Download Conditional Inactivation of the Mouse Model

11/12/2014
Strategy for Vhl conditional knockout:
Conditional inactivation of the von Hippel–Lindau
tumor suppressor gene: Mouse model
Two independent Hoxb7-Cre lines: Expressed early in collecting ducts
and distal tubules; not in proximal tubules.
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)
Tien Hsu
Boston University School of Medicine
National Central University, Taiwan
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
2014 VHL Symposium, Madrid
Conditional Vhl knockout in kidney tubule cells (Hoxb7-Cre)
Appearance of clear cells with abnormal nuclei and
basement membrane breakdown
w.t.
Periodic acidSchiff's stain
Vhlhdel/del
w.t.
A
200X B
Vhlhdel/del
C
Vhlhdel/del
D
*
Vhlhdel/del
E
*
100 m
*
*
100 m
H & E stain
Vhlhdel/del
Vhlhdel/del
*
Gl
f
Dilated tubules (minicysts)
Infiltration of interstitial cells
Increased density of vasculature
Appearance of transformed clear cells
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
*
*
Vhlhdel/del
Abnormal clear cells
Basement membrane breakdown
Proteinaceous cast in dilated tubules
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
1
11/12/2014
A majority of abnormal tubules resides in
the ascending loops of Henle
a 1
Penetrance is 100% at 3 months.
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)
Cre driver
*
2
pVHL
*
3
LT
DBA
(Proximal tubule)
4
5
NCC
(Collecting duct)
TH
(Ascending Loop
of Henle)
(Distal tubule)
*
*
*
b % of distorted tubules positive for tubule markers (n=# of tubules examined)
TH
(n=460)
DBA
(n=406)
44.70%
1.97%
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
NCC
(n=399)
LT
(n=227)
Negative
(n=60)
2.51%
18.94%
18.33%
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Hyperplasia in Vhlhdel/del kidney tubules
100 m
H&E
mitotic figures
A
wt
Aa
wt
Ab
wt
VHL-/- Bb
VHL-/-
# Ki67 positive cells / 10x field of view
(from 4 kidneys)
VHL-/-
P<0.001
Ki67
B
100 m
VHL-/- Ba
proliferating cells
Mut
w.t.
(n=11)
(n=11)
10.00±5.081.00±1.48
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
2
11/12/2014
Systemic phenotypes 1 – Liver
Systemic phenotypes 2 - hematopoiesis
Vhlhdel/del
Liver hemangioma
Clear cell lesions in the liver
w.t.
Unpublished
Unpublished
F4/80
Vhl-/-
SMA
w.t.
# of positive cells
per 20x field
Infiltration of immune cells — inflammation
Fibrosis in Vhl KO kidney
# of positive cells
per 20x field
# of positive cells
per 20x field
B220
CD3
Sirius red
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
Macrophages
+
mut
18±7.48238±78.9
50m
50 m
250 n=3
200 p=0.003*
150
100
50
100
80
60
40
20
n=3
p<0.001*
B cells
wt
mut
0.5±159.7±11.6
250
200
150
100
50
n=3
p<0.001*
T cells
wt
mut
12±9.38150±27.5
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
3
11/12/2014
VHL patient kidney shows pre-cancerous
inflammation and fibrotic lesions
A
Vhlhdel/del mouse
VHL patient
uninvolved kidney
Primary Vhl mutant tubule cells show increased ERK
signaling, CTGF, TGF and nuclear NFkB.
100 m
B
Uninvolved
region
CAIX
Neighboring sections
Sirius red
ccRCC
*
*
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
wt
A
Vhlhdel/del
F
A
F
1
2
3
4
5
6
7
# lesions
a
Fold change
(KO/wt)*
1
TIMP-1
9.0
tissue inhibitor of
metalloproteinases
2
M-CSF
2.1
macrophage colonystimulating factor
3
CCL2/
MCP-1
8.7
4
CXCL9/
MIG
2.01
5
IL-16
1.5
monocyte
chemotactic protein 1
monokine induced by
gamma interferon; T
cell attractant
chemotactic for CD4+ cells
6
IL-1ra
8.8
IL-1 antagonist
7
CCL5/
RANTES
2.2
chemotactic for T
cells, eosinophils, and
basophils
20
15
10
5
0
c Collagen deposit
20
15
10
5
0
b
p=0.0452*
p=0.0020*
% Ki67+ epithelial nuclei
per 20X field
24
Identity
# lesions per
4X sagittal section
1
Spot
% collagen deposition
10X sagittal section
b
a
Vhl KO phenotypes can be rescued by cytokine
signaling inhibitor
p=0.0440*
p=0.3447
p=0.0002*
p=0.0433*
p=0.0001*
Ki67+ cells
d # macrophages
# macrophages
20X sagittal section
Cytokine array profile of the 3 months old Vhl
KO kidney
400
p=0.0530
300
200
100
0
Rux: Ruxolitinib, a JAK1/2 inhibitor
*Average of two independent samples
Inflammatory response in the microenvironment is important for the initiation
of tubule defects and hyperplasia.
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
4
11/12/2014
The Vhl mutant phenotypes are Hif1-dependent but not Hif2-dependent
% Ki67+ epithelial nuclei
per 20X field
a
p=0.3447
Tumor-host interaction — Induction of inflammatory
microenvironment
p=0.0002*
p=0.0001*
Ki67+ cells
FGFR
VHL mutant TGF
NFkB
 mesenchymal characteristics
# lesions
30
20
10
0
c
p=0.8883
p=0.0034*
p=0.0034*
p=0.0440*

