Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

Crina Tiron¹, Hallvard Haugen¹, Christine Gjerdrum¹, Torill Høiby¹, Ingunn Stefansson¹ Tone Sandal¹, Karin Collet¹, Emmet McCormack¹, Bjørn Tore Gjertsen¹, David
Micklem¹, Lars Akslen¹, Carlotta Glackin², James Lorens¹. ¹University of Bergen, Bergen, Norway; ²Beckman Center, City of Hope, Duarte, CA

Metastasis underlies the majority of cancer-related deaths. Hence, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transition
(EMT) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignat progression. We show that the presence of the receptor tyrosine kinase Axl in mammography-detected primary breast cancers
independently predicts strongly reduced overall patient survival, and mached patient metastatic lesion show enhanced Axl expression. We demonstrate that Axl is strongly induced by epithelial-to-mesenchymal transition in pre-
malignant mammary epithelial cells that establishes an autocrine signaling loop with its ligant, Gas6. Using epi-allelic RNA interference analysis in metastatic breast cancer cells we deliniated a distinct threshold of Axl expression
for mesenchymal-like in vitro cell invasiveness, and to form tumors in foreign tissue engineered microenvironments in vivo. Importantly, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells
from the mammary gland to lymph nodes and several major organs, and increased overall survival, in two diffrent optical imaging-bassed experimental breast cancer models. Thus, Axl represent a novel downstream effector of
tumor cell EMT that is required for breast cancer metastasis. The detection and targeted of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Axl expression is a strong negative prognostic factor for human breast cancer survival
A B Weak AXL expression (90/6) C D
1.0 (A) Axl expression in human breast tumors. (B)
Variable Categories HR P
Probability of survival

Univariate survival analysis (Kaplan-Meier method)

0.8
Histologic grade Grade 1 of breast cancer patients. (C) Multivariate survival
analysis of human breast patients. (D) Axl expres-
0.6
Strong AXL expression (64/11)
Grade 2-3 3.79 0.040 sion in primary breast carcinoma and metastases.
0.4 Lymph node status 0-1
>1 9.16 0.001
0.2
AXL expression
Log Rank test, P=0.035
AXL expression Low(0-4)
0.0 High (6-9) 3.27 0.021
20 40 60 80 100
Months after primary treatment

Axl is required for breast cancer cell invasiveness Axl is upregulated by EMT inducers in breast epithelial cells In vivo epi-allelic analysis reveals a distinct Axl expression thresh-
A B A
MCF10a
B C old required for breast tumor formation
i ii i iii MCF10a

uc x
MDA-MB-231
9 8 0
l27 xl27 xl28 xl2
MDA-MB-231
sh279 shAxl2 o
c T
t
Z S
ol
ntr wis eb2 lug nai
S
l A B
L A hA hA A
sh sh s s sh
Twist

control

Twist
h Axl Gas6
shAxl278
Zeb2 1.8
% of Max

shAxl280

Total Photon/mm²/sec
h Actin Tubulin 1.6
% of Max
% of Max

sh279 shAxl2
lysate IP Slug 1.4 Therapeutic
shLuc Slug 1.8
Std
c xl2 c xl2
shLu shA shLu shA shAxl279 ii MDA-MB-231 shAxl2 Snail Snail
1.2 threshold
shAxl278 1 Ineffective shRNA 1.6 Ineffective
Ineffective

Normalized tumor growth

sh279 shAxl2 Benefit

Snail

Slug
Twist
shAxl280
h Axl
shAxl2 Axl Zeb2 0.8 *
control
Gas6 * control
control 0.6 * * * * 1.4 Benefit
anti-hAxl anti-pAxl 10 10 * *
10¹ 102 103 104 105 E-cadherin 101 10² 10³ 0.4 **
1.2

Tumor growth
Gas6
Axl
EPI

Axl
Axl 0.2
markers

β-catenin 1.0
1.2
C D E
Zeb2
N-cadherin

Tumor Diameter (mm)

0.8
MES

MDA-MB-231 MDA-MB-231 1 Ineffective shRNA

*
shLuc shAxl2 *
1.4 Vimentin Vimentin 0.8 **
** 0.6
shLuc
Actin 0.6 0.4
Relative invasion

1.2
Serum

shAxl279
shLuc

1 shAxl278 0.4 0.2

shAxl280 0.2
0.8
* shAxl2
100x 400x
D 0.0
0 25 50 75 100
0.6
i MCF10a iii 0 1 2 3 4
Axl Knockdown Level
l (A) MCF10a cells transduced with retroviral vectors en-
shAxl2
SDF-1

0.4 * ro Time (weeks)

nt i st ug ail
0.2 co Tw Sl Sn coding Twist, Zeb2, Slug, Snail. (B) Axl surface expres-
100x 400x Gas6 sion in MCF10a cells encoding Twist, Zeb, Slug, Snail.
% of Max

0
Tubulin Slug (C) Mesenchymal morphology at 72 hours post-seeding.
ii (D) Gas6 expression in MCF10a cells (i, total lysate; ii,
(A) Graded Axl surface expression in MDA-MB 231 cells (i, western blots; ii, anti-Axl flow cytometry analysis). (B) Gas6 expres- MCF10a Snail conditioned medium; iii, anti-Gas6 6 flow cytometry (A) Image analysis of tumor growth (total photon) and radial infiltration (signal diameter) by subcutaneous Axl epi-
l allelic MDA-MB-231/GFP-Luc xenografts in NOD/SCID mice comprising shAxl278, shAx280, shAxl2 normalized
sion in MDA-MB-231 cells (i, total lysate; ii, conditioned medium; iii, anti-Gas6 flow cytometry analysis). Invasiveness of ro st ug ail
nt i analysis).
co Tw Sl Sn control to shAxl279 (ineffective shRNA). (B) Tumor growth (normalized 28 day total photon measurements) plotted versus
MDA-MB 231 cells correlates with Axl expresion level (C) serum and (D) SDF-1,*p<0.05. (E) Growth of MDA-MB-231 cells in 10 10 10
Gas6 * Axl knockdown (geometric mean fluorescence). (N = 6mice/group), *p<0.05, **p<0.005.
3D-matrigel. Gas6

Tissue engineered breast tumors require Axl expression Axl is required for human breast carcinoma to metastasize Axl is essential for post-immune response recurrence and
i ii Ai B i metastasis of syngeneic breast carcinoma cells in BALB/c
Weeks post implantation Photon counts Weeks post implantation Photon
counts/mm2/s
mice
0 1 2 3 4 mm²/s 0 2 4 6 8 9 116970 5
control 30

2231 *
MDA-MB 231/D3H2LN GFP-Luc

shAxl2
Total photon (10¹)sec¹¹

1.3 control + EC + SMC

Total photon (10¹)sec¹¹

control
control

A
-

87728 4
shAxl2 + EC + SMC
Tumor diameter (mm)

shAxl2
control + EC + SMC
Primary tumor

***
6

i ii
shAxl2 + EC + SMC 20
3
control

**
1.0 * Weeks postimplantation
MDA-MB 231 GFPLuc

54830 Photon
4
5

2 day1 1 2 3 4 5 6 7 8 counts/mm²/sec
1673 shAxl279

Total Pho ton (x10 5 ) sec-1

10 2590
Ventral scan

0.7 **
shAxl2

32898
sh-mAxl2
4T1-GFP-Luc-shAxl279

1 *
**

0.5 10
0
2 4 6 8 9 0 2 4 6 8 9 1950 3
1045 ii ii Time (weeks) Time (weeks)

** Weeks post implantation

Dorsal scan

Photon
0.2
Metastasis to Lymph node

9
Total photon (10¹)sec¹¹

4 6 8 9 counts/mm2/s
-

24 53 133 526
shAxl2 control
shAxl2

*** 1470 2
4

** 6

627 3
990
Ventral scan

15
4T1-GFP-Luc-shmAxl2

10 10 10 10 1
Tumor diameter (mm)

4 6 8 9

10 Time (weeks)
* *
510

iii ** C Detected metastases D 0

*
Dorsal scan

1.0 shAxl2 (N=5)

** Cell line
10 MDA-MB
In vivo
Organ
Ex vivo
0.8
270 0 1 2 3 4 5 6 7 8
Time (weeks)
Probability of survival

231/D3H2LN
Control shAxl2 GFP-Luc Lymph node Lungs Kidneys Ovaries Liver 0.6 10
5/6 4/6 3/6 2/6 0/6
5 control
0.4
shAxl2 0/6 0/6 1/6 0/6 0/6
iv N (number of animals/group) = 6 mice/group 0.2
Vector (N=10)
B Detected metastases
0.0 Log Rank, P=0.001
Ex vivo
65 70 75 80 85 90 95 100
120 50μm 50μm 0 1 2 3 4 E Number of days alive Cell line Organ
Time (weeks) i Ex vivo imaging ii H/E staining (10x) Lungs Bone Rib cage
Lymph
Ovaries Kidneys Liver Brain Intestine Spleen
MDA-MB 231/D3H2LN GFP Luc MDA-MB 231/D3H2LN GFP Luc nodes
control shAxl2 control shAxl2
shAxl279
Mammary pad

5/7 5/7 5/7 4/7 4/7 3/7 2/7 2/7 2/7 1/7
Mammary pad

1192 1192
v (A) Temporal in vivo bioluminiscence imaging sh-mAxl2 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7
Microvascular Density

100 20 *
10
117
10
117
of (i) mamary fat pad growth (ii) axillary lymph N (number of animals/group) = 7 mice/group
node metastasis. (B) Biphotonic quantitation of
Vessel Diameter

Lung
Lung
(no.vessels/mm²)

80 16 Axl is required for growth of tricellular tissue im- 10 10 (i) primary mammary tumor (total photon) and
C
Axillary tissue

plants in NOD-SCID mice. (i) Representative in radial infiltration (signal diameter) and (ii) total
Axillary tissue

832 832
60 12
(μm)

vivo bioluminescence images. (ii) Images analysis photon from thoracic lymph node area of
10 10 1
(total photon, upper and tumor diameter, lower). (iii) NOD/SCID mice. (C) Ex vivo detected metas- 1
40 8 Aspect of excised tumor tissue engineering im-
511 511
tasis. (D) Univariate survival analysis (Kaplan-
Ovary

Ovary

plants (28 days). (iv) Anti-human CD31-staining. (v) Meier method) between control and shAxl2 (E)
20 4 Intrascaffold human vessel diameter (left) and mi-
10
1220
10
1220 Ex vivo bioluminiscence images and histologi- 2
6
Kdiney

Kdiney

crovascular density (right) in tissue engineered cal analysis of excised organs showing the
0 0 (tricellular) tumors. (N=7mice/group), *p<0.05, 10 10 presence of metastasis (arrow) in several Anatomical location 7
Control shAxl2 Control shAxl2 **p<0.005, ***p<0.0005. 9 9
organs. (N=6mice/group), *p<0.05,
3 6
1.Brain 3
Liver

Liver

2.Axillary lymph node

0 0
**p<0.005, ***p<0.0005. 7 3.Lungs
4.Ovaries 8
5.Femur and Tibia
4 6.Rib cage 4
8 7.Liver
Conclusions 8.Kidneys

5
shAxl279 5 shmAxl2
● Axl is a strong independent predictor of breast cancer patient overall survival
(A) Temporal in vivo bioluminescence imaging (i) of orthotropic (mammary fat pad) injected synge-
nic 4T1-GFP-Luc cells expressing mouse Axl shRNA (4T1-GFP-Luc-shmAxl2) or negative control
● Autocrine Axl-signaling is induced by EMT human Axl shRNA (4T1-GFP-Luc-shAxl279) into BALB/c mice. (ii) Quantitation of whole-body bio-
luminescence (total photon) injected BALB/c mice. (B) Survey of metastasis sites (8-weeks post-
orthotopic implantation) monitored by ex vivo bioluminescence detection of 4T1-GFP-Luc cells in
● Axl is critical for malignant invasiveness and required tumor formation in foreign microenvironments excised organs. (C) Bioluminescence images from representative female BALB/c mice acquired at
8 weeks post-orthotopic injection. (N=7mice/group), *p<0.05.

● Axl is essential for breast cancer cell metastasis