Jurnal Metastasis Paru 2

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Cell. Author manuscript; available in PMC 2013 August 17.
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Cell. 2012 August 17; 150(4): 764–779. doi:10.1016/j.cell.2012.06.035.
The BMP Inhibitor Coco Reactivates Breast Cancer Cells at Lung

Metastatic sites
Hua Gao1, Goutam Chakraborty1, Ai Ping Lee-Lim1, Qianxing Mo2, Markus Decker1,5, Alin
Vonica3, Ronglai Shen2, Edi Brogi4, Ali H. Brivanlou3, and Filippo G. Giancotti1,*
1Cell Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065
2Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New

York, NY 10065
3Laboratory of Molecular Embryology, The Rockefeller University, New York, NY 10065
4Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
SUMMARY
The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A
gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-β ligands, induces
dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that
Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the
manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco
induces a discrete gene expression signature, which is strongly associated with metastatic relapse
to the lung but not to the bone or brain in patients. Experiments in mouse models suggest that
these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-
initiating cells need to overcome organ-specific anti-metastatic signals in order to undergo
reactivation.
INTRODUCTION
Metastatic relapse of breast cancer and other carcinomas frequently occurs several years
after initial surgery. Increasing evidence suggests that tumor cells that have disseminated
from early lesions, including ductal carcinomas in situ, undergo an extended period of
dormancy in the stroma of target organs (Nguyen et al., 2009; Valastyan and Weinberg,
2011). It is currently unclear if tumor cells become dormant as a consequence of intrinsic

defects or in response to inhibitory signals that they encounter in foreign
microenvironments.
© 2012 Elsevier Inc. All rights reserved.

*
Correspondence: f-giancotti@ski.mskcc.org.
5Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
ACCESSION NUMBERS
Gene expression data of MDA231-sh-control, sh-Coco #2, and sh-Coco #4 cells are deposited at Gene Expression Omnibus
(GSE28049).
SUPPLEMENTAL INFORMATION
Supplemental Data include seven figures, nine tables, and Supplemental Experimental Procedures can be found with this article online
at http://www.cell.com/supplemental/
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Gao et al. Page 2
Many malignancies, including breast cancer, are fuelled by a limited, although not
necessarily small, number of cancer stem cells, which undergo self-renewal as well as
generate rapidly proliferating progenitors and aberrantly differentiated post-mitotic cells
(Clevers, 2011; Gupta et al., 2009). The metastatic capacity of human pancreatic and
colorectal cancers is restricted to a subpopulation that includes cancer stem cells (Hermann
et al., 2007; Pang et al., 2010). Furthermore, the Epithelial to Mesenchymal Transition
(EMT) that facilitates tumor dissemination produces cells endowed with the capacity to self-
renew, suggesting that these two cellular processes are intimately linked (Mani et al., 2008).
Finally, the Id1/3 transcription factors and the miR200 and miR335 microRNAs promote the
colonization phase of breast cancer metastasis at least in part by modulating cancer stem cell
function (Gupta et al., 2007; Korpal et al., 2011; Shimono et al., 2009; Tavazoie et al.,
2008). These results suggest that the cancer stem cells possess the migratory and self-
renewal capabilities necessary to colonize distant organs, whereas the remaining tumor cells
lack metastatic capacity.
The ability of metastasis-initiating cells to enter into, and eventually exit from, proliferative
quiescence suggests an additional commonality with adult tissue stem cells. However, the
relationship between cancer stem cell behavior and dormancy at metastastic sites is poorly
understood. In this paper, we provide evidence that Coco, a secreted antagonist of TGF-β
ligands, induces dormant metastasis-initiating cells to undergo reactivation in the lung.
Mechanistic studies suggest that Coco exerts this function by blocking paracrine BMP
signalling and thereby enhancing the self-renewal capability of metastasis-initiating cells.
RESULTS
A Gain-of-function Screen for Genes that Mediate the Post-dissemination Phase of
Metastasis
We designed a gain-of-function cDNA screen that uses the mouse as a filter to isolate genes
that mediate metastasis (Figure 1A) and applied it to a previously described series of
mammary carcinoma cell lines, which appear to be arrested at defined steps of metastasis
(Aslakson and Miller, 1992). Upon orthotopic injection, the 67NR cells give rise to non-
invasive tumors, the 168FARN cells colonize locoregional lymphnodes but do not gain
access to the vasculature, and the 4TO7 cells are able to disseminate but do not produce
macroscopic metastases. In contrast, the 4T1 cells produce macroscopic metastases in the
lung (Figure 1B). Upon transduction with cDNA libraries derived from 4T1 cells, the 67NR
or 168FARN cells did not acquire the capability to give rise to lung metastases in 8 weeks,
suggesting that the introduction of a single gene did not enable these cells to penetrate into
the bloodstream and acquire the additional capabilities required for metastatic colonization.
In contrast, the 4TO7 cells infected with the 4T1 libraries produced a total of 8 lung nodules
in multiple mice (Figure 1B). After proviral rescue and re-introduction in 4TO7 cells, 3 of
the 8 cDNAs isolated from individual lesions promoted lung metastasis without affecting
primary tumor growth (Figures S1A; not shown). In contrast, 4TO7 cells infected with
empty vector did not produce macroscopic lesions upon injection in 30 mice. This screening
strategy can thus be used to identify mediators of the homing and outgrowth step of
metastasis.
Coco Promotes Lung Colonization

We focused on cDNA1 because it encoded an N-terminally truncated but potentially active
version of Coco, a secreted inhibitor of TGF-β ligands (Bell et al., 2003) (Figure S1B).
Rossant and colleagues had isolated the same transcript and termed it Dante (Pearce et al.,
1999). Studies on frog development had shown that Coco binds directly to BMP and Nodal
proteins, blocking their ability to bind to their cognate receptors, and interferes with Wnt

Gao et al. Page 3
signaling (Bell et al., 2003). Experiments in Xenopus embryos and 4TO7 cells indicated that
cDNA1 possesses all the biological activities of full-length Coco (Figure S1C-F).
Conversely, expression of full-length Coco induced the 4TO7 cells to colonize the lung after
orthotopic injection, confirming that Coco promotes spontaneous metastasis similar to

cDNA1 (Figure S1G).
The expression of Coco correlated with metastatic capability in the 4T1 progression series
(Figure 1C, top). Of note, a large fraction of Coco remained associated with the cell layer
rather than diffusing in the medium (Figure 1C, top). Treatment of live cells with modest
amounts of highly purified trypsin led to the disappearance of Coco from the cell layer
(Figure 1C, bottom), indicating that this inhibitor associates with the pericellular matrix and
might therefore accumulate at high concentrations near the cell surface.
Interestingly, neither expression nor silencing of Coco modified the ability of tumor cells to
proliferate in vitro (Figures 4D and S1H; not shown) or to form primary tumors when 1 ×
105 cells were injected in the mammary fat pad (Figure 1E and 1F). Furthermore, Coco did
not disrupt epithelial adhesion in normal murine mammary epithelial cells (Figure S1I) or
induce completion of an EMT program and enhance invasion in 4TO7 cells (Figures S1J
and S1K). These results suggest that Coco does not promote primary tumor growth or
invasion.
To examine if Coco promotes the colonization step of metastasis, we performed tail-vein

injection experiments. Expression of Coco enabled the 4TO7 cells to metastasize efficiently
to the lung under these conditions (Figure 1G). Conversely, silencing of Coco using two
distinct shRNAs suppressed the ability of the highly metastatic 4T1 cells to colonize the
lung (Figure 1H). Similar inhibitory effects were observed upon depletion of Coco in 66cl4
cells, which were derived from the same spontaneous tumor as the 4T1 cells but are less
aggressive (Aslakson and Miller, 1992) (Figure S1L; >99% inhibition), and in ErbB2-
transformed mammary tumor cells isolated from MMTV-Neu(YD) mice (Guo et al., 2006)
(Figure S1M; >90% inhibition). We concluded that Coco mediates lung colonization.
Coco Induces Exit from Dormancy

