CD24 Expression Causes the Acquisition of Multiple Cellular Properties Associated with Tumor Growth and Metastasis Petra Baumann, 1 Natascha Cremers, 1 Frans Kroese, 2 Gertraud Orend, 3 Ruth Chiquet-Ehrismann, 4 Toshi Uede, 5 Hideo Yagita, 6 and Jonathan P. Sleeman 1 1 Forschungszentrum Karlsruhe, Institut fu ¨r Toxikologie und Genetik, Karlsruhe, Germany; 2 Department Cell Biology, University of Groningen, Groningen, the Netherlands; 3 Institute of Biochemistry and Genetics, University of Basel; 4 Friedrich Miescher Institute for Biomedical Research, Novartis Forschungsstiftung, Basel, Switzerland; 5 Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Kita, Sapporo, Japan; and 6 Department of Immunology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan Abstract The glycosylphosphatidylinositol-anchored membrane protein CD24 functions as an adhesion molecule for P-selectin and L1 and plays a role in B-cell development and neurogenesis. Over the last few years, a large body of literature has also implicated CD24 expression in tumorigenesis and progression. Here, we show that ectopic CD24 expression can be sufficient to promote tumor metastasis in experimental animals. By developing a doxycycline-inducible system for the expression of CD24 in breast cancer cells, we have also analyzed the cellular properties that CD24 expression influences. We found that CD24 expression increased tumor cell proliferation. Furthermore, in addition to promoting binding to P-selectin, CD24 expression also indirectly stimulated cell adhesion to fibronectin, collagens I and IV, and laminin through the activation of A 3 B 1 and A 4 B 1 integrin activity. Moreover, CD24 expression supported rapid cell spreading and strongly induced cell motility and invasion. CD24-induced prolifera- tion and motility were integrin independent. Together, these observations implicate CD24 in the regulation of multiple cell properties of direct relevance to tumor growth and metastasis. (Cancer Res 2005; 65(23): 10783-93) Introduction CD24, also known as heat-stable antigen in the mouse, is a glycosylphosphatidylinositol-anchored membrane protein of het- erogeneous molecular weight ranging from 30 to 70 kDa (1). The mature protein is only 27 to 30 amino acids long, and most of the molecular weight of the protein consists of extensive N- and O-linked glycosylation. CD24 is expressed in cells of the hematopoietic system, such as B-cell precursors and neutrophils, in neuronal tissue, and in certain epithelial cells, such as keratinocytes and renal tubular epithelium. CD24 is thought to function as an adhesion molecule. It is known to bind to P-selectin, a protein expressed on thrombin-activated platelets and endothelial cells (2), and to L1, a member of the immunoglobulin superfamily that is expressed on neural and lymphoid cells (3, 4). CD24-deficient mice display defects in B-cell development (5) consistent with the reported role for CD24 in cell- cell interactions of B cells (6). CD24-deficient mice also display increased neurogenesis (7), in agreement with the observation that CD24 inhibits neurite outgrowth (8, 9). An expanding body of literature points to a role for CD24 in the tumorigenesis and progression of a number of types of cancer. CD24 expression is a prognostic indicator of poor survival in breast cancer (10), non–small cell lung carcinomas (11), and ovarian (12) and prostate tumors (13). Enhanced CD24 expression in comparison to matched nonmalignant tissue has also been reported for a number of other types of cancer, including B-cell lymphoma (14), renal cell carcinoma (15), small cell lung carcinoma (16), nasopharyngeal carcinoma (17), hepatocellular carcinoma (18), Merkel cell carcinoma (19), pancreatic carcinoma (20), and neural tumors (21). Additionally, CD24 has been repeatedly identified in gene expression profiling screens to identify genes whose expression correlates with tumorigenesis and tumor progression (22–26). In cellular and animal assays, CD24 has been reported to support the rolling of tumor cells on endothelial monolayers (27) and to promote tumor cell invasive- ness in vivo (28). In previous studies, we isolated CD24 in suppression subtractive hybridization screens to identify genes whose expression is up- regulated in metastatic breast and pancreatic carcinoma cells (23). Here we show that CD24 expression can be sufficient to promote metastasis in vivo , and report studies which identify properties that CD24 expression confers on tumor cells. By establishing a system in which we can induce expression of CD24 in mammary carcinoma cells, we show that CD24 expression stimulates tumor cell proliferation, can promote tumor cell binding to P-selectin, fibronectin, and other extracellular matrix components, and also stimulates cell motility and invasion. These properties are highly relevant for tumor growth and progression and suggest that CD24 is a pleiotropic stimulator of these processes. Materials and Methods Cell culture, antibodies, and peptides. The rat carcinoma lines 1AS and MTLy were cultivated as previously described (23). The anti-rat a 3 integrin antibody was obtained from Chemicon (Temecula, CA). The other anti-rat integrin subunit antibodies (29) and the anti-rat CD24 antibody HIS50 (30) have been described. Anti-focal adhesion kinase (FAK) polyclonal antibodies (C903) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phosphotyrosine antibodies conjugated to horseradish peroxidase (PY20H) were purchased from BD Transduction Laboratories (San Diego, CA). CS-1 and RGD peptides were bought from Bachem (Weil am Rhein, Germany). Construction of the CD24 expression plasmids. A full-length rat CD24 cDNA corresponding to bases 21 to 320 of Genbank accession no. NM_012752 was amplified by reverse transcription-PCR (RT-PCR) using HF polymerase (Clontech, Palo Alto, CA). The PCR product was cloned into the pTRE2 vector (Clontech) to create the pTRE-CD24 plasmid and into the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Jonathan P. Sleeman, Forschungszentrum Karlsruhe, Institut fu ¨r Toxikologie und Genetik, Postfach 3640, 76021 Karlsruhe, Germany. Phone: 49-7247-82-6089; Fax: 49-7247-82-3354; E-mail: sleeman@itg.fzk.de. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0619 www.aacrjournals.org 10783 Cancer Res 2005; 65: (23). December 1, 2005 Research Article Research. on May 30, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from