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 lowdensity 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. (Blood. 2008;112:1730-1739) IntroductionFour-pass transmembrane proteins of the tetraspanin family regulate cell migration, fusion, and signaling events by functioning as organizers of a multimolecular membrane complex called the tetraspanin web or tetraspanin-enriched microdomains. 1,2 The CD9 protein is a member of the tetraspanin family and has been identified as a suppressor of cancer spread. 3 Numerous previous studies have shown reduced CD9 expression in various cancers, which correlated with the presence of distant metastasis and a poorer prognosis. [4][5][6][7][8][9][10][11] The clinical importance of CD9 in the diagnosis, staging, and prognosis of tumors has been growing. 12 Recently, it was reported that adenoviral transduction of CD9 inhibited lymph node metastasis in an orthotopic lung cancer model. 13 The investigators evaluated metastatic growth in the mediastinal lymph nodes using gene delivery of CD9. These findings strongly suggest that CD9 plays indispensable roles in malignant tumor progression and shows the feasibility of gene therapy to prevent metastasis. However, we cannot say that the therapeutic application of CD9 is now available. Functions of tetraspanins, to a great extent, may depend on expression patterns of their counterparts or other molecules on the tetraspanin web. Actually, there are reports that overexpression of CD9 does not affect metastatic properties, 14 or that CD9 expression is downregulated at the primary site but reexpressed at the metastatic site. 15 So, the precise mechanisms of the metastasis suppression should be elucidated more clearly.Aggrus/podoplanin, a type-I transmembrane glycoprotein, has been shown to be up-regulated in a number of different cance...

Podoplanin, a mucin-like transmembrane sialoglycoprotein, promotes platelet aggregation and may be involved in cancer cell migration, invasion, metastasis, and malignant progression. Podoplanin/aggrus is highly expressed in testicular seminoma, suggesting that it may be a sensitive marker for testicular seminomas. Here we investigated the expression of podoplanin in central nervous system (CNS) germ cell tumors (GCTs) by immunohistochemical staining of tumor samples from 62 patients. In 40 of 41 (98%) germinomas (including germinomatous components in mixed GCTs), podoplanin was diffusely expressed on the surface of germinoma cells; lymphocytes, interstitial cells, and syncytiotrophoblastic giant cells were negative for podoplanin. Except for immature teratomas (12/17; 71%), podoplanin expression was absent in non-germinomatous GCTs, including seven teratomas, seven embryonal carcinomas, seven yolk sac tumors, and seven choriocarcinomas. In immature teratomas, focal podoplanin staining was observed in fewer than 10% of immature squamous and columnar epithelial cells. Thus, podoplanin expression may be a sensitive immunohistochemical marker for germinoma in CNS GCTs. As such, it may be useful for diagnosis, for monitoring the efficacy of treatment, and as a potential target for antibody-based therapy.

The platelet aggregation-inducing factor, Aggrus (also known as podoplanin), is reported to contribute to cancer metastasis by mediating cancer cell-platelet interaction. Aggrus has been shown to be upregulated in many different types of cancers. Thus, not only the functional inhibition of Aggrus, but also its application as a cancer-specific antigen has therapeutic potential. Among a series of anti-Aggrus mAb established previously, no mouse anti-human Aggrus mAb exists that possesses the ability to neutralize platelet aggregation. For precise preclinical examinations of mouse and monkey models, the establishment of Aggrus-neutralizing mouse mAb and their chimeric Abs is needed. In this study, we established two mouse anti-human Aggrus mAb, P2-0 and HAG-3. A precise analysis of their epitopes revealed that P2-0 recognized the conformation near the bioactive O-glycosylation site at the Thr 52 residue. In contrast, HAG-3 recognized the amino-terminus side at a short distance from the conformation recognized by P2-0. We observed that only P2-0 attenuated Aggrus-induced platelet aggregation and Aggrus binding to its platelet receptor, that is, the C-type lectin-like receptor-2. Consistent with these data, only P2-0 prevented the experimental metastasis of human Aggrusoverexpressing CHO cells. Subsequently, we cloned the complementary determining region of P2-0 and produced the murine ⁄ human chimeric P2-0 antibody. This chimeric antibody maintained its inhibitory activity of Aggrus-induced platelet aggregation and experimental metastasis. Thus, P2-0 and its chimeric antibody are expected to aid the development of preclinical and clinical examinations of Aggrus-targeted cancer therapy. (Cancer Sci 2011; 102: 2051-2057

Understanding metastasis is integral to curative cancer treatments. Using a mouse genetic screening model, we identified Merm1/Wbscr22 as a novel metastasis promoter that includes a methyltransferase fold in its structure. Merm1 showed high levels of expression in invasive breast cancer. Ectopic expression of Merm1 in nonmetastatic cells enhanced metastasis formation without affecting cell growth and motility. The intact methyltransferase fold of Merm1 was required for metastasis formation. Interestingly, Merm1 expression promoted cell survival after entrapment in the lung microvasculature. Consistent with these results, knockdown of endogenous Merm1 in tumor cells reduced lung retention and metastasis formation. On the basis of comparative transcriptome analysis, Merm1 expression was negatively correlated with the expression of tumor suppressor Zac1. We confirmed that Merm1 suppressed Zac1 expression with histone H3 methylation at Lys 9 in the Zac1 promoter region. Zac1 can induce apoptosis through its ability to transcriptionally coactivate p53, which regulates apoptosis in the vasculature and is often downregulated in metastasis. We found that Zac1 knockdown reduced the p53-dependent apoptosis that was enhanced by Merm1 knockdown, thereby increasing lung retention of metastatic cells. Our findings show that Merm1 enhances cancer cell survival in the vasculature by suppressing Zac1/p53-dependent apoptosis, thereby enhancing metastasis. Cancer Res; 71(3); 1146-55. Ó2010 AACR.