Confocal imaging of lung sections revealed that the 4TO7 cells extravasate in the stroma of
the lung within 1 day after injection in the tail vein (Figures 2A and 2B). Coco did not
enhance the ability of 4TO7 cells to infiltrate the lung. However, whereas control 4TO7
cells remained solitary and seemingly quiescent in the stroma of this organ, a small fraction
of 4TO7-Coco cells started to proliferate from around day 14 and gave rise to metastatic
outgrowths (Figures 2A and 2B). Most of the lesions were relatively small and had not yet
undergone neoangiogenesis at day 21 but became large and vascularized by day 35 (Figure
2A). While the number of tumor cells within outgrowing micrometastases was comparable
to that of solitary tumor cells at day 21, the tumor cells within macro-metastases far
outnumbered the solitary tumor cells at day 35 (>9 fold). Anti-Ki67 staining suggested that a
large majority of solitary 4TO7 and 4TO7-Coco cells were not actively cycling (>97%). In
contrast, a large fraction of the 4TO7-Coco cells within metastatic lesions were actively
proliferating (Figures S2A and S2B). The solitary 4TO7 and 4TO7-Coco cells were not
apoptotic at any time point examined (Figure S2C), in agreement with the hypothesis that
they had become dormant.
To confirm that the solitary tumor cells detected in the lung were quiescent, we performed
5-ethynyl-2’-deoxyuridine (EdU) incorporation experiments (Figure 2C). Confocal imaging
after fluorescent conversion of EdU and anti-GFP staining indicated that > 95% of solitary
4TO7 and 4TO7-Coco cells did not enter into or traverse the S-phase over each of 3 distinct
EdU incorporation periods. In contrast, a large fraction of 4TO7-Coco cells within

Gao et al. Page 4
outgrowing micrometastic lesions and overt metastases transited through the S phase over
the same times (Figures 2D and 2E). Therefore, the 4TO7 cells undergo a protracted period
of solitary tumor dormancy in the lung, but expression of Coco enables a fraction of these
cells to exit from dormancy and produce metastatic outgrowths.
To examine if silencing of Coco induces dormancy, we examined the lungs of mice injected
with control or Coco-silenced 4T1 cells. As anticipated, silencing of Coco did not inhibit
extravasation or decrease the number of solitary tumor cells present on lung sections at day
14 and 28 (Figure S2D). Furthermore, whereas a fraction of control 4T1 cells initiated
proliferation to give rise to metastatic outgrowths, virtually all Coco-silenced cells remained
quiescent (Figures 2F and S2D), suggesting that depletion of Coco suppresses lung
colonization by preventing the reactivation of dormant cells.
Coco Inhibits BMP Signaling in Metastasis-initiating Cells

To define the molecular underpinnings of the pro-metastatic activity of Coco, we examined
its ability to regulate BMP, Nodal, and Wnt signaling in mammary tumor cells. We excluded
Nodal because the 4TO7 and 4T1 cells do not express nodal receptors or Cripto and do not
respond to Nodal in reporter assays (Figures S2E and S2F; not shown). However, they
express two type I and type II BMP receptors as well as Frizzled7 and LRP5 and 6 (Figures
2G and S2G). As anticipated, BMP4 induced robust C-terminal phosphorylation of BMP-
responsive Smad proteins (1, 5, 8) in 4TO7 cells, and expression of Coco reversed this
process (Figure 2G). In contrast, Coco did not inhibit but partially enhanced Wnt signaling
(Figure S2H-K). Coco may exert this latter effect by alleviating the intracellular inhibitory
crosstalk that BMP receptors exert on β-catenin (He et al., 2004; Kobielak et al., 2007).
Immunostaining of lung sections indicated that virtually all the solitary, dormant 4TO7 cells
displayed strong nuclear accumulation of P-Smad over 35 days of observation, suggesting
that BMP signaling was robustly activated in these cells (Figures 2H and 2I).
Semiquantitative RT-PCR indicated that the 4TO7 and 4T1 cells express low levels of BMP
proteins, whereas the cellular elements of the normal lung express significant levels of
several BMP proteins (Figure S2L), in general agreement with prior data showing that both
epithelial and mesenchymal cells in the lung produce BMP (Danesh et al., 2009). Metastatic
seeding by 4TO7 or 4TO7-Coco cells did not modify the levels of BMP mRNAs in recipient
lungs (Figure S2M). We thus inferred that the 4TO7 cells underwent protracted proliferative
quiescence in response to BMP proteins that had been produced by both epithelial and
mesenchymal cells and deposited in the stroma between alveoli.
Intriguingly, a small fraction (~2%) of the dormant tumor cells present in the lungs of mice
injected with GFP-tagged 4TO7-Coco cells displayed no nuclear accumulation of P-Smad
(Figures 2H and 2I). We speculate that these cells accumulated more Coco in their
pericellular matrix or were exposed to lower amounts of BMP as compared to other cells. In
agreement with the hypothesis that these rare P-Smad− cells were fated to produce
metastatic outgrowths, both the incipient lesions and the macrometastases displayed no
nuclear accumulation of P-Smad (Figures 2H and 2I). The solitary 4TO7 and 4TO7-Coco
cells as well as the outgrowing 4TO7-Coco cells did not exhibit accumulation of β-catenin
in the nucleus, suggesting that β-catenin signaling does not contribute to reactivation
(Figures S2N and S2O).
In a reciprocal set of experiments, we evaluated if silencing of Coco induced reactivation of

Smad signaling in solitary 4T1 cells that would have been destined to give rise to metastatic
lesions. A small but sizeable fraction of solitary 4T1 cells (~ 5%) and all of those within
outgrowing metastases did not display nuclear accumulation of P-Smad suggesting that the
metastases arise from P-Smad-negative cells (Figure 2J). This subpopulation of P-Smad-

Gao et al. Page 5
negative cells was not detected in the lungs of mice injected with Coco-silenced 4T1 cells.
In fact, > 99.3% of dormant Coco-silenced 4T1 cells displayed strong nuclear accumulation
of P-Smad (Figure 2J). Together, these findings suggest that Coco promotes exit from
dormancy by alleviating the ability of stromal BMP to enforce a dormant state.
Coco Promotes Tumor Sphere Formation In Vitro

