Cancer-Stimulated Mesenchymal Stem Cells Create a Carcinoma Stem Cell Niche via Prostaglandin E2 Signaling

Abstract

Unlabelled:
Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell-derived interleukin-1 (IL-1) induces prostaglandin E(2) (PGE(2)) secretion by MSCs. The resulting PGE(2) operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE(2) and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE(2) and cytokines.

Significance:
Although PGE2 has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE(2) axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells.

Full text

Cancer-Stimulated Mesenchymal Stem
Cells Create a Carcinoma Stem Cell
Niche via Prostaglandin E
2 Signaling
Hua-Jung Li 1 , Ferenc Reinhardt 1 , Harvey R. Herschman 4 , and Robert A. Weinberg 1 – 3
RESEARCH ARTICLE
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ABSTRACT Mesenchymal cells of the tumor-associated stroma are critical determinants of
carcinoma cell behavior. We focus here on interactions of carcinoma cells with
mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able
to infl uence the phenotype of the carcinoma cells. We fi nd that carcinoma cell–derived interleukin-1
(IL-1) induces prostaglandin E
2 (PGE 2 ) secretion by MSCs. The resulting PGE
2 operates in an autocrine
manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines
by the MSCs. The PGE
2 and cytokines then proceed to act in a paracrine fashion on the carcinoma cells
to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indi-
cate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via
release of PGE
2 and cytokines.
SIGNIFICANCE: Although PGE
2 has been implicated time and again in fostering tumorigenesis, its
effects on carcinoma cells that contribute specifi cally to tumor formation are poorly understood. Here
we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E
synthase-1 (mPGES-1)/PGE
2 axis in MSCs recruited to the tumor-associated stroma by releasing IL-1,
which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells. Cancer Discov;
2(9); 840–55. ©2012 AACR.
Authors’ Affi liations: 1 Whitehead Institute for Biomedical Research;
2 Department of Biology, Massachusetts Institute of Technology;
3 MIT
Ludwig Center for Molecular Oncology, Cambridge, Massachusetts; and
4 Departments of Biological Chemistry and Pharmacology, Molecular Biol-
ogy Institute, and Jonsson Comprehensive Cancer Center, UCLA, Los Ange-
les, California
Note: Supplementary data for this article are available at Cancer Discovery
Online ( http://cancerdiscovery.aacrjournals.org/ ).
Corresponding Author: Robert A. Weinberg, Whitehead Institute for Bio-
medical Research, 9 Cambridge Center, Cambridge, MA 02142. Phone:
617-258-5159; Fax: 617-258-5230; E-mail: weinberg@wi.mit.edu
doi: 10.1158/2159-8290.CD-12-0101
©2012 American Association for Cancer Research.
INTRODUCTION
Carcinoma cells recruit mesenchymal cells into the tumor-
associated stroma; these mesenchymal cells then proceed to
modify the stroma, helping to establish a tissue microen-
vironment that favors tumor progression. Paracrine signals
emanating from the resulting tumor-associated stroma can
subsequently modulate the behavior of the carcinoma cells ( 1 ).
Among the recruited stromal cells are bone marrow–derived
mesenchymal stem cells (MSC), which are known to exhibit
multipotent differentiation potential ( 2 ). In the context of
cancer pathogenesis, MSCs contribute to the formation of
fi broblast and myofi broblast populations in the tumor-asso-
ciated stroma ( 3, 4 ) and promote the growth, progression, and
metastasis of tumors ( 3 , 5 , 6 ). Precisely how MSCs infl uence
tumor progression is, however, poorly understood.
Elevated COX-2 mRNA and protein levels are found in many
malignant tissues and are often associated with poor clinical
outcome ( 7 ). The tumor-enhancing effects of COX-2 are gener-
ally ascribed to its role in producing prostaglandin E
2 (PGE 2 ),
which has pleiotropic effects on cell proliferation, survival,
angiogenesis, motility, and invasiveness ( 8 ). In addition to
the neoplastic cells themselves, cells of the tumor- associated
stroma contribute to elevated COX-2 expression ( 9, 10 ). How-
ever, it has been unclear whether the PGE
2 that promotes
tumor progression derives from neoplastic cells, fi broblasts,
macrophages, or some combination of these cell types.
Independent of these questions is the issue of heterogeneity
of the neoplastic cells within carcinomas. Observations of a vari-
ety of human cancer types have revealed the existence of tumor-
initiating cells (TIC), often called cancer stem cells (CSC), which
coexist as minority populations within tumors, together with a
majority population of cancer cells that lack tumor-initiating
ability ( 11 ). Passage by neoplastic epithelial cells through an
epithelial–mesenchymal transition (EMT) allows these cells to
approach the stem cell state ( 12, 13 ). Moreover, EMT programs
are known to be induced by heterotypic signals that epithelial
cells receive from the microenvironment ( 14 ). However, the
nature of these heterotypic signals and the identities of the stro-
mal cells that release them remain poorly understood.
We show here that, in response to stimulation by carci-
noma cells, MSCs express greatly elevated levels of PGE
2 . The
resulting PGE
2 , together with cytokines also induced in the
MSCs, contribute to entrance of nearby carcinoma cells into
a stem cell–like state.
RESULTS
PGE 2 Induction in MSCs following Interaction
with Carcinoma Cells
We initially studied the interactions in culture of LoVo
and SW1116 human colorectal carcinoma cells with MSCs.
Minimal PGE
2 accumulation was observed in pure LoVo,
SW1116, or MSC cultures monitored over a 72-hour period
( Fig. 1Aa and Supplementary Fig. S1A). However, PGE
2 levels
increased by approximately 6.5-fold when the LoVo cells were
cocultured with twice the number of MSCs for 48 hours
and increased by approximately 60-fold after 72 hours of
co culture. Correspondingly, levels of the COX-2 enzyme were
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Li et al.
RESEARCH ARTICLE
***
***
***
***
9
LoVo/MSC
LoVo
MSC
SW1116MSC
SW1116
PGE2 (ng/mL)
8
4
3
2
1
0
24 48 7260 663612
Time (h)
*
*
*
*
**
PGE2 (ng/mL)
0
DME
LoVoCM
LoVoCM + anti-IL-1α
LoVoCM + anti-IL-1β
LoVoCM + anti-IL-1αβ
IL-1α 1 ng/mL
IL-1α 0.1 ng/mL
IL-1β 1 ng/mL
IL-1β 0.1 ng/mL
4
8
12
16
20
28
48
68
***
PGE2 (ng/mL)
LoVoshsc
LoVoshIL1β-1
LoVoshIL1β-2
LoVoshIL1αβ-1
LoVoshIL1αβ-2
0
3
6
9
12
15
18
***
***
***
PGE2 (ng/mL)
MSCshsc + DME
MSCshsc + LoVoCM
MSCshsc + LoVoCM
+10 ng/mL IL-1ra
MSCshsc + LoVoCM
+1,000 ng/mL IL-1ra
0
4
8
12
16
20 **
*
LoVo
HCC1806
BT549
SUM149
SUM159
MDA-MB-453
MDA-MB-231
SW1116
0
IL-1α or IL-1ra level in CM (pg/mL)
IL-1β level in CM (pg/mL)
20
40
60
80
100
120
140
IL-1α
IL-1β
IL-1ra
160
0
6
12
18
24
30
PGE2 (ng/mL)
M
LoVo
HCC1806
*, P < 0.05; **, P < 0.001; ***, P < 0.005
BT549
SUM149
SUM159
MB453
MB231
SW1116
LoVoM
HCC1806M
BT549M
SUM149M
SUM159M
MB453M
MB231M
SW1116M
***
***
***
***
***
0
4
8
12
16
20
24
28
32
36
40
44
48
52
LoVo
MSC
LoVo+MSC
LoVo/MSC
SW1116
Sw1116+MSC
SW1116MSC
COX-2
GAPDH
Aab
B
C
a
ab
bc
Figure 1. Carcinoma cell–secreted IL-1 induces PGE
2 production in MSCs. A, PGE
2 (a) and COX-2 (b) were measured in the indicated conditioned
medium or cultures. PGE
2 data are means ± SE, n = 3. ***, P < 0.005 (vs. that in LoVo medium). LoVo + MSC; LoVo lysate and MSC lysate mixed in equal
amounts; LoVo/MSC, lysate of LoVo/MSC coculture. The same nomenclature applies to the SW1116 cells. B, a, MSCs were treated with LoVoCM, LoVoCM
with neutralizing antibodies (500 ng/mL), or IL-1 only. b, MSCs were cocultured with LoVo cells expressing shRNAs against IL1α + IL1β (LoVoshIL1αβ-1,
LoVoshIL1αβ-2), IL1β (LoVoshIL1β-1, LoVoshIL1β-2), or a scrambled sequence (LoVoshsc).c, MSCs were treated with DME, LoVoCM, or LoVoCM+IL-1ra.
