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.     

Contortrostatin,a Homodimeric Disintegrin,Actively Disrupts Focal Adhesion and Cytoskeletal Structure and Inhibits Cell Motility Through a Novel Mechanism

Irttegrins play a major role in the regulation of cell motility. They physically link the extracellular environment to the cytoskeleton and participate in large protein complexes known as focal adhesions. In this report, it is demonstrated that treatment of tumor cells with the homodimeric disintegrin contortrostatin induces integrin-mediatcd tyrosine phosphorylation events and causes severe disruptions in the actin cytoskeleton and disassembly of focal adhesion structures without affecting cellular adhesion to a reconstituted basement membrane. Included in this disruption is the tyrosine phosphorylation and altered subcellular localization of FAK. Through use of transfected 293 cells with specific integrin expression profiles and anti-αvβ3 mAbs, we demonstrate that these events are mediated exclusively by the αvβ3 integrin and are likely the result of contortrostatin-mediated crosslinking of this receptor at the cell surface, since monovalent disintegrins, flavoridin or echistatin do not induce such effects. Further, it is shown that contortrostatin potently inhibits motility in cells expressing the αvβ3 integrin. The results of this study describe a novel integrin-mediated mechanism by which cell motility can be inhibited and suggest an alternative approach to therapeutic intervention for cancer invasion and metastasis.     

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.     

Glycosylation of the laminin receptor (α3β1) regulates its association with tetraspanin CD151: Impact on cell spreading,motility, degradation and invasion of basement membrane by tumor cells

Invasion is the key requirement for cancer metastasis. Expression of β1,6 branched N-oligosaccharides associated with invasiveness, has been shown to promote adhesion to most Extra Cellular Matrix (ECM) and basement membrane (BM) components and haptotactic motility on ECM (fibronectin) but attenuate it on BM (laminin/matrigel) components. To explore the mechanism and to evaluate the significance of these observations in terms of invasion, highly invasive B16BL6 cells were compared with the parent (B16F10) cells or B16BL6 cells in which glycosylation was inhibited. We demonstrate that increased adhesion to matrix components induced secretion of MMP-9, important for invasion. Further, both the subunits of integrin receptors for fibronectin (α5β1) and laminin (α3β1) on B16BL6 cells were shown to carry these oligosaccharides. Although, glycosylation of receptors had no effect on their surface expression, it had same differential effect on cell spreading as haptotactic motility. Absence of correlation between invasiveness and expression of most tetraspanins (major regulators of integrin function) hints at an alternate mechanism. Here we show that glycosylation on α3β1 impedes its association with CD151 and modulates spreading and motility of cells apparently to reach an optimum required for invasion of BM. These studies demonstrate the complex mechanisms used by cancer cells to be invasive.     

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n?=?20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.     

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.     

Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways

The tetraspanin membrane protein CD151 has been suggested to regulate cancer invasion and metastasis by initiating signaling events. The CD151-mediated signaling pathways involved in this regulation remain to be revealed. In this study, we found that stable transfection of CD151 into MelJuSo human melanoma cells lacking CD151 expression significantly increased cell motility, matrix metalloproteinase-9 (MMP-9) expression, and invasiveness. The enhancement of cell motility and MMP-9 expression by CD151 overexpression was abrogated by inhibitors and small interfering RNAs targeted to focal adhesion kinase (FAK), Src, p38 MAPK, and JNK, suggesting an essential role of these signaling components in CD151 signaling pathways. Also, CD151-induced MMP-9 expression was shown to be mediated by c-Jun binding to AP-1 sites in the MMP-9 gene promoter, indicating AP-1 activation by CD151 signaling pathways. Meanwhile, CD151 was found to be associated with alpha(3)beta(1) and alpha(6)beta(1) integrins in MelJuSo cells, and activation of associated integrins was a prerequisite for CD151-stimulated MMP-9 expression and activation of FAK, Src, p38 MAPK, JNK, and c-Jun. Furthermore, CD151 on one cell was shown to bind to neighboring cells expressing CD151, suggesting that CD151 is a homophilic interacting protein. The homophilic interactions of CD151 increased motility and MMP-9 expression of CD151-transfected MelJuSo cells, along with FAK-, Src-, p38 MAPK-, and JNK-mediated activation of c-Jun in an adhesion-dependent manner. Furthermore, C8161 melanoma cells with endogenous CD151 were also shown to respond to homophilic CD151 interactions for the induction of adhesion-dependent activation of FAK, Src, and c-Jun. These results suggest that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through FAK-Src-MAPKs pathways in human melanoma cells, leading to enhanced cell motility and MMP-9 expression.     