Since the metastasis-initiating cells share functional properties with cancer stem cells
(Nguyen et al., 2009; Valastyan and Weinberg, 2011), we considered the possibility that
Coco promotes the manifestation of cancer stem cell traits. Phenotypic analysis indicated
that the 4TO7 and 4T1 cells express high levels of the mammary epithelial stem cell markers
CD24, CD29, and CD49f but do not express markers found in bi-potential progenitors or
cells differentiated along the luminal or myoepithelial lineage (Figure S3A; Table S1). In
addition, both types of cells are endowed with a program of gene expression similar to that
of normal mammary epithelial stem cells (Table S2). Tumor-initiating cells isolated from
mouse models of ErbB2-mediated mammary tumorigenesis exhibit a similar phenotype (Lo
et al., 2011).
The cancer stem cells are defined by an inherent capability to undergo self-renewal, to give
rise to an aberrantly differentiated progeny, and to seed tumors in vivo (Clevers, 2011;
Gupta et al., 2009). To examine if Coco affects cancer stem cell function, we first examined
its ability to influence self-renewal in vitro by using the mammosphere assay (Dontu et al.,
2003). The 4TO7-Coco cells form about twice as many tumor spheres as compared to 4TO7
cells at each of three subsequent passages (Figure 3A and S3B). Administration of
exogenous Coco produced a similar and dose-dependent effect in naïve 4TO7 cells (Figure
3B). In agreement with the observation that 4TO7 cells produce small amounts of BMP2
and 5 (Figure S2L), treatment of naïve 4TO7 cells with 10 ng/ml of exogenous BMP4
caused a profound inhibition of tumor sphere formation, which was reversed by concurrent
administration of Coco (Figures 3C and 3D). Neither Coco nor BMP affected the ability of
4TO7 cells to survive or progress through the cell cycle under the conditions of the assay
(Figure S3C; not shown). We infer that Coco expands their ability of 4TO7 cells to give rise
to tumor spheres by blocking the small amounts of BMP that they produce.
We next evaluated if BMP can cause partial or aberrant differentiation of mammary tumor
cells. Notably, treatment of 4TO7 cells with BMP4 induced expression of the transcription
factor GATA3, a master regulator of luminal differentiation, whereas concurrent
administration of recombinant Coco reversed this process (Figure S3F). In addition, whereas
the 4TO7 cells that became dormant upon extravasation in the lung stroma displayed strong
reactivity for GATA3 irrespective of whether they expressed Coco, the metastatic
outgrowths formed by 4TO7-Coco cells were uniformly negative for GATA3 (Figure S3G).
These results suggest that the high levels of BMP present in the lung stroma induce the
4TO7 cells to express GATA3, and they further imply that expression of Coco reverses this
process in tumor cells fated to give rise to metastatic outgrowths. However, BMP4 did not
induce expression of markers associated with differentiated luminal cells under standard
culture conditions or in 3D Matrigel (Table S1; not shown). Thus, BMP upregulates
GATA3, seemingly poising breast cancer cells toward luminal differentiation, but is
insufficient to initiate partial or aberrant differentiation en face of oncogenic signaling.
Prior studies have suggested that transgenic ErbB2 mammary tumors follow a cancer stem
cell model. Staining with the lipophilic dye PKH-26, which is diluted after each cell
division, has indicated that the cells that retain the dye during the mammosphere assay
possess the highest self-renewal capacity in vitro and the highest tumor initiation capacity in
vivo (Cicalese et al., 2009). To examine if Coco affects self-renewal in vitro, we stained
Coco-silenced and control primary tumor cells from MMTV-Neu(YD) mice with PKH-26

Gao et al. Page 6
and subjected them to serial tumor sphere assay (Figure 3E). As previously reported,
replating of the PKHHIGH, PKHLOW, and PKHNEG subsets led to tumor sphere formation in
all cases, albeit with decreasing efficiency. Notably, knock down of Coco inhibited tumor
sphere formation at each passage (Figure 3F). Treatment of naïve ErbB2-transformed cells
with BMP4 exerted the same effect and Coco reversed it (Figure S3D). Silencing of Coco
did not reduce the ability of ErbB2-transformed cells to survive or proliferate (Figure S3E)
nor induced them to express differentiated genes in 3D Matrigel (not shown). These results
suggest that Coco selectively sustains the ability of ErbB2-transformed cells to undergo self-
renewal in vitro.
Coco Promotes Tumor Initiation In Vivo

Prompted by the observation that Coco increases clonogenic outgrowth under standard
culture conditions as well as in soft agar (Figures S3H and S3I), we examined if it also
enhanced tumor initiation in vivo. We found that Coco significantly increases the ability of
4TO7 cells to seed tumors in vivo (Figure 3G). As anticipated, this effect was evident only
when limiting numbers of tumor cells were injected in the mammary fat pad. Conversely,
silencing of Coco inhibited the tumor-initiation capacity of 4T1 cells (Figure 3H).
Semiquantitative RT-PCR experiments indicated that while the normal cell types present in
the mammary fat pad express BMP7, the primary tumors generated by 4TO7 or 4T1 cells
injected at this site did not express any of the BMP genes (Figure S2L). These results
suggest that Coco promotes tumor initiation in the mammary gland as well as reactivation in
the lung because it opposes the ability of stromal BMP to block clonogenic outgrowth.
Coco Sustains Expression of Stem Cell Transcription Factors

The transcriptional program regulated by the embryonic stem cell transcription factors Sox2,
Nanog, and Oct4 is often reactivated in aggressive and metastatic breast cancers (Ben-Porath
et al., 2008; Wong et al., 2008). Furthermore, the transcriptional coactivator Taz, which is
inhibited by the Hippo tumor suppressor pathway, has been recently implicated in breast
cancer stem cell maintenance (Cordenonsi et al., 2011). Q-PCR experiments indicated that
the 4TO7 cells express high levels of Sox2 and lower levels of Taz and Nanog, whereas the
ErbB2-transformed cells express high levels of Oct4 and lower levels of the remaining three
transcriptional regulators (Figure S3J and S3K). Intriguingly, Coco increased the levels of
expression of Nanog, Sox2, and Taz in 4TO7 cells (Figure 3I). Conversely, BMP4
completely suppressed their expression (Figure 3J). Consistently, silencing of Coco
significantly reduced the level of expression of Nanog, Oct4, and Taz and completely
ablated expression of Sox2 in ErbB2-transformed cells (Figure S3L). Since Nanog, Sox2,
and Oct4 are part of a self-sustaining circuit that powers stem cell maintenance (Young,
2011) and Taz appears to be specifically required in breast tumor progenitor cells
(Cordenonsi et al., 2011), Coco may contribute to the manifestation of breast cancer stem
cell traits by influencing the expression of these transcription factors.
Coco Promotes Metastatic Reactivation by Inhibiting BMP Signaling

To examine the mechanism through which Coco enhances the capability of mammary tumor
cells to outgrow in the mammosphere assay and during lung colonization, we transduced
4TO7 cells with Smad6, which inhibits canonical BMP signalling (Hata et al., 1998), with
the activated β-catenin mutant S4A, or with both Smad6 and β-catenin-S4A (Figure 4A,
left). These mutant proteins exerted the anticipated signalling effects (Figures S4A-D).
Whereas Smad6 increased tumor sphere formation by approximately twofold, similar to
Coco, activated β-catenin did not induce this effect, either alone or with Smad6 (Figures 4A
and 4B). In addition, Smad6 attenuated the induction of GATA3 in response to BMP (Figure
S4E). These results suggest that Coco promotes self-renewal in vitro predominantly by
inhibiting BMP signaling.