After incubation, media were collected and assayed for PGE
2 . Data are means ± SE, n = 3. C, a, IL-1α, IL-1β, and IL-1ra protein levels in conditioned
medium of the carcinoma cells. Data are means ± SE, n = 3. b, PGE
2 levels in carcinoma cells, MSCs (M), and cocultures of the carcinoma cells with MSCs.
Data are means ± SE, n = 3. ***, P < 0.005 (vs. that in MSC culture).
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PGE2 and the MSC-Derived Cancer Stem Cell Niche RESEARCH ARTICLE
also increased in the coculture ( Fig. 1Ab ). In contrast, there
was no PGE
2 increase in SW1116/MSC cocultures. Of note,
PGE
2 production was induced equally strongly in [LoVo]
[MSC] transwell cocultures, which only permitted their
intercommunication via soluble factors (Supplementary Fig.
S1B) and in MSCs treated with LoVo-conditioned medium
(LoVoCM; Supplementary Fig. S1C and S1D). Hence, soluble
factors secreted by LoVo cells were responsible for inducing
PGE
2 production by MSCs.
Other work (in Supplementary Data, Section 1; Supple-
mentary Fig. S1E) showed that the secretion of interleukin-1β
(IL-1β) and IL-1α by carcinoma cell populations was correlated
with their respective abilities to elicit PGE
2 production in
cocultured MSCs. Thus, when MSCs were treated with recom-
binant IL-1β or IL-1α, the induced PGE
2 levels were similar
to or higher than those induced by LoVoCM ( Fig. 1Ba ). Con-
versely, IL-1α– and IL-1β–neutralizing antibodies attenuated
by 60% the production of LoVoCM-induced PGE
2 ( Fig. 1Ba ).
In addition, short hairpin RNAs (shRNA) directed against
IL-1α and IL-1β mRNAs expressed in LoVo cells decreased by
84% the ability of their conditioned media to induce PGE
2 in
MSCs ( Fig. 1Bb ) and recombinant IL-1ra, a natural antagonist
of the IL-1 receptor, attenuated by 73% the LoVoCM-induced
PGE
2 production by MSCs ( Fig. 1Bc ). IL-1 α/β secreted by the
carcinoma cells was largely responsible for the PGE
2 produc-
tion by MSCs, whereas IL-1ra could antagonize this induction.
Relationship of IL-1 Production by Carcinoma
Cells to Their Ability to Induce PGE
2
The levels of the IL-1α, IL-1β, and IL-1ra mRNAs and
secreted proteins were quantifi ed in 6 human breast carcinoma
cell lines and the SW1116 colorectal carcinoma cell line, in
addition to the LoVo cells examined above. Cells of the LoVo,
HCC1806, BT549, SUM149, and SUM159 lines expressed ele-
vated levels of IL-1α and/or IL-1β, but relatively low levels of
IL-1ra; conversely, MDA-MB-453, MDA-MB-231, and SW1116
cells secreted little or no detectable IL-1α/β and/or relatively
high levels of IL-1ra (Supplementary Fig. S1F and Fig. 1Ca ).
When cultured individually, the carcinoma cell lines
expressed low or undetectable PGE
2 levels ( Fig. 1Cb ). However,
upon coculture with MSCs, the IL-1α/β–secreting carcinoma
cells induced approximately 80- to 500-fold increases of COX-2
and PGE
2 production, whereas the carcinoma cells secreting
low levels of IL-1α/β failed to stimulate COX-2 or PGE
2 for-
mation (Supplementary Fig. S1G and Fig. 1Cb ). In addition,
IL-1α-/IL-1β–neutralizing antibodies and IL-1ra attenuated
PGE
2 production induced in MSCs by the conditioned media
from the various IL-1–secreting carcinoma cells (Supplemen-
tary Fig. S1H). Hence, the ability to stimulate PGE
2 in MSCs
seemed to be a frequent but not universal property of breast
and colon carcinoma cells and was directly correlated with
their abilities to signal via secreted IL-1α/β. Because IL-1α and
IL-1β were both capable of PGE
2 induction, we used the term
“IL-1” to refer to both IL-1α and IL-1β in the text that follows.
Induction of Cytokines in MSCs following Their
Interaction with Carcinoma Cells
In addition to PGE
2 , GRO-α, IL-6, IL-8, and regulated upon
activation, normal T-cell expressed, and secreted (RANTES)
in the culture media of LoVo/MSC cocultures increased by
34-, 10-, 79-, and 21-fold, respectively, following 120 hours
of coculture ( Fig. 2A ). In [LoVo][MSC] transwell cultures,
in which direct contact between the LoVo cells and MSCs
was prevented, PGE
2 , GRO-α, IL-6, and IL-8 were induced to
comparable levels; in contrast, RANTES expression was not
elevated ( Fig. 2A ). Following direct coculture, RANTES pro-
duction occurred far more rapidly than did the accumulation
of the other cytokines or PGE
2 (Supplementary Fig. S2A).
We also found that GRO-α, IL-6, and IL-8, like PGE
2 , were
induced by LoVoCM in MSCs (Supplementary Fig. S1B). To
determine whether IL-1 was able, by itself, to induce con-
comitant production in MSCs of the 3 cytokines and PGE
2 , we
assessed mRNAs levels in MSCs that had been treated for 48
hours with vehicle or recombinant IL-1 ( Fig. 2B ). The resulting
10-fold and 4-fold increases in MSCs of COX-2 and microsomal
prostaglandin-E synthase-1 [mPGES-1 (a second PGE
2 biosyn-
thetic enzyme)] mRNAs, the increase of COX-2 protein, and
the 10- to 100-fold decrease of 15-hydroxyprostaglandin dehy-
drogenase (15-PGDH) mRNA (encoding the PGE
2 -degrading
enzyme) confi rmed the key role of IL-1 in modulating the lev-
els of enzymes governing PGE
2 production and accumulation
in MSCs. Moreover, IL-1 treatment alone elicited substantial
increases (36- to 440-fold) of IL-6, IL-8, and GRO-α mRNAs
in MSCs. COX-2, IL-6, IL-8, and GRO-α mRNA induction was
detectable within 30 minutes of IL-1 exposure and reached
a maximum at 1 to 2 hours thereafter (Supplementary
Fig. S2B). This key role of IL-1 was further confi rmed by
knocking down IL-1α/β mRNAs in LoVo cells, resulting in a
60% to 90% decrease in the induced levels of IL-6, IL-8, and
GRO-α mRNAs (Supplementary Fig. S2C). Thus, IL-1 was
both necessary and suffi cient to induce PGE
2 , IL-6, IL-8, and
GRO-α production in MSCs.
We also confi rmed that LoVoCM and IL-1 could induce
comparable levels of PGE
2 (Supplementary Fig. S2D) and
cytokines (Supplementary Fig. S2E) in other types of mesen-
chymal cells that may arise from the differentiation of MSCs
( 4 ), including human breast MSCs (bMSCs) isolated from a
breast cancer patient, human colonic myofi broblasts (CCD-
18co), and primary human mammary stromal fi broblasts.
Autocrine PGE 2 Cooperation with IL-1
Paracrine Signaling Leading to PGE
2 and
Cytokine Production by MSCs
IL-8 and IL-6 are known to be induced in certain cells by PGE
2
( 15 ). To determine whether PGE
2 played a role in the induction
of these cytokines in MSCs, PGE
2 production by MSCs was
blocked with indomethacin, which inhibits the COX-1 and
COX-2 enzymes. In LoVo/MSC cocultures, GRO-α, IL-6, and
IL-8 protein induction was reduced by 85% to 98% by indometh-
acin; moreover, this induction could be partially rescued by
providing PGE
2 to indomethacin-treated cocultures ( Fig. 2C ).
Although PGE
2 treatment alone could not induce GRO-α, IL-6,
or IL-8 expression in LoVo cells or MSC cells, additional PGE
2
potentiated the cytokine induction in LoVo/MSC cocultures
and in IL-1–treated MSCs ( Fig. 2C and Supplementary Fig. S2F).
MSCs expressed 2 distinct PGE
2 cell-surface receptors, EP2
and EP4 (Supplementary Fig. S2G). To support the notion
that MSC-derived PGE
2 acted in an autocrine fashion, the
EP2 and EP4 receptor antagonists AH6809 and GW627368X
were added to MSC cultures, along with either LoVoCM
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Li et al.
