2001 CTOS Annual Meeting Posters— Biology

EVALUATION OF EZRIN, A NOVEL DETERMINANT FOR METASTASIS IN OSTEOSARCOMA: A COMPARATIVE APPROACH.
Chand Khanna¹,  Seuli Bose¹,  Sung-Hyeok Hong¹,  Ryan Cassaday¹,  Stephen Hewitt²,  Richard Gorlick³,  Lee Helman^1
1Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health,  ²Laboratory of Pathology Tissue Array Research Project, National Cancer Institute, National Institutes of Health,  ³Memorial Sloan-Kettering

METHODS: Using cDNA microarrays and a metastasis based methodology for array evaluation we have defined 11 genes (out of 3899 cDNAs) most likely to explain differences in the behavior of a high (K7M2) and low (K12) metastatic model of murine osteosarcoma. Ezrin, a gene not previously described in osteosarcoma, was selected for further evaluation based on its pivotal role in linking the actin cytoskeleton to the cell membrane. Evaluation of ezrin in the murine osteosarcoma models included Northern, Western, and immunocytochemistry. The function of ezrin was studied by generating K7M2 cells with low expression of ezrin through the stable transfection of these cells with a full-length antisense ezrin construct. Preliminary evaluation of ezrin function included transwell® matrigel invasion and heterotypic adhesion assays. The relevance of ezrin in osteosarcoma was examined in a canine osteosarcoma tissue array consisting of 75 canine osteosarcoma primary tumors and 11 pulmonary metastases. The expression of ezrin in human osteosarcoma was examined by immunohistochemistry.

RESULTS: Differential expression of ezrin between K7M2 and K12 models by Northern was concordant with microarray (2.9:1). Evidence for differential post-transcriptional regulation came from Western analysis where an 8.0 fold increase in ezrin protein was seen in highly metastatic K7M2 compared to poorly metastatic K12. Immunostaining was consistent with Western results and supported greater membrane (active) localization of ezrin in the more aggressive K7M2 cells. Stable antisense ezrin transfection of the K7M2 cells resulted in clones with low (K7M2-AS1.42 and K7M2-AS1.56) and intermediate (K7M2-AS13) ezrin protein levels. Functional studies compared the antisense ezrin transfectants with an empty vector control (K7M2-EV) and the wild-type K7M2 and K12 cells. K7M2-AS1.42 and K7M2-AS1.56 had notably decreased invasiveness through matrigel compared to K7M2-AS13 and the K7M2-EV control (p=0.06 and p=0.05 respectively). K7M2-AS1.42, K7M2-AS1.56 and K7M2-AS13 had heterotypic adherence kinetics similar to the less metastatic K12 cells. Immunohistochemistry for ezrin using the canine osteosarcoma tissue array demonstrated high ezrin expression in 100%(11/11) of pulmonary metastases compared to 30% (42/70) of primary tumors (p=0.0001). Furthermore dogs without primary tumor ezrin had longer median survival than dogs with ezrin staining (Ezrin(-) 280 days vs Ezrin(+) 136 days; p=0.10). In frozen pediatric osteosarcoma ezrin expression was seen in 8/12 tissues by immunohistochemistry. High intensity staining was seen in 0/7 primary tumors and 2/5 pulmonary metastases.

CONCLUSION: This comparative approach has demonstrated the relevance and potential importance of ezrin in the biology of osteosarcoma metastasis. Preliminary results suggest a role of ezrin during cellular invasion and heterotypic adhesion. Ongoing efforts will define the role of ezrin in vivo and through all steps of the metastatic cascade. Staining of a recently constructed pediatric osteosarcoma tissue array has been completed and will define the relevance of ezrin in pediatric osteosarcoma.