Dissecting the diverse functions of the metastasis suppressor CD82/KAI1

The recent identification of metastasis suppressor genes, the products of which inhibit metastasis but not primary tumor growth, distinguishes oncogenic transformation and tumor suppression from a hallmark of malignancy, the ability of cancer cells to invade sites distant from the primary tumor. The metastasis suppressor CD82/KAI1 is a member of the tetraspanin superfamily of glycoproteins. CD82 suppresses metastasis by multiple mechanisms including inhibition of cell motility and invasion, promotion of cell polarity as well as induction of senescence and apoptosis in response to extracellular stimuli. A common feature of these diverse effects is CD82 regulation of membrane organization as well as protein trafficking and interactions, which affects cellular signaling and intercellular communication.     

Controlling cell surface dynamics and signaling: how CD82/KAI1 suppresses metastasis

The recent identification of metastasis suppressor genes, uniquely responsible for negatively controlling cancer metastasis, are providing inroads into the molecular machinery involved in metastasis. While the normal function of a few of these genes is known; the molecular events associated with their loss that promotes tumor metastasis is largely not understood. KAI1/CD82, whose loss is associated with a wide variety of metastatic cancers, belongs to the tetraspanin family. Despite intense scrutiny, many aspects of how CD82 specifically functions as a metastasis suppressor and its role in normal biology remain to be determined. This review will focus on the molecular events associated with CD82 loss, the potential impact on signaling pathways that regulate cellular processes associated with metastasis, and its relationship with other metastasis suppressor genes.     

Anti-miR-197 inhibits migration in HCC cells by targeting KAI 1/CD82

Aim

To investigate the metastatic effects and mechanisms of miR-197 in hepatocellular carcinoma (HCC).

Methods and results

The levels of miR-197 increased in HCC cells and tissues compared with a normal hepatic cell line (LO2) and adjacent nontumorous liver tissues, respectively. miR-197 expression negatively correlated with CD82 mRNA expression in these cell lines and tissues. Dual luciferase reporter assay and Western blot confirmed a direct interaction between miR-197 and CD82 3′UTR sequences. After miR-197 was silenced in HCC cells, CD82 expression increased. In the presence of human hepatocyte growth factor (HGF), cells silenced for anti-miR-197 exhibited elongated cellular tails and diminished lamellipodia due to reductions in both ROCK activity and the levels of Rac 1 protein. Downregulation of miR-197 along with the upregulation of CD82 in HCC cells resulted in the inhibition of HCC migration and invasion in vitro and in vivo.

Conclusion

Taken together, these data suggest that anti-miR-197 suppresses HCC migration and invasion by targeting CD82. The regulation of the miR-197/CD82 axis could be a novel therapeutic target in future HCC effective therapy.     

Role of a metastatic suppressor gene KAI1/CD82 in the diagnosis and prognosis of breast cancer

Globally, breast cancer is the most common type of cancer in females and is one of the leading causes of cancer death in women. The advancement in the targeted therapies and the slight understanding of the molecular cascades of the disease have led to small improvement in the rate of survival of breast cancer patients. However, metastasis and resistance to the current drugs still remain as challenges in the management of breast cancer patients. Metastasis, potentially, leads to failure of the available treatment, and thereby, makes the research on metastatic suppressors a high priority. Tumor metastasis suppressors are several genes and their protein products that have the capability of arresting the metastatic process without affecting the tumor formation. The metastasis suppressors KAI1 (also known as CD82) has been found to inhibit tumor metastasis in various types of solid cancers, including breast cancer. KAI1 was identified as a metastasis suppressor that inhibits the process of metastasis by regulating several mechanisms, including cell motility and invasion, induction of cell senescence, cell–cell adhesion and apoptosis. KAI1 is a member of tetraspanin membrane protein family. It interacts with other tetraspanins, chemokines and integrins to control diverse signaling pathways, which are crucial for protein trafficking and intracellular communication. It follows that better understanding of the molecular events of such genes is needed to develop prognostic biomarkers, and to identify specific therapies for breast cancer patients. This review aims to discuss the role of KAI1/CD82 as a prognosticator in breast cancer.     