RESEARCH ARTICLE
0
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***
0
5
10
15
20
00
1
2
3
4
5
6
7
8
15
25
10
15
× 10
× 10
60
100
0IL-6
20
40
60
80
100 LoVoCM
+EP2/4 antagonist
IL-1
+EP2/4 antagonist
2
1
3
4
5
6
10−9Control IL1-α
1 ng/mL
IL1-β
1 ng/mL
IL1-α
+
IL1-β
10−8
10−7
10−6
10−5
10−4
10−3
10−2
10−1
100
101
LoVo/MSC
[LoVo][MSC]
SW1116MSC
MSC
LoVo
SW1116
20
40
60
80
***
**
**
**
**
**
*
***
***
*** ***
***
*
*
**
***
***
*** ***
***
***
***
*** ***
***
**
** ***
***
***
***
*** ***
100
Relative expression
of soluble factor
Cytokine level (ng/mL)
mRNA induction
(percent compared to the level
in MSC treated with LoVoCM or IL-1)
mRNA level by qPCR
(Target gene/GAPDH)
120
PGE2
GRO-α
IL-6
IL-8
GRO-α
IL-6
IL-8
RANTES
PGE2
LoVO
MSC
Indo
–
+
–
–
–
–
+
–
–
+
+
–
–
+
+
+
+
+
+
+
+
+
–
–
+
–
+
–
+
+
+
–
COX-2
GRO-α
IL-6
mPGES1
15-PGDH
IL-8
β-Actin
COX-2
140
160
AB
C
E
D
**, P < 0.01; ***, P < 0.005, compared with MSC culture
***, P < 0.005
EP4 EP4
IL-6
IL-8
PGE2
EP2/4
GRO-α
EP4
EP2/4 EP2/4
PGE2
PGE2
AA
COX-2
mPGES1
IL-1
IL-1
IL-1R IL-1R
IL-1R
MSC
LoVo cell
*, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with control
*, P < 0.05; **, P < 0.01, MSC treated with
LoVoCM or IL-1 only.
IL-8 GRO-α
Figure 2. IL-1 and PGE 2 mediate GRO-α, IL-6, and IL-8, but not RANTES induction in carcinoma cell–MSC coculture. A, levels of PGE
2 and the cytokines
were measured in conditioned media from the cultures. Data are means ± SE, n = 3. **, P < 0.01; ***, P < 0.005 (vs. that in MSC culture). B, mRNA levels
of the enzymes governing PGE
2 production and the cytokines (top) and COX-2 protein levels (bottom) in MSCs treated with IL-1 as indicated. Data are
means ± SE, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (vs. control). C, cytokine levels in conditioned media from MSCs, LoVo cells, and LoVo/MSC cocul-
tures, in the presence of indomethacin (indo; 100 μmol/L), PGE
2 (100 nmol/L), or Indo + PGE
2 . Data are means ± SE, n = 3. D, IL-6, IL-8, and GRO-α mRNA
induction in MSCs either by LoVoCM or by IL-1 in the presence of EP2 and EP4 antagonists (AH6809 15 μmol/L + GW627368X 20 μmol/L). mRNA levels
are set as 0% for vehicle-treated MSCs and 100% for LoVoCM-treated or IL-1–treated MSCs. Data are means ± SE, n = 3. *, P < 0.05; **, P < 0.01 (vs. MSC
treated with LoVoCM or IL-1 without inhibitors). E, the proposed interactions for PGE
2 and cytokine induction from MSCs. AA, arachidonic acid.
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PGE2 and the MSC-Derived Cancer Stem Cell Niche RESEARCH ARTICLE
or IL-1. LoVoCM- and IL-1–induced IL-6, IL-8, and GRO-α
mRNA was suppressed by 60% to 80% by these EP receptor
antagonists ( Fig. 2D ). Figure 2E summarizes our model that
(i) COX-2, mPGES1, and PGE
2 are initially induced in MSCs
by IL-1 released by carcinoma cells and that (ii) the resulting
PGE
2 , acting in an autocrine manner on MSCs, then cooper-
ates with ongoing IL-1 paracrine signaling to trigger IL-6,
IL-8, and GRO-α production by MSCs ( Fig. 2E ).
Effects of MSCs on Carcinoma Cell
Mesenchymal and Invasive Traits
Before further analyzing the interactions between the car-
cinoma cells and MSCs, we confi rmed that, as reported by
others ( 5 , 16 ), MSCs are indeed recruited to IL-1–secreting
tumors in vivo (described in Supplementary Data, Section 2;
Supplementary Fig. S3A and B). Having done so, we further
investigated the infl uence of MSCs on carcinoma cell behav-
ior, more specifi cally by analyzing the effects of MSC cocul-
ture on the expression by the tumor cells of markers of EMT,
a cell–biological program that imparts motility, invasiveness,
and self-renewal to carcinoma cells ( 14 ). After culturing, either
alone or together with tandem dimer (td) Tomato-MSCs
(5 days), LoVo and HCC1806 cells were isolated by fl uores-
cence-activated cell sorting (FACS) and analyzed for E-cad-
herin, vimentin, fi bronectin, and β-actin protein expression
( Fig. 3A ). E-cadherin, a key epithelial marker, was decreased
by 98% to 100% in both LoVo and HCC1806 cells cocultured
with MSCs. Conversely, vimentin and fi bronectin proteins—
both mesenchymal markers—were robustly induced in both
carcinoma cells ( Fig. 3A ). Moreover, expression of the SNAIL
protein, an EMT-inducing transcription factor (EMT-TF),
was increased 5- to 69-fold in the MSC/carcinoma cocultures
( Fig. 3A ).
We then determined whether PGE
2 and/or the cytokines
produced by MSCs in LoVo/MSC cocultures could elicit an
EMT-like response in LoVo cells. PGE
2 was able, on its own,
to cause a decrease in E-cadherin protein in LoVo cells ( Fig.
3B , ∼70% decrease) but failed to elicit concomitant robust
increases of mesenchymal markers, that is, vimentin and the
ZEB1, SNAIL, and TWIST1 EMT-TFs ( Fig. 3B ).
In contrast to PGE
2 , IL-6 alone induced ZEB1 (7-fold),
SNAIL (3-fold), and vimentin (3-fold) protein expression in
LoVo cells, but was unable to decrease E-cadherin protein.
However, treatment of LoVo cells with PGE
2 together with
the 4 cytokines induced both a decrease of E-cadherin protein
expression (∼80%) and increases of vimentin (9-fold), ZEB1
(14-fold), SNAIL (13-fold), and TWIST1 (10-fold) protein
expression ( Fig. 3B ). Hence, activation of a more complete
EMT program in the carcinoma cells required the concomi-
tant activation of multiple signaling pathways, specifi cally
those triggered in these cells by PGE
2 acting together with the
indicated cytokines.
Signifi cantly more carcinoma cells invaded in LoVo/MSC
cultures than in cultures of LoVo cells alone ( Fig. 3C ). To
examine whether PGE
2 played a critical role in the MSC-
induced carcinoma cell invasiveness, LoVo/MSC cocultures
were treated with NS398, a COX-2 inhibitor; this treatment
resulted in an 80% reduction of MSC-induced LoVo cell inva-
siveness ( Fig. 3C ). Greater than 60% of this inhibition could
be reversed by adding PGE
2 to these cocultures.
Also, LoVo cell invasion was significantly reduced by
antibodies that neutralize either IL-6, GRO-α, or RANTES
( Fig. 3D ). Accordingly, the MSC-induced carcinoma cell
invasiveness seemed to derive from a confluence of PGE
2 ,
IL-6, GRO-α, and RANTES signals impinging on the LoVo
cells.
We also examined possible effects of MSCs on carcinoma
cell invasion in vivo . The carcinoma cells injected on their own
formed reasonably well-encapsulated tumors ( Fig. 3E , a, b, c).
In contrast, the carcinoma cells coinjected with MSCs formed
extensive invasive fronts that extended into adjacent muscle
layers ( Fig. 3E , d, e, f). In addition, we observed the intrava-
sation of carcinoma cells into nearby microvessels ( Fig. 3E ,
insert). To summarize, these data indicated that MSCs facili-
tated LoVo cell invasion both in vitro and in vivo .
Effects of MSCs on Tumor Initiation
by Carcinoma Cells
Due to the fact that transformed epithelial cells that have
undergone EMT contain larger subpopulations of TICs
( 12, 13 , 17 ), we determined whether EMT induction of carci-
noma cells by MSCs was similarly accompanied by an increase
in tumor-initiating ability. When populations of 5 × 10
4 IL-1–
secreting carcinoma cells were coinjected with 2 × 10
5 MSCs,
their ability to give rise to palpable tumors was measurably
increased (1/6 to 6/6 for LoVo, 1/6 to 6/6 for HCC1806, 0/6
to 5/6 for SUM159 and 1/6 to 5/6 for SUM149, Fig. 3F ). In
contrast, tumor initiation by 5 × 10
4 MDA-MB-231 or MDA-
MB-453 cells, neither of which secrete IL-1, was not increased
by coinjection with 2 × 10
5 MSCs.
We quantifi ed these interactions more precisely by implant-
ing limiting dilutions of LoVo cells at 4 dosages, together
with 5 × 10
5 admixed MSCs. The presence of admixed MSCs
increased the tumor-initiating frequencies of 5 × 10
5 , 5 × 10
4 ,
and 5 × 10
3 implanted LoVo cells from 4/6, 1/6, and 0/6 to
6/6, 6/6, and 6/6. ( Fig. 3G ). As judged by the extreme limit-
ing dilution analysis [ELDA (18 )], the frequency of TICs in
cultured LoVo cells was 9 × 10
−7 to 6 × 10
−5 ; in the presence
of admixed MSCs, this frequency increased to 1 × 10
−3 to 1 ×
10
−2 ( Fig. 3H ).