# macrophages
per 20x field
# lesions per
4x sagittal section
b
# macrophages
Cytokines/
chemokines
200
150
100
50
Recruitment
Inflammation


X
X

X
X
X
0
Cytokines/
chemokines
X
X
ccRCC
Immune cells
Myofibroblasts
Inflammation is suspected as an inducer of cancer.
Oncogene. 2014 Jul 14;0. [Epub ahead of print]
The anti-inflammatory property of statins is protective
against kidney cancer
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.
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.
Acknowledgements
Boston University:
Tracy Pritchett
Hannah Bader
Gouthami Nallamothu
Richard Near
Rong Chen
Collaborators:
Prof. Giuseppe Gargiulo
University of Bologna, Italy
Dr. Boris Adryan
Cambridge University
National Central University:
Chanyan Kuo
Yu-Hui Shih
Yuki Hagiwara
Peter Fang
Chi-chien wu
5
11/12/2014
Mouse knockout strategy
Vhl knockout kidney over-produces pro-inflammatory cytokines
Vhl-/-
Wild-type
Vhl-/-
Proximal
convoluted tubule
Bowman's
capsule
Where is the origin of ccRCC?
Proximal tubules or distal tubules?
Distal convoluted
tubule
IL-1
Glomerulus
Consideration for mouse knockout
strategy:
1. Proximal tubules may not be the only
origin of ccRCC:
Use KO in distal tubules
100 m
Cortex
TGF1
Medulla
Loop of Henle
2. Most of the knockouts were in mixed
genetic background:
Use a purified genetic background
Collecting
duct
to ureter
Penetrance of kidney phenotype is 100% at
3 months
Basement membrane break down
Periodic acid-Schiff's stain (basement membrane)
wt
Vhlhdel/del
d
p
w.t.
200X
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-GFP
0 (0/11)
10.4±1.7 (11/11)
200X
Vhl-/-
200X
6
11/12/2014
Mutant cells unassociated with the epithelia are
detected in Vhl knockout kidneys
Dysplastic Vhl mutant cells
GFP stain
Hoxb7-Cre-EGFP/VhlloxP/loxP
VhlloxP/loxP
VHL patient kidney shows pre-cancerous
inflammation and fibrotic lesions
A
Vhlhdel/del mouse
Mutant cells unassociated with the epitheliua are
detected in Vhl knockout kidneys
VHL patient
uninvolved kidney
Vimentin stain
VhlloxP/loxP
Hoxb7-Cre; VhlloxP/loxP
100 m
VHL patient kidney sections
B
a
RCC
b
Neighboring tissues
c
H&E
a'
Sirius red
200x
200x
Infiltration of
leukocytes
b'
c'
Fibrosis
Epithelial cells
Interstitial cells
7
11/12/2014
Vhl KO kidney and human pre-cancer lesion show
increased infilatration of myofibroblasts
A
v
Hoxb7-Cre is expressed in collecting ducts, ascending loops of
Henle and convoluted distal tubules
Hoxb7-Cre-EGFP driver:
PDGFR
v
B
t
GFP
Tubule marker
MOUSE
Mouse w.t.
Mouse
Dolichos biflorus
agglutinin (DBA)
[Collecting ducts]
Vhl-/-
C
D
DBA
1
v
HUMAN
Human normal
Merge
2
1
Human pre-cancer
E
TH
2
Human ccRCC
NCC
Tamm-Horsfall
(TH) protein
[Ascending loop
of Henle]
Thiazide sensitive
NaCl Cotransporter
(NCC)
[Convoluted distal
tubules]
8