hSMC2, novel transcriptional target of Wnt signaling pathway
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Transcript of hSMC2, novel transcriptional target of Wnt signaling pathway
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hSMC2, novel transcriptional target of Wnt signaling pathway
Lucía Suárez-López Drug delivery and targeting unit
CIBBIM-Nanomedicine VHIR meeting 2012
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Adapted from Davies, R. J., et al. 2005.
Colon cancer progression model
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Pathway members altered in 90% of CRC
Adapted from Davies, R. J., et al. 2005.
Colon cancer progression model
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Wnt pathway along the colon crypt
Wnt ligands
WNT ON
WNT OFF
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Wnt pathway along the colon crypt
Aberrant crypt foci
Tumorogenesis initiation
Wnt ligands
WNT ON
WNT OFF
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Wnt pathway OFF
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Wnt pathway OFF ON
C-MYC, Cyclin D
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ATPases highly conserved along evolution
Arqueobacteria Mammals
Responsible for Higher-order chromosome organization and dynamics
Tatsuya Hirano, 2010
SMC family: Structural Maintenance of Chromosomes
SMC2 & SMC4 = Condensin Complex
SMC2 SMC4
Non-SMC regulatory subunits
SMC core members
• Condensin I • Condensin II
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ATPases highly conserved along evolution
Arqueobacteria Mammals
Responsible for Higher-order chromosome organization and dynamics
Tatsuya Hirano, 2010
SMC family: Structural Maintenance of Chromosomes
SMC2 & SMC4 = Condensin Complex
SMC2 SMC4
Non-SMC regulatory subunits
SMC core members
• Condensin I • Condensin II
Introduces positive supercoilings into DNA
DNA
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Condensin complex is upregulated in CRC
SMC2 CAP-G CAP-G2 CAP-H
• QPCR on CRC samples
*** *** *** ***
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Condensin complex is upregulated in CRC
SMC2 CAP-G CAP-G2 CAP-H
• QPCR on CRC samples
*** *** *** ***
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SMC2 is upregulated in CRC
31 35 36 38 85 86 N T N T N T N T N T N T
SMC2
ACTIN
Case
150 kDa
37 kDa
• Western Blot on CRC paired samples
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SMC2 is upregulated in CRC
31 35 36 38 85 86 N T N T N T N T N T N T
SMC2
ACTIN
Case
150 kDa
37 kDa
• Western Blot on CRC paired samples
SMC2 is up-regulated in 20 out 29 tumor samples: 69%
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• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
SMC2 is upregulated in CRC
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• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
SMC2 is upregulated in CRC
Colon adenocarcinoma
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• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
SMC2 is upregulated in CRC
Normal mucosa
Colon adenocarcinoma
Wnt-target genes expression
pattern
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SMC2 expression is linked to β-catenin
Wnt signaling activation Nuclear accumulation of β-catenin
Membrane β-catenin
Nuclear β-catenin
β-catenin SMC2
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SMC2 expression is linked to β-catenin
Wnt signaling activation Nuclear accumulation of β-catenin
Membrane β-catenin
Nuclear β-catenin
β-catenin
SMC2 is up-regulated in tumors where β-catenin is nuclear
SMC2
Fisher exact test p=0,04, N=43
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Is SMC2 a target of Wnt/β-catenin pathway?
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SMC2 is down-regulated upon Wnt inhibition
TCF-4
SMC2
C-MYC
ACTIN
- + - + - + - +
Time 24h 48h 72h 96h
Dox
Ls174T/dnTCF4 Dominant negative form of TCF4
Ls174T/pTER-bCAT siRNA against β-catenin
- + - + - + - +
Time 24h 48h 72h 96h
Dox
SMC2
C-MYC
ACTIN
β-catenin
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SMC2 is down-regulated upon Wnt inhibition
TCF-4
SMC2
C-MYC
ACTIN
- + - + - + - +
Time 24h 48h 72h 96h
Dox
Ls174T/dnTCF4 Dominant negative form of TCF4
Ls174T/pTER-bCAT siRNA against β-catenin
- + - + - + - +
Time 24h 48h 72h 96h
Dox
SMC2
C-MYC
ACTIN
β-catenin
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SMC2 is down-regulated upon Wnt inhibition
TCF-4
SMC2
C-MYC
ACTIN
- + - + - + - +
Time 24h 48h 72h 96h
Dox
Ls174T/dnTCF4 Dominant negative form of TCF4
Ls174T/pTER-bCAT siRNA against β-catenin
- + - + - + - +
Time 24h 48h 72h 96h
Dox
SMC2
C-MYC
ACTIN
β-catenin
SMC2 is under β-catenin/TCF4 regulation, but is it direct or indirect?
