Caveolin-1 Reduces Osteosarcoma Metastases by Inhibiting c-Src Activity and Met Signaling Lara Cantiani, 1 Maria Cristina Manara, 1 Cinzia Zucchini, 2 Paola De Sanctis, 2 Monia Zuntini, 1 Luisa Valvassori, 2 Massimo Serra, 1 Martina Olivero, 3 Maria Flavia Di Renzo, 3 Mario Paolo Colombo, 4 Piero Picci, 1 and Katia Scotlandi 1 1 Laboratory of Oncology Research, Rizzoli Orthopaedic Institute; 2 Center for Molecular Genetic Research ‘‘Fondazione CARISBO’’ at the Istituto di Istologia ed Embriologia Generale, University of Bologna, Bologna, Italy; 3 Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, University of Turin, Candiolo (Turin), Italy; and 4 Immunotherapy and Gene Therapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy Abstract Caveolin-1 (Cav-1) is highly expressed in normal osteoblasts. This article reports that Cav-1 down-regulation is part of osteoblast transformation and osteosarcoma progression and validates its role as oncosuppressor in human osteosarcoma. A survey of 6-year follow-up indicates a better overall survival for osteosarcoma expressing a level of Cav-1 similar to osteoblasts. However, the majority of primary osteosarcoma shows significantly lower levels of Cav-1 than normal osteoblasts. Accordingly, Met-induced osteoblast transforma- tion is associated with Cav-1 down-regulation. In vitro , osteo- sarcoma cell lines forced to overexpress Cav-1 show reduced malignancy with inhibited anchorage-independent growth, migration, and invasion. In vivo , Cav-1 overexpression abrogates the metastatic ability of osteosarcoma cells. c-Src and c-Met tyrosine kinases, which are activated in osteosar- coma, colocalize with Cav-1 and are inhibited on Cav-1 over- expression. Thus, Cav-1 behaves as an oncosuppressor in osteosarcoma. Altogether, data suggest that Cav-1 down- modulation might function as a permissive mechanism, which, by unleashing c-Src and Met signaling, enables osteosarcoma cells to invade neighboring tissues. These data strengthen the rationale to target c-Src family kinases and/or Met receptor to improve the extremely poor prognosis of metastatic osteosarcoma. [Cancer Res 2007;67(16):7675–85] Introduction The frequency of primary malignant bone tumors ranks osteosarcoma second after multiple myeloma. Osteosarcoma is a high-grade malignant tumor composed of mesenchymal cells producing osteoid and immature bone with a peak incidence in the second decade of life (1). Although current treatment modalities, which include surgery and neoadjuvant multidrug chemotherapy, significantly improved the 5-year disease-free survival from 10% to 60%–70% (2, 3), no significant new drugs or treatment approaches have been developed during the last 10 years, and the percentages of cure remain unacceptably low for high-risk patients (4). Drug resistance and metastasis are the major causes of treatment failure. Differently from carcinomas, molecular genetics of osteosarcoma progression is not documented and the most accepted prognostic factors are based on response to treatment (percentage of necrosis or P-glycoprotein expression; refs. 5–8) rather than on genetic signature characterizing the development and progression, with few exceptions (9–11). To search for such signature, we did a microarray analysis of genes involved in modulation of osteosarcoma malignancy by comparing osteosarcoma cell lines with different metastasis behavior and identified caveolin-1 ( Cav-1 ) gene whose expression correlates with a lower metastasis potential (12, 13). Cav-1 is the major protein component of caveolae (14), which are abundant in terminally differentiated mesenchymal cells, including adipocytes, endothelial cells, fibroblasts (15), and osteoblasts (16, 17). Caveolae provide physical interaction and compartmentalization of several mem- brane receptors. As part of receptor signaling pathways, Cav-1 plays functions that are important in tumorigenesis and metastasis (see refs. 18–20 for reviews). However, despite significant studies have been done over the last 10 years, its functions in malignancy are very complex and still unclear, as Cav-1 has been described as either a tumor suppressor or a tumor promoter, depending on tumor type and/or stage. Although overexpression of Cav-1 is associated with, and in fact causal in metastasis and progression in some types of carcinomas, particularly prostate cancer (21, 22), in breast cancer as well as fibrosarcoma, Cav-1 has been character- ized as oncosuppressor (23–25). In this article, we validate the role of Cav-1 as oncosuppressor in human osteosarcoma, as we show its down-modulation in more aggressive tumors and show Cav-1 ability to suppress the invasive-metastatic ability of osteosarcoma cells in functional assays in vitro and in vivo . Materials and Methods Cell lines and transfection. The osteosarcoma cell lines U-2 OS, Saos-2, and MG63 were obtained from the American Type Culture Collection. IOR/OS-7 and IOR/OS-9 were obtained at the Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli (Bologna, Italy) and previously characterized (26). Human osteoblasts (HOS) and their Met-transformed variants were obtained at the Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, University of Turin [Candiolo (Turin), Italy] and previously characterized (27). pcDNA3 vector was used to construct plasmids expressing mouse Cav-1 (28), which differs from human Cav-1 at only two amino acids and is functionally indistinguishable from the human protein (15), and human antisense Cav-1 (21). Stable transfectants expressing Cav-1 were obtained from U-2 OS, whereas Cav-1–deficient clones were derived from U-2 OS and Saos-2 by using calcium-phosphate transfection method. Cells transfected with the empty vector pcDNA3 were used as negative controls. Transfectants were selected in Iscove’s modified Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). L. Cantiani and M.C. Manara contributed equally to this work. Requests for reprints: Katia Scotlandi, Laboratory of Oncology Research, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna, Italy. Phone: 39-051-63-66- 760; Fax: 39-051-6366-761; E-mail: katia.scotlandi@ior.it. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4697 www.aacrjournals.org 7675 Cancer Res 2007; 67: (16). August 15, 2007 Research Article Research. on January 3, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from