Expression and Roles of Nestin in Pancreatic Cancer

Departments of Pathology and Integrative Oncological

Pathology, Nippon Medical School, Tokyo, Japan

Nestin: Neuroepithelial Stem Cell Marker (Cell, 1990)

Class Intermediate filament protein

I Acidic keratin

II Neutral keratin, Basic keratin

III Vimentin, Desmin, GFAP, Peripherin

IV Neurofilaments (L, M, H chains)

V Lamin

VI Nestin, Tanabin, Transitin, Paranemin, Synemin

Nestin is a member of the class VI intermediate filament protein.

Structure of Intermediate Filament Proteins

Intermediate filament

Monomer

Heterodimer

10 nm

Tetramer

N-terminalC-terminal

α-helical rod domain

Nestin forms heterodimers

with vimentin or internexin.

Nestin forms heterodimers with other types of intermediate filament proteins, and these heterodimers combine to form intermediate filaments.

Foregut

endoderm

Pancreatic

progenitors

Endocrine

progenitors

Exocrine

progenitors

Islets

Acini

Ducts

Nestin(+)

p48(+)

p48(+)

Pdx1(+)

Pdx1

p48

Expression of Nestin in Stem/Progenitor Cells

Pancreas

Nestin is also expressed in progenitor cells of brain, heart, muscle, testis and

blood vessels.

Expression of Nestin in Tumors

Overexpression of nestin has been reported in various types of malignant tumors, and our group has found a higher expression of nestin in glioblastoma, cervical cancer, melanoma and pancreatic cancer.

Nestin

Gastrointestinal tract tumor

Pancreatic ca, GIST

Lung tumor

Non small cell ca,

Small cell ca

Endocrine or metabolic tumorBreast ca, Thyroid ca

Otorhinolaryngological tumorSalivary gland ca

Brain tumor

Glioblastoma

Urinary tumor

Prostate ca

Orthopedic tumor

Osteosarcoma

Gynecological tumor

Ovarian ca, Uterine ca

Skin tumor

Melanoma, SCC

Expression of Nestin in Pancreatic Cancer

Kawamoto et al. Human Pathol 2009

Immunohistochemistry of Nestin in

Pancreatic Cancer

6

Nestin was strongly expressed in 30% of

pancreatic cancer cases.

Expression Levels of Nestin

PK- 45HPANC-1

0

1.0

Sc-1 Sc-4 Sh-1 Sh-10

0.5

**

GAPDH

Sc-16 Sh-7Sc-13 Sh-80

0.5

1.0

**

1. Wild

2. Sc13

3. Sc16

4. Sc17

5. Sh7

6. Sh8

7. Sh9

250

150

100

75

β-actin 42KD

1 2 3 4 5 6 7

β-actin

GAPDH

Nestin

GAPDH

Nes

tin

/18S

rR

NA

Nestin shRNA–transfected clones (Sh) showed lower expression levels of nestin than

sham cells (Sc). Matsuda et al., Cancer Biol Ther. 2011

Phase Contrast Images

PK- 45HPANC-1

Sc

Sh

Sc

Sh

Sham cells showed round or short spindle-like appearance with loose cell adhesion, while Nestin

shRNA-transfected cells showed a sheet-like appearance with tight cell adhesion.

PK- 45HPANC-1

Sc

Sh

Sc

Sh

The Nestin shRNA-transfected cells showed a markedly higher expression of F-actin

(arrows) than sham cells.

Expression of F-actin

Expression Levels of E-cadherin

PK- 45HPANC-1

E-cadherin

GAPDH

1. Wild

2. Sc13

3. Sc16

4. Sc17

5. Sh7

6. Sh8

7. Sh9

250

150

100

75

β-actin 42KD

1 2 3 4 5 6 7

β-actin

GAPDH

0

1

2

3

4

E-c

ad

he

rin

/18

S r

RN

A

*

*

0

0.5

**

Expression levels of E-cadherin were higher in nestin shRNA-transfected cells than in

sham cells.

Sc ScSh Sh

Matsuda et al., Cancer Biol Ther. 2011

Scratch Assay (Wound Healing Assay)

0

10

20

30

Sc-13 Sc-16 Sh-7 Sh-8

* *

PK- 45HPANC-1

0

10

20

Sc-1 Sc-4 Sh-1 Sh-10

% o

f w

ou

nd

ed

are

a f

ille

d

**

0 h

8 h

0 h

12 h

Sc Sh Sc Sh

The percentage of the wounded area filled was statistically lower in nestin shRNA-

transfected PANC-1 and PK-45H cells than in sham cells.

Boyden Chamber AssayM

igra

ted

cells

0

500

Sc-1 Sc-4 Sh-1 Sh-10

* *

Sc-1 Sc-4 Sh-1 Sh-10

**

Inv

ad

ed

cell

s

**

0

50

100

0

100

200

300

*

*

Sc-13 Sc-16 Sh-7 Sh-8

0

100

200

Sc-13 Sc-16 Sh-7 Sh-8

*

*

PK- 45HPANC-1

Migrated and invaded cells were fewer in nestin shRNA-transfected cells than sham

cells.

In vivo Metastasis Assay

Sh

There was less metastasis to the liver by nestin shRNA-transfected cells (Sh) compared

with sham cells (Sc) in NOG mice.

Sc

Matsuda et al., Cancer Biol Ther. 2011

Immunohistochemical Analysis of Nestin in

Pancreatic Cancer Tissue

Nestin is strongly expressed in vascular endothelial cells adjacent to pancreatic cancer

cells.

Ca

Analysis of Nestin or CD34-positive Blood Vessels

using Software (WinROOF ver. 6.1)

Nestin

CD34 B

Proliferation Assay of Blood Vessels using

PCNA-positive Endothelial Cells

Nestin

PCNA

**

Yamahatsu et al. Ｉnt J Oncol,

2012

Inhibition of Nestin in Mouse Endothelial Cells (TKD2)

siNestinsiNegativeTKD2

0

1

2

3

4

5

6

Cell

s/w

ell

×105

*

TKD2

Cell Proliferation Assay in vitro

*

siNestinsiNegative

(Day)

siNestinsiNegative

siNestin

Injection of siRNA targeting Mouse Nestin mRNA in Subcutaneous Human

PDAC Tumors in Nude Mice

Yamahatsu et al.Ｉnt J Oncol, 2012

Nestin positive tumor vessels（tumor angiogenesis）

(Metastasis)

Liver and Lung metastases

Conclusion

siRNA targeting nestin

Inhibition of nestin using siRNA may be a novel therapeutic option for PDAC patients.

Nestin positive PDAC cells

Thank you for your attention