11/12/2014
1
Conditional inactivation of the von Hippel–Lindau tumor suppressor gene: Mouse model
Tien HsuBoston University School of MedicineNational Central University, Taiwan
2014 VHL Symposium, Madrid
Hoxb7-Cre x VhlloxP/loxP: 18 mutant (Hoxb7-Cre; VhlloxP/loxP); 13 wild-type (Hoxb7-Cre or VhlloxP/loxP)
Hoxb7-Cre-EGFP x VhlloxP/loxP: 17 mutant (Hoxb7-Cre-EGFP; VhlloxP/loxP); 10 wild-type (Hoxb7-Cre-EGFP or VhlloxP/loxP)
Strategy for Vhl conditional knockout:
Two independent Hoxb7-Cre lines: Expressed early in collecting ducts and distal tubules; not in proximal tubules.
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
200XA C
Gl
H & E stain
Conditional Vhl knockout in kidney tubule cells (Hoxb7-Cre)
Dilated tubules (minicysts) Infiltration of interstitial cellsIncreased density of vasculature Appearance of transformed clear cells
w.t.EB
**
*
*
*
DVhlhdel/del Vhlhdel/del Vhlhdel/del Vhlhdel/del
100 m
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Periodic acid-Schiff's stain
*
*
Vhlhdel/del
f
w.t.
Vhlhdel/del Vhlhdel/del
Appearance of clear cells with abnormal nuclei and basement membrane breakdown
Abnormal clear cells Basement membrane breakdownProteinaceous cast in dilated tubules
100 m
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
11/12/2014
2
Cre driver
wt # of
lesions/section(penetrance)
Vhlhdel/del
# of lesions/section(penetrance)
Hoxb7-Cre 0 (0/6) 10.2±1.2 (6/6)
Hoxb7-Cre-EGFP 0 (0/11) 10.4±1.7 (11/11)
Penetrance is 100% at 3 months.
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
a DBA
NCC
LTpVHL
TH
**
*
1 2 3
4 5
**
b % of distorted tubules positive for tubule markers (n=# of tubules examined)
TH (n=460)
DBA (n=406)
NCC (n=399)
LT(n=227)
Negative (n=60)
44.70% 1.97% 2.51% 18.94% 18.33%
A majority of abnormal tubules resides in the ascending loops of Henle
(Ascending Loop of Henle)(Distal tubule)
(Collecting duct)(Proximal tubule)
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
VHL-/-
Hyperplasia in Vhlhdel/del kidney tubules
A
B Ba Bb
wt
VHL-/- VHL-/- VHL-/-
Aa Ab
Ki67
wt wt
H&E
100 m
100 m
mitotic figures
proliferating cells
Oncogene. 2014 Jul 14;0. [Epub ahead of print]#
Ki6
7 po
sitiv
e ce
lls /
10x
field
of v
iew
(from
4 k
idne
ys)
Mut (n=11)
w.t.(n=11)
10.00±5.081.00±1.48
P<0.001
11/12/2014
3
Systemic phenotypes 1 – Liver
Clear cell lesions in the liverLiver hemangiomaVhlhdel/del
w.t.
Unpublished
Systemic phenotypes 2 - hematopoiesis
Unpublished
Siriu
s re
d
Fibrosis in Vhl KO kidney
SM
A
w.t. Vhl-/-
50 m
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Infiltration of immune cells — inflammation
50100150200250
# of
pos
itive
cel
lspe
r 20x
fiel
d
+18±7.48
mut238±78.9
n=3p=0.003*
20406080
100
wt0.5±1
mut59.7±11.6
n=3p<0.001*
wt12±9.38
mut150±27.5
n=3p<0.001*
50100150200250
# of
pos
itive
cel
lspe
r 20x
fiel
d#
of p
ositi
ve c
ells
per 2
0x fi
eld
B22
0F4
/80
CD
3
50m
Macrophages
B cells
T cells
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
11/12/2014
4
VHL patient kidney shows pre-cancerous inflammation and fibrotic lesions
VHL patient uninvolved kidneyVhlhdel/del mouse
A
100 m
ccRCCUninvolved
region C
AIX
Siriu
s re
d
**
Nei
ghbo
ring
sect
ions
B
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Primary Vhl mutant tubule cells show increased ERKsignaling, CTGF, TGF and nuclear NFkB.
Cytokine array profile of the 3 months old VhlKO kidney
2.2CCL5/
RANTES7
8.8IL-1ra6
1.5IL-165
8.7CCL2/MCP-1
3
2.1M-CSF2
9.0TIMP-11
Fold change(KO/wt)*
IdentitySpot
2.01CXCL9/
MIG4
*Average of two independent samples
1 2 3 567
A
F
1 24
wt
Vhl
hdel
/del A
F
4
a b
monocyte chemotactic protein 1
tissue inhibitor of metalloproteinases
monokine induced by gamma interferon; T cell attractant
chemotactic for T cells, eosinophils, and basophils
chemotactic for CD4+ cells
macrophage colony-stimulating factor
IL-1 antagonist
Vhl KO phenotypes can be rescued by cytokine signaling inhibitor
a
# le
sion
s pe
r4X
sag
ittal
sec
tion
0
5
10
15
20 p=0.0020*p=0.0452* b
% K
i67+
epith
elia
l nuc
lei
per 2
0X fi
eld
p=0.3447p=0.0002*
p=0.0001* p=0.0433*
c
% c
olla
gen
depo
sitio
n10
X sa
gitta
l sec
tion p=0.0440*
0
5
10
15
20
� �
� �
�
�
��
��
# m
acro
phag
es20
X sa
gitta
l sec
tion p=0.0530
0
100
300
400
�
�
�
�
�
��
�
200
d
Rux: Ruxolitinib, a JAK1/2 inhibitor
Inflammatory response in the microenvironment is important for the initiation of tubule defects and hyperplasia.