MSC-Induced Increases in ALDH
high CSC-Enriched
Population and Tumor Initiation
CSCs, which have some characteristics associated with
normal stem cells, are cells defi ned operationally by their
tumor-initiating ability ( 19 ). We validated the use of alde-
hyde dehydrogenase (ALDH) as a marker of tumor-initiating
cells and thus CSCs (refs. 20, 21 ; described in Supplemen-
tary Data, Section 3; Supplementary Figs. S4A–S4C and
S5A–S5B). We observed that a 5-day coculture of LoVo or
HCC1806 cells with MSCs resulted in approximately 7-fold
and 20-fold increases, respectively, in ALDH1 protein levels
in the carcinoma cells ( Fig. 4A and Supplementary Fig. S5C).
To extend this observation, we cultured an unfractionated
LoVo cell population or already sorted ALDH
high or ALDH
low
LoVo cell subpopulations (Supplementary Fig. S4Ba), either
alone or in the presence of a 2-fold excess of tdTomato-
labeled MSCs, for 5 days. All 3 LoVo cell populations, when
cocultured with MSCs, developed larger subpopulations of
ALDH
high cells (59%, 38%, and 18%) than when cultured
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Li et al.
RESEARCH ARTICLE
Figure 3. MSCs elicit EMT, invasion of carcinoma cells, and increased tumor initiation of xenografts. A, E-cadherin, vimentin, fi bronectin, SNAIL, and
β-actin protein expression in carcinoma cells cultured either alone or with MSCs. The numbers indicate relative protein levels. B, EMT markers and EMT-
TFs were measured in LoVo cells treated with vehicle, PGE
2 (100 nmol/L), or the cytokines (100 ng/mL IL-6, 100 ng/mL IL-8, 100 ng/mL GRO-α, and/or
10 ng/mL RANTES) as indicated for 6 days. C, left: LoVo-tdTomato cells were cultured either alone or with MSCs in the upper wells of Boyden chambers.
The presence or absence of NS398 (50 μmol/L) or NS-398 + PGE
2 (100 nmol/L) is indicated for each panel. The images show the LoVo-tdTomato cells
that migrated through the Matrigel-coated membranes in 72 hours. Scale bar, 100 μm. Right, data are means ± SE, n = 3. D, LoVo cell migration in LoVo/
MSC coculture treated with cytokine-neutralizing antibodies (Ab) as indicated. Data are means ± SE, n = 5. E, MSCs increase invasion of LoVo tumors.
LoVo cells (5 × 10
5 cells per injection) were injected subcutaneously into SCID mice either alone (a, b, c) or with MSCs (5 × 10
5 cells per injection, d, e, f).
After 8 weeks, tumors of comparable size were isolated. Hematoxylin and eosin staining was carried out on the tumor sections. Scale bar, 100 μm. F and
G, weights of tumors derived from (F) carcinoma cells (5 × 10
4 cells per injection) and (G) LoVo cells injected into SCID mice, either alone or with MSCs.
Filled circles indicate individual tumor weights; open circles indicate no tumor grew at the site of injection. Bars are means ± SE. H, the ranges of the
estimated tumor-initiating frequencies in panel G evaluated by ELDA (with 95% confi dence).
Cultured
with MSC
E-cadherin
Vimentin
Fibronectin
SNAIL
β-Actin
Vimentin
E-cadherin
ZEB1
SNAIL
TWIST1
β-Actin
LoVO
–+ – MSC
MSC+
NS398+
PGE2
MSC
NS398
PGE2
+
MSC
Ab
–
–
+
–
+
IL-6
+
IL-8
+
Gro-α
+
RAN
++
++
+
MSC+
NS398
HCC1806
AC D
B
FG
H
E
–+
1 0 1 0.02
0
Migrated cell/545292 μm2
Migrated cell/545292 μm2
1 9.4 1 102
1 1009 1 90
1
1 0.27 0.23
abc
de f
13 9
17 14
13 13
110
1/6
3
2
1
*, P < 0.05; **, P < 0.001; ***, P < 0.005
Tumor weight (g)
Tumor weight (g)
LoVo
LoVo+MSC
HCC1806
HCC1806+MSC
SUM159
SUM159+MSC
SUM149
SUM149+MSC
MB-231
MB-231+MSC
MB-453
MB-453+MSC
MSC
0
6/6
***
1/6 6/6
***
0/6 5/6
***
1/6 5/6 0/6 0/6 0/6 0/6 0/6
4/6 1/6 0/6 6/6
Tumor-initiating rate
Tumor-initiating rate P = 2.14 × 10–217
105104103105104103102
105105105105
LoVo (5×)
9 × 10–7 2 × 10–6 6 × 10–6
LoVo
LoVo +
MSC
1 × 10–3 4 × 10–3 1 × 10–2
MSC (5×)
Group Lower Estimate Upper
6/6 6/6 5/6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
***
–
PGE2
IL-6
IL-8
GRO-α
RANTES
AII 5
LoVoLoVo + MSC
5169
0
20
40
60
80
100
120
140
160
180
20
40
60
80
100
120 **
**
****
*
**
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PGE2 and the MSC-Derived Cancer Stem Cell Niche RESEARCH ARTICLE
Cultured
with MSC
ALDH1
LoVo
A
C
D
a
b
Ba
b
–+
16.8
ALDH
6/6
4
3
0
0
*, P < 0.05 LoVo
LoVo
(after 5-day culturing
with MSC)
0.1
0.2
0.3
0.4
0.1
0.2
Tumor weight (g)
0.3
0.4
0.5
Tumor weight (g)
0.6
0.7
0.8
0.9
1.0
1.1
6/6 2/6 1/6 6/6 6/6 6/6
Tumor-initiating rate
1/6 5/6
Tumor-initiating rate
*
4/6 0/6 0/6 6/6 6/6 5/6 5/6
FSC
Cell density
High
Low
Cultured alone for another 5 days
after sorting out the MSCs
LoVo
cultured with MSCs
7%
21%
19%
18%
LoVo
cultured alone
LoVo
cultured
5 d alone + 5 d alone
LoVo
cultured
5 d with MSCs + 5 d alone
ALDHlow
20%
38%
Whole
ALDHhigh
Whole
30%
32%
38%
59%
ALDHhigh
β-Actin
P = 4.45 × 10
–8
P = 2.29 × 10–9
ALDHhigh (5×)
ALDHlow (5×)
MSC (5×)
105104103102104
105105105
103
105104103
105
105
105
104
105
103
105
102
102
P = 1.68 × 10–246
4 × 10–5 4 × 10–4 3 × 10–4
ALDHhigh
7 × 10–7 2 × 10–6 5 × 10–6
ALDHlow
2 × 10–3 11ALDHhigh
+MSC
3 × 10–4 8 × 10–4 2 × 10–3
ALDHlow
+MSC
95% CI
Group Lower Estimate Upper
Figure 4. MSC-induced increase in tumor initiation is refl ected in an increase in ALDH
high CSC-enriched populations. A, ALDH1 protein expression in
LoVo cells cultured either alone or with MSCs. The numbers indicate relative protein levels. B, unsorted (whole) ALDH
high and ALDH
low LoVo cells were
cultured either alone or with tdTomato-MSCs. After 5 days, ALDH activity of LoVo cells was analyzed by fl ow cytometry (a) and tdTomato-MSCs were
removed from the cultures by fl ow sorting. After removing MSCs, the LoVo cells were cultured alone for another 5 days. The ALDH activities were
again analyzed by fl ow cytometry (b). The percentages indicate the percentage of ALDH
high LoVo cells; that is, percent of LoVo cells with ALDH activity
beyond the indicated thresholds. C, a, weights of tumors derived from ALDH
high or ALDH
low LoVo cells injected into SCID mice, either alone or with MSCs.
Solid-fi lled and hash-fi lled circles indicate individual tumor weights; open circles indicate no tumor grew at the site of injection. Red circles: injection of
ALDH
high LoVo cells. Blue circles: injection of ALDH
low LoVo cells. Hash-fi lled circles: injection of LoVo cells, solid-fi lled circles: injection of LoVo cells and
MSCs. Bars are means ± SE. b, the ranges of the estimated tumor-initiating frequencies evaluated by ELDA. D, LoVo cells cultured with tdTomato-MSCs
have increased numbers of TICs. Cells were cultured as in B. After isolating LoVo cells by sorting, cells (5 × 10
4 cells per injection) were injected into mice.
After 6 weeks, the tumors were isolated and weighed. Filled circles indicate individual tumor weights. Open circles indicate no tumor grew at the site of
injection. Bars are means ± SE.
