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?