Gao et al. Page 7
To examine if inhibition of BMP signaling promotes metastatic reactivation, we injected

4TO7 cells expressing Smad6 or activated β-catenin intravenously. Whereas Smad6
promoted lung colonization as efficiently as Coco, activated β-catenin induced this process
to a modest extent (Figure 4C). Expression of a dominant negative form of BMPR-IB

induced both 4TO7 cells and Coco-silenced 4T1 cells to colonize the lung. However, this
construct inhibited BMP signaling and therefore promoted lung metastasis less efficiently as
compared to Smad6 (Figures S4A, S4B, S4F and S4G). These results indicate that inhibition
of BMP signaling rescues 4TO7 cells and Coco-silenced 4T1 cells from tumor dormancy.
As an alternative approach, we tested if BMP signaling decreased tumor initiation. 4T1 cells
co-expressing an activated form of BMPR-IB (Q203D) together with BMPR-II (jointly
termed CA-BMPR) were significantly less tumorigenic upon injection in the mammary fat
pad as compared to controls (Figures 4D and S4H). These results indicate that BMP
signaling suppresses the tumor initiating capacity of 4T1 cells.
To further study the connection between tumor initiating capacity and metastatic outgrowth,
we examined the ability of 4T1 cells expressing CA-BMPR to metastasize to the lung upon
tail vein injection. Whereas control 4T1 cells were highly metastatic in this assay, those
expressing CA-BMPR were unable to colonize the lung, confirming that BMP signaling
opposes metastatic colonization (Figure 4E). CA-BMPR exerted a similar effect in 4TO7-
Coco cells (Figure S4I). These findings suggest that Coco induces metastasis-initiating cells
to exit from dormancy by alleviating the capacity of lung-derived BMP proteins to activate
canonical Smad signalling.
Coco Promotes Human Breast Cancer Metastasis

To explore the role of Coco in human breast cancer metastasis, we first examined a panel of
12 human breast cancer cell lines (Figure 5A). Immunoblotting indicated that the non-
tumorigenic or non-invasive cells as well as the ER+ cells capable of colonizing the bone
upon intracardiac injection exhibit low or undetectable levels of Coco, whereas the MDA-
MB231 cells, which can colonize efficiently the lung and belong to the basal B gene
expression cluster associated with a stem cell phenotype, express Coco (Figure 5A).
Although, as revealed by additional experiments, the MDA-MB231 cells express the BMP
inhibitors Noggin, DAN and Chordin-like 1 at levels comparable to those of Coco (Figure
S5A-C), silencing of Coco was sufficient to suppress their ability to colonize the lung
(Figures 5B and S5B). Monitoring for 6 additional weeks revealed that the Coco-silenced
cells eventually gave rise to micrometastases (Figure S5D, left). These lesions did not arise
as a result of re-expression of Coco but presumably through the acquisition of genetic or
epigenetic modifications able to bypass its requirement (Figures S5D, right and S5E).
Depletion of Coco also suppressed the lung colonization ability of CN34.2a cells, which
were derived from the pleural effusion of a patient affected by metastatic breast cancer but
are not as aggressive as the MDA-MB231 cells (Padua et al., 2008), suggesting that Coco is
a general mediator of lung colonization.
Sorting according to the surface expression of CD44 and CD24 has been successfully used
to identify cancer stem cells within primary breast tumors or mammary cell lines, including
the MDA-MB231 cells (Al-Hajj et al., 2003; Cordenonsi et al., 2011). As anticipated, the
large majority of control MDA-MB231 cells were CD44HIGH/CD24LOW/−, consistent with a
cancer stem cell phenotype. Silencing of Coco led to the appearance of a large
subpopulation of cells that did not express CD44, suggesting that expression of Coco is
necessary to maintain the expression of this marker in MDA-MB231 (Figure 5C). In
consonance with this observation, depletion of Coco profoundly inhibited the ability of
MDA-MB231 cells to form tumor spheres in vitro without affecting their survival or
proliferation (Figures 5D, S5G and S5H). Finally, silencing of Coco inhibited the capacity

Gao et al. Page 8
of MDA-MB231 cells to initiate tumorigenesis upon orthotopic injection in NOD-SCID-

IL2γR−/− mice (Figure 5E). These results provide evidence that Coco promotes maintenance
of various stem cell traits by MDA-MB231 cells.
To study the expression of Coco in human breast cancer, we stained 3 distinct TMAs
comprising 15 normal or dysplastic glands and 126 breast cancers with affinity-purified
antibodies reacting selectively with Coco (Table S3 and Experimental Procedures). Whereas
normal or displastic mammary epithelial cells did not express detectable levels of Coco, the
tumor cells and scattered stromal cells in a large fraction of breast tumors produced variable
amounts of Coco (Figures 5F, 5G and S5I). Statistical analysis did not reveal any correlation
between the intensity of staining for Coco and pathological grade, clinical stage, ER, or
HER2 status in these samples (not shown). Interestingly, Kaplan Meyer analysis of the
MSKCC TMA dataset, which is annotated for metastatic relapse, indicated that the patients
with primary tumors exhibiting high levels of Coco had a significantly shorter overall
survival (46.2 ± 7.7 months) as compared to the remaining patients (104.9 ± 23.2 months)
(Figure 5H). We were not able to uncover other correlations, possibly because of the limited
number of cases present in this TMA and the paucity of paired samples. These observations
suggest that Coco confers a selective advantage during both tumor initiation and
progression.
Coco Expression Signatures Predict Metastatic Relapse to the Lung

DNA microarray analysis was used to examine if Coco’s activity correlates with metastatic
relapse in human breast cancer. Many signaling pathways, including the BMP pathway, are
restrained by negative feedback loops, which ensure expression of target genes only after a
defined threshold of signaling has been reached. We thus reasoned that a Coco-dependent
signature of gene expression may have been more reflective of Coco’s activity than the level
of expression of Coco mRNA. Furthermore, the Affymetrix HG-U133A and Agilent
platforms do not contain probes for Coco.
DNA microarray analysis of control and Coco-silenced MDA-MB231 cells led to the
definition of a signature comprising 56 genes (Figure 6A). By using a leave-one-out cross
validation method, we identified the 14 genes most relevant in predicting overall metastatic
relapse in the NKI295 dataset (Figure 6B, left), which comprises early stage, lymphnode
negative cases (van de Vijver et al., 2002) (Table S4), and validated their predictive power
on the EMC286 dataset (Figure S6A), which comprises early stage tumors from patients,
who did not undergo adjuvant therapy after surgery (Wang et al., 2005) (Table S4). Finally,
we tested the ability of the 14-gene signature to predict overall metastatic relapse in a
combined dataset comprising the MSK82, EMC192, and EMC286 cohorts. This large
dataset comprises patient populations with distinct clinical, pathologic and treatment
characteristics, in proportions similar to those occurring in the breast cancer population at

large (Bos et al., 2009) (Table S4). Interestingly, the 14-gene signature was strongly
associated with overall metastatic relapse in this cohort (P=2.9E-4; n=560) (Figure 6B,
right).
Further distillation of the 14-gene signature led to the identification of 2 of its component
genes, KIAA1199 and NDRG1, whose combined overexpression predicted overall relapse
in the EMC286 dataset with efficiency similar to that of the 14-gene signature (Figure S2B).
Although both genes encode for proteins of unknown cellular function, prior studies have
suggested that KIAA1199 sustains Wnt signaling in colorectal cancer (Birkenkamp-
Demtroder et al., 2011) and NDRG1 is activated by HIF-1α, through inactivation of Myc-
mediated repression, and by AP-1 (Ellen et al., 2008). Interestingly, expression of the 2
genes strongly correlated with poor prognosis in the largest cohort comprising the MSK82,
EMC192, EMC286, and NKI295 datasets (P=2.9E-7; n=855) (Figure S6B).

