Proffered Papers— Basic Science/Biology

DIFFERENTIAL EXPRESSION OF EZRIN IN A HIGH AND LOW METASTATIC OSTEOSARCOMA MODEL

Khanna C, Prehn J, Khan J, Nguyen P, Trepel J, Meltzer P, Helman L., (Pediatric Oncology and Medicine Branches, National Cancer Institute, and Cancer Genetics Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892)

Osteosarcoma (OSA) is the most common primary tumor of bone. Despite successful control of the primary tumor and adjuvant chemotherapy, relapse of OSA in the lungs occurs in over 30% of patients within five years. In order to understand the complex process of metastasis from appendicular tumors to the lungs, we have used cDNA microarray to define differences in gene expression between clonally related murine model variants of osteosarcoma that differ in pulmonary metastatic potential. The murine osteosarcoma models are characterized by orthotopic growth at appendicular sites in balb/c mice, spontaneous pulmonary metastasis to the lungs, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential.

A 4000 gene cDNA microarray was used to compare gene expression between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) models. Differentially expressed genes were defined by a red to green ratio not equal to 1.0 with 99% confidence (> or < 1.0 +- 99% CI), a mean maximum signal intensity greater than 2000, and concordant differential expression in two separate microarray hybridizations. Eighty genes were defined as differentially expressed between K7M2 and K12. Forty-two genes were over-expressed in K7M2 compared to K12 and 38 genes were over-expressed in K12 compared to K7M2.

A functional approach to the analysis of the differentially expressed genes was taken, by assigning each gene to six6 non-mutually exclusive metastasis-associated categories (using the PubMed and OMIM databases): proliferation and apoptosis, motility and cytoskeleton, invasion, immune-surveillance, adherence and angiogenesis. The high and low metastatic variants were then evaluated within each of these metastasis-associated processes. Functional studies demonstrated significant differences in motility, adherence, and angiogenesis that favored the aggressive behavior in K7M2 compared to K12. For this reason, the differentially expressed genes with motility, adherence,and angiogenesis associated functions were considered more likely to explain differences in the metastatic behavior of K7M2 and K12 than genes associated with proliferation and apoptosis, invasion and immune-surveillance.

This approach brought attention to ezrin, a motility and adherence gene not previously described in OSA, that was found over-expressed in K7M2 compared to K12 tumors. We have confirmed differential expression of ezrin by Northern analysis. Immunocyostaining for ezrin has confirmed increased levels of ezrin protein and demonstrated the enhanced localization of ezrin at the cell membrane in K7M2 compared to K12 cells. Using Northern analysis, we have demonstrated the expression of ezrin in dogs with naturally occurring osteosarcoma and in 5/5 human osteosarcoma cell lines. Our ongoing work will attempt to define the biological role of ezrin in osteosarcoma and the relevance of ERM proteins (ezrin, radixin, and moesin) in human cases of osteosarcoma.