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