hSMC2, novel transcriptional target of Wnt signaling pathway Lucía Suárez-López Drug delivery and targeting unit CIBBIM-Nanomedicine VHIR meeting 2012
hSMC2, novel transcriptional target of Wnt signaling pathway
Lucía Suárez-López Drug delivery and targeting unit
CIBBIM-Nanomedicine VHIR meeting 2012
Adapted from Davies, R. J., et al. 2005.
Colon cancer progression model
Pathway members altered in 90% of CRC
Adapted from Davies, R. J., et al. 2005.
Colon cancer progression model
Wnt pathway along the colon crypt
Wnt ligands
WNT ON
WNT OFF
Wnt pathway along the colon crypt
Aberrant crypt foci
Tumorogenesis initiation
Wnt ligands
WNT ON
WNT OFF
Wnt pathway OFF
Wnt pathway OFF ON
C-MYC, Cyclin D
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
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
Condensin complex is upregulated in CRC
SMC2 CAP-G CAP-G2 CAP-H
• QPCR on CRC samples
*** *** *** ***
Condensin complex is upregulated in CRC
SMC2 CAP-G CAP-G2 CAP-H
• QPCR on CRC samples
*** *** *** ***
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 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%
• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
SMC2 is upregulated in CRC
• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
SMC2 is upregulated in CRC
Colon adenocarcinoma
• 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
SMC2 expression is linked to β-catenin
Wnt signaling activation Nuclear accumulation of β-catenin
Membrane β-catenin
Nuclear β-catenin
β-catenin SMC2
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
Is SMC2 a target of Wnt/β-catenin pathway?
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 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 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?
TCF4 is bound to SMC2 promoter in vivo
TSS
+1 -595
TATA
-561
Sp1
-301
Sp1
+219
Sp1 TATA
-12
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
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
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
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
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
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
SMC2
SMC4
NCAPH
GAPDH
sc SMC2 sc SMC2 sc SMC2
24 48 72Time (h):
siRNA:
SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
SMC2
SMC4
NCAPH
GAPDH
sc SMC2 sc SMC2 sc SMC2
24 48 72Time (h):
siRNA:
SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA SMC2
siRNA scrambled
DLD1 cells Mice injection 48h 24h
1st transfection
2nd transfection
SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA SMC2
siRNA scrambled
DLD1 cells Mice injection 48h 24h
1st transfection
2nd transfection
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
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
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