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TCF4 is bound to SMC2 promoter in vivo
TSS
+1 -595
TATA
-561
Sp1
-301
Sp1
+219
Sp1 TATA
-12
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TCF4 is bound to SMC2 promoter in vivo
TSS
+1 -595
TATA
-561
Sp1
-301
Sp1
+219
Sp1 TATA
-12
TBE 2 TBE 3
Hs 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGGTGTTGA 74
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pt 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGGTGTTGA 74
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||
Mmt 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGG---TGA 71
.|.|.||...|||||||||||||||||||||||||||||||||||| |||.|||||||||.|||.|||||
Rn 1 CAGACGCGTCGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAG----AAACAGTTCCGGCACGGTTGTTG- 69
.|.|.||...||||.|||||||||||||||||||||||||||||||| ||.|||||||||.|||.|||||
Mms 1 CAGACGCCTCGGAGTTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGG----AACAGTTCCGGCACGGTTGTTG- 69
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TCF4 is bound to SMC2 promoter in vivo
TSS
+1 -595
TATA
-561
Sp1
-301
Sp1
+219
Sp1 TATA
-12
TBE 2 TBE 3
Hs 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGGTGTTGA 74
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pt 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGGTGTTGA 74
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||
Mmt 1 AATAAGCAATGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGGAATAAATAGTTCCGGCGCGGG---TGA 71
.|.|.||...|||||||||||||||||||||||||||||||||||| |||.|||||||||.|||.|||||
Rn 1 CAGACGCGTCGGAGGTGGGGTCCTTTGCTCGCGCCGAAATTCAAAG----AAACAGTTCCGGCACGGTTGTTG- 69
.|.|.||...||||.|||||||||||||||||||||||||||||||| ||.|||||||||.|||.|||||
Mms 1 CAGACGCCTCGGAGTTGGGGTCCTTTGCTCGCGCCGAAATTCAAAGG----AACAGTTCCGGCACGGTTGTTG- 69
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Identification of the TCF4 responding element in SMC2 promoter
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
• Luciferase reporter assays
WT
2Mut
3Mut
1/2/4/5 Mut
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Identification of the TCF4 responding element in SMC2 promoter
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
• Luciferase reporter assays
DLD-1 cells HCT116 cells
pgl3b WT 2Mut 3MUT 1/2/4/5Mut pgl3b WT 2Mut 3MUT 1/2/4/5Mut
WT
2Mut
3Mut
1/2/4/5 Mut
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Identification of the TCF4 responding element in SMC2 promoter
• Luciferase reporter assays
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
DLD-1 cells
pgl3b WT 3MUT 1/2/4/5Mut
HCT116 cells
pgl3b WT 3MUT 1/2/4/5Mut
WT
2Mut
3Mut
1/2/4/5 Mut
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Identification of the TCF4 responding element in SMC2 promoter
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
Luc+ 1 2 3 4 5
• Luciferase reporter assays
TBE3 is responsible for
β-catenin/TCF4 transactivation of SMC2 promoter
DLD-1 cells HCT116 cells
pgl3b WT 3MUT 1/2/4/5Mut pgl3b WT 3MUT 1/2/4/5Mut
WT
2Mut
3Mut
1/2/4/5 Mut
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SMC2
SMC4
NCAPH
GAPDH
sc SMC2 sc SMC2 sc SMC2
24 48 72Time (h):
siRNA:
SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
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SMC2
SMC4
NCAPH
GAPDH
sc SMC2 sc SMC2 sc SMC2
24 48 72Time (h):
siRNA:
SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
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SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA SMC2
siRNA scrambled
DLD1 cells Mice injection 48h 24h
1st transfection
2nd transfection
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SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA SMC2
siRNA scrambled
DLD1 cells Mice injection 48h 24h
1st transfection
2nd transfection
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SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA SMC2
siRNA scrambled
DLD1 cells Mice injection 48h 24h
1st transfection
2nd transfection
SMC2 is a new potential
therapeutic target
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Summary
1. Condensin complex is up-regulated in colon cancer
2. SMC2 is under direct regulation of β-catenin/TCF4 complex
3. SMC2 is proposed as new potential therapeutic target for CRC treatment
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Acknowledgments
DRUG DELIVERY AND TARGETING GROUP Verónica Dávalos Julio Castaño Anthea Messent Simó Schwartz Navarro
FUNCTIONAL VALIDATION AND PRE-CLINICAL RESEARCH Yolanda Fernández Ibane Abásolo