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Li et al.
RESEARCH ARTICLE
alone (38%, 20%, and 7%), that is, 1.6- to 2.6-fold increases in
ALDH
high cells ( Fig. 4Ba ).
We also used FACS analysis to remove the tdTomato-MSCs
from LoVo/MSC cocultures, then further propagated the
LoVo cells for 5 days in the absence of MSCs. We found that
the levels of the ALDH
high LoVo cell subpopulations that had
previously been increased by coculture with MSCs reverted
to levels comparable with those observed in LoVo cells that
had never experienced MSC coculture ( Fig. 4Bb ). Our data
indicated that maintenance of elevated numbers of ALDH
high
LoVo cells depended on continuous interactions of the LoVo
cells with MSCs.
Use of the ALDH marker as the sole stem cell marker is
likely to have led to an underestimate in the increase of the
number of CSCs, as ALDH
high cells are a CSC-enriched popu-
lation rather than being a pure CSC population. To refi ne the
markers used to identify CSCs, we determined that a LoVo
subpopulation more enriched for CSCs could be identifi ed by
concomitant use of the ALDH
high and CD133
+ markers, which
have been used to defi ne CSCs in various cancer cell popula-
tions (refs. 22–25 ; described in Section 3, Supplementary
Data; Supplementary Fig. S6A–C). Indeed, in the LoVo cells
cocultured for 5 days with MSCs, the ALDH
high /CD133 + LoVo
cells were increased from 1.4% to 16.4% of the overall cell pop-
ulation, that is, a 11.7-fold increase (Supplementary Fig. S6C).
We also determined whether the MSC-induced increase of
ALDH
high LoVo cells observed in culture was accompanied
by an increase of TICs in LoVo cells. Limiting dilutions of
sorted ALDH
high and ALDH
low LoVo cells were injected sub-
cutaneously into severe combined immunodefi cient (SCID)
mice, either alone or together with MSCs ( Fig. 4Ca ). As calcu-
lated using ELDA, the TIC frequency of ALDH
high LoVo cells
injected on their own was 4 × 10
−5 to 3 × 10
−4 , whereas that of
ALDH
high LoVo coinjected with MSCs was 2 × 10
−3 to 1 ( Fig.
4Cb ). For ALDH
low LoVo cells injected alone, the TIC frequency
was 7 × 10
−7 to 5 × 10
−6 ; coinjection with MSCs increased this
frequency to 3 × 10
−4 to 2 × 10
−3 ( Fig. 4Cb ). TIC frequencies of
both ALDH
high and ALDH
low LoVo cells were increased by sev-
eral orders of magnitude when coinjected with MSCs.
The continued presence of MSCs in the implanted cell popu-
lations complicates interpretation of the direct effects of MSC
on the tumor initiation of carcinoma cells, as the MSCs might
affect tumor initiation by a number of different mechanisms.
To address this issue, LoVo cells or HCC1806 cells were isolated
by FACS analysis from 5-day carcinoma cell/tdTomato-MSC
cocultures and were then immediately injected subcutaneously
into SCID mice, in parallel with LoVo cells previously cul-
tured alone, to determine the effect on the tumor initiation of
the cell culture interactions ( Fig. 4D and Supplementary Fig.
S7A). Accordingly, TIC frequencies of LoVo cells were increased
by one order of magnitude following a 5-day coculture with
MSCs invitro (Supplementary Fig. S7B). An increase of tumor
initiation by prior coculture with MSCs was also observed on
HCC1806 cells (Supplementary Fig. S7C).
Infl uence of PGE
2 Signaling
on the ALDH
high CSC State
To understand more precisely how the signals exchanged
between carcinoma cells and MSCs led to increases in ALDH
high
CSCs, we fi rst ascertained whether PGE
2 and/or cytokines
produced by MSCs in LoVo/MSC cocultures could elicit
ALDH1 expression. Only PGE
2 was able, on its own, to elicit an
increase in ALDH1 expression in LoVo cells ( Fig. 5A ). Moreo-
ver, when PGE
2 was combined with the 4 cytokines, there was
no further increase in ALDH1 expression.
EP4 is the only PGE
2 receptor highly expressed by LoVo
cells (Supplementary Fig. S2G). To elucidate in more depth
the effects of PGE
2 on LoVo cells, we treated these cells with
vehicle or PGE
2 for 5 days. The vehicle-treated cultures con-
tained 10.3% ALDH
high LoVo cells, whereas the LoVo cells
treated with PGE
2 contained approximately 25% ALDH
high
LoVo cells ( Fig. 5B ). We added GW627368X (the EP4 recep-
tor antagonist) to LoVo cells to determine whether the basal,
unperturbed levels of ALDH
high cells depended on ongoing
PGE
2 autocrine signaling; this treatment reduced by approxi-
mately 60% the basal level of ALDH
high LoVo cells ( Fig. 5B ).
These data suggested that within LoVo cells, subpopulations
of cells are maintained in an ALDH
high state, in part, through
ongoing, low-level autocrine PGE
2 signaling. Moreover, this
signaling and associated entrance into the ALDH
high state
could be enhanced by exogenously supplied PGE
2 .
The observed increase in ALDH1 expression induced
by PGE
2 was not confi ned to LoVo cells. PGE
2 treatment
induced elevated ALDH1 expression (2.9- to 12.9-fold) in
LoVo, SUM149, SUM159, and BT549 cells ( Fig. 5C ), all of
which were previously found to elicit increased PGE
2 produc-
tion from cocultured MSCs.
The LoVo cells that had been treated ex vivo with vehicle
or PGE
2 for 5 days were implanted subcutaneously in SCID
mice. Tumors derived from control LoVo cells occurred in
3 of 16 injected hosts, whereas the corresponding PGE
2 -
treated cells formed tumors in 18 of 24 hosts ( Fig. 5D ).
Hence, a substantial increase in ALDH
high cells and TICs
could be achieved by PGE
2 treatment of LoVo cells ex vivo .
Moreover, treating LoVo cells with the cocktail of cytokines
(IL-6, IL-8, GRO-α, and RANTES) in addition to PGE
2 did
not signifi cantly increase the tumor initiation beyond that
observed for PGE
2 treatment alone (Supplementary Fig. S8).
In addition to its effects on TICs, PGE
2 is likely to contribute
to the maintenance of CSCs in vivo by increasing tumor ang-
iogenesis (Supplementary Fig. S9, discussed in Supplemen-
tary Data, section 4).
Role of PGE
2 Signaling in the
MSC-Induced ALDH
high CSC-Enriched
Population and Tumor Initiation
To confi rm that the above-described role of PGE
2 could
explain the ability of MSCs to induce CSC formation, we
inhibited PGE
2 synthesis with NS398, or PGE
2 signaling
with GW627368X, in cocultures of LoVo cells and tdTomato-
MSCs. Blocking PGE
2 signaling by either route prevented
most of the increases in ALDH
high LoVo CSCs by MSCs
(described in Section 5, Supplementary Data; Supplementary
Fig. S10). LoVo cells that had been cocultured under the
various conditions with MSCs for 5 days were sorted by FACS
analysis to eliminate tdTomato-MSCs and were then injected
subcutaneously into SCID mice. The increase in TIC fre-
quencies resulting from a 5-day coculture with MSCs (from
4/20 to 14/19) was prevented by introducing either NS398
or GW627368X into the cocultures (from 14/19 to 3/17 or
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ALDH1
A
D
Fab
E
BC
β-Actin
ALDH1
1 3.5
1 0.8
1 3.8
1 1.4
β-Actin
ALDH1
β-Actin
ALDH1
1 12.9
1 0.6
1 2.9
11
β-Actin
ALDH1
β-Actin
1 0.6 0.9 0.9 1.7
–
PGE2
PGE2GW627368X
10.3%
24.9%
LoVo
4.27%
IL-6
IL-8
GRO-α
RANTES
AII 5
Vehicle
PGE2
Veh ic l e
PGE2
Vehicle
SUM149 LoVo
BT549 SUM159
ALDH activity
Tumor-initiating rate
*
3/16
0.0
0
4
8
12
16
20 ***
LoVo
LoVo
MSCshsc+DME
MSCshcox-2+LoVoCM
MSCshsc+LoVoCM
LoVo LoVo+
MSCshcox-2
*, P < 0.05; ***, P < 0.005
LoVo+
MSCshsc
LoVo
(MSC)
LoVo
(MSC+NS)
LoVo
(MSC+GW)
LoVo
(MSC+NS+PGE2)
PGE2 (ng/mL)
0
0.5
1.0
1.5
2.0
2.5
Tumor weight (g)
LoVo
PGE2-pretreated
0.1
0.2
Tumor weight (g)
0.3
0.4
0.5
0.0
0.1
0.2
Tumor weight (g)
0.3
0.4
0.6
0.5
18/24
Tumor-initiating rate
*
***
4/20 14/19 3/17 7/8 1/12
Tumor-initiating rate
******
1/12 10/12 3/12
0 100
102103104105
200 300
Count
400 500 600
12
Figure 5. COX-2–PGE 2 signaling is required for MSC-induced increase in ALDH
high CSC-enriched population and tumor initiation. A, ALDH1 protein
expression in LoVo cells treated as indicated for 5 days. B, ALDH activity of LoVo cells treated with vehicle, PGE
2 (100 nmol/L), or GW627368X
(20 μmol/L) was analyzed by fl ow cytometry. The percentages indicate the percentage of ALDH
high LoVo cells; that is, LoVo cells with ALDH activity
beyond the indicated thresholds. The gray line at the right side of the plot indicates the threshold of the high ALDH activity. C, ALDH1 protein levels in
various carcinoma cells treated with vehicle or PGE
2 . The numbers indicate relative protein levels. D, PGE
2 increases LoVo TICs. LoVo cells pretreated
with vehicle or PGE
2 (100 nmol/L) were injected into SCID mice (5 × 10
4 cells per injection). After 6 weeks, tumors were isolated and weighed. Bars are
means ± SE. E, LoVo cells were cultured with tdTomato-MSCs, PGE
2 , NS398, or GW627368X, as indicated. After 5 days, the LoVo cells were isolated by
cell sorting and injected into SCID mice (5 × 10
4 cells per injection). After 7 weeks, the tumors were isolated and weighed. Filled circles indicate individual
tumor weights; open circles indicate no tumor grew at the site of injection. Bars are means ± SE. F, a, levels of PGE
2 secreted by LoVoCM-treated MSC-
shsc and MSCshcox-2. Data are means ± SE, n = 3. b, weights of tumors derived from LoVo cells (5 × 10
4 cells per injection) injected into SCID mice either
alone, with MSCshsc, or with MSCshcox-2 (2 × 10
5 cells per injection). Filled circles indicate individual tumor weights; open circles indicate no tumor grew
at the site of injection. Bars are means ± SE.
