Annexin 2 expression is reduced in human osteosarcoma metastases. Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adults. The development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. We used representational difference analysis to isolate genes differentially expressed between primary human osteosarcoma tumors and subsequent metastatic lung lesions to identify genes potentially involved in metastatic potential. Several genes were differentially expressed between the two tumor populations, including annexin2. The levels of annexin2 mRNA and protein inversely correlated with metastatic potential in a subset of human osteosarcoma tumor specimens, as well as in a human osteosarcoma cell line selected for increased metastatic potential. Annexin2 has been described in several cellular localizations with various functional implications, many of which may be relevant to metastatic potential. Therefore, the subcellular localization of endogenous annexin2 protein was evaluated biochemically by subcellular fractionation and immunologically by flow cytometry and immunofluorescence in osteoblastic cells. Annexin2 was localized to the cytoplasm and intracellular aspect of the plasma membrane, excluded from the nucleus and undetectable on the cell surface or in the conditioned medium. Overexpression of annexin2 in osteosarcoma cells did not alter several in vitro phenotypes often used to assess metastatic potential including motility, adhesion, and proliferation. However, our previous data have implicated annexin2 in the mineralization process of osteoblastic cells in vitro. Consistent with an increase in differentiation-induced mineralization, there was diminished tumorigenicity and experimental metastatic potential of osteosarcoma cells overexpressing annexin2. These data suggest that annexin2 may downregulate osteosarcoma aggressiveness by inducing a more differentiated state in osteoblastic cells.Copyright 2004 Wiley-Liss, Inc.