928

CD9

CD9 molecule;CD9;CD9 molecule

5H9 antigen|BA-2/p24 antigen|CD9 antigen|CD9 antigen (p24)|cell growth-inhibiting gene 2 protein|leukocyte antigen MIC3|motility related protein-1|tetraspanin-29

12p13.3

protein-coding

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental MDA-MB-231 breast cancer cell line. Here, we investigated the putative relationship between CD9 expression and the osteotropic phenotype. MATERIALS AND METHODS: Overexpression of CD9 was analyzed by immunoblotting in different cell lines. Immunohistochemistry was used to assess CD9 expression in primary tumors and metastatic lesions. In vivo experiments were conducted in mice using a monoclonal antibody against CD9. RESULTS: CD9 overexpression was confirmed in osteotropic cells. CD9 was significantly overexpressed in bone metastases versus primary tumors and visceral metastatic lesions. Finally, in vivo experiments showed that an antibody against CD9 delays homing of B02 cells in bone marrow, slowing down bone destruction. CONCLUSION: Our study reveals a potential implication of CD9 in the formation of bony metastases from breast cancer cells.

Motility-related protein-1 (CD9), a type of cell surface glycoprotein comprising a four-pass transmembrane domain that forms multimeric complexes with other cell surface proteins, belongs to the tetraspanins family. From previous studies, we know that CD9 is considered to function primarily as a progression and metastasis suppressor in a variety of cancers, including breast, non-small cell lung colon and myeloma. However, an expanding body of literature has shown the contradictory outcome that tetraspanin CD9 is also vital in promoting cancer progression in several types of cancer. This review summarizes the recent studies on CD9 and concludes that it does not always act as a progression and metastasis suppressor. Conversely, in specific cases, CD9 may promote tumor progression through the following three aspects: Facilitating tumor cell transmigration, increasing tumor cell motility and hastening the growth of some cancers. In addition, CD9 appears to be an important marker of cancer stem cells in certain types of tumor.

Recently, we established that a murine monoclonal antibody (MAb) MH8-4 inhibits the motility of the colon cancer cell line RPMI4788 and that it recognizes integrin alpha3. In addition, we have also cloned the motility-related protein-1 (MRP-1)/cluster of differentiation 9 (CD9) as a metastasis suppressor molecule. We investigated integrin alpha3 expression in 114 resected colon cancers using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate whether these experimental results are of relevance in the prognosis of actual colon cancers. Furthermore, we investigated the correlation of integrin alpha3 with MRP-1/CD9 and KAI1/CD82. Sixty patients (52.6%) were evaluated as integrin alpha3-positive and 54 patients (47.4%) as integrin alpha3-negative. Integrin alpha3 expression was associated with tumor status, lymph node status and pathologic stage. The overall and disease-free survival rates for patients whose tumors were positive for integrin alpha3 were significantly higher than for those with integrin alpha3-negative tumors (p < 0.001 and p < 0.001, respectively). This same tendency was observed in node-negative patients (p = 0.007 and p = 0.001, respectively). Integrin alpha3 was found to be the significant prognostic factor in a multivariate analysis using the Cox proportional hazards model (p = 0.036). A correlation was found between integrin alpha3 with MRP-1/CD9 and KAI1/CD82 for stage I tumors. However, no correlation was found in stage III tumors. Our data seem to suggest that low expression of integrin alpha3 is a useful indicator of a poor prognosis for colon cancer patients and that colon cancer progresses following collapse of the complex formed by integrin alpha3 with MRP-1/CD9 and KAI1/CD82.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. Juxtacrine activity of proHB-EGF (the membrane-anchored form of HB-EGF) has been shown to be significantly potentiated when it is coexpressed with CD9 in vitro. The purpose of our study was to investigate the issue of whether proHB-EGF and CD9 are coexpressed in gastric cancer. HB-EGF gene expression and protein production in human gastric cancers was investigated, and EGF receptor and CD9 expressions were also evaluated. HB-EGF mRNA levels in gastric cancers were elevated, compared with normal gastric tissues, especially in the intestinal type. ProHB-EGF immunoreactivity was detected primarily in the cytoplasm and plasma membrane of gastric cancer cells. Of 66 patients, 40 (60.6%) exhibited proHB-EGF immunoreactivity and the level of its expression was significantly associated with tumor status (p < 0.01) and histological differentiation (p < 0.001). In addition, proHB-EGF mRNA was detected at high levels in the intestinal type by in situ hybridization. CD9 immunoreactivity was found to be preserved in 26 of 36 patients (72.2%) and CD9 protein expression was inversely associated with lymph node status (p < 0.05). A significant correlation between its expression and histological differentiation (p < 0.01) was found, and the association of CD9 with proHB-EGF was increased in the intestinal type, as evidenced by an immunoprecipitation method. These results indicate that the coexpression of proHB-EGF and CD9 may be involved in the tumorigenesis and/or proliferation of gastric cancers in a juxtacrine manner.

Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit appear to occur in the development of metastasis. We sought to identify the genes involved in the metastatic process of gastric GIST. Microarray analysis was performed to compare gene expressions between three gastric GIST and four metastatic liver GIST. expression levels were higher for 165 genes and lower for 146 genes in metastatic liver GIST. The upregulation of five oncogenes and downregulation of four tumor suppressor genes including versican and CD9 were confirmed by quantitative reverse transcriptional PCR. Immunohistochemistry in 117 GIST revealed that protein levels of versican and CD9 were higher and lower, respectively, in metastatic GIST. High expression of versican and low expression of CD9 in 104 primary gastric GIST correlated with poor disease-free survival (P = 0.0078 and P = 0.0018). In addition to the c-kit gene mutation, genetic or epigenetic changes other than c-kit play important roles in the metastatic process. In particular, versican and CD9 are potential prognostic markers in gastric GIST.

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (P<0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with beta1 integrins and distributed at the tumor cell periphery and cell-cell contacts, suggesting that CD9 modifies beta1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

CD9 has been reported to play a role in tumor metastasis suppression. However, it is not fully understood how CD9 affects the hematogenous spread of tumor cells. To clarify a new mechanism (or mechanisms), we generated HT1080 cells that had been transfected with a CD9-expressing plasmid. Ectopic expression of CD9 in HT1080 cells actually reduced their metastatic ability. CD9 expression reduced lung retention and platelet aggregation activity of the transfectants. Because HT1080 cells express the metastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplanin on their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with Aggrus via transmembrane domains 1 and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that each CD9 and Aggrus interacted homophilically, and that they colocalized in low-density membrane fractions. Deleting TM1 and TM2 attenuated the ability of CD9 to interact homophilically or to localize in low-density membrane fractions. The expression of CD9-wild-type (WT), but not CD9 lacking TM1 and TM2, attenuated the platelet aggregation and metastasis induced by forced expression of Aggrus in CHO cells. Therefore, CD9 may act as a metastasis suppressor, at least in part, by neutralizing Aggrus-mediated platelet aggregation.

OBJECTIVE: To study the expression of neutral endopeptidase (CD10) and motility-related protein-1 (CD9) in malignant melanoma and their clinical significance. METHODS: Immunohistochemical study for CD10 and CD9 using Streptavidin-biotin complex technique was carried out in 48 cases of primary cutaneous malignant melanoma (CMM), 23 cases of metastatic melanoma and 23 cases of benign nevus. RESULTS: The positivity rate of CD10 was highest in metastatic melanoma and lowest in benign nevus (P < 0.01). In contrast, the positivity rate of CD9 in metastatic melanoma was lower than that in CMM (P < 0.05). The expression of CD9 was inversely correlated with that of CD10 in malignant melanoma (CMM: r = -0.40, P = 0.005; metastatic MM: r = -0.44, P = 0.034). The expression of CD10 and CD9 in CMM also correlated with tumor histology, Clark's level of invasion and presence of nodal metastasis. A similar relationship was also observed for CD10 and CD9 expression in stromal fibroblasts of CMM (r = -0.43, P = 0.007). CONCLUSIONS: CD10 and CD9 expression correlates with the invasiveness and metastatic potential of malignant melanoma; both factors may demonstrate a counteracting effect. These two markers have potential implications in prognostic assessment of CMM. Stromal fibroblasts may also play an important role in the progression of CMM.