Gao et al. Page 9
To examine if expression of Coco correlates specifically with metastasis to the lung, we first
compared the levels of Coco in lung and bone metastatic variants of MDA-MB231 cells
(Zhang et al., 2009). Notably, Coco was upregulated in the lung metastatic variants
LM2-4180 and LM2-4175 but not in the bone metastatic variants Bo-1833 and Bo-2287
(Figure 6C). None of 9 additional secreted BMP inhibitors had a similar pattern of
upregulation in lung metastatic variants (Figure S5A). To corroborate the hypothesis that
expression of Coco underlies organ-specific metastasis to the lung, we evaluated if the 14-
gene and 2-gene signatures are able to selectively predict lung metastasis. Preliminary
analyses indicated that both signatures were able to predict relapse to the lung but not to the
bone or brain in the MSK82, EMC192, and EMC286 datasets (Figure S6C). To avoid the
potentially confounding effect of patients with multi-site metastases, we repeated the
analysis in the large combined dataset (MSK82 + EMC192 + EMC286) after exclusion of
such patients. The results indicated that both signatures were strongly associated with lung
but not bone or brain metastasis (Figures 6D and 6E) and predicted overall survival as well
as lung metastasis independently of transcriptomic subtype (Table S5), tumor size,
lymphnode positivity, ER status, HER2 status, pathological grade or expression of the NKI
70-gene poor survival signature (Tables S6 and S7). Finally, although the original Coco
signature comprising 56 genes displayed target organ-specificity similar to that of the
previously described Lung Metastasis Signature (Figure S6D) and was able to predict
overall survival with similar efficiency (Table S8), the two signatures only shared 3 genes
(Table S9), consistent with the involvement of biologically distinct mechanisms (Minn et al.,
2005).
To examine if NDRG1 and KIAA1199 participated in lung colonization, we silenced each

of the two genes, singly or in combination, in MDA-MB231 cells (Figure S6E-G). Although
single silencing of NDRG1 or KIAA1199 did not inhibit lung colonization (Figures 6F, 6G,
S6E, and S6F), simultaneous downregulation of both genes led to a significant inhibition of
lung colonization (>80% inhibition at 10 weeks; Figure 6H and S6G), suggesting that
NDRG1 and KIAA1199 are Coco-regulated genes involved in lung colonization.
Coco is not Required for Colonization of Bone or Brain

To assess if Coco participates in metastasis to the bone or brain, we performed intracardiac
injection experiments. Bioluminescent imaging showed that mice injected with control 4T1
cells develop bone, brain and adrenal gland metastases (Figures 7A and 7B). The bone
lesions occurred in >80% of the mice and were osteolytic (Figures 7A and S7A). Notably,
silencing of Coco did not reduce the rate of metastasis to bone, brain or adrenal gland or
inhibit the growth of individual lesions (Figures 7A and 7B), suggesting that Coco is not
required for colonization of these organs.
We hypothesized that Coco may mediate organ-specific metastasis to the lung because the
stroma of this organ contains particularly high levels of bioactive BMP proteins. Since the
action of BMP proteins is regulated by multiple secreted inhibitors, some of which can
neutralize each other or also function as direct activators, as well as by complex additional
mechanisms (Walsh et al., 2010), it is impossible to estimate the amount of bioavailable
BMP present in a given tissue by using direct methods. Therefore, to gauge the amount of
active BMP present in the bone marrow stroma, we examined P-Smad signaling in tumor
cells that had infiltrated this microenvironment. Analysis of bone sections from control mice
indicated strong nuclear accumulation of BMP-responsive P-Smad proteins in chondrocytes
in the growth plate but not in most hematopoietic cells (Figure S7B-D). Intriguingly, a large
fraction of the solitary 4T1 cells present in the bone marrow 7 days after intracardiac
injection did not display nuclear accumulation of P-Smad (Figure 7C). Furthermore,
virtually all the constituent cells in all micrometastases and osteolytic lesions detected at 5
weeks were similarly P-Smad-negative (Figure 7D), suggesting that these outgrowths had

Gao et al. Page 10
originated from P-Smad-negative solitary tumor cells. Similar results were obtained from
examining the micrometastatic lesions that arose in the bone of 2 out of 5 mice injected with
4TO7 cells 7.5 weeks earlier (Figure 7D). Finally, also all of the solitary tumor cells
detected in the brain parenchyma of mice injected 7 days earlier with 4T1 cells did not
display nuclear accumulation of BMP-responsive P-Smad proteins (Figure 7C). In contrast,
we had found that all the 4T07 and more than 94% of the 4T1 cells that had undergone
dormancy in the lung were P-Smad-positive (Figure 2H and 2J). These results suggest that
the levels of bioactive BMP proteins capable of engaging their cognate receptors on tumor
cells are low in at least a subset of stromal microenvironments in the bone and brain.
Together, these results suggest that Coco selectively mediates colonization of the lung
because it enables tumor cells to overcome the inhibitory action of BMP proteins that they
encounter upon infiltrating this organ.
DISCUSSION
We have found that breast cancer cells that have successfully extravasated in the lung and
survived initial attrition remain dormant for an extended period because stroma-derived
BMP proteins limit their ability to proliferate. Production of Coco enables a fraction of these
cells to overcome inhibitory BMP signaling and to outgrow into macroscopic metastases.
Thus, although most disseminated tumor cells may possess intrinsic defects that preclude
them from surviving or undergoing active proliferation in the lung, those that are fated to
give rise to clinical metastases, the metastasis-initiating cells, face strong anti-metastatic
signals originating from the parenchyma of this organ (Figure 7E).
It is plausible that only a subpopulation of tumor cells – the so called cancer stem cells –
possess the extensive self-renewal capability necessary for successful colonization of target
organs (Clevers, 2011; Valastyan and Weinberg, 2011). Our results suggest that BMP
enforces tumor dormancy by repressing two key cancer stem cell traits: self-renewal in vitro
and tumor initiation in vivo. They further suggest that Coco induces exit from dormancy by
reversing the ability of BMP to inhibit cancer stem cell function. This model is consistent
with previous studies indicating that the BMP pathway inhibits self-renewal and promotes
differentiation in pluripotent stem cells and various adult stem cells (Varga and Wrana,
2005). In addition, it is in general agreement with the observation that BMP signaling
contributes to inhibit mesenchymal and stem cell states in human mammary epithelial cells
(Scheel et al., 2011).
Of interest, BMP profoundly inhibited the expression of the transcription factors Nanog,
Sox-2, and Oct-4, which comprise the core regulatory circuit that sustains embryonic stem
cells (Young, 2011), and of the Hippo transducer Taz, which confers stem cell-related traits
on breast cancer cells (Cordenonsi et al., 2011). In contrast, Coco enhanced the expression
of these transcriptional regulators by reversing the effect of BMP. We presume that the
interrelationships between these transcriptional regulators are complex, and that each
component contributes to sustain a cancer stem cell program of gene expression, underlying
the functions necessary for metastatic reactivation. In fact, the transcriptional modules that
include as core components Nanog, Sox-2 and Oct-4 as well as Taz/Yap are overexpressed
in aggressive and metastatic solid tumors (Ben-Porath et al., 2008; Wong et al., 2008).
The observation that Coco promotes colonization of the lung, but not of the bone or brain,
reveals the existence of organ-specific barriers to metastatic reactivation. A large fraction of
breast cancer cells that infiltrated the bone marrow stroma and virtually all of those that
lodged in the brain parenchyma to give rise to metastatic outgrowths did not display nuclear
accumulation of BMP-responsive P-Smads, suggesting that they had not been exposed to
high levels of bioactive BMP. Thus, although they may face additional barriers, many tumor