Regulation of Endothelial Cell Motility by Complexes of Tetraspan Molecules CD81/TAPA-1 and CD151/PETA-3 with α3β1 Integrin Localized at Endothelial Lateral Junctions

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.     

The adenosine deaminase-binding region is distinct from major anti-CD26 mAb epitopes on the human dipeptidyl peptidase IV(CD26) molecule

CD26 or dipeptidyl peptidase IV (DPP-IV) is a cell surface protease involved in T cell activation. Monoclonal antibodies (mAbs) directed against the CD26 molecule are able to stimulate CD26-expressing T cells. Although many different CD26-specific mAbs exist which are able to provide a triggering signal in T cells, little is known about their specific epitopes on the CD26 molecule. Whereas some mAbs were shown to compete with each other and to inhibit the association of adenosine deaminase (ADA) and human immunodeficiency virus 1 (HIV-1)-derived Tat protein with CD26, other CD26-specific mAbs obviously bind to distinct regions on DPP-IV. In the present study we have generated truncated versions of the human CD26 molecule and expressed them in COS-1 cells to study the binding pattern of a panel of 14 CD26-specific mAbs in confocal microscopy and, thus, correlated the CD26-specific mAbs epitopes with the binding region of ADA. We show that the majority of anti-CD26 mAbs is directed against the glycosylation-rich region of the molecule whereas the ADA-binding site could be located in the cysteine-rich region of DPP-IV. In contrast to binding experiments with purified ADA, which revealed a specific association with CD26 on CD26-positive Jurkat cells, HIV-derived Tat protein did not interact specifically with CD26 on transfected Jurkat cells, nor could Tat binding be competed by anti-CD26-specific mAbs.     

Integrin α-3 ß-1’s central role in breast cancer,melanoma and glioblastoma cell aggregation revealed by antibodies with blocking activity

Breast cancer, melanoma and glioblastoma cells undergo cell-mediated aggregation and aggregate coalescence in a transparent 3D Matrigel environment. Cells from normal tissue and non-tumorigenic cell lines do not exhibit these behaviors. Here, 266 monoclonal antibodies (mAbs) demonstrated to interact with a wide variety of membrane, secreted and matrix proteins, have been screened for their capacity to block these tumorigenic cell-specific behaviors in a 3D environment. Remarkably, only six of the 266 tested mAbs exhibited blocking activity, four targeting integrin ß-1, one targeting integrin α-3 and one targeting CD44. Colocalization of integrins ß-1 and α-3 in fixed cells and in live aggregates suggests that the integrin α-3 ß-1 dimer plays a central role in cancer cell aggregation in the 3D environment provided by Matrigel. Our results suggest that blocking by anti-integrin and anti-CD44 mAbs involves interference in cell-cell interactions.     

CD151 对甲状腺癌细胞迁移及侵袭能力的影响

目的:构建CGTHW-1/pGenesil-1-CD151shRNA稳转细胞系和空载体细胞系CGTHW-1/pGenesil-1,探讨CD151对人甲状腺癌CGTHW-1细胞迁移及侵袭的影响及其作用机制。方法:应用Lipofectamine2000将真核干扰载体pGenesil-1-CD 151 shRNA和空载体pGenesil-1导入甲状腺滤泡癌细胞CGTHW-1,经卡那霉素抗性筛选得到稳定的克隆并扩大培养成细胞系,Western blot检测CD151在CGTHW-1细胞中的表达;划痕实验和Transwell实验分别观察CD151对细胞迁移和侵袭能力的影响。结果:成功建立了CGTHW-1/pGenesil-1-CD151shRNA稳转细胞系和空载体细胞系CGTHW-1/pGenesil-1,Western blot结果显示CD151在CGTHW-1细胞中表达,干扰CD151基因后,CGTHW-1细胞的迁移和侵袭能力明显降低。结论:将CD151基因干扰后可明显抑制甲状腺癌细胞的迁移和侵袭能力。CD151可能是影响甲状腺癌侵袭转移的重要因子之一。     

CD151对甲状腺癌细胞迁移及侵袭能力的影响

目的：构建CGTHW-1／pGenesil．1-CDl51shRNA稳转细胞系和空载体细胞系CGTHW-1／pGenesil-1,探讨CD151对人甲状腺癌CGTHW—1细胞迁移及侵袭的影响及其作用机制。方法：应用Lipofectamine2000将真核干扰载体pGenesil-1-CD151shRNA和空载体pGenesil－1导入甲状腺滤泡癌细胞CGTHW-1,经卡那霉素抗性筛选得到稳定的克隆并扩大培养成细胞系,Westernblot检测CDl51在CGTHW-1细胞中的表达;划痕实验和Transwell实验分别观察CD151对细胞迁移和侵袭能力的影响。结果：成功建立了CGTHw-1／pGenesil-1-CDl51shRNA稳转细胞系和空载体细胞系CGTHW-1／pGenesil—1,Westernblot结果显示CD151在CGTHW-1细胞中表达,干扰CD151基因后,CGTHW-1细胞的迁移和侵袭能力明显降低。结论：将CDl51基因干扰后可明显抑制甲状腺癌细胞的迁移和侵袭能力。CD151可能是影响甲状腺癌侵袭转移的重要因子之-。     

Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.     