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Li et al.
RESEARCH ARTICLE
1/12, respectively, Fig. 5E ). Adding PGE
2 to the cocultures
along with NS398 restored the tumor-initiating frequency
from 3/17 to 7/8.
To validate the role of COX-2 in these properties, we
knocked down COX-2 in MSCs. The ability of the resulting
MSCshcox-2 cells to produce PGE
2 in response to LoVoCM
was reduced by 90% ( Fig. 5Fa ). The tumor-initiating fre-
quency of 5 × 10
4 LoVo cells was increased from 1/12 to 10/12
by coinjection with MSCs expressing a control, scrambled
shRNA (MSCshsc cells; Fig. 5Fb ). In contrast, in mice coin-
jected with MSCshcox-2 cells, the tumor-initiating frequency
of LoVo cells was increased from 1/12 to only 3/12, support-
ing the notion that COX-2-dependent PGE
2 induced in MSCs
was required for the observed robust increases of LoVo TICs.
PGE 2 Induces
b
-Catenin Nuclear Localization
and Transactivation
The β-catenin signaling pathway has been implicated in
maintaining stem cell and CSC homeostasis in most epi-
thelial tissues ( 26, 27 ). Relevant here is the fi nding that
PGE
2 treatment leads to Akt activation; activated Akt subse-
quently stimulates β-catenin signaling in several ways ( 28, 29 ).
Accordingly, we examined activation of Akt/glycogen synthase
kinase-3 (GSK-3)/β-catenin signaling axis in LoVo cells treated
with vehicle, PGE
2 , GW627368X, or GW627368X + PGE
2 . We
found that PGE
2 treatment led to a 12-fold increase in Akt
phosphorylation at Thr473 ( Fig. 6A ), which indicates func-
tional Akt activation ( 30 ). Conversely, inhibiting PGE
2 signal-
ing with GW627368X in PGE
2 -treated LoVo cells blocked 50%
of the PGE
2 -induced Akt phosphorylation ( Fig. 6A ). Moreo-
ver, β-catenin activity is positively affected by Akt-mediated
phosphorylation of its Ser552 residue ( 31, 32 ). PGE
2 treat-
ment of LoVo cells, which caused a 12-fold increase of Akt
phosphorylation, also increased β-catenin phosphorylation
at Ser552 (11-fold), an increase that was blocked entirely by
GW627368X treatment ( Fig. 6A ).
Akt also enhances β-catenin by phosphorylating and
thereby inactivating GSK-3; this prevents inactivation of
β-catenin by unphosphorylated GSK-3, as Ser21-unphos-
phorylated GSK-3α and Ser9-unphosphorylated GSK-3β
phosphorylate β-catenin at its Ser33/Ser37/Thr41 residues,
leading to its degradation ( 33, 34 ). PGE
2 strongly increased
the phosphorylation of GSK-3α at Ser21 (56-fold above basal
level, Fig. 6A ). Although Ser9 of GSK-3 was already phospho-
rylated before PGE
2 treatment, this basal level of GSK-3β Ser9
phosphorylation was reduced (70%) by adding GW627368X
( Fig. 6A ), presumably by blocking basal autocrine PGE
2 sign-
aling. Inhibition of PGE
2 signaling by GW627368X prevented
the inactivating phosphorylation of GSK-3α and GSK-3β
and, conversely, increased phosphorylation of β-catenin at
Ser33/Ser37/Thr41 residues that lead to its degradation by
15-fold ( Fig. 6A ). These data indicated that inhibition of
PGE
2 signaling leads to β-catenin phosphorylation, a prelude
to its proteasomal degradation.
A strong inhibition of β-catenin signaling is also achieved
by E-cadherin, which recruits β-catenin to adherens junctions
associated with the plasma membrane, thereby preventing its
nuclear localization and its actions in promoting transcription
( 35 ). PGE 2 decreased the E-cadherin and ZO-1 protein located
at the cell junctions ( Fig. 6B and C ). Correspondingly, in PGE
2 -
treated LoVo cells, β-catenin was found in cell nuclei, rather
than being sequestered by E-cadherin in adherens junctions
( Fig. 6B and C ). PGE
2 caused a 5.3-fold increase of β-catenin
in the nuclear fraction; this increase was blocked by adding
the EP4 antagonist during PGE
2 treatment ( Fig. 6D ). We also
found, by analyzing expression in LoVo carcinoma cells of a
number of β-catenin/TCF-regulated genes, that PGE
2 -induced
nuclear β-catenin was functionally active ( Fig. 6E ).
PGE 2 -Induced Effects on ALDH
high Cancer Cells
Are Mediated by
b
-Catenin Signaling
PGE 2 induces Akt phosphorylation in carcinoma cells
in a phosphatidylinositol 3-kinase (PI3K)-dependent man-
ner ( 28 ). To determine whether the activation of the Akt/
GSK-3/β-catenin signaling axis was required for PGE
2 -
induced ALDH
high LoVo CSCs, we treated ALDH
high LoVo
cells with vehicle, PGE
2 , GW627368X, LY294002 (a PI3K
inhibitor), FH353 (a β-catenin/TCF inhibitor), or carda-
monin (a β-catenin inhibitor) for 5 days at concentrations
that did not cause signifi cant cell death.
LY294002 functioned as effi ciently as the EP4 antagonist
by completely blocking the exogenous PGE
2 -induced increase
of ALDH
high LoVo cells and by decreasing the endogenous
PGE
2 -maintained basal ALDH
high LoVo cells ( Fig. 6F, a and d ).
Although the 2 β-catenin inhibitors FH353 and cardamonin
blocked the PGE
2 -induced increase of ALDH
high LoVo cells
( Fig. 6Fb and Fc ), these inhibitors did not function as effec-
tively as the EP4 antagonist or the PI3K inhibitor. PGE
2 /EP4
signaling, acting through the Akt/GSK-3/β-catenin signaling
axis ( 28 ), contributes to induction of the ALDH
high LoVo cell
phenotype.
Contribution of MSCs in Tumor Stroma
to the Stem Cell Niche of ALDH
high CSCs
PGE 2 is metabolically unstable and is thought to act within
tissues over short distances, doing so in both an autocrine and
a paracrine manner. Wishing to pursue this notion further, we
examined whether ALDH
high CSCs were located near MSCs (or
their mesenchymal derivatives) in tumors that arose follow-
ing the coinjection of these 2 cell types. ALDH
high LoVo cells
( Fig. 7Aa , red signal) were often surrounded by tdTomato-
labeled MSCs ( Fig. 7Aa , green signal) or their derivatives
in tissue sections of LoVo/MSC xenografts; in addition,
many of these mesenchymal cells expressed COX-2 ( Fig. 7Aa ,
cyan signal). Moreover, the mesenchymal cells associated
with the ALDH
high tumor cells expressed FSP, the fi broblast
marker ( Fig 7Ab , red signal; discussed in Supplementary
Data, section 6; Supplementary Fig. S11), indicating that
the MSC-derived fi broblasts rather than their MSC precur-
sors were largely responsible for forming the stromal micro-
environment of these ALDH
high cells. The conclusion that
PGE
2 induces the formation of ALDH
high CSCs was further
supported by human colorectal adenocarcinoma studies in
which we found the juxtaposition within tumors of such
CSCs with stroma expressing COX-2, the likely source of
PGE
2 production (discussed in Supplementary Data, section
7; Supplementary Fig. S12A and B). These observations lead
us to propose that MSCs (or their differentiated derivatives)
create a niche within tumors, leading to induction and/or
maintenance of CSC subpopulations.