Gao et al. Page 11
cells that infiltrate the bone or the brain do not need to neutralize locally produced BMP in
order to outgrow. Since pre-treatment with BMP blocks the ability of MDA-MB231 cells to
colonize the bone after intracardiac injection (Buijs et al., 2011), we speculate that breast
cancer cells that infiltrate the bone are sensitive to the inhibitory action of BMP. They
simply do not need to avert it through production of Coco because they lodge within
sanctuaries devoid of the cytokine.
Several lines of evidence indicate that Coco is a particularly potent mediator of metastatic
reactivation and that it exerts its effect by inhibiting lung-derived BMP (Supplementary
Discussion). In particular, Coco was the only BMP inhibitor whose expression correlated
with lung metastatic capacity. Furthermore, although the MDA-MB231 cells expressed
Chordin-like 1, DAN, and Noggin at levels similar to those of Coco, knock down of Coco
was sufficient to block the lung metastatic capacity of these cells. Although all secreted
BMP inhibitors function as ligand traps, some have additional functions. Furthermore,
individual members differ in oligomerization state, affinity for individual BMP proteins, and
ability to bind to the extracellular matrix (Walsh et al., 2010). Therefore, Coco might be a
particularly potent mediator of lung colonization because it has a very high affinity for BMP
proteins or because it binds to the pericellular matrix and therefore reaches a very high
concentration near the cell surface. However, we cannot exclude the possibility that Coco
induces metastatic reactivation in the lung also through BMP-independent mechanisms,
which remain to be defined.
The lag phase that separates the entry of tumor cells producing Coco into the lung stroma
and their outgrowth suggests that additional adaptive mechanisms mediate lung
colonization. Signals initiated by β1 integrins enable the initial outgrowth of tumor cells that
have established productive interactions with the interstitial matrix of this organ (Shibue and
Weinberg, 2009). In addition, the matrix proteins Tenascin C and Periostin organize niches
that nurse outgrowing micrometastases by modulating Notch and Wnt signalling,
respectively (Malanchi et al., 2012; Oskarsson et al., 2011). Although these signaling
mechanisms have not been specifically linked to the reactivation of solitary tumor cells, it is
possible that they cooperate with Coco to drive this process. Alternatively, they may act
following initial outgrowth to foster the expansion of micrometastatic lesions, similar to the
role of VCAM-1 in osteolytic bone lesions (Lu et al., 2011). Our observation that activated
β-catenin does not mediate exit from solitary tumor cell dormancy is consistent with the
hypothesis that Periostin falls in this latter class of prometastatic entities.
Attesting to the clinical relevance of our findings, patients with breast carcinomas
expressing high levels of Coco exhibited reduced overall survival. Furthermore, expression
of a 14-gene Coco signature predicted relapse to the lung but not to the bone or brain, in
agreement with the notion that Coco specifically promotes colonization of the lung.
Provocatively, further distillation of the signature led to the identification of 2 genes that
maintained an intact ability to predict relapse to the lung and, in fact, participated in this
process in a mouse model. Based on these observations, we suggest that the 14-gene and 2-
gene signatures may be used to identify patients with a significant risk to develop lung
metastases. Furthermore, we propose that monoclonal antibodies or other biological agents
blocking Coco may exhibit anti-metastatic activity.
Coco has emerged from a gain-of-function retroviral cDNA screen in a mouse model of lung
metastatic dormancy. The identification of Coco demonstrates that such screens could be
used to define the genes that mediate the exit of solitary tumor cells from dormancy in the
lung. Furthermore, similar screens using genome-wide shRNA libraries might enable the
identification of genes that promote dormancy. Finally, both cDNA and shRNA screens
could be applied to other mouse models to identify the mechanisms that regulate tumor

Gao et al. Page 12
dormancy at other metastatic sites or to investigate other steps of the metastatic cascade. By
affording the advantage of rapid biological validation of single entities that are able to
enforce dormancy or mediate exit from it, such gain-of-function genetic screens should lead
to the identification of additional potential therapeutic targets for the treatment of metastatic
disease.
EXPERIMENTAL PROCEDURES
Retroviral cDNA screen
Size-fractionated retroviral cDNA libraries were constructed essentially as described
previously (Koh et al., 2004). 67NR, 168FARN, or 4TO7 cells were infected independently
with the libraries at an MOI of 3:1 and injected in the mammary fat pad of BALB/c mice.
Clonogenic tumor cells isolated from macroscopic metastases were expanded in selective
medium (Aslakson and Miller, 1992). Proviral DNA was rescued and sequenced as
described previously (Koh et al., 2002).
Animal Studies
Tumorigenesis and metastasis assays were performed as previously described (Pylayeva et
al., 2009). Animal studies were conducted in accordance with protocols approved by the
Institutional Animal Care and Use Committee of MSKCC.
Tumor sphere assays

Mammosphere assays were performed essentially as previously described (Dontu et al.,
2003). ErbB2 cells were stained with PKH-26 dye and subjected to sequential tumor sphere
assays as described earlier (Cicalese et al, Cell 2009).
Human metastasis samples

TMAs containing primary breast tumors and lung metastases were generated by the
MSKCC Department of Pathology in compliance with protocols approved by the MSKCC
Institutional Review Board and after the subjects gave their informed consent. Coco
immunoreactivity was evaluated and scored by a clinical pathologist (E.B.). Overall survival
was examined as a function of Coco abundance in breast tumor samples.
Statistical Analysis
Comparisons between two groups were performed using an unpaired two-sided t test (p <
0.05 was considered significant). Results are reported as mean ± SD or ± SEM unless
otherwise noted. All in vitro experiments were performed at least three times.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank G. Daley, J. Massagué, and F. Miller for reagents, X. Zhang and X. Jing for help with X-ray imaging, M.
Akram for Coco staining of human TMAs, members of the Giancotti laboratory for discussions, and the staff of the
Genomics and the Molecular Cytology Facilities for their help. This work was supported by the Geoffrey Beene
Cancer Center at MSKCC and NIH grant P01 CA094060.