Blockade of CD200 in the presence or absence of antibody effector function: implications for anti-CD200 therapy

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.     

miR-9 inhibits the metastatic ability of hepatocellular carcinoma via targeting beta galactoside alpha-2,6-sialyltransferase 1

Glycosylation of cell surface proteins regulates critical cellular functions, including invasion and metastasis in cancer cells. Emerging evidence has shown that microRNAs (miRNAs) are involved in regulating both the glycosylation modifications on cell surface and the progression of cancer. In this study, we investigated the role of miR-9 in α-2,6-linked sialylation and the metastasis of mouse hepatocellular carcinoma (HCC). According to array-based miRNA expression profiling data of HCC cell lines Hepa1–6, Hca-P, and Hca-F with different lymphatic metastatic capacities, reverse correlation was found between miR-9 expression levels and the metastatic potential in these HCC cells. Additionally, β-galactoside α-2,6-sialyltransferase 1 (St6gal1) expression level is associated negatively with miR-9 and positively with metastatic potential. Bioinformatics analysis indicated that miR-9 could target St6gal1, which was verified by luciferase reporter assays. miR-9 overexpression reduced expression of St6gal1, which subsequently suppressed HCC cells metastatic potential. Moreover, upregulation of miR-9 could inhibit integrin-β1/FAK-mediated cell motility and migration signaling in mouse HCC cells. Together, our results suggest that miR-9 could act as a tumor suppressor and regulate mouse HCC cells migration and invasion by inhibiting the α-2,6-linked sialylation. This finding may provide insight into the relationship between abnormal miRNA expression and aberrant cell surface glycosylation during tumor lymphatic metastasis.     

Plant lectin can target receptors containing sialic Acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.     

The HGF/SF-induced phosphorylation of paxillin, matrix adhesion, and invasion of prostate cancer cells were suppressed by NK4, an HGF/SF variant

Hepatocyte growth factor/scatter factor (HGF/SF) plays a crucial role in cancer cell migration, matrix adhesion, invasion, and angiogenesis, via the phosphorylation of the c-met tyrosine kinase. This study examined the ability of NK4, a recently discovered HGF/SF variant, to inhibit the influence of HGF/SF on cell-matrix interaction, paxillin phosphorylation, and invasion of prostate cancer cells. HGF/SF was shown to dramatically enhance tumour cell motility, invasion, cell-matrix adhesion, together with an increase in the degree of paxillin phosphorylation and formation of focal adhesion complexes. However, these HGF/SF-induced effects were suppressed by the presence of NK4. NK4 effectively inhibited the degree of HGF/SF-induced paxillin phosphorylation and matrix adhesion. As a consequence, the matrix invasion of these prostate cancer cells was also suppressed by NK4. In conclusion, this study shows that these HGF/SF-enhanced events, which are critical steps in metastasis, can be inhibited through the addition of NK4, thus warranting further in vivo studies on the implication of NK4 as a potential antimetastasis agent in prostate cancer.     

CD151 regulates HGF-stimulated morphogenesis of human breast cancer cells

We previously demonstrated that CD151 forms a functional complex with c-Met and integrin α3/α6 in human salivary gland cancer cells. In the current study, we investigated the involvement of CD151, c-Met, and integrin α3/α6 in the cellular morphogenesis of human breast cancer cells. Knockdown of CD151, integrin α3, or integrin α6 expression abolished branching morphogenesis. Decreased c-Met expression in these cells led to the formation of rudimentary networks and prevented their conversion. Furthermore, hepatocyte growth factor (HGF) promoted cellular morphogenesis by accelerating network reorganization. Immunoprecipitation revealed a specific association between CD151 and c-Met. The involvement of CD151 and integrin α3/α6 in HGF-dependent signaling was confirmed by the decreased Akt phosphorylation in cells lacking CD151, integrin α3, or integrin α6. Hence, the regulation of CD151 expression might contribute to changes in HGF/c-Met signaling and thereby modulate the phenotypic characteristics of cancer cells.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.