Preconditioning of the tumor vasculature and tumor cells by intermittent hypoxia: implications for anti-cancer therapies

(1)

This work is supported by grants from the FNRS (FRSM - Télévie) , the J. Maisin Fundation and the Belgian Federation against Cancer.

Influence of intermittent hypoxia on the vascular and tumor cell

compartments: implications for anti-cancer therapies.

P. Martinive

1*

_{, F. Defresne}

1 _{, C. Bouzin}

1 _{, F. Lair}

1 _{, V. Grégoire}

2 _{, C. Michiels}

3 _{, C. Dessy}

1 _{and O. Feron}

1

1 _{Unit of Pharmacology and Therapeutics (FATH5349) [*current affiliation: Ulg] and}

2 _{Center for Molecular Imaging and}

Experimental Radiotherapy, UCL Medical School, Brussels and

3 Biochemistry and Cellular Biology Lab, FUNDP, Namur

.

INTRODUCTION

1. Hypoxia is a common feature in tumors associated with an increased resistance to anti-cancer therapies.

2. In addition to O

₂

diffusion-limited hypoxia, another form of tumor hypoxia, named Intermittent Hypoxia (IH), is described:

IH is characterized by fluctuating changes in pO

₂

due to heterogeneities in red blood cell flux within the tumor vascular network.

3. Peculiarities of IH are that tumor vasculature itself may be directly influenced by the hypoxic episodes and that re-oxygenation phases

complicate the usual hypoxia-induced phenotypic pattern.

Nor mox ia Hyp oxia 3h Hyp oxia 6h Inte rm. hypo xia 0 50 100 150

**

*

S

u

rv

iv

a

l

(%

)

Nor mox ia Nor mox ia-R X Hyp oxia 3 h-RX Hyp oxia 6 h-RX Inte rm. h ypox ia-R X

0

50

100

150 *

**

*

S

u

rv

iv

a

l

(

%

)

No

rm

ox

ia

Hy

po

xi

a

Hy

po

xi

a

HIF1α

α

siRNA

+

-0 H1 R1 H2 R2 H3 R3 H3 R3

HIF-1α

α

p-Akt

Akt

LY294002 15 µM

1. IH promotes EC survival

C lo n o g e n ic S u rv iv a l A s s a y

2. Activation of HIF-1α

α

and Akt during Intermittent Hypoxia

Serum Free

Radiotherapy

5. Inhibition of HIF-1α

α

reverses IH preconditioning

6. IH reduces the extent of apoptosis in both vascular and tumor cells in vivo

CONCLUSIONS

1. Intermittent hypoxia “preconditions” endothelial cells and tumor cells in such a way that they become more resistant to

apoptosis (i.e. radiotherapy) and more prone to participate in tumor progression.

2. The stimulation of the mitochondrial respiration and the activation of the PI3K/Akt pathway during intermediary

reoxygenation periods act as the major triggers of the stabilization of HIF-1α.

3. The accumulation of HIF-1α plays a key role in the IH preconditioning, underscoring the importance of drugs targeting

HIF-1α to resensitize the tumor vasculature to anticancer treatments.

0 H1

R1

H2

R2

H3

R3

HIF-1α

α

p-Akt

Akt

Western Blotting Assay (from EC)

siRNA

_HIF1-α

α

pAkt

Stabilization

HIF1-α

α

LY294002

C

on

tr

ol

_1x

1h

_2x

1h

_3x

1h

3h

c

hr

on

ic

1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4

∆∆∆∆

O

2 /

∆∆∆∆

t

(µ

M

/m

in

)

4. IH stimulates

the respiratory mitochondrial

chain and is associated with HIF-1α

α

stabilization

MODELS

Billups-Rothenberg incubator

7% O

₂

Endothelial Cells

Hypoxia

1h

Hypoxia

1h

Hypoxia

1h

Reoxygenation

30 min

Reoxygenation

30 min

Reoxygenation

60 min

Radiotherapy

Hypoxic chamber

EC

TLT Carcinoma

< 1% O

₂

Intermittent hypoxia = 3 cycles of hypoxia/reoxygenation

E P R R e s p ir a to ry A s s a y EC O2 consumption rate

Western Blotting Assay

10µm TLT hepatocarcinoma

Normoxia

200µ m

TLT hepatocarcinoma

Intermittent Hypoxia

200µ m

TLT hepatocarcinoma

1

2

3

4

5

6

7

8

9 10 11 12

100

150

200

250

300 Days

T

u

m

o

r

D

ia

m

e

te

r

Tunel-positive apoptotic nuclei (pink) within the CD31-immnostained (green) tumor vasculature or tumor section counterstained with DAPI (blue).

Untreated

Inter. Hyp. + 10Gy

Normoxia + 10Gy

Tumor growth delay when radiotherapy (10Gy) is administrated to mice prechallenged by intermittent hypoxia (red) vs maintained in normoxia (green); p<0.01; and tumor growth in untreated mice (black).

TLT liver tumor-bearing mice were exposed to either Intermittent Hypoxia (7%O₂), chronic hypoxia or ambient air and were locally irradiated.

Western Blotting Assay (from EC)

C lo n o g e n ic S u rv iv a l A s s a y

No

rm

ox

ia

In

te

rm

. H

yp

ox

ia

Ch

ro

ni

c

Hy

po

xi

a

0 50 100 150 200 250

T

U

N

E

L

-p

o

si

ti

ve

va

sc

u

la

r

st

ru

ct

u

re

s

(%

)

**

Normoxia IH IH+LY 0 10 20 30 40 50 60 70 80 90 100 110

*******

S u rv iv a l (% )

3. HIF-1α

α

is a key actor in IH preconditioning

C lo n o g e n ic S u rv iv a l A s s a y 0 25 50 75 100 125

S

u

rv

iv

a

l

(%

)

Interm. hypoxia:

HIF-1a-siRNA:

-+

-

+

-

-**

ns

**

HIF-1α

α

_-siRNA:

0 25 50 75 100 s u rv iv a l (% )

Interm . Hypoxia :

--

-

+

--

+

**

Rad. (2Gy):

-

+

HIF-1 a -siRNA :

*

HIF-1α

α

_-siRNA:

H3

+Rotenone

(2 µM)

H3

HIF-1α

α

H3 H3+Rotenone 0 10 20 30 40 50 60 70 80 90 100 110 H IF -1 αααα R e la ti v e E x p re s s io n ( % )

RESULTS

AIM OF THE STUDY

To examine whether IH may promote endothelial and tumor cell survival and thereby induce resistance to pro-apoptotic treatments.