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Merlin Airik , PhD Post-doctoral fellow

SERUM DEPRIVATION INDUCES EWING SARCOMA CELLS TO ADOPT A MORE INVASIVE PHENOTYPE AND TO UP REGULATE RHO-MKL TRANSCRIPTIONAL SIGNALING Merlin Airik , Melanie Krook , Christopher Scannell , Andrew Haak , Richard R. Neubig , Elizabeth R. Lawlor

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Merlin Airik , PhD Post-doctoral fellow

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  1. SERUM DEPRIVATION INDUCES EWING SARCOMA CELLS TO ADOPT A MORE INVASIVE PHENOTYPE AND TO UP REGULATE RHO-MKL TRANSCRIPTIONAL SIGNALING Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard R. Neubig, Elizabeth R. Lawlor Departments of Pediatrics, Pathology and Pharmacology University of Michigan, Ann Arbor, Michigan, USA

  2. Merlin Airik, PhD Post-doctoral fellow Chris Scannell Graduate student Melanie Krook Graduate student Andrew Haak Graduate student Richard R. Neubig, MD, PhD Dept of Pharmacology Sunbeam Foundation, Russell G. Adderley Endowment UM SARC Sarcoma SPORE (NIH), Liddy Shriver Sarcoma Initiative

  3. Ewing Sarcoma: Metastasis • Can be an early or late event • Many patients with localized disease relapse at metastatic sites months and even years following an initial clinical remission • Phenotypic plasticity of cancer cells is well-characterized in epithelial malignancies: cells transition back and forth between metastatic and non-metastatic phenotypes • Epithelial to mesenchymal transition (EMT) is implicated in the initiation of metastasis of epithelial malignancies Does phenotypic plasticity exist in Ewing sarcoma? What molecular pathways drive metastasis of Ewing sarcoma cells?

  4. Serum deprivation induces cell cycle arrest & morphologic change in Ewing sarcoma cells A. B. 10% 0% + 10% x 2.5hr A673 A673 10%FBS 0%FBS C. D. 10% 0% + 10% x 2.5hr CHLA9 CHLA9

  5. Serum deprived cells exhibit enhanced motility and migration A. B. C. D. xCelligence CIM-plate migration assay 24hr end-point migration assay 10% 0% 10% 0%

  6. Rho-Transcriptional Signaling • Rho-GTPases contribute to cell movement and cancer metastasis • Change so actin cytoskeleton directly in cytoplasm • Activation of downstream transcriptional signaling via translocation of MKL/MRTF to nucleus • Rho-MKL transcriptional axis is required for breast cancer and melanoma metastasis (Medjkane, Nature Cell Biol 2009) • MYL9 and MYH9 implicated as metastasis effectors MKL Nature Reviews Molecular Cell Biology 11, 353-365 (May 2010)

  7. Serum starved Ewing sarcoma cells show dramatic changes in arrangement of the actin cytoskeleton 10% FBS 0% FBS A. B. DAPI Phalloidin

  8. Nuclear localization of MKL & upregulationof MYL9in serum starved Ewing sarcoma cells A. B. C. Normalized ratio Normalized ratio 10% 0% 10% 0% 10% 0% +10% x24h MYL9 MYH9 MYL9

  9. Inhibition of the Rho-MKL transcriptional axis restores morphology & actincytoskeleton A. B. DAPI Phalloidin 10% FBS 0% FBS MKL 0% FBS + 800nM CCG-1423 CCG-1423 CCG-1423: a small molecule inhibitor of RhoA transcriptional signaling Evelyn,…Neubig. Molecular Cancer Therapy 2007

  10. Pharmacologic inhibition of the Rho-MKL transcriptional axis blocks Ewing sarcoma cell motility A673 SFM + 800nM CCG-1423 A673 SFM 0h 12h 24h

  11. Summary • Phenotypic plasticity exists in Ewing sarcoma cells in vitro • Serum starvation induces a rapid and reversible switch to a more migratory phenotype • The migratory phenotype is associated with increased nuclear localization of transcriptional co-activator MKL and increased expression of the metastasis-associated MKL target gene MYL9 • Small molecule inhibitors of Rho-MKL transcriptional axis block Ewing cell migration Activation of Rho-MKL signaling Metastatic Relapse Opportunity for metastasis prevention? Growth factor deprivation Diminished blood supply? Rapid tumor growth? Altered microenvironment? Post-chemotherapy?

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