Mutagenesis analysis suggested the presence of a positive element located within the [-170, -154] region. Gel shift experiments using HEL extracts were compatible with the binding of the transcriptional factor Sp1 to the [-237, -205] region and indicated that a non-identified protein binds to the 3' end of the [-205, -154] region.

Upregulation of CD9 expression during TPA treatment of K562 cells.

The CD9 antigen, a major platelet glycoprotein, is a member of the tetraspan superfamily. We show that treatment of K562 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces megakaryocytic differentiation, leads to a seven-fold increase in CD9 expression, which becomes associated with the integrin beta1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of the megakaryocytic-specific marker GPIIb (CD41) as well as integrins beta3 (GPIIIa/CD61), alpha v (CD51) and VLA-2 (CD49b). Both GPIIb/IIIa expression and CD9 upregulation are dependent on protein kinase C (PKC) activation since they are blocked by the specific inhibitor GF109203X. Steady-state levels of CD9 and GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to accumulate during TPA treatment. However, the increase of the CD9 mRNA was detected much earlier than the increase of GPIIb mRNA (1-2 h vs 24-48 h). Using different constructs of the 5'-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregulation in the megakaryocytic lineage could occur at early stages of differentiation.

Proteomics-based identification of RS/DJ-1 as a novel circulating tumor antigen in breast cancer.

PURPOSE: We used a proteomics-based approach to identify tumor proteins that elicit a humoral response in breast carcinoma and that may occur as circulating antigens. EXPERIMENTAL DESIGN: The breast cell line SUM-44 was used as a source of tumor cell proteins for two-dimensional PAGE (2-D PAGE) and for Western blot analysis in which individual sera were analyzed for primary antibodies. RESULTS: Sera from 30 newly diagnosed patients with breast cancer were screened for IgG antibodies to tumor cell proteins. Sera from 116 patients with other cancers and from 25 healthy subjects served as controls. Restricted reactivity against a set of three proteins, identified by mass spectrometry as isoforms of a novel oncogenic protein that regulates RNA-protein interaction (designated RS/DJ-1), was observed in four patients with breast cancer, but not in healthy subjects. The identity was further confirmed by Western blotting with specific antibodies. RS/DJ-1 was found to be secreted in the breast cell line SUM-44, which led us to determine whether RS/DJ-1 was found in circulation in breast cancer. Interestingly, unlike in controls, RS/DJ-1 was readily detectable in sera from 37% of newly diagnosed patients with breast cancer. CONCLUSION: The presence of autoantibodies and/or circulating RS/DJ-1 protein in sera from patients with breast cancer may have clinical utility.

CD9 gene deficiency does not affect smooth muscle cell migration and neointima formation after vascular injury in mice.

The hypothesis that CD9, a member of the tetraspanin family, plays a role in smooth muscle cell (SMC) migration was tested with the use of a vascular injury model in wild-type (CD9+/+) and CD9-deficient (CD9-/-) mice. Neointima formation 3 weeks after electric injury of the femoral artery was not significantly different in CD9+/+ and CD9-/- mice (area of 0.019 +/- 0.0034 mm2 versus 0.013 +/- 0.0036 mm2; mean +/- SEM, n = 6). The medial areas were also comparable, resulting in intima/media ratio's of 1.3 +/- 0.15 and 0.90 +/- 0.22, respectively. Nuclear cell counts in cross-sectional areas of the injured region were comparable in media (33 +/- 5 versus 27 +/- 2) and neointima (135 +/- 16 versus 97 +/- 17) of CD9+/+ and CD9-/- arteries. Immunocytochemical analysis revealed expression of CD9 in the endothelium, by SMC in the media and by some fibroblasts in the adventitia of non-injured femoral arteries. Three weeks after injury, there appeared to be a gradient of increased CD9 expression from the adventitia to the neointima, in which SMC are abundantly present. Immunogold labeling and electron microscopy with non-injured femoral arteries of CD9+/+ mice confirmed the presence of CD9 at the surface of adventitial fibroblasts and in SMC or pericytes, as well as in the endothelium. Thus, in this model CD9 is highly expressed by migrating SMC, but deficiency of CD9 does not affect SMC migration or neointima formation after perivascular injury.

Contribution of proteomics to tumor immunology.