CD9, a member of the tetraspanin family, functions as an organizer in "tetraspanin webs," through interacting with other cell adhesion molecules. It plays a role in differentiation, fertilization, and cell migration. We investigated the expression and function of CD9 in melanoma. CD9 protein expression in B16 mouse melanoma and six human melanoma cell lines was decreased compared to normal melanocytes. B16F1 clones stably overexpressing CD9 had reduced ability to form colonies in soft agar; however, paradoxically these overexpressing clones had increased ability to invade Matrigel. Similarly, transient overexpression of CD9 in the human metastatic melanoma cell line WM9 dramatically decreased anchorage-independent growth, while transient overexpression of CD9 in the radial growth phase cell line SbCl2 resulted in the gain of Matrigel invasion activity. DNA sequencing of CD9 cDNA from all six human melanoma cell lines did not show deletions, insertions, or mutations. Treatment of all six human melanoma cell lines with the histone deacetylase inhibitor trichostatin A increased CD9 levels. The DNA methylation inhibitor 5-aza-cytidine also increased CD9 protein levels with greater increases seen in cell lines derived from more malignant melanomas.

Motility-related protein-1 (CD9), a type of cell surface glycoprotein comprising a four-pass transmembrane domain that forms multimeric complexes with other cell surface proteins, belongs to the tetraspanins family. From previous studies, we know that CD9 is considered to function primarily as a progression and metastasis suppressor in a variety of cancers, including breast, non-small cell lung colon and myeloma. However, an expanding body of literature has shown the contradictory outcome that tetraspanin CD9 is also vital in promoting cancer progression in several types of cancer. This review summarizes the recent studies on CD9 and concludes that it does not always act as a progression and metastasis suppressor. Conversely, in specific cases, CD9 may promote tumor progression through the following three aspects: Facilitating tumor cell transmigration, increasing tumor cell motility and hastening the growth of some cancers. In addition, CD9 appears to be an important marker of cancer stem cells in certain types of tumor.

CD9 has been reported to play a role in tumor metastasis suppression. However, it is not fully understood how CD9 affects the hematogenous spread of tumor cells. To clarify a new mechanism (or mechanisms), we generated HT1080 cells that had been transfected with a CD9-expressing plasmid. Ectopic expression of CD9 in HT1080 cells actually reduced their metastatic ability. CD9 expression reduced lung retention and platelet aggregation activity of the transfectants. Because HT1080 cells express the metastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplanin on their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with Aggrus via transmembrane domains 1 and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that each CD9 and Aggrus interacted homophilically, and that they colocalized in low-density membrane fractions. Deleting TM1 and TM2 attenuated the ability of CD9 to interact homophilically or to localize in low-density membrane fractions. The expression of CD9-wild-type (WT), but not CD9 lacking TM1 and TM2, attenuated the platelet aggregation and metastasis induced by forced expression of Aggrus in CHO cells. Therefore, CD9 may act as a metastasis suppressor, at least in part, by neutralizing Aggrus-mediated platelet aggregation.

A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5-8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT-PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were significant factors for prognosis (P<0.0001, P=0.0083 and P=0.0004, respectively). Based on these results, we classified the patients into three groups according to their MRP-1/ CD9 and K-ras status. Patients with both MRP-1/CD9 positive and wild K-ras tumors were defined as group A, patients with either reduced MRP-1/CD9 or mutant K-ras tumors were defined as group B and patients with both reduced MRP-1/CD9 and mutant K-ras tumors were designated as group C. This new classification was significantly correlated with the tumor status and pathological stage (P=0.0098 and P=0.0017, respectively). The overall survival rate of the group A patients was significantly better than the group B patients (59.6% vs 27.9%, P=0.0001) and also that of group B patients was better than the group C patients (27.9% vs 20.0%, P=0.0378). This tendency was also found in patients with 110 node-negative NSCLCs (A vs B vs C=75.8% vs 34.9% vs 0.0%, P<0.0001). A Cox multivariate regression analysis in NSCLC patients demonstrated that an evaluation for both MRP-1/CD9 expression and K-ras mutations had a significant prognostic effect as well as nodal status (P<0.0001).