# lesionsKi67+ cells
Collagen deposit # macrophages
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
11/12/2014
5
# le
sion
s pe
r4x
sag
ittal
sec
tion
p=0.8883
0
10
20
30 p=0.0034*p=0.0034*
b
# m
acro
phag
espe
r 20x
fiel
d0
50
100
200
p=0.0440*
�
�150�
�
�
�
��
c
� ��
a
% K
i67+
epith
elia
l nuc
lei
per 2
0X fi
eld
p=0.3447p=0.0002*
p=0.0001*
The Vhl mutant phenotypes are Hif1-dependent but not Hif2-dependent
# lesions
Ki67+ cells
# macrophages
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Tumor-host interaction — Induction of inflammatory microenvironment
FGFRTGFNFkB
mesenchymal characteristics
Immune cells
VHL mutant
XXX
X
X XX
ccRCC Myofibroblasts
RecruitmentInflammation
Inflammation is suspected as an inducer of cancer.
Cytokines/chemokines
Cytokines/chemokines
A retrospective study of 483,733 veterans (US) has shown that statins is protective against the development of renal cell carcinoma1. Statin use was significantly associated with a risk reduction of renal cell carcinoma of 48%, irrespective of age, sex, and the presence of obesity and smoking.
Also, simvastatin can inhibit renal cancer cell growth and metastasis via AKT/mTOR, ERK and IL6-induced JAK2/STAT3 pathway2.
The anti-inflammatory property of statins is protective against kidney cancer
1. Khurana V, Caldito G, Ankem M. Statins might reduce risk of renal cell carcinoma in humans: case-control study of 500,000 veterans. Urology 2008 71:118–122.
2. Fang Z, Tang Y, Fang J, et al. Simvastatin inhibits renal cancer cell growth and metastasis via AKT/mTOR, ERK and JAK2/STAT3 pathway. 2013 PLoS ONE 8:e62823.
AcknowledgementsBoston University:Tracy PritchettHannah BaderGouthami NallamothuRichard NearRong Chen
National Central University:Chanyan KuoYu-Hui ShihYuki HagiwaraPeter FangChi-chien wu
Collaborators:
Prof. Giuseppe GargiuloUniversity of Bologna, Italy
Dr. Boris AdryanCambridge University
11/12/2014
6
IL-1
Wild-type Vhl-/- Vhl-/-TG
F1
Vhl knockout kidney over-produces pro-inflammatory cytokines
100 m
Proximal convoluted tubule Bowman's
capsule
Glomerulus
Distal convoluted tubule
Collecting duct
to ureter
Cortex
Medulla
Loop of Henle
Mouse knockout strategy
Consideration for mouse knockout strategy:
1. Proximal tubules may not be the only origin of ccRCC: Use KO in distal tubules
2. Most of the knockouts were in mixed genetic background:Use a purified genetic background
Where is the origin of ccRCC?Proximal tubules or distal tubules?
wt Vhlhdel/del
Cre driverwt
# of lesions/section(penetrance)
Vhlhdel/del
# of lesions/section(penetrance)
Hoxb7-Cre 0 (0/6) 10.2±1.2 (6/6)
Hoxb7-Cre-GFP 0 (0/11) 10.4±1.7 (11/11)
Penetrance of kidney phenotype is 100% at 3 months
Periodic acid-Schiff's stain (basement membrane)
w.t.
Vhl-/-
d
p
200X
200X
200X
Basement membrane break down
11/12/2014
7
Mutant cells unassociated with the epithelia are detected in Vhl knockout kidneys
GFP stain
VhlloxP/loxP Hoxb7-Cre-EGFP/VhlloxP/loxP
Dysplastic Vhl mutant cells
VHL patient kidney shows pre-cancerous inflammation and fibrotic lesions
RCC Neighboring tissues
H&E
Siriu
s re
d
B
a b c
a' b' c'
VHL patient kidney sections
VHL patient uninvolved kidneyVhlhdel/del mouse
A
Fibrosis
Infiltration of leukocytes
100 mVhlloxP/loxP Hoxb7-Cre; VhlloxP/loxP
Vimentin stain
200x 200x
Interstitial cells
Mutant cells unassociated with the epitheliua are detected in Vhl knockout kidneys
Epithelial cells
11/12/2014
8
PDGFRv v t
A B
1
2
2v
1
C DMouse w.t. Mouse Vhl-/-
Human normal Human pre-cancer
Human ccRCC
E
Vhl KO kidney and human pre-cancer lesion show increased infilatration of myofibroblasts
MOUSE
HUMAN
GFP MergeTubule marker
DBA
NCC
TH
Hoxb7-Cre-EGFP driver:
Thiazide sensitive NaCl Cotransporter (NCC)[Convoluted distal tubules]
Dolichos biflorus agglutinin (DBA)[Collecting ducts]
Tamm-Horsfall(TH) protein[Ascending loop of Henle]
Hoxb7-Cre is expressed in collecting ducts, ascending loops of Henle and convoluted distal tubules
Top Related