Gao et al. Page 13
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The secreted BMP inhibitor Coco mediates breast cancer colonization of the lung
Coco induces exit from dormancy and reactivation by blocking paracrine BMP
signaling
BMP signaling inhibits cancer stem cell traits and lung colonization
Coco-dependent gene expression signatures predict relapse to the lung in patients

Gao et al. Page 17
Figure 1. Coco Mediates Lung Colonization

(A) Gain-of-function retroviral cDNA screen.
(B) Metastatic capabilities of the cell lines and results of the first screen.
(C) Proteins precipitated from supernatants (4 ml) or extracted from cell layers (30 μg) were
subjected to immunoblotting (top). Cells were treated with or without trypsin for 30 minutes
before immunoblotting (bottom).
(D) 4TO7-TGL cells transduced with myc-Coco or empty vector (top) and 4T1 cells
expressing a control shRNA (sh-Control) or 2 shRNAs targeting Coco (sh #1 and #3) were
subjected to immunoblotting.
(E, F) 4TO7-TGL cells expressing Coco or not (E) and control and Coco-silenced 4T1-TGL
cells (F) were inoculated orthotopically in syngeneic mice. The graphs show mean tumor
volumes.
(G, H) 4TO7-TGL cells expressing Coco or not (G) and control and Coco-silenced 4T1-
TGL cells (H) were inoculated i.v.. Lung metastasis was measured by bioluminescent
imaging. The panels show representative images (left) and the graph shows the normalized
photon flux (right).
In all panels, error bars represent SD.
See also Figure S1.

Gao et al. Page 18
Figure 2. Coco Induces Reactivation in the Lung

(A) Mice were injected i.v. with 4TO7-TGL cells expressing Coco or not and sacrificed at
the indicated times. Lung sections were stained as indicated.
(B) The graph shows the number of GFP-positive solitary tumor cells per microscopic field
(left vertical axis) and of metastatic lesions per lung section (right vertical axis) at the
indicated times. Metastases were detected only in the lungs of mice injected with 4TO7-
Coco cells (red bars; P < 5.6E-4 at 21 days and < 3.1E-05 at 35 days).
(C) Outline of the EdU incorporation experiment.
(D) Representative images of lung sections stained as indicated.
(E) The graph shows the percentage of EdU+ cells at the indicated times. Cells expressing
Coco were categorized as indicated. **p<0.01.
(F) Control (sh-Control) and Coco-silenced (sh-Coco #1 and #3) 4T1-TGL cells were
inoculated i.v.. Lung sections were stained with anti-Ki-67 and anti-GFP. The graph shows
the percentage of Ki-67+ tumor cells at the indicated times. Control cells were categorized as
solitary (Sol.) or outgrowing (Out.). **p<0.01.
(G) Semiquantitative RT-PCR analysis of BMP receptors (top). Cells were left untreated or
exposed to 1 ng/ml BMP4 for 30 minutes and subjected to immunoblotting (bottom).
(H, I) 4TO7 and 4TO7-Coco cells were inoculated i.v. and lung sections were stained with
anti-P-Smad 1, 5, 8 and anti-GFP. The graph on the left shows the percentage of P-Smad−

Gao et al. Page 19
solitary tumor cells and the graph on the right the percentage of P-Smad− metastatic
outgrowths at the indicated times. Metastases were only detected in mice injected with
4TO7-Coco cells (red bars) (H). The panels show the positive staining pattern generated by
anti-P-Smad antibodies on virtually all dormant 4TO7 cells and the lack of reactivity of the
same antibodies with a subpopulation of dormant 4TO7-Coco cells. Metastases arising in
mice injected with 4TO7-Coco were counterstained with hematoxylin (bottom right).
(J) Control and Coco-silenced 4T1 cells were inoculated i.v. and lung sections were stained
with anti-P-Smad and anti-GFP. The graph shows the percentage of P-Smad− cells at the
indicated times. Control cells were categorized as solitary (Sol.) or outgrowing (Out.).
See also Figure S2.

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Figure 3. Coco Promotes Cancer Stem Cell Traits

(A) The graph shows the number of tumor spheres at each of three subsequent passages per
103 cells seeded.
(B) Coomassie Blue staining of recombinant Coco (left) and number of tumor spheres
formed by cells plated with the indicated concentrations (right).
(C, D) Cells were plated with the indicated concentrations of Coco and BMP. The graph
shows the efficiency of tumor sphere formation as compared to control cells. ***p<0.001
(C). Representative images (Coco: 0.9 μg/ml, BMP4: 10 ng/ml) (D).
(E, F) Outline of PKH-26 staining and serial tumor sphere assay. ErbB2-transformed cells
were stained with PKH-26 (left) and subjected to tumor sphere assay. The primary spheres
were dissociated and their constituent cells were sorted according to staining intensity
(PKHHIGH, PKHLOW, and PKHNEG) (center) and subjected to secondary tumor sphere
assays. Finally, the PKHHIGH cells from secondary tumor spheres were subjected to the
same protocol to derive tertiary tumor spheres (E). The graph show the results obtained with
control and Coco-silenced ErbB2-transformed cells. *p<0.05, **p<0.01, ***p<0.001, NS-
not significant.
(G, H) 4TO7 and 4TO7-Coco cells (G) and control and Coco-silenced 4T1 cells (H) were
inoculated orthotopically at the indicated doses. The graphs show mean tumor volumes.
(I, J) Q-PCR of indicated genes in 4TO7 and 4TO7-Coco cells (I) and in 4TO7 cells left
untreated or exposed to 10 ng/ml BMP4 for 2 days (J). In panel A, B, C and F, error bars
represent SD.
In panel G, H, I and J, error bars represent SEM.
See also Figure S3 and Table S1, S2.

Gao et al. Page 21
Figure 4. Inhibition of BMP Signaling Enhances Cancer Stem Cell Traits and Lung Colonization
(A) Cells were transduced with the indicated constructs were subjected to immunoblotting
(left) and tumor sphere assay (right).
(B) Representative images.
(C) 4TO7 cells expressing the indicated constructs were inoculated i.v.. The panels show
representative images (top) and the graph the normalized photon flux at the indicated times
(bottom).
(D) 4T1 cells transduced as indicated were injected orthotopically at the indicated doses.
The graph shows the mean tumor volumes.
(E) Cells were inoculated i.v. and lung metastasis was assessed as in C.
See also Figure S4.

