Dickkopf-1 inhibits thyroid cancer cell sur-vival and migration by regulating -catenin/E-cadherin signaling

Sun Wook Cho, Eun Jung Lee, Young A Kim, Kyung Won Kim,

Jung Ah Lim, Won Sang Yoo, Hoon Sung Choi, Hwa Young Ahn,

Ka Hee Yi, Do Joon Park, Bo Youn Cho, Young Joo Park

Department of Internal Medicine, Seoul National University College of Medicine Department of Pathology, Seoul National University Boramae Medical CenterDepartment of Internal Medicine, Seoul National University Boramae Medical Center Department of Internal Medicine, Chung-Ang University Hospital

Wnt/-catenin/E-cadherin signal-ing in oncogenesis

Role of Dkk-1 in oncogenesis in human cancers

Pancreatic cancer1

Esophageal cancer2

NSCLC2

Colon cancer3

Hepatocellular carcinoma4

ONCOGENE

TUMOR SURPRESSOR GENE

1Takahashi et al., 20102Yamabuki et al., 2007

3Kuphal et al., 20064Qin et al., 2007

Tissue microarray (1997-1999, SNUH)

PTC (n=150), ATC (n=15)

PTC, membranous PTC, cytoplasmic

PTC, nucleus ATC, nuclear

A subset of PTC patient showed aberrant expression of -catenin

2009, Korean Endocrine Socitey

Aim of study

• To evaluate the associations between the expression of -catenin and E-cadherin with clinical characteristics in human PTC.

• To investigate the therapeutic potential of Dkk-1 in human PTC in vitro and in vivo

Localization of -catenin

Normal membranous

(n=135)

DecreasedMembranous

(n=7)

Cytoplasmic dominant

(n=7)P-values

E-cadherin  P<0.001

Normal 130 (98.5) 1 (25.0) 2 (20.0)

Aberrant 5 (1.5) 6 (75.0) 5 (80.0)

Cyclin D1        P=0.003

Negative 9 (6.7) 4 (57.1) 1 (14.3)

Positive 125 (93.3) 3 (42.9) 6 (85.7)

The correlation between -catenin and E-cadherin in human PTC

Tissue microarray (1997-1999, SNUH), PTC (n=150)

Comparisons of clinical characteristics ac-cording to the -catenin/E-cadherin status

P-value

E-cadherin

P-value Membrane dominant All aberrant Normal Loss

(n=134) (n=16) (n=133) (n=17)

Age at diagnosed (mean ± SD) 50.26 ± 15.9 48.6 ± 18.5 0.702 49.7 ± 15.6 53.1 ± 19.8 0.062

Female, n (%) 22 (16.4) 4 (25.0) 0.412 22 (16.5) 4 (23.5) 0.489

Tumor size (cm) 2.43 ± 1.44 2.59 ± 0.99 0.674 2.44 ± 1.47 2.49 ± 0.97 0.326

Extrathyroidal invasion, n (%) 96 (71.6) 10 (62.5) 0.457 95 (71.4) 11 (64.7) 0.572

LN metastasis, n (%) 64 (66.7) 11 (78.6) 0.357 61 (64.9) 14 (87.5) 0.054

Recurrence or metastasis, n (%) 8 (11.8) 5 (45.5) 0.013 8 (11.6) 5 (50.0) 0.007

Median follow-up duration: 14.4 (1315 ) years

-catenin

hDKK-1

hGAPDH

P<0.05

Dkk

-1/G

AP

DH

Normal Cancer

Dkk-1, as a tumor suppressor gene in human PTC

H-tori

B-CPA

P

BHP10-3

SNU790

1 2 3 40

50

100

150

200

250

Dkk

-1/G

AP

DH

hDKK-1

hGAPDH

H-tori

B-CPA

P

BHP10-3

SNU790

Human PTC tissue Human PTC cell-lines

Normal Cancer

SNU-790

1 2 3 40.00

0.25

0.50

0.75

1.00

1.25

BHP10-3

Cyclin D1

SNU790 BCPAP

-actin

Cleaved Caspase-3

Fre

qu

enc

y

Intensity of Annexin V

VEH Dkk-1

+Dkk-1

Dkk-1 inhibited tumor cell survivals in human PTC cells

VEH Dkk-1

1 20

10

20

30

40

50

SNU-790 BHP10-3

VehicleDkk-1 (20nM)

An

nex

in V

+ C

ell

(%)

+Dkk-1

BHP10-3SNU-790

Treatment of Dkk-1 rescue an aber-rant expression of -catenin

SN

U7

90B

HP

103

Vehicles Dkk-1

Anti--catenin Ab

NS

Vehicle

Dkk-1

NS

Na/K-ATPase

GAPDH

E-cadherin

DKK-1 (nM) 0 20 50Cytosol Membrane

0 20 50

Treatment of Dkk-1 rescue the loss of E-cadherin expression

Effects of Dkk-1 on E-cadherin expression

Vehicles Dkk-1

Anti-E-cadherin Ab

Mig

rati

on

(%

)

Effects of Dkk-1 on cell migration

VEH Dkk-1

Vehicle Dkk-1

Vehicle Dkk-1

Invading cellsMatrigel

Matrigel invasion assay

Dkk-1

Wound healing assay

Tumors from Xenotransplantation of BHP10-3 cells overexpressing Dkk-1

200

150

100

50

0

(Days) 5 10 14 21

Control

Dkk-1

pMSCVIRESGFP pMSCVDkk-1IRESGFP

Retrovirus-mediated Dkk-1 over-expression in BHP10-3 cells

Control

Control

Dkk-1

Dkk-1

Control Dkk-1

Tum

or

volu

me

(mm

3)

-catenin E-cadherin

Co

ntr

ol

Dkk

-1Tumors from Xenotransplantation of BHP10-3 cells overexpressing Dkk-1

H&E

Summaries

• Loss of membranous E-cadherin expression was significantly correlated with aberrant loca-tions of -catenin.

• Patients with loss of E-cadherin expression and aberrant locations of -catenin showed higher rates of LN or distant metastasis than those with normal membranous staining.

Summaries

• Dkk-1 treatment inhibited PTC cell survival and rescued the aberrant expression of -catenin from cytoplasm to membrane.

• Dkk-1 also recovered the loss of membranous E-cadherin expression in PTC cells, inhibiting cell migration and invasion.

• Xenotransplantation of BHP10-3 cells overex-pressing Dkk-1 showed delayed tumorigenesis with relocations of the aberrant expression of -catenin and E-cadherin.

Conclusions

Dkk-1 might have a therapeutic po-tential in PTC through regulation of

-catenin/E-cadherin system