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PGE2 and the MSC-Derived Cancer Stem Cell Niche RESEARCH ARTICLE
Figure 6. PGE 2 induces ALDH
high cancer cells through the Akt/GSK-3/β-catenin signaling axis. A, activation of Akt/GSK-3/β-catenin signaling in LoVo
cells treated as indicated for 1 hour was analyzed by Western blots for phosphorylated Akt, total Akt, phosphorylated GSK-3α, phosphorylated GSK-3β,
total GSK-3β, phosphorylated β-catenin, total β-catenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The numbers indicate relative protein
levels. B, the distribution of E-cadherin (red), ZO-1 (green), and β-catenin (red) in LoVo cells treated with vehicle or PGE
2 (100 nmol/L) for 48 hours was
analyzed by immunofl uorescence. Cell nuclei were stained with DAPI (4′6-diamidino-2-phenylindole, in blue). The graphs show the fl uorescence intensi-
ties along the dashed lines in the images of β-catenin staining. β-Catenin intensities are in red lines and DAPI intensities are in blue lines. C, quantifi cation
of the levels of E-cadherin, ZO-1, and β-catenin associated with membrane and of nuclear β-catenin. The fl uorescence intensities for the staining of these
proteins in randomly selected cells in images (e.g., the cells crossed by dashed lines in (B) were quantifi ed. Bars are means ± SE, n > 50 for each bar. M
β-cat, membrane-bound β-catenin; N β-cat, nuclear β-catenin. D, nuclear/cytosolic distribution of β-catenin in LoVo cells treated as indicated was ana-
lyzed by Western blot of β-catenin, GAPDH, and lamin B1, a nuclear envelope marker, in nuclear and cytosolic fractions. E, mRNA expression of selected
β-catenin/TCF-dependent genes in LoVo cells treated with vehicle or PGE
2 for 7 hours. The mRNA levels of these genes were normalized to GAPDH
mRNA. Data are fold induction by PGE
2 (vs. that of vehicle-treated LoVo cells). Data are means ± SE, n = 3. F, ALDH activities of ALDH
high LoVo cells
treated with vehicle, PGE
2 , PGE 2 + GW627368X (GW; 20 μmol/L), PGE
2 + LY294002 (0.5 μmol/L), or PGE
2 + FH535 (6 μmol/L) for 5 days (a–c). Percent-
ages of ALDH
high LoVo cells are presented in the table (d). The gray lines at the right side of the plots indicate the thresholds of the high ALDH activity.
GAPDH
0
2
Fold induction (mRNA level)
4
6
8
10
12
14
Self-renewal
Invasiveness/EMT a
b
d
c
Cell signaling
NANOG
Oct4
SOX2
SOX9
MMP2
MMP7
MMP9
TWIST1
TGFβ3
Jag1
Wnt3a
FGF4
PDGFRA
ALDH activity
Count
0
4
Vehicle
8
12
16
20
1
20
450
350
250
150
50
102103104106
450
350
250
150
50
450
350
250
150
50
40 60 80 20 40 60 80
2
3
GSK-3β
p9-GSK-3β
p21-GSK-3α
Akt
p473-Akt
–
1 11.8 5.8 0
1 56 8.3 0.1
1 0.9 0.3 0.3
1 0 11.7 15
1 11.1 0 0
PGE2GW
PGE2
+ GW
PGE2
Vehicle
A
EF
BC
D
PGE2
***, P < 0.005
Vehicle PGE2PGE2
+ GW
PGE2
+ LY
PGE2
+ FH
PGE2
+ CA
Vehicle
PGE2
PGE2 + GW
PGE2 + LY
PGE2 + FH
PGE2 + CA
29.6
37.6
17.8
22.5
28.9
25.8
ALDHhigh
cell %
p33/37/41-
p552-
β-Catenin
GAPDH
CN
1 5.3 0.9 1.2
CNC NCN
Lamin B1
β-Catenin
E-cadherin
***
***
***
***
ZO1
M β-cat
N β-cat
β-Catenin
β-Catenin DAPI
ZO1
Gray (×103)
Fluorescence intensity (Gray ×103)
E-cadherin
β-Catenin
β-Catenin
– PGE2GW
PGE2
+ GW
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Li et al.
RESEARCH ARTICLE
COX-2
IL1α
IL1β
5.30
2.06
2.09
8.43E-6
5.54E-5
4.27E-4
COX-2
Gene Colona
a
A
B
C
Breastb
(1)
(4)
EP4 EP2/4
AA
PGE2
PGE2
mPGE1
COX-2
MSC
LoVo cell
EMT
Invasion
Stemness
PI3K
Akt
GSK-3 IL-1
IL-1R
IL-8
IL-6
β-cat
GRO-α
β-cat
Least expressed
(1) 22 colon mucinous carcinoma
(2) 101 colon carcinoma
(3) 19 normal colon samples
(4) 25 human triple-negative breast carcinoma
(5) 153 nontriple-negative breast carcinoma samples
Most expressed
(5)
(2) (3)
Fold
change P-value
5.35 7.77E-9
9.57 4.60E-11
4.66 6.23E-8
IL1α
IL1β
tdTomato-MSC ALDH1 COX-2 Merge
btdTomato-MSC FSP COX-2 Merge
Figure 7. MSCs in tumor stroma serve as an ALDH
high cancer stem cell niche. A, immunofl uorescence analyses were carried out on tumors derived from
LoVo cells (5 × 10
4 cells per injection) injected along with tdTomato-MSCs (5 × 10
5 cells per injection), using antibodies against tdTomato-RFP (in green),
ALDH1 (in red, panel a), fi broblast surface protein FSP (in red, panel b), and COX-2 (cyan). Panels a and b are serial sections from one tumor. Scale bar,
100 μm. B, IL-1 and COX-2 mRNA expression in human colon and breast carcinoma. The “fold change” indicates the average mRNA levels of the 3 genes in
colon mucinous carcinoma samples, compared with that of normal colon samples (panel a) and in TNBC samples, compared with that of non-TNBC sam-
ples (panel b). C, the proposed interactions for induction and maintenance of EMT, cancer cell stemness, and invasiveness by MSCs. AA, arachidonic acid.
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PGE2 and the MSC-Derived Cancer Stem Cell Niche RESEARCH ARTICLE
Correlation of COX-2 Expression with
CSC Properties and a More Aggressive
Tumor Phenotype
To determine whether elevated IL-1 production correlates
with COX-2/PGE
2 expression in human primary carcino-
mas, we compared the normalized IL-1α, IL-1β, and COX-2
mRNA levels across 19 human normal colon and 123 human
colon carcinoma samples and across 178 human invasive
breast carcinoma samples (Cancer Genome Atlas analyzed
by Oncomine). IL-1α and IL-1β were expressed at signifi -
cantly higher levels (9.6-fold and 4.7-fold in colon carcinoma
and 2.1-fold and 2.1-fold in breast carcinoma) in aggressive
subtypes of samples—colon mucinous carcinoma and triple-
negative breast carcinoma (TNBC), respectively ( Fig. 7B ).
Moreover, the COX-2 mRNA levels were correlated with the
IL-1α and IL-1β mRNA levels; COX-2 mRNA was elevated
5.4-fold and 5.3-fold in colon mucinous carcinoma and
TNBC when compared with normal colon samples and other
breast cancers ( Fig. 7B ). These correlations suggested that the
signaling mechanisms described above ( Fig. 7C ), involving
IL-1–activated expression of COX-2/PGE
2 , may be relevant
to understanding the pathogenesis of colon mucinous car-
cinomas and TNBCs.
DISCUSSION
Elevated IL-1 expression has been correlated with increased
malignant progression and more aggressive phenotypes in
many types of cancer ( 36 ). However, the mechanism(s) under-
lying this correlation have been unclear. We describe here a
bidirectional, reciprocal interaction between carcinoma cells
and the MSCs ( Fig. 7C ). The signaling is initiated through the
release of IL-1 by carcinoma cells. We present evidence that
seems to explain this connection between IL-1 production
and increased tumor aggressiveness in many, and perhaps all,
IL-1–secreting carcinomas.
