serum deprivation induces ewing sarcoma cells to adopt a more invasive phenotype and to up regulate...
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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 PharmacologyUniversity of Michigan, Ann Arbor, Michigan, USA
Merlin Airik, PhDPost-doctoral fellow
Melanie KrookGraduate student
Chris ScannellGraduate student
Andrew HaakGraduate student
Richard R. Neubig, MD, PhDDept of Pharmacology
Sunbeam Foundation, Russell G. Adderley Endowment UM SARC Sarcoma SPORE (NIH), Liddy Shriver Sarcoma Initiative
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?
Serum deprivation induces cell cycle arrest & morphologic change in Ewing sarcoma cells
10%FBS0%FBS
A673
CHLA9
10% 0%
10% 0%A.
C.
B.
D.
A673
CHLA9
+ 10% x 2.5hr
+ 10% x 2.5hr
A. B.
Serum deprived cells exhibit enhanced motility and migration
10%0%
10% 0%
C. D.xCelligence CIM-plate migration assay 24hr end-point migration assay
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
Nature Reviews Molecular Cell Biology 11, 353-365 (May 2010)
MKL
Serum starved Ewing sarcoma cells show dramatic changes in arrangement of the actin cytoskeleton
A.
B.
10% FBS 0% FBS
DAPI
Phalloidin
Nuclear localization of MKL & upregulation of MYL9 in serum starved Ewing sarcoma cells
MYL910% 0% +10% x24h
Nor
mal
ized
ratio
MYL9 MYH910% 0% 10% 0%
Nor
mal
ized
ratio
A.
C.
B.
Inhibition of the Rho-MKL transcriptional axis restores morphology & actin cytoskeleton
MKL
CCG-1423
CCG-1423: a small molecule inhibitor of RhoA transcriptional signalingEvelyn,…Neubig. Molecular Cancer Therapy 2007
A. B.
10% FBS
0% FBS
0% FBS+800nM CCG-1423
DAPIPhalloidin
0h
24h
12h
A673SFM
A673SFM + 800nM CCG-1423
Pharmacologic inhibition of the Rho-MKL transcriptional axis blocks Ewing sarcoma cell motility
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
Metastatic Relapse
Activation of Rho-MKL signaling
Opportunity for metastasis prevention?
Growth factor deprivation Diminished blood supply? Rapid tumor growth? Altered microenvironment? Post-chemotherapy?
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