Lucía Suárez' predoctoral presentation at the 6th VHIR Scientific Session. Watch the video of the presentation after the last slide.

1. hSMC2, novel transcriptional
target of Wnt signaling pathway
Lucía Suárez-López
Drug delivery and targeting unit
CIBBIM-Nanomedicine
VHIR meeting 2012

2. Colon cancer progression model
Adapted from Davies, R. J., et al. 2005.

3. Colon cancer progression model
Pathway members altered in 90% of CRC
Adapted from Davies, R. J., et al. 2005.

4. Wnt pathway along the colon crypt
WNT OFF
WNT ON
Wnt ligands

5. Wnt pathway along the colon crypt
WNT OFF
WNT ON Aberrant crypt foci
Tumorogenesis initiation
Wnt ligands

6. Wnt pathway
OFF

7. Wnt pathway
OFF ON
C-MYC,
Cyclin D

8. SMC family: Structural Maintenance of Chromosomes
 ATPases highly conserved along evolution
Arqueobacteria  Mammals
 Responsible for Higher-order chromosome organization and dynamics
SMC2 & SMC4 = Condensin Complex
SMC2 SMC4
SMC core members
Non-SMC regulatory subunits
• Condensin I
• Condensin II
Tatsuya Hirano, 2010

9. SMC family: Structural Maintenance of Chromosomes
 ATPases highly conserved along evolution
Arqueobacteria  Mammals
 Responsible for Higher-order chromosome organization and dynamics
SMC2 & SMC4 = Condensin Complex
SMC2 SMC4
SMC core members
Non-SMC regulatory subunits
• Condensin I
• Condensin II
Introduces positive supercoilings into DNA
DNA
Tatsuya Hirano, 2010

10. Condensin complex is upregulated in CRC
• QPCR on CRC samples
SMC2 CAP-G CAP-G2 CAP-H
*** *** *** ***

11. Condensin complex is upregulated in CRC
• QPCR on CRC samples
SMC2 CAP-G CAP-G2 CAP-H
*** *** *** ***

12. SMC2 is upregulated in CRC
• Western Blot on CRC paired samples
Case 31 35 36 38 85 86
N T N T N T N T N T N T
SMC2 150 kDa
ACTIN 37 kDa

13. SMC2 is upregulated in CRC
• Western Blot on CRC paired samples
Case 31 35 36 38 85 86
N T N T N T N T N T N T
SMC2 150 kDa
ACTIN 37 kDa
SMC2 is up-regulated in 20 out 29 tumor samples: 69%

14. SMC2 is upregulated in CRC
• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.

15. SMC2 is upregulated in CRC
• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
Colon adenocarcinoma

16. SMC2 is upregulated in CRC
• IHC on CRC paraffin embebed tissues: SMC2 up-regulated in tumoral counterparts.
Normal mucosa
Wnt-target genes
expression
pattern
Colon adenocarcinoma

17. SMC2 expression is linked to β-catenin
Wnt signaling activation Nuclear accumulation of β-catenin
β-catenin SMC2
Membrane
β-catenin
Nuclear
β-catenin

18. SMC2 expression is linked to β-catenin
Wnt signaling activation Nuclear accumulation of β-catenin
β-catenin SMC2
Membrane
β-catenin
Nuclear
β-catenin
Fisher exact test p=0,04, N=43
SMC2 is up-regulated in tumors where β-catenin is nuclear

19. Is SMC2 a target of Wnt/β-catenin pathway?

20. SMC2 is down-regulated upon Wnt inhibition
Ls174T/dnTCF4 Ls174T/pTER-bCAT
Dominant negative form of TCF4 siRNA against β-catenin
Time 24h 48h 72h 96h Time 24h 48h 72h 96h
Dox - + - + - + - + Dox - + - + - + - +
TCF-4 β-catenin
C-MYC C-MYC
SMC2 SMC2
ACTIN ACTIN

