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