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Unpublished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Unpublished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

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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

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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