Requirement of the p130CAS-Crk coupling for metastasis suppressor KAI1/CD82-mediated inhibition of cell migration

KAI1/CD82 protein is a member of the tetraspanin superfamily and has been rediscovered as a cancer metastasis suppressor. The mechanism of KAI1/CD82-mediated suppression of cancer metastasis remains to be established. In this study, we found that migration of the metastatic prostate cancer cell line Du145 was substantially inhibited when KAI1/CD82 was expressed. The expression of focal adhesion kinase (FAK) and Lyn, a Src family tyrosine kinase and substrate of FAK, was up-regulated at both RNA and protein levels upon KAI1/CD82 expression. The activation of FAK and Lyn, however, remained unchanged in Du145-KAI1/CD82 cells. As a downstream target of FAK-Lyn signaling, the p130CAS (Crk-associated substrate) protein was decreased upon the expression of KAI1/CD82. Consequently, less p130CAS-CrkII complex, which functions as a "molecular switch" in cell motility, was formed in Du145-KAI1/CD82 cells. To confirm that the p130CAS-CrkII complex is indeed important for the motility inhibition by KAI1/CD82, overexpression of p130CAS in Du145-KAI1/CD82 cells increased the formation of p130CAS-CrkII complex and largely reversed the KAI1/CD82-mediated inhibition of cell motility. Taken together, our studies indicate the following: 1) signaling of FAK-Lyn-p130CAS-CrkII pathway is altered in KAI1/CD82-expressing cells, and 2) p130CAS-CrkII coupling is required for KAI1/CD82-mediated suppression of cell motility.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

KAI1/CD82 decreases Rac1 expression and cell proliferation through PI3K/Akt/mTOR pathway in H1299 lung carcinoma cells

Although the KAI1/CD82 protein has been reported to inhibit cell metastasis in many studies, its mechanism of action has not yet been fully elucidated. In the present study, we investigated the possible effects of KAI1/CD82 on the metastatic phenotype in H1299 lung carcinoma cells. These studies were based on the pivotal role that the acquisition of motile phenotype plays on the initial steps of metastasis. KAI1/CD82‐mediated morphological changes were observed using phase contrast microscopy. We report here, that a KAI1/CD82‐induced phenotypic change was involved in the decrease of Rac1 expression and GTPase activity. However, we found that KAI1/CD82 did not regulate Rac1 mRNA levels. This suggests the existence of another regulatory mechanism of Rac1 protein maturation or activation. To identify the signaling pathway of Rac1 regulation, we investigated the PI3K/Akt/mTOR pathway, since the PI3K/Akt pathway regulates Rac1 activation and mTOR is known to play a regulatory role in protein translation. H1299/CD82‐transfectants showed lower mTOR expression and cell growth than the control group. The data obtained from this study suggested that KAI1/CD82 decreased the metastatic phenotype of H1299 lung carcinoma cells by down‐regulating Rac1 expression through the PI3K/Akt/mTOR pathway. Copyright © 2008 John Wiley & Sons, Ltd.     

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。     

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.     

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.     

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。     

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.     

Tetraspanin CD82 Inhibits Protrusion and Retraction in Cell Movement by Attenuating the Plasma Membrane-Dependent Actin Organization

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.     

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.     

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.     

An improved method for isolating Schwann cells from postnatal rat sciatic nerves

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro. This work was supported by Tsinghua-Yue-Yuen Medical Sciences Fund, the National Natural Science Foundation of China (contract grant numbers: 30670528, 30700848, 30772443), Beijing Municipal Science & Technology Commission (BMSTC, contract grant number: H060920050430), National Basic Research Program of China (also called the 973 Program, contract grant number: 2005CB623905), and the National Natural Science Foundation of Beijing (contract grant number: 7082090).     

Phenolic Compounds from the Fruits of Prunus davidiana (Rosaceae) and Their Antioxidant Activities

This research was supported by Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ014204032019) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1A6A3A01100042).     

Novel strategies to mine alcoholism-related haplotypes and genes by combining existing knowledge framework

High-throughout single nucleotide polymorphism detection technology and the existing knowledge provide strong support for mining the disease-related haplotypes and genes. In this study, first, we apply four kinds of haplotype identification methods (Confidence Intervals, Four Gamete Tests, Solid Spine of LD and fusing method of haplotype block) into high-throughout SNP genotype data to identify blocks, then use cluster analysis to verify the effectiveness of the four methods, and select the alcoholism-related SNP haplotypes through risk analysis. Second, we establish a mapping from haplotypes to alcoholism-related genes. Third, we inquire NCBI SNP and gene databases to locate the blocks and identify the candidate genes. In the end, we make gene function annotation by KEGG, Biocarta, and GO database. We find 159 haplotype blocks, which relate to the alcoholism most possibly on chromosome 1∼22, including 227 haplotypes, of which 102 SNP haplotypes may increase the risk of alcoholism. We get 121 alcoholism-related genes and verify their reliability by the functional annotation of biology. In a word, we not only can handle the SNP data easily, but also can locate the disease-related genes precisely by combining our novel strategies of mining alcoholism-related haplotypes and genes with existing knowledge framework. Supported by the National Natural Science Foundation of China (Grant Nos. 30570424, 60601010 and 30600367), the National High-Tech Research and Development Program of China, (Grant No.2007AA02Z329), the Key Science and Technology Program of Heilongjiang Province(Grant No.GB03C602-4), Natural Science Foundation of Heilongjiang Province (Grant No. F2008-02), Youth Science Foundation of Harbin Medical University (Grant No. 060045) and Science Foundation of Heilongjiang Province Education Department (Grant Nos. 11531113 and 1152hq28).     

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.     

中美生物医学合作研究计划及其前景

中美生物医学合作研究计划实施3年来,收到了来自中美双方科学家共657项申请.这些申请在研究内容上涉及肿瘤、感染性疾病(包括HIV感染和艾滋病相关研究)、过敏性疾病、医学免疫学、精神健康及神经性疾病(中风和帕金森病).经过国家自然科学基金委员会和美国国立卫生研究院的联合评审程序,有109项申请项目获得了资助.联合评审的机制和获资助的109个研究项目的合作研究为今后中美双方的进一步合作奠定了基础.     

The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation

Metastasis is the primary cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. In particular, relatively little is known about metastasis in cancers of mesenchymal origins, which are known as sarcomas. Approximately ten proteins have been characterized as 'metastasis suppressors', but how these proteins function and are regulated is, in general, not well understood. Gp78 (also known as AMFR or RNF45) is a RING finger E3 ubiquitin ligase that is integral to the endoplasmic reticulum (ER) and involved in ER-associated degradation (ERAD) of diverse substrates. Here we report that expression of gp78 has a causal role in the metastasis of an aggressive human sarcoma and that this prometastatic activity requires the E3 activity of gp78. Further, gp78 associates with and targets the transmembrane metastasis suppressor, KAI1 (also known as CD82), for degradation. Suppression of gp78 increases KAI1 abundance and reduces the metastatic potential of tumor cells, an effect that is largely blocked by concomitant suppression of KAI1. An inverse relationship between these proteins was confirmed in a human sarcoma tissue microarray. Whereas most previous efforts have focused on genetic mechanisms for the loss of metastasis suppressor genes, our results provide new evidence for post-translational downregulation of a metastasis suppressor by its ubiquitin ligase, resulting in abrogation of its metastasis-suppressing effects.     

Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.