KAI1/CD82 suppresses hepatocyte growth factor-induced migration of hepatoma cells via upregulation of Sprouty2

We conducted a study concerning the suppressive mechanism of KAI1/CD82 on hepatoma cell metastasis. Hepatocyte growth factor (HGF) induces the migration of hepatoma cells through activation of cellular sphingosine kinase 1 (SphK1). Adenovirus-mediated gene transfer of KAI1 (Ad-KAI1) downregulates the SphK1 expression and suppresses the HGF-induced migration of SMMC-7721 human hepatocellcular carcinoma cells. Overexpression of KAI1/CD82 significantly elevates Sprouty2 at the protein level. Ablation of Sprouty2 with RNA interference can block the KAI1/CD82-induced suppression of hepatoma cell migration and downregulation of SphK1 expression. It is demonstrated that KAI1/CD82 suppresses HGF-induced migration of hepatoma cells via upregulation of Sprouty2.     

胆管癌组织中KAI1/CD82基因的表达及意义

目的:探讨肿瘤转移抑制基因KAI1/CD82在胆管癌组织中的表达情况及临床病理意义。方法:应用免疫组织化学技术检测48例胆管癌组织及8例正常胆管组织中的KAI1/CD82蛋白表达。结果:KAI1/CD82蛋白在胆管癌组织中阳性表达率31.3%,明显低于正常胆管组织(87.5%,P<0.01)。KAI1/CD82蛋白的表达与肿瘤分化程度、转移相关(P<0.05),而与胆管癌患者年龄、性别、肿瘤部位和病理类型无关。结论:KAI1/CD82蛋白低表达可能参与了胆管癌的发生、发展,并对肿瘤转移的判断有一定指导意义。     

Attenuation of EGF receptor signaling by a metastasis suppressor, the tetraspanin CD82/KAI-1

The 'metastasis suppressor' CD82/KAI-1, a member of the tetraspanin superfamily of transmembrane proteins, is widely distributed in normal tissues [1], and has been shown to be suppressed in the advanced stages of various epithelial malignancies [2-6]. Although the physiological relevance of this change is unknown, in vitro data show that ectopically expressed CD82/KAI-1 can suppress tumor cell migration, a process underlying the dissemination of tumor cells in vivo [5]. The function of CD82/KAI-1 is not known and it has been proposed that association of CD82/KAI-1 with other cell-surface proteins may be pivotal in directing its biological activities [7,8]. We show here that the CD82/KAI-1 tetraspanin is directly associated with the EGF receptor (EGFR), and that ectopic expression of CD82/KAI-1 in epithelial cells specifically suppresses EGF-induced lamellipodial extensions and cell migration. In cells expressing CD82/KAI-1, the initial activation of EGFR is not affected, but subsequent desensitization of EGF-induced signaling occurs more rapidly. This attenuation is correlated with an increased rate of receptor endocytosis. These results identify CD82/KAI-1 as a new regulator of EGF-induced signaling and show that the association of EGFR with the tetraspanin is critical in EGFR desensitization.     

KAI1/CD82 在早孕小鼠子宫内膜组织的表达研究

目的：观察KAI1／CD82 mRNA和蛋白在小鼠妊娠D1-D8子宫内膜组织的表达。方法：以胚胎与肿瘤同源性为理论基础,胚胎植入与肿瘤侵袭转移相似为切入点,采用免疫组化和RT-PCR技术。结果：KAI1／CD82 mRNA和蛋白在早孕子宫中,KAI1／CD82mRNA的表达渐增多,蛋白表达的量和范围也渐增强。结论：KAI1／CD82mRNA和蛋白在早孕子宫组织中的动态表达,提示它在胚胎精确侵袭子宫内膜的调节中发挥作用,是滋养层细胞精确侵袭调控的分子机制之一。     

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.     

A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration

Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, we screened for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppressed the migration defect. Moreover, loss-of-function mutations in th caused migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescued RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility.     

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.     

KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line

We conducted a study on the mechanism of KAI1/CD82-mediated suppression of tumor invasiveness and metastasis, and examined its effect on MMP-9 activity and the TIMP1 levels in H1299 human non-small cell lung carcinoma cells. The H1299 human lung carcinoma cells were transfected with pcDNA3.1-CD82 and stable transfectant clones that had a high KAI1/CD82 expression were obtained. We performed Western blot analysis, cell invasion assay, gelatin zymography, and RT-PCR to assess the KAI1/CD82 expression and tumor invasiveness, the MMP-9 activity, the MMP-9 mRNA and protein levels, and the TIMP1 levels in the H1299/CD82 transfectant cells and compared the results with those of the control groups. The H1299/CD82 transfectants exhibited significant suppression of cell invasion, reduced MMP9 enzyme activity, elevated MMP9 mRNA and MMP-9 protein levels, and elevated TIMP1 levels. It may be postulated that KAI1/CD82 over-expression in the H1299 non-small cell lung carcinoma cells suppresses the tumor invasiveness and metastatic potential by inducing MMP9 inactivation via the up-regulation of TIMP1.     

Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas

We previously showed that activation of the small GTPase Cdc42 promotes breast cell migration on a collagen matrix. Here we further define the signaling pathways that drive this response and show that Cdc42-mediated migration relies on the adaptor molecule p130(Cas). Activated Cdc42 enhanced p130(Cas) phosphorylation and its binding to Crk. Cdc42-driven migration and p130(Cas) phosphorylation were dependent on the Cdc42 effector Ack1 (activated Cdc42-associated kinase). Ack1 formed a signaling complex that also included Cdc42, p130(Cas), and Crk, formation of which was regulated by collagen stimulation. The interaction between Ack1 and p130(Cas) occurred through their respective SH3 domains, while the substrate domain of p130(Cas) was the major site of Ack1-dependent phosphorylation. Signaling through this complex is functionally relevant, because treatment with either p130(Cas) or Ack1 siRNA blocked Cdc42-induced migration. These results suggest that Cdc42 exerts its effects on cell migration in part through its effector Ack1, which regulates p130(Cas) signaling.     

Role of tumor metastasis suppressor gene KAI1 in digestive tract carcinomas and cancer cells

The KAI1 gene is identified as a tumor metastasis suppressor gene in many types of cancer. We examined KAI1 gene and its protein KAI1/CD82 expression by in situ hybridization and immunohistochemical analysis, and found that KAI1 mRNA and protein expression were inversely correlated with lymph node and distant metastasis in digestive tract carcinomas, but not with age and gender of the patient, or with tumor differentiation. Moreover, KAI1/CD82 protein expression positively reflected the survival outcome of patients. Western blot analysis showed that VP-16 increased KAI1/CD82 protein expression obviously in various cancer cell lines, especially in those that were highly metastatic. This increased KAI1/CD82 expression was associated with its translocation from the cytomembrane to the nucleus, in which it interacted with nuclear p53 protein, forming a strong complex, observed by confocal microscopy and co-immunoprecipitation, respectively. In nude mice, after feeding with VP-16, the number of tumors metastasized from spleen to liver was obviously reduced, and KAI1/CD82 protein expression became stronger in those metastatic tumors. Accordingly, this demonstrated that KAI1 might be used as an indicator for predicting the clinical outcome, and VP-16 may be clinically considered as a promising candidate for anti-metastasis with regard to its potential to upregulate KAI1 expression.The study was supported by the Key Project of Science & Technology of the Ministry of Education (00073), the National High Technology Research and Development Program of China (Program 863, grant 2001AA620401), the National Natural Science Foundation of China (grants 39880015, 30170477), and the National Outstanding Youth Science Foundation of China (grant 39825502).     

N-Glycosylation pattern of recombinant human CD82 (KAI1), a tumor-associated membrane protein

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn₁₅₇ for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.     

The interaction of CFLAR with p130Cas promotes cell migration

CASP8 and FADD Like Apoptosis Regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis mediated by Fas and TRAIL. In addition to its anti-apoptotic function, CFLAR is also an important mediator of tumor growth. High level of CFLAR expression correlates with a more aggressive tumor. However, the mechanism of CFLAR signaling in malignant progression is not clear. Here we report a novel CFLAR-associated protein p130Cas, which is a general regulator of cell growth and cell migration. CFLAR-p130Cas association is mediated by the DED domain of CFLAR and the SD domain of p130Cas. Immunofluorescence observation showed that CFLAR had the colocalization with p130Cas at the focal adhesion of cell membrane. CFLAR overexpression promoted p130Cas phosphorylation and the formation of focal adhesion complex. Moreover, the enhancement of cell migration induced by CFLAR overexpression was obviously inhibited by p130Cas siRNA. In silico analysis on human database suggests high expressions of CFLAR or/and p130Cas are associated with poor prognosis of patients with lung cancer. Together, our results suggest a new mechanism for CFLAR involved in tumor development via association with p130Cas.