Gao et al. Page 22
Figure 5. Coco Promotes Human Breast Cancer Metastasis

(A) Human breast cancer cells classified as indicated (Supplementary Experimental
Procedures) were subjected to immunoblotting (Bo: bone only; L*: predominantly lung;
BaA and BaB: basal A and B; Lu: luminal).
(B) Control and Coco-silenced MDA-MB-231 cells were subjected to immunoblotting (top
left) and lung colonization assay. The graph shows the normalized photon flux (bottom left,
±SD) and the panels show representative images (right).
(C, D) Control and Coco-silenced MDA-MB-231 cells were subjected to FACS (C) and
tumor sphere assay (D, left, ±SD). Viability at the end of the assay was measured by MTT
staining (D, right, ±SEM).
(E) Control and Coco-silenced MDA-MB-231 cells were inoculated orthotopically at the
indicated doses in NOD/SCID/IL2Rγ−/− mice. The graphs show mean tumor volumes (±
SEM. *p<0.05). **p<0.01, n.s.-not significant.
(F) Normal breast tissue and a case of invasive ductal carcinoma were stained with
monospecific antibodies to Coco and counterstained with Hematoxylin. Note the positivity
of nests of tumor cells and scattered stromal cells (high magnification inset).
(G) TMAs comprising 126 primary breast tumors were stained with anti-Coco. The pictures
show images of cases exhibiting varying levels of positivity. The graph shows the
distribution of staining intensity across samples.
(H) Kaplan-Meier analysis of overall survival based on Coco expression in primary tumors.
See also Figure S5 and Table S3, S4.

Gao et al. Page 23
Figure 6. Coco Predicts Specifically Relapse to the Lung

(A) Hierarchical clustering of genes upregulated or downregulated (> 2 fold) in Coco-
silenced MDA-MB231 cells as compared to control cells. NDRG1 and KIAA119 are
underlined.
(B) Kaplan-Meier analysis of metastasis-free survival in the NKI295 (left) or the indicated
combined dataset (right). Patients were divided according to expression of the 14-gene Coco
signature (red line, positive; blue line, negative).
(C) MDA-MB231 cells and their lung and bone metastatic derivatives were subjected to
immunoblotting.
(D, E) Kaplan-Meier analysis of lung-only metastasis-free survival (left), bone-only
metastasis-free survival (middle), and brain-only metastasis-free survival (right) in the
MSK82, EMC192 and EMC286 combined dataset. Patients were divided according to
expression of the 14-gene (D) or 2-gene Coco signature (E).
(F, G) MDA-MB-231 cells expressing a control sh-RNA (sh-Control) and two sh-RNAs
targeting NDRG1 (sh-NDRG #3 and #5) (F) or two sh-RNAs targeting KIAA1199 (sh-
KIAA #1 and #5) (G) were subjected to lung colonization assay.
(H) MDA-MB-231 cells expressing a control sh-RNA (sh-Control) or sh-RNAs targeting
NDRG1 and KIAA1199 (sh-KIAA #1/ NDRG #3 and KIAA #5/ NDRG #5) were subjected

Gao et al. Page 24
to lung colonization assay. The graph shows the normalized photon flux (left) and the panels
show representative images (right).
See also Figure S6 and Table S4-S7.


Gao et al. Page 25
Figure 7. Coco-mediated Blockage of BMP is Not Required for Metastasis to the Bone or Brain
(A, B) Mice inoculated i.c. with control or Coco-silenced 4T1-TGL cells were subjected to
bioluminescent imaging. Bone metastases were confirmed by X-ray radiography. The panels
show representative images. Yellow dotted lines delineate the boundaries of osteolytic
lesions in the radiograms (A). The graph shows the photon flux signal of metastases in the
hind limbs, brains and adrenal glands of mice (B, ± SEM).

(C) Mice were inoculated i.c. with 4T1-TGL cells and sacrificed 7 days later. Bone and
brain sections were subjected to staining with anti-P-Smad and anti-GFP. The graph shows
the percentage of P-Smad+ and P-Smad− tumor cells in the bone and the brain. The images
show representative images.
(D) Mice were inoculated i.c. with 4T1 and 4TO7 cells and sacrificed at the indicated times.
Bone sections were stained with H&E and anti-P-Smad. The graph shows the percentage of
P-Smad+ and P-Smad− tumor cells in micrometastases and macroscopic metastases
generated by 4T1 cells and in micrometastatic clusters produced by 4TO7 cells (top left).
The pictures show representative images.
(E) Model of Coco’s function.
See also Figure S7.

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Cell. 2012 August 17; 150(4): 764–779. doi:10.1016/j.cell.2012.06.035.

The BMP Inhibitor Coco Reactivates Breast Cancer Cells at Lung

2Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New

2011). It is currently unclear if tumor cells become dormant as a consequence of intrinsic

© 2012 Elsevier Inc. All rights reserved.

signalling and thereby enhancing the self-renewal capability of metastasis-initiating cells.

Coco Promotes Lung Colonization

Cell. Author manuscript; available in PMC 2013 August 17.

orthotopic injection, confirming that Coco promotes spontaneous metastasis similar to

To examine if Coco promotes the colonization step of metastasis, we performed tail-vein

Coco Induces Exit from Dormancy

Cell. Author manuscript; available in PMC 2013 August 17.

cells to exit from dormancy and produce metastatic outgrowths.

Coco Inhibits BMP Signaling in Metastasis-initiating Cells

In a reciprocal set of experiments, we evaluated if silencing of Coco induced reactivation of

Cell. Author manuscript; available in PMC 2013 August 17.

dormancy by alleviating the ability of stromal BMP to enforce a dormant state.

Coco Promotes Tumor Sphere Formation In Vitro

Cell. Author manuscript; available in PMC 2013 August 17.

Coco Promotes Tumor Initiation In Vivo

Coco Sustains Expression of Stem Cell Transcription Factors

Coco Promotes Metastatic Reactivation by Inhibiting BMP Signaling

Cell. Author manuscript; available in PMC 2013 August 17.

To examine if inhibition of BMP signaling promotes metastatic reactivation, we injected

to a modest extent (Figure 4C). Expression of a dominant negative form of BMPR-IB

Coco Promotes Human Breast Cancer Metastasis

Cell. Author manuscript; available in PMC 2013 August 17.

of MDA-MB231 cells to initiate tumorigenesis upon orthotopic injection in NOD-SCID-

Coco Expression Signatures Predict Metastatic Relapse to the Lung

characteristics, in proportions similar to those occurring in the breast cancer population at

Cell. Author manuscript; available in PMC 2013 August 17.

To examine if NDRG1 and KIAA1199 participated in lung colonization, we silenced each

Coco is not Required for Colonization of Bone or Brain

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Tumor sphere assays

Human metastasis samples

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Figure 1. Coco Mediates Lung Colonization

Cell. Author manuscript; available in PMC 2013 August 17.

Figure 2. Coco Induces Reactivation in the Lung

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Figure 3. Coco Promotes Cancer Stem Cell Traits

Cell. Author manuscript; available in PMC 2013 August 17.

Cell. Author manuscript; available in PMC 2013 August 17.

Figure 5. Coco Promotes Human Breast Cancer Metastasis

Cell. Author manuscript; available in PMC 2013 August 17.

Figure 6. Coco Predicts Specifically Relapse to the Lung

Cell. Author manuscript; available in PMC 2013 August 17.

See also Figure S6 and Table S4-S7.

Cell. Author manuscript; available in PMC 2013 August 17.

hind limbs, brains and adrenal glands of mice (B, ± SEM).

Cell. Author manuscript; available in PMC 2013 August 17.