1. EXPRESSION OF EZRIN mRNA AND PROTEIN IN OSTEOSARCOMA: RELATIONSHIP TO PATIENT CLINICAL CHARACTERISTICS Taiqiang Yan, Rita Kandel, Nalan Gokgoz, Robert Bell, Irene Andrulis & Jay Wunder University Musculoskeletal Oncology Unit, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada

2. Khanna C et al. Cancer Research 2001, 61: 3750-3759 Khanna C et al. Nature Medicine 2004, 10: 182-186 Ezrin was overexpressed in a highly metastatic murine Osteosarcoma cell line Ezrin overexpression was associated with enhanced motility, adherence In an orthotopic murine tumor model, metastases were reduced by Ezrin suppression and increased by transfection of full-length Ezrin constructs Ezrin overexpression was associated with poor outcome in canine and pediatric Osteosarcoma Ezrin

3. Background Ezrin is the best characterized ERM (ezrin- radixin-moesin) protein family member ERM proteins act as linkers between the cell membrane and the actin cytoskeleton ERM proteins are involved in signal transduction through growth factor receptors and adhesion molecules

4. Study Goals Examine level of Ezrin mRNA and protein in biopsy specimens from patients with newly diagnosed Osteosarcoma Compare Ezrin mRNA and protein levels Correlate Ezrin expression with patient clinicopathologic data and outcome

5. Patients and Tumors 39 patients with high grade intramedullary Osteosarcoma 14 patients with mets at diagnosis 25 patients with localized tumors operative biopsy before chemotherapy fresh frozen tumor from biopsy paraffin embedded tissue from biopsy patient follow-up

6. Quantitative Real-Time RT-PCR ABI prism 7900HT system TaqMan probe House keeping gene asparagine synthetase as internal control mRNA levels normalized to a control cell line

7. Immunohistochemistry Paraffin-embedded tissue blocks from Osteosarcoma biopsy specimens prior to chemotherapy Avidin-biotinylated peroxidase complex method Sigma 3C12 monoclonal antibody Assessed % positive staining cells Assessed localization- membrane - cytoplasmic

8. Patient Characteristics CharacteristicsMetastasis at diagnosis No metastasis at diagnosis P value GenderMale: 8 Female: 6 Male: 16 Female: 9 0.31 Mean Age (Years)24.227.50.51 Tumor LocationProximal: 11 Distal: 3 Proximal: 12 Distal: 13 0.09 Mean Tumor Size (cm) 14.29.10.0001

9. Relative value (ezrin/AS)Number of tumors 0.3-0.9912 1.0-1.9910 2.0-2.996 3.0-8.811 1. Ezrin mRNA expression in Osteosarcoma by Real-Time RT-PCR

10. Ezrin mRNA expression by Real-Time RT-PCR and patient characteristics at diagnosis No correlation between Ezrin mRNA expression and patient clinical characteristics Level of Ezrin mRNA was significantly lower in tumors from patients with metastases at diagnosis compared to patients who presented with localized disease (p=0.039)

11. Ezrin mRNA expression and clinical outcome for “curable” patients For 25 patients with localized Osteosarcoma at diagnosis, there was no difference in the level of Ezrin mRNA expression between 9 patients who developed metastases during follow-up and 16 patients who remain free of disease (p=0.28)

12. Ezrin: Cellular Localization Ezrin protein exists in the cytoplasm in an Inactive form Phosphorylation leads to Ezrin Activation and membrane translocation

13. Diffuse Ezrin IHC staining Cytoplasm (arrow) and Membrane staining (Low power) Membrane Ezrin IHC staining (High power) 2. Ezrin Immunohistochemisty

14. Ezrin Immunohistochemisty Ezrin staining (> 10% cells) Metastasis at Diagnosis No Metastasis at Diagnosis Membrane only4/144/25 Membrane + Cytoplasm 2/141/25 Cytoplasm only1/148/25 23/39 (59%) tumors had positive Ezrin IHC staining

15. Ezrin protein expression by IHC and clinical outcome in “curable” patients Patients with localized tumors at diagnosis Ezrin Membrane IHC was positive (>10% cells) in 2/9 (22%) tumors which later metastasized vs 3/16 (19%) tumors without systemic spread We did not identify any correlation between Ezrin IHC and patient characteristics at diagnosis or outcome - 10% IHC staining - any staining - membrane, cytoplasm or both

16. 3. Ezrin mRNA vs protein expression the level of Ezrin mRNA expression did not correlate with protein by IHC 6 of 12 tumors with Ezrin Cytoplasm staining also had mRNA overexpression None of 8 tumors with Ezrin Membrane staining had mRNA overexpression p=0.042

17. Discussion Ezrin Membrane and Cytoplasm staining by IHC in Osteosarcoma A distinctive Membranous Ezrin IHC pattern was seen more commonly in tumors from patients with metastases at diagnosis Ezrin activation and binding to the cell membrane may facilitate early invasion and metastasis in Osteosarcoma Cytoplasmic Ezrin staining was more common in tumors localized at diagnosis Ezrin mRNA expression only correlated with cytoplasmic staining

18. Discussion Lack of Ezrin mRNA – Ezrin protein correlation: Different mechanisms of transcription/translation Overactivation of post-translational modification in tumors with metastases at diagnosis Different Ezrin isoforms due to alt. splice variants - Original cDNA cloning paper (Gould et al, EMBO 1989) - Genomic structure

19. Acknowledgements Ontario Cancer Research Network (OCRN) National Cancer Institute of Canada (NCIC) Canadian Institutes of Health Research (CIHR) Interdisciplinary Health Research Team (IHRT) in Musculoskeletal Neoplasia Rubinoff-Gross Chair in Orthopaedic Oncology at Mount Sinai Hospital, University of Toronto

22. Hunter K. Ezrin, a key component in tumor metastasis. Trends in Molecular Medicine, 2004, 10: 201-204 Regulate cellular activities, such as survival, adhesion, migration and invasion Cell-cell interaction Cell-matrix interaction MAPK signal transduction Rho GTPase signal transduction Akt mediated cellular apoptosis

23. There is a significant correlation between increased ezrin immunoreactivity and higher histological grade in astrocytoma. Geiger KD, etc. Ezrin immunoreactivity is associated with increasing malignancy of astrocytic tumors but is absent in oligodendrogliomas. Am J Pathol 2000, 157:1785-93. Ezrin expression correlates with tumor thickness and level of invasion in primary cutaneous melanoma. Ilmonen S, etc. Ezrin in primary cutaneous melanoma. Mod Pathol 2005, 18:503-10.

24. Ezrin has been reported to be involved in dissemination of soft tissue sarcoma, and could be valuable as an additional prognostic marker. Weng WH, etc. Prognostic impact of immunohistochemical expression of ezrin in highly malignant soft tissue sarcomas. Clin Cancer Res, 2005, 11: 6198-6204. Ezrin was also indicated as a key metastatic regulator in rhabodomysarcoma and breast cancer. Yu Y, etc. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 2004, 10:175-81. Elliott BE, etc. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res 2005, 7:R365-73.

25. Future Directions Ezrin isoform characterization Growth inhibition by siRNA to confirm whether Ezrin is involved in cell proliferation Antibody to phosphorylated Ezrin to determine functional status of protein subcellular distribution

26. Khanna C, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis, Nature Medicine, 2004, 10: 182-186 Canine OSA (n=83) Pediatric OSA (n=19)

27. No Mets at Diagnosis Mets at Diagnosis Microarray Analysis of Osteosarcoma Ezrin Yan et al, CTOS 2004 Khanna et al, Cancer Res 2001 Leonard et al, Br J Cancer 2003