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EWS/FLI Regulates NKX2.2 via GLI EWS/FLI Regulates NKX2.2 via GLI in a Non-SHH-Dependent Mannerin a Non-SHH-Dependent Manner
Savita SankarSavita Sankar11, Leah A. Owen, Leah A. Owen11 and andStephen L. LessnickStephen L. Lessnick1,21,2
11Department of Oncological Sciences, Center for Department of Oncological Sciences, Center for Children at Huntsman Cancer Institute, and Children at Huntsman Cancer Institute, and 22Division of Division of
Pediatric Hematology/Oncology, University of Utah Pediatric Hematology/Oncology, University of Utah School of Medicine, Salt Lake City, UT School of Medicine, Salt Lake City, UT
EWS/FLI is an Aberrant EWS/FLI is an Aberrant Trancription FactorTrancription Factor
EWS/FLI ChIP-chipEWS/FLI ChIP-chip
EWS
FLIFormaldehyde crosslinking, DNA shearing
EWS
FLI
Recover DNA, hybridize to Agilent promoter
microarray
~900 “direct” EWS/FLI target
promoters/genes
high-affinity siteE-value = 4.7x10-43
repetitive elementE-value = 5x10-175
EWS/FLI Response ElementsEWS/FLI Response Elements
EWS/FLI responseelement in NR0B1promoter
NKX2.2 is Necessary forNKX2.2 is Necessary forOncogenic TransformationOncogenic Transformation
Regulation of NKX2.2Regulation of NKX2.2
NKX2.2 was not on ChIP-chip listNKX2.2 was not on ChIP-chip list NKX2.2 promoter does not have obvious NKX2.2 promoter does not have obvious
EWS/FLI binding siteEWS/FLI binding site Implies NKX2.2 is not a direct target of Implies NKX2.2 is not a direct target of
EWS/FLIEWS/FLI Reporter assays also suggest NKX2.2 is Reporter assays also suggest NKX2.2 is
not a direct EWS/FLI targetnot a direct EWS/FLI target How is NKX2.2 regulated?How is NKX2.2 regulated?
NKX2.2NKX2.2
Involved in CNS developmentInvolved in CNS development Involved in pancreatic islet developmentInvolved in pancreatic islet development Involved in oligodendrocyte developmentInvolved in oligodendrocyte development Regulated by Sonic Hedgehog in CNSRegulated by Sonic Hedgehog in CNS Is NKX2.2 regulated by Sonic Hedgehog in Ewing’s Is NKX2.2 regulated by Sonic Hedgehog in Ewing’s
sarcoma?sarcoma?
Sonic Hedgehog PathwaySonic Hedgehog Pathway
cyclopamine
Cyclopamine treatment of Ewing’s sarcoma cell lines did not reduce NKX2.2 expression (with appropriate controls).
Other possibilities?
GLI1 as a potential NKX2.2 GLI1 as a potential NKX2.2 regulatorregulator
GLI consensus human: -1966-CCCGGGTCCGACCACCCAGGACGCGCC-1939mouse: -1976-CCCGGGTCCGACCACCCAGGACGCGCC-1949zebrafish: -2323-CCCGGGTCCGACCACCCAGGACGCGCC-2296
1) GLI1 is upregulated by EWS/FLI (on expression microarray dataset)2) GLI1 was on ChIP-chip list for EWS/FLI (likely direct target)3) GLI consensus binding site in NKX2.2 promoter, conserved across species
EWS
FLIGLI1 NKX2.2
GLI1
Working model
GLI1 is Regulated by EWS/FLIGLI1 is Regulated by EWS/FLI
GLI1-Knockdown Reduces GLI1-Knockdown Reduces NKX2.2 ExpressionNKX2.2 Expression
GLI1 Expression is Required for GLI1 Expression is Required for Oncogenic TransformationOncogenic Transformation
Still to do...Still to do...
Is NKX2.2 the only critical GLI1-regulated Is NKX2.2 the only critical GLI1-regulated gene, or are others required?gene, or are others required?
More detailed analysis of NKX2.2 More detailed analysis of NKX2.2 promoterpromoter
More detailed analysis of GLI1 promoterMore detailed analysis of GLI1 promoter
Lessnick labSam FrancisKunal GangwalMichelle Kinsey Ashley KowalewskiWen LuoSavita SankarRichard SmithTish Toomey
Chi BraunreiterJeff HancockLeah OwenDeb Trem
FundingNational Cancer Institute
American Cancer SocietyTerri Anna Perine Sarcoma Fund
Liddy Shriver Sarcoma InitiativeSunbeam Foundation
Department of Pediatrics, U of UtahHuntsman Cancer Foundation
Huntsman Cancer Institute CCSG
Barbara GravesPete Hollenhorst
Cheryl CoffinLor RandallKen BoucherCody HaroldsenAtul ShahLynn JordeScott Watkins
Todd GolubKim StegmaierAndrew Kung
Email: [email protected]: International Ewing’s Sarcoma
Research Forum