Transmembrane 4 superfamily (TM4SF) is a recently described gene family, and TM4SF members are known to play roles in the signal transduction pathways and to regulate cell activation, development, proliferation, and motility. MRP-1/CD9, KAI1/CD82, and ME491/CD63, members of the TM4SF, have been reported to suppress tumor progression or metastasis. Previously, we showed that MRP-1/CD9 suppressed cell motility and metastatic potential to lungs. Moreover, reduction of MRP-1/CD9 and KAI1/CD82 gene expression was found to be a factor in a poor prognosis for patients with non-small cell lung cancer. However, among TM4SF, CD151 is identical to an existing gene, PETA-3, which may promote tumor metastasis of malignant cells, and its expression may be involved in the malignant progression of cancer. The function of CD151 is opposite that of the metastasis suppressor genes, MRP-1/CD9 and KAI1/CD82. On the basis of these results, we used reverse transcription-PCR and immunohistochemical techniques for a retrospective study of CD151 gene expression in tumor tissues from 145 lung cancer patients; 72 tumors were stage I, 29 stage II, 27 stage IIIA, and 17 stage IIIB. Whereas 86 patients had tumors positive for the CD151 gene, 59 had tumors that were negative for the CD151 gene. The overall survival rate of patients with CD151-positive tumors was much lower than that of CD151-negative patients (51.9% versus 73.1%; P = 0.013). Our findings suggest that high CD151 gene expression in lung cancer may be associated with a poor prognosis. Assessment of CD151 could be instrumental for improvements in lung cancer diagnosis and therapies.

Transmembrane 4 superfamily (TM4SF) is a recently described gene family, and TM4SF members are known to play roles in the signal transduction pathways and to regulate cell activation, development, proliferation, and motility. MRP-1/CD9, KAI1/CD82, and ME491/CD63, members of the TM4SF, have been reported to suppress tumor progression or metastasis. Previously, we showed that MRP-1/CD9 suppressed cell motility and metastatic potential to lungs. Moreover, reduction of MRP-1/CD9 and KAI1/CD82 gene expression was found to be a factor in a poor prognosis for patients with non-small cell lung cancer. However, among TM4SF, CD151 is identical to an existing gene, PETA-3, which may promote tumor metastasis of malignant cells, and its expression may be involved in the malignant progression of cancer. The function of CD151 is opposite that of the metastasis suppressor genes, MRP-1/CD9 and KAI1/CD82. On the basis of these results, we used reverse transcription-PCR and immunohistochemical techniques for a retrospective study of CD151 gene expression in tumor tissues from 145 lung cancer patients; 72 tumors were stage I, 29 stage II, 27 stage IIIA, and 17 stage IIIB. Whereas 86 patients had tumors positive for the CD151 gene, 59 had tumors that were negative for the CD151 gene. The overall survival rate of patients with CD151-positive tumors was much lower than that of CD151-negative patients (51.9% versus 73.1%; P = 0.013). Our findings suggest that high CD151 gene expression in lung cancer may be associated with a poor prognosis. Assessment of CD151 could be instrumental for improvements in lung cancer diagnosis and therapies.

CD9, a transmembrane protein known as motility-related protein-1, plays a pivotal role in regulating cell adhesion, motility, and proliferation, and has been regarded as an important metastasis-inhibitory factor of various human cancers. However, little information has been obtained regarding the highly metastatic human small-cell lung cancer (SCLC). In the present study, an SCLC cell line (OS3-R5), lacking CD9 expression, was transfected with human CD9 gene to assess the role of CD9 on the metastatic potential of SCLC. CD9 gene transfection into OS3-R5 cells resulted in cell proliferation and motility in vitro. Parental and mock-transfected OS3-R5 cells developed liver metastasis and malignant ascites when they were intravenously inoculated into NK cell-depleted SCID mice. CD9 gene transfection into OS3-R5 cells caused suppression of the liver metastasis and malignant ascites. Immunohistochemical analysis revealed that the number of proliferating tumor cells was significantly fewer in liver lesions produced by CD9 gene-transfected OS3-R5 cells than those produced by parental or mock control OS3-R5 cells. In addition, no detectable levels of CD9 were expressed in metastatic tumor cells in mice bearing CD9 gene-transfected OS3-R5 cells, as well as those in mice bearing parental or mock control OS3-R5 cells. These results suggest that the restored expression of CD9 in SCLC cells may reduce the metastatic spread of SCLC cells via the inhibition of cell proliferation and motility.

A majority of small cell lung cancer (SCLC) cells lack a metastasis suppressor, tetraspanin CD9, and CD9 expression promotes their apoptosis. By a proteomics-based approach, we compared an SCLC cell line with its CD9 transfectant and found that a calcium-binding neuronal protein, calretinin, is upregulated in CD9-positive SCLC cells. Ectopic or anticancer drug-induced CD9 expression upregulated calretinin, whereas CD9 knockdown down-regulated calretinin in SCLC cells. When calretinin was knocked down, CD9-positive SCLC cells revealed increased Akt phosphorylation and decreased apoptosis. These results suggest that CD9 positively regulates the expression of calretinin that mediates proapoptotic effect in SCLC cells.