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High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma

High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma. Kartik Krishnan, Gaurav Khanna, Stephen Hewitt, Chand Khanna, and Lee J. Helman. Pediatric Oncology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland USA. Mice. 1. 0.

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High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma

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  1. High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma Kartik Krishnan, Gaurav Khanna, Stephen Hewitt, Chand Khanna, and Lee J. Helman Pediatric Oncology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland USA

  2. Mice 1 . 0 Low Ezrin 0 . 8 0 . 6 Intermediate Ezrin 0 . 4 High Ezrin 0 . 2 0 . 0 0 2 5 5 0 7 5 1 0 0 1 2 5 D a y s Dogs Humans Ezrin Expression Correlates with Survival in Osteosarcoma Khanna C, et al., Nature Medicine, 10 (2); 2004

  3. Ezrin Expression in Ewings Sarcoma

  4. LD VW WV 5838 KAD TC71 RDES Primary Cultures Cell Lines 202 IB: Mouse MAb a-Ezrin (Sigma) 96 66 45 VW 18 y.o. female presented in 1/01 with widely disseminated disease involving multiple bony sites including femurs, vertebrae, and skull. Diagnosed with Ewing’s sarcoma with Type IV translocation. Progressed on chemo and radiation therapy. Died 8/01. VWCell line Primary xenograft developed tumors rapidly. Stable tissue culture growth established at approximately one year, confirmed presence of type IV EWS-Fli1 translocation. In Mice, orthotopic implantation resulted in tumors with a latency between 21 and 56 days. Experimental metastases developed with intravenous injection between 45 and 80 days.

  5. T567 Y143 Y353 Band 4.1 homology a-helix F-actin Binding N C EZRIN • Membrane-cytoskeleton linker protein • Initially identified as a determinant of cell shape • Plays a critical role in cell motility and attachment • Activatation initiated by threonine phosphorylation at position 567 • Subsequent tyrosine phosphorylation allows for interaction with signal transduction pathways

  6. GFP CMV Selected Clones Unselected Batch VW-GFP VW-A VW EzrinT567A-GFP T567A WT WT Ezrin WB: a-Ezrin DominantNegativeClones EZRIN T567A

  7. Ezrin Bjornsti and Houghton Nat Rev Cancer Cyclin D1 MYC HIF 1a

  8. VW-GFP Ezrin T567A AKT VW-GFP Serum: - + - + IB: a-P-Ser473 AKT IB: a-AKT

  9. 4EBP1 VW-GFP Ezrin T567A VW-GFP Serum Stimulation (minutes) 0 30 120 0 30 120 IB: a-P-Thr37-Thr46-4EBP1 IB: a-4EBP1

  10. p42/44 MAPK VW-GFP Ezrin T567A VW-GFP Serum: - + - + IB: a-P-Thr202Tyr204 p42/44 IB: a-p42/44

  11. Mice 1 X 106 cells injected IV Assessed for development of experimental metastases At Day 70: VW-GFP 3 / 6 VW-GFP-EzrinT567A 0 / 6

  12. Conclusions Ezrin is highly and ubiquitously expressed in Ewing’s sarcoma. Expression of a Ezrin containing a non-phosphorylatable mutation at threonine 567 functions as dominant negative. These dominant negative effects are observed on AKT, as well as 4EBP1. Ewing’s sarcoma cells expressing a dominant negative Ezrin form experimental metastases at a lower rate than wild type.

  13. Future Directions • Expression of Ezrin anti-sense to completely remove Ezrin expression and evaluate growth, survival, and signaling. • Analysis of effect of dominant negative and anti-sense Ezrin in other Ewing’s sarcoma cell lines, each with a different base line level of Ezrin expression. • Orthotopic implantation of dominant negative expressing cell lines, followed by amputation in mice to evaluate metastatic potential of these cells. • Further molecular studies: • What other signal transduction pathways involve Ezrin? • What are the Ezrin kinases? Are they suitable targets for small molecule inhibition? c. Proteomics

  14. Pediatric Oncology Branch National Cancer Institute, NIH Lee Helman Choh Yeung Melissa Burgos Shaan Gandhi Tumor and Metastasis Biology Section Pediatric Oncology Branch National Cancer Institute, NIH Chand Khanna Gaurav Khanna Arnulfo Mendoza Tissue Array Research Project National Cancer Institute, NIH Stephen Hewitt Acknowledgments

  15. Highly Metastatic K7M2 Primary Tumor VS Less Metastatic K12 Primary Tumor K7M2 Wild type K12 Wild type Murine Microarray Analysis: EZRIN Khanna C, et al., Cancer Research, 61 ;2001

  16. K7M2/AS Ezrin 1.46 K7M2/AS Ezrin 2.12 K7M2/AS Ezrin 2.13 K7M2/AS Ezrin 2.15 K7M2/AS Ezrin 1.52 K7M2/AS Ezrin 13 K7M2 wt K12 wt Ezrin Tubulin Relative Expression 12.4 1.0 1.1 1.1 12.2 15.9 2.8 21.3

  17. mTOR Signaling Pathway (PIK-related kinase, related to ATM, TRRAP etc.) Bjornsti and Houghton Nat Rev Cancer

  18. Osteosarcoma Ezrin

  19. CD44, PDGFRA F-Actin Threonine 567 Phosphorylation Tyrosine Phosphorylation P P P p85 PI3K P AKT Growth, Survival RhoGDI

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