21. SMC2 is down-regulated upon Wnt inhibition
Ls174T/dnTCF4 Ls174T/pTER-bCAT
Dominant negative form of TCF4 siRNA against β-catenin
Time 24h 48h 72h 96h Time 24h 48h 72h 96h
Dox - + - + - + - + Dox - + - + - + - +
TCF-4 β-catenin
C-MYC C-MYC
SMC2 SMC2
ACTIN ACTIN

22. SMC2 is down-regulated upon Wnt inhibition
Ls174T/dnTCF4 Ls174T/pTER-bCAT
Dominant negative form of TCF4 siRNA against β-catenin
Time 24h 48h 72h 96h Time 24h 48h 72h 96h
Dox - + - + - + - + Dox - + - + - + - +
TCF-4 β-catenin
C-MYC C-MYC
SMC2 SMC2
ACTIN ACTIN
SMC2 is under β-catenin/TCF4 regulation, but is it direct or indirect?

23. TCF4 is bound to SMC2 promoter in vivo
TATA Sp1 Sp1 TATA TSS Sp1
-595 -561 -301 -12 +1 +219

24. TCF4 is bound to SMC2 promoter in vivo
TATA Sp1 Sp1 TATA TSS Sp1
-595 -561 -301 -12 +1 +219
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

25. TCF4 is bound to SMC2 promoter in vivo
TATA Sp1 Sp1 TATA TSS Sp1
-595 -561 -301 -12 +1 +219
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

26. Identification of the TCF4 responding element in SMC2
promoter
• Luciferase reporter assays
WT 1 2 3 4 5 Luc+
2Mut 1 2 3 4 5 Luc+
3Mut 1 2 3 4 5 Luc+
1/2/4/5 Mut 1 2 3 4 5 Luc+

27. Identification of the TCF4 responding element in SMC2
promoter
• Luciferase reporter assays
WT 1 2 3 4 5 Luc+
2Mut 1 2 3 4 5 Luc+
3Mut 1 2 3 4 5 Luc+
1/2/4/5 Mut 1 2 3 4 5 Luc+
DLD-1 cells HCT116 cells
pgl3b WT 2Mut 3MUT 1/2/4/5Mut pgl3b WT 2Mut 3MUT 1/2/4/5Mut

28. Identification of the TCF4 responding element in SMC2
promoter
• Luciferase reporter assays
WT 1 2 3 4 5 Luc+
2Mut 1 2 3 4 5 Luc+
3Mut 1 2 3 4 5 Luc+
1/2/4/5 Mut 1 2 3 4 5 Luc+
DLD-1 cells HCT116 cells
pgl3b WT 3MUT 1/2/4/5Mut pgl3b WT 3MUT 1/2/4/5Mut

29. Identification of the TCF4 responding element in SMC2
promoter
• Luciferase reporter assays
WT 1 2 3 4 5 Luc+
2Mut 1 2 3 4 5 Luc+ TBE3 is responsible for
β-catenin/TCF4
3Mut 1 2 3 4 5 Luc+ transactivation of SMC2 promoter
1/2/4/5 Mut 1 2 3 4 5 Luc+
DLD-1 cells HCT116 cells
pgl3b WT 3MUT 1/2/4/5Mut pgl3b WT 3MUT 1/2/4/5Mut

30. SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
Time (h): 24 48 72
siRNA: sc SMC2 sc SMC2 sc SMC2
SMC2
SMC4
NCAPH
GAPDH

31. SMC2 role in tumorogenesis
• siRNA mediated Knockdown of SMC2
Time (h): 24 48 72
siRNA: sc SMC2 sc SMC2 sc SMC2
SMC2
SMC4
NCAPH
GAPDH

32. SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA siRNA
1st 2nd scrambled SMC2
transfection transfection
DLD1 cells Mice injection
48h 24h

33. SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA siRNA
1st 2nd scrambled SMC2
transfection transfection
DLD1 cells Mice injection
48h 24h

34. SMC2 role in tumorogenesis
• Tumor Xenografts
siRNA siRNA
1st 2nd scrambled SMC2
transfection transfection
DLD1 cells Mice injection
48h 24h
SMC2 is a new
potential
therapeutic
target

35. 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

36. 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