In the postgenome era, the global analysis of gene and protein expression is allowed at RNA and protein level by microarrays and proteomics. The application of these complementary approaches provides new opportunities for tumor immunology investigation. Indeed, applied to the study at the molecular level of the differentiation and maturation of dendritic cells which are the most potent antigen presenting cells, microarray analysis has identified important changes in a large number of genes, whereas the proteomic analysis provided information that could not be obtained at the RNA level, such as the separation of different isoforms and the characterization of post-translational modifications. On the other hand, proteomics allows serological screening of tumor antigens. Indeed, two-dimensional (2-D) polyacrylamide gel electrophoresis allows simultaneously separation several thousand individual proteins from tumor tissue or tumor cell lines. Proteins eliciting humoral response in cancer are identified by 2-D Western blot using cancer patient sera, followed by mass spectrometry analysis and database search. Applied to different types of cancer, the proteome based approach has allowed us to define several tumor antigens. The common occurrence of autoantibodies to certain of these proteins in different cancers may be useful in cancer screening and diagnosis as well as for immunotherapy.

CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins.

CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.

CD9, but not other tetraspans, associates with the beta1 integrin precursor.

The molecules of the tetraspan superfamily have two unequal extracellular domains separated by four transmembrane (TM) domains. These molecules are associated on the cell surface with each other and with other partner molecules, in particular beta1 integrins. We now show that CD9 associates with the precursor of the beta1 integrin (pre beta1). This association is detected as early as 15 min after metabolic labeling, and the use of Brefeldin A demonstrates that it does not require Golgi modifications of either CD9 or integrin beta1. The specificity of this interaction is demonstrated by the fact that other tetraspans, CD63, CD81, and CD82, do not associate with pre beta1, and CD9 does not associate with immature human histocompatibility leukocyte antigen class I. In order to localize the region of CD9 responsible for the association with the beta1 integrin, we have generated two reciprocal chimeric CD9/CD82 molecules with the junction localized just after the third TM region. The large extracellular loop of CD9 or the fourth TM domain, or both, appear to be sufficient to mediate an association with the mature integrin with a high efficiency, compared to CD82. By contrast, association with pre beta1 requires at least two regions of the molecule. Mutation of CD9 at the consensus site of the tetraspan superfamily, localized between the second and the third TM domain, did not impair the co-precipitation of pre beta1. Finally, because pre beta1 associates with calnexin, we have investigated a possible association of CD9 with this chaperone molecule. CD9 associates with calnexin independently of its association with the beta1 integrin, suggesting that calnexin could be involved in the processing of CD9.

CD9 antigen is an accessory subunit of the VLA integrin complexes.

The CD9 antigen is a cell surface glycoprotein of unknown function which belongs to the tetraspans family. We demonstrate here, by precipitation, Western blotting and co-capping experiments, that this molecule is associated with a large fraction of beta 1 integrins in two cell lines, the pre-B cell line NALM-6 and the megakaryocytic cell line HEL. In HEL cells, CD9 antigen is only associated with VLA-4. In contrast, in NALM-6 cells, CD9 antigen is associated with both VLA-4 and VLA-5. On the other hand, only the beta 1 chain is co-precipitated with the CD9 antigen in transfected L cells. These data show that the CD9 antigen is associated with the beta 1 chain rather than with a particular integrin. CD9 monoclonal antibodies (mAb) did not modify the binding of HEL and NALM-6 cells to fibronectin, laminin or collagen. The association of CD9 antigen to VLA integrins is strengthened by the fact that both CD9 and anti-VLA mAb induce aggregation of the two cell lines and inhibit their migration in Transwell chambers. Because the aggregating effect, but not the inhibition of migration, is observed in CEM or CD9-transfected CEM cells, these two effects are likely to be mediated by different mechanisms.

The major CD9 and CD81 molecular partner. Identification and characterization of the complexes.

By associating with specific partner molecules and with each other, the tetraspanins are thought to assemble multimolecular complexes that may be especially relevant with respect to metastasis. We have previously identified a 135-kDa molecule (CD9P-1) as a major molecular partner of CD9 in cancer cell lines. This molecule was identified, after immunoaffinity purification and mass spectrometry analysis, as the protein encoded by the KIAA1436 gene and the human ortholog of a rat protein known as FPRP. Cross-linking experiments detected a complex of the size of CD9 plus CD9P-1, showing that these glycoproteins directly associate with each other, probably in the absence of any other molecule. The use of chimeric CD9/CD82 molecules revealed the role of the second half of CD9, comprising the large extracellular loop and the fourth transmembrane domain. CD9P-1 was also shown to form separate complexes with CD81 and with an unidentified 175-kDa molecule. It also associated with other tetraspanins under conditions maintaining tetraspanin/tetraspanin interactions. The identification of a protein strongly linked to the tetraspanin web and the production of a specific monoclonal antibody will help to further characterize the role of this "web" under physiological and pathological conditions.

Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions.

The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151.

Functional analysis of four tetraspans, CD9, CD53, CD81, and CD82, suggests a common role in costimulation, cell adhesion, and migration: only CD9 upregulates HB-EGF activity.

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics.

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.