IL-1 secreted by carcinoma cells induces COX-2 and
mPGES1 expression in MSCs. The 2 enzymes collaborate in
MSCs to generate PGE
2 levels, which can increase by 80- to
500-fold ( Fig. 1Cb ). Of note, we show these responses oper-
ate equally well in MSCs and in their more differentiated
descendants (Supplementary Fig. S2D and S2E). We note
that others recently reported that IL-1 secreted by head and
neck squamous cell carcinoma cells induce PGE
2 from fi brob-
lasts ( 37 ). Consequently, fi broblasts and myofi broblasts, both
of which accumulate in large numbers in the stroma, may
also represent sources of the PGE
2 and the subsequently pro-
duced cytokines described here.
Importantly, although COX-2 is highly expressed in both neo-
plastic and stromal cells in tumors, not all COX-2-expressing
cells can produce PGE
2 . Thus, despite large variations in
COX-2 expression, the colorectal carcinoma cell lines that
we examined produced only about 10 to 40 pg/mL of PGE
2
(Supplementary Fig. S1A). These levels are dwarfed by the
15,000 to 40,000 pg/mL of PGE
2 produced by the IL-1–stimu-
lated MSCs studied here ( Fig. 1Cb ). This failure by COX-2-
expressing carcinoma cells to produce signifi cant levels of
PGE
2 may be due to the absence in many carcinoma cells
of signifi cant levels of mPGES1 expression (Supplementary
Fig. S1A and S1D). Hence, COX-2 expression, on its own, is
unlikely to provide an accurate indication of PGE
2 production
by carcinomas.
MSC-produced PGE 2 acts in 2 ways within such tumors—in
an autocrine fashion on the MSCs that produced it and in
a paracrine fashion on the nearby IL-1–releasing carcinoma
cells. The MSC autocrine signaling elicits a second wave of
signaling responses: In collaboration with ongoing paracrine
IL-1 signaling from carcinoma cells, the autocrine PGE
2
induces IL-6, IL-8, GRO-α, and RANTES cytokines in the
MSCs. Together, these MSC-derived molecules induce a third
wave of responses that profoundly alter the carcinoma cells
that initiated this signaling cascade ( Fig. 2E and 7C ).
The changes induced in carcinoma cells by the confl u-
ence of PGE
2 and cytokine signals are all components of the
complex cell–biological program termed EMT. Although this
program has been increasingly implicated in the acquisi-
tion of phenotypes associated with high-grade malignancy
( 14 ), major questions concerning EMT have remained unan-
swered. Among them are the paracrine signals, ostensibly of
stromal origin, that trigger EMT in carcinoma cells. Here we
present a scenario that explains how EMT can be induced in
carcinoma cells by a reactive stroma.
This work also addresses another longstanding puzzle about
EMT: Is it usually activated as a single, coherent program or,
alternatively, are distinct components of this program acti-
vated separately, each by a distinct set of heterotypic signals?
Our observations indicate that the latter scenario is more
likely. For example, PGE
2 caused a decrease of E-cadherin
expression in carcinoma cells, whereas cytokines were required
to induce the vimentin and ZEB1 expression that is usually
depicted as intrinsic components of the EMT program
( Fig. 3B ). Such responses suggest the possibility that, during
the course of spontaneous tumor progression, some carcinoma
cells may receive only a subset of these signals and accordingly
only activate portions of the EMT program, whereas the others
receiving the complete suite of heterotypic signaling molecules
may pass through an entire EMT program.
Research by ourselves and others has connected EMT
with entrance into a stem cell–like state, both in normal and
neoplastic epithelial cells ( 12, 13 , 17 ). These fi ndings are also
echoed by this work, in which we observed a concomitant
entrance into the mesenchymal and stem cell states in response
to MSC-derived heterotypic signals. PGE
2 , which activated
portions of the EMT program, was able to increase both
the number of CSCs and the frequency of tumor initiation
( Fig. 5A–D ). Our fi ndings here further clarify the connection
of EMT with entrance into a stem cell–like state by showing
that the partial EMT induced by PGE
2 , which represses cell–
cell junctions without inducing mesenchymal traits, suffi ces
to increase CSCs. The observation of MSC-induced increases
in CSCs is consistent with the recent fi nding of prostaglan-
din-induced increases in the number of CD44
+ tumor cells
( 38, 39 ). The unique contribution of PGE
2 is underscored
by the observation that other MSC-derived cytokines, when
combined with PGE
2 , had only marginal effects on further
increasing the TIC frequency (Supplementary Fig. S8).
In earlier work ( 27 ), we documented an alternative means of
activating the EMT program that involves canonical and non-
canonical WNTs, together with TGFβ. Those fi ndings echoed
the present observations, as both studies showed that multiple
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854 | CANCER DISCOVERYSEPTEMBER 2012 www.aacrjournals.org
Li et al.
RESEARCH ARTICLE
distinct heterotypic signals, acting in concert, are required to
activate an EMT in carcinoma cells. These earlier fi ndings left
open the possibility that other EMT-inducing signals beyond
those documented at the time may converge on the WNT and
TGF-β signaling pathways to activate EMT programs. We note
here that PGE
2 -activated signals do, indeed, converge on one
of these signaling cascades by activating β-catenin signaling,
the same pathway that represents the main signaling channel
lying downstream of canonical WNT signaling.
In tumors that arise from IL-1–producing carcinoma cells,
we fi nd that this interleukin plays a critical role in the tumor
cell–induced COX-2/mPGES1/PGDH/PGE
2 response in
MSCs that is required for tumor progression. IL-1 blockage
has been used in thousands of patients to control infection
and infl ammatory disease and has a remarkable safety record
( 40 ). On the basis of our fi ndings and the existing clinical use
of IL-1 inhibitors, IL-1 inhibition may present a promising
alternative to COX-2 inhibitors for cancer therapy. In addi-
tion, limited therapeutic options are currently available for
TNBCs, which produce higher levels of IL-1 ( Fig. 7B ), because
they are unresponsive to standard receptor–mediated treat-
ments. Accordingly, our fi ndings suggest a possible option for
treating these aggressive subtypes of breast and colon cancer.
METHODS
Cell Culture
Human carcinoma cell lines HCC1806, BT549, MDA-MB-231,
MDA-MB-453, SW1116, and LoVo were obtained from American
Type Culture Collection and human bone marrow–derived MSCs
(Sciencell) were obtained from Sciencell. SUM149 and SUM159 cells
were provided by S.P. Ethier (Wayne State University, Detroit, MI).
The human carcinoma cell lines SUM149, SUM159, BT549, MDA-
MB-231, MDA-MB-453, SW1116, and LoVo were authenticated by
microarray analysis. HCC1806 and MSC were not passaged more
than 6 months after receipt.
Animal Experiments
All research involving animals complied with protocols approved
by the MIT Committee on Animal Care. In experiments evaluating
tumor initiation and growth, the tumors were isolated and weighed
at the end of each experiment. To measure TIC frequency, serial dilu-
tions of cancer cell suspensions were injected subcutaneously into
nude mice. TIC frequencies of the samples were determined using
the ELDA webtool ( 18 ).
PGE 2 and Cytokine Assays
The concentrations of PGE
2 and cytokines were determined by
ELISA as described in the manufacturers’ protocols. PGE
2 levels were
measured using a PGE
2 direct Biotrack assay kit (GE Healthcare).
Human cytokine levels were measured using Quantikine kits (R&D
Systems).
Invasion Assay
Cell invasion was evaluated by using BD Matrigel Invasion Cham-
bers, 8.0 μm (BD Biosciences). The cells that migrated through the
membrane during the incubation period were counted in 5 randomly
selected regions.
See Supplementary Materials and Methods for more information.
Disclosure of Potential Confl icts of Interest
No potential confl icts of interest were disclosed.
Authors’ Contributions
Conception and design: H.-J. Li, H.R. Herschman, R.A. Weinberg
Development of methodology: H.-J. Li, H.R. Herschman
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): H.-J. Li, F. Reinhardt
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): H.-J. Li, H.R. Herschman
Writing, review, and/or revision of the manuscript: H.-J. Li, H.R.
Herschman, R.A. Weinberg
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): H.-J. Li, H.R. Herschman
Study supervision: H.R. Herschman, R.A. Weinberg
Acknowledgments
The authors thank members of the Weinberg laboratory (Wai
Leong Tam, Michael Hwang) and the Herschman laboratory (Tomo-o
Ishikawa, Art Catapang) for discussion and technical support; the
Whitehead Flow Cytometry Core for technical support; and Sarah
Dry and the UCLA Translational Pathology Core Laboratory for
providing colon adenocarcinoma samples.
Grant Support
The work was supported by Breast Cancer Research Founda-
tion (R.A. Weinberg), Susan G. Komen for the Cure (H.-J. Li), NIH
(R.A. Weinberg: U54CA163109 and H.R. Herschman: R01CA123055
and P50CA086306), and Ludwig Center for Molecular Oncology
(R.A. Weinberg).
Received March 9, 2012; revised June 21, 2012; accepted June 22,
2012; published OnlineFirst July 3, 2012.
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2012;2:840-855. Published OnlineFirst July 3, 2012.Cancer Discