1) The study examines how and why certain breast cancer cells metastasize to bone through a "seed pre-selection" process. 2) It finds that high Src activity (denoted by a "Src activity signature" or SRS) in primary tumors associates with bone metastasis. 3) In ER-/ERBB2- breast cancers, cytokines CXCL12 and IGF1 in the tumor microenvironment activate Src which promotes cell survival and bone metastasis. Long term exposure to these cytokines in vitro selects for breast cancer cells with higher Src activity and bone metastatic ability.

1. A “Seed Pre-Selection” Hypothesis of
Breast Cancer Metastasis
Xiang (Shawn) Zhang
Lester and Sue Smith Breast Center
Baylor College of Medicine

2. Metastasis: a multi-step biological process
Primary tumor growth
Invasion and intravasation
Survive in circulation
Extravasation
Colonization
Primary tumor Metastasis
A sequence of Darwinian selection processes?

3. Metastasis organ tropism and latency
Metastasis behavior can be encoded in primary tumors – A conundrum:
How and why do pro-metastasis traits arise in primary tumors?

4. We set out to understand the basis of metastasis organ-
tropism and latency using integrative approaches.
Bone metastasis
Clinical samples Years to decades
Lung metastasis
Brain metastasis
Primary tumor
transcriptional profile Pathways linked to clinical outcome:
1. Is there metastasis?
KRAS SRC TGFß 2. To where?
3. When?
WNT MYC E2F
STAT3 NFkB
…
Signaling pathway
signatures obtained
from cell line models  Experimental validation: are these pathways
functionally driving metastasis?

5. A gene expression signature denoting Src activity (SRS)
Parental
1833
4175
1834
831
Bone pTyr416-Src
Lung Total c-Src
1
Brain
Tubulin
Unselected
Parental-A
Parental-B
2295
1833
2293
1834
4175
4173
4180
2280
4142
2287
923
836
725
831
0
Adrenal

6. Association of SRS
with overall and late-onset bone relapse
Bone relapse by SRS status
15 *
1.0
SRS
Hazard ratio
ER
0.8 ** *
SRS+ breast tumors: 10
Probability
0.6 ~90% of ER+ tumors
~50% of HER2+ tumors
0.4 ~25% of TN tumors 5 **
*** ***
0.2 SRS+
SRS-
0.0 0
>0 >12 >24 >36 >48 >60
0 40 80 120 160 bone metastasis onset
Bone metastasis-free survival
(months)
The association is unique between SRS and bone metastasis, and is
independent of other conventional clinical parameters (treatment, stage,
genomic grades …).

7. Src knock-down in aggressively metastatic cells inhibits bone metastasis
(MDA-MB-231 xenograft model)
Intracardiac
Bone
injection metastasis
35 days
flux at hind limbs x10–5
10 p=0.002
Normalized photon
Control 8
* * 6
4
Src RNAi
2
0
Src RNAi +
Src RNAi
Control
Src Rescue
Src-Rescue
*
MDA231-BoM1833
No effect of Src RNAi on mammary tumor growth or on lung metastasis.
Knock-down of Fyn and Yes did not have similar effects.

8. Dasatinib, a drug that inhibits Src, suppresses bone colonization
Day 35 after injection Day 35 after injection
Normalized photon flux at
Normalized photon flux at p=0.002 p=0.61
hind limb (x103)
hind limbs (x103)
10 6
p=0.054
8
4
p=0.001
p=0.011
6
4 2
2
0 0
   
Day 14
Day 0
Day 7
Mock
Dasatinib
Src rescue Wild type Dasatinib
c-Src resistant c-Src
Dasatinib Start
MDA231-BoM1833
MDA231-BoM1833 Src RNAi
No effect of dasatinib on lung-metastasis.

9. Src knock-down in aggressively metastatic cells
compromises their survival in the bone marrow
Control Src RNAi Dasatinib Src Rescue
p=0.02
TUNEL
20 p=0.005 p=0.003
Percent TUNEL+ cells
15
10
5
Ki67
0
Control
Src RNAi
Dasatinib
Rescued
Bone met
microenvironment
Cancer
Cells Bone
Src inhibition has no effect on
degradation
osteoclast mobilization.
Osteoclasts

10. Cytokines enriched in the human bone metastasis
microenvironment
58 Human breast cancer metastasis samples
Bone Lung Liver Brain
BMP2: bone morphogenetic protein 2
IGF2: insulin-like growth factor 2
CLEC11A: C-type lectin domain family 11A; SCGF
CXCL12: chemokine C-X-C motif 12; SDF1
CXCL14: chemokine C-X-C motif 14
GMFG: glia maturation factor, gamma
IGF1: insulin-like growth factor 1
JAG1: jagged 1
NOV: nephroblastoma expressed; IGFBP9
PDGFA: platelet-derived growth factor alpha
PGF: placental growth factor
PXDN: peroxidasin homolog
SPP1: secreted phosphoprotein 1; osteopontin
TGFB1: transforming growth factor, beta 1
TGFB3: transforming growth factor, beta 3
TNFSF10: tumor necrosis factor family 10; TRAIL
VEGFC: vascular endothelial growth factor C
-2 0 2 4
The enrichment of many of these cytokines are recapitulated in
xenografted metastases.

11. Src mediates CXCL12 and IGF1-induced survival and antagonizes
TRAIL-induced apoptosis
Control
Src RNAi
Rescue ns **
* **
**
Percentage Viable (Day 5)
60 ** ** 60
Percent Viable (Day 3)
50 50
40 40
30 30
20 20
10 10
0 0
No serum No serum + No serum + No Serum No Serum +
CXCL12 IGF1 TRAIL

12. Model: Src confers survival advantages to cancer cells in the bone
microenvironment.
Bone marrow survival factors
IGF1 CXCL12 TRAIL
IGF1R CXCR4 DR4/5
Src
.
.
. Survival
.
.
.
SRS+ breast cancer cell Zhang et al., 2009
Competence Competence
to infiltrate Competence to colonize
to survive
Breast Bone
carcinoma marrow Bone macrometastasis
(years to decades) CXCR4, PTHrP, IL11,
MMP1, OPN
How and why is Src activated in primary tumors?
Whether can we find a better indicator of the
existence of latent bone metastasis?

13. ER+ and ERBB2+ subtypes: Src activity inborn with
tumorigenesis process?
Survival and
Src
proliferation MCF7 T47D
Estradiol (10nM) − 5’ 15’ 30’ 1h 6h − 5’ 15’ 30’ 1h 6h
Estrogen Cytoplasm
pY215-
Src
pY416-
Src
a-Tubulin
transcription
Nucleus
SKBR3 HCC1954 SKBR3 HCC1954
Src RNAi − + − + NRG1 (10ng/mL) − 5’ 15’ − 5’ 15’
pY877-ERBB2 pY215-Src
pY1221/2-ERBB2 pY416-Src
pY1289-ERBB3
ERBB2/ c-Src
ERBB3 a-Tubulin a-Tubulin

14. SRS associates with ER and ERBB2 statuses
Bone relapse
SRS
ER
ERBB2
Negative Positive
25 ER+
ER-/ERBB2+
Percentage of tumors (%)
ER-/ERBB2-
20
15
10
5
0
SRS score
(scaled between -1 and 1)

15. To search for Src activation mechanism in the ER-
/ERBB2- subtype
Approach: compare SRS+ tumors with SRS- tumors and examine the
genes that associate with SRS (but not part of the SRS itself).
EMC-MSK-615 TRANSBIG UPPSALA STOCKHOLM
ER-/ERBB2-
tumors
3 9 7 4
0
4 3 1
1
4 1 8
5
8 SRS-
v.s.
SRS+
CXCL12
CXCL14
IGF1
IGF2
15
10
-log10p
5
0

16. A same group of cytokines enriched in SRS+ primary
tumors and bone metastases
ER-/SRS+ ER-/SRS+
+4 58 Human breast cancer metastasis samples
IGF1
Bone Lung Liver Brain
+2 IGF2 CXCL14
CXCL12 CXCL12
0 IGF1
CXCL14 IGF2
-2
overexpressed in bone
ERBB2
Other cytokines
metastases
IGF1
IGF2
CXCL12
CXCL14
ERBB2
ER-/ERBB2- ER-/ERBB2+

17. ER-/ERBB2- subtype: CXCL12 and IGF1 activate AKT in a Src-
dependent manner
MDA231 CN34
MDA231 CN34
CXCL12          
          
Control Rescued Src RNAi Control Src RNAi Rescued
IGF1
CXCL12             Dasatinib
         
P-AKT pAKT
AKT
pSrc
c-Src
AKT
CXCR4
Src
Tubulin
Tubulin
1. CXCL12 and IGF1 do NOT directly activate Src.
2. CXCL12 and IGF1 promote cell survival in a Src dependent manner.
3. Cancer cells do NOT express CXCL12 and IGF1.

18. ER-/ERBB2- Subtype: the hypothesis of “Metastasis seed pre-selection”
58 Human breast cancer metastasis samples
ER-/SRS+ ER-/SRS+ Bone Lung Liver Brain
Primary tumors Bone marrow
Survival upon
arrival
v Colonization
CXCL12 IGF1
CXCL12
A IGF1
CXCR4 IGF1R
B
Src
C PI3K/AKT
…
Enrichment of
Bone marrow survival factors
Src activity
IGF1 CXCL12 TRAIL
IGF1R CXCR4 DR4/5
Src
.
.
* *
. Survival
* * * .
.
.
* *
SRS+ breast cancer cell

19. Long term incubation with CXCL12/IGF1 causes a colony
expansion process in vitro
Low serum with
CXCL12/IGF1
MDA-MB-231 cells
Low serum
only
Parental cells Low serum Colony expansion Subpopulations
+ CXCL12, IGF1 Regular medium
3 ~ 9 weeks 1 ~ 2 weeks

20. Long term incubation with CXCL12/IGF1 expanded
colonies with higher Src activity
MDA-MB-231 (Passage: 3)
Parental
IGF1 (ng/ml): 0 3 10 30 100 P
CXCL12 (ng/ml): 0 10 30 100 300 P IGF1(10ng/ml)
CXCL12 (30ng/ml)
0.4
intensity
Relative
0.2
0
pTyr416-Src:
Total-Src:
Tubulin:
0 101 102 103 104 105
pY416-Src
Western Phospho-Flow

21. Long term incubation with CXCL12/IGF1 led to more bone
Normalized photon flux at hind limbs metastasis
100 Parental
p = 0.0003 p = 0.021
p < 0.0001
0/0
10
10/30
1
P 0 10 100
P 0 30 300 100/300
IGF1 (ng/ml)
CXCL12 (ng/ml)
Bioluminescence Hind limbs Vertebrae
H&E H&E
 No increase in lung metastasis  bone specific
 The effect on bone metastasis was also seen in
another cell line: CN34

22. In vitro selected MDA231 derivatives have not
acquired osteolytic competence
Parental
0-0
10-30
10 - 30
BoM-1833
BoM-1833
10-30: MDA231 derivatives selected by in vitro cultivation with
IGF1 and CXCL12 (10ng/ml and 30ng/ml, respectively).
Bone metastasis genes identified
through in vivo selection (Kang et BoM-1833: MDA231 derivatives extracted from established bone
al., 2003) lesions (Kang et al., 2003)

23. Seed pre-selection in the indolent metastasis systems
ER- pleural effusion samples
CN37 CN34
0+0 10 + 30 0 + 0 10 + 30
P P
pTyr416-Src
Tubulin
p = 0.014
25
(Human B2M / Mouse Actb)
101
Relative mRNA expression
20 Mock
Normalized photon
Dasatinib
flux at hind limbs
100
15
×10-5
p = 0.0007
10 10-1
5
10-2
0
0-0 10-30
0 10 20 30
CN34
Days after injection

24. ER-/ERBB2- Subtype: the hypothesis of “Metastasis seed pre-selection”
Primary tumors Bone marrow
Survival upon
? ? arrival
v Colonization
CXCL12 IGF1
CXCL12
A IGF1
CXCR4 IGF1R
B
Src
C PI3K/AKT
…
Enrichment of
Src activity

25. Carcinoma-associated fibroblasts (CAFs) are
enriched in SRS+ tumors
*
CAF over-expressing genes
Breast cancer metastases
1.0
0.8
CAF SRS
Probability
0.6 p = 0.025 signature 6 associated
(77) genes (44)
0.4
CAF+
0.2 CAF- 1
SPARCL
IGFBP4
IGF1
FOS
CXCL14
CXCL12
0
0 50 100 150
Bone metastasis free survival
(Month)

26. MSCs differentiate into CAFs and provide CXCL12/IGF1 in
mammary tumors
MSCs
MDA231
MDA231 alone
MDA231 and hMSC
co-injection
aSMA IGF1 CXCL12

27. Cancer cells purified from mammary tumors with
abundant CAFs exhibit higher Src activity
MSCs
± Inhibitors:
AMD3100  CXCR4
MDA231 BMS754807  IGF1R
MSCs: + − + + − +
Inhibitors: − − + − − +
Individual tumors: 1 2 3 4 5 1 2 3 1 2 3 6 7 8 9 10 4 5 6 4 5 6
pTyr416-Src:
Tubulin:
p = 0.029 p = 0.019 p = 0.024 p < 0.0001
15
0.8
Bone metastasis
0.6
Src Activity
10
0.4
0.2 5
0.0
0
+ + +
MDA231 -
MSC + +
Inhibitors - - +

28. The hypothesis of “Metastasis seed pre-selection”
Primary tumors Bone marrow
CAF Survival upon arrival
v MSC Colonization
CXCL12 IGF1
CXCL12
A IGF1
CXCR4 IGF1R
B
Src
C PI3K/AKT
…
Enrichment of
Src activity

29. Summary
• The chance that a patient carries latent bone metastasis may be
predictable based on primary tumor profiles (stromal content and
Src activity).
• We provide one possible answer to the metastasis progression
puzzle:
– When the microenvironment of a primary tumor resembles that of a distant
organ, it will pre-select cancer cells predisposed to colonize that organ.

30. Acknowledgement
• PI: Joan Massagué • Don Nguyen
• Qiongqing Wang • Paula Bos
• Xin Jin
• David Padua
• Johansson Baez
• Larry Norton
• Clifford Hudis • Karen Xi
• William Gerald • Qing Chen
• Neal Rosen
• Weiping Shu
• John Foekens
• Marcel Smid • And other Massagué Lab
members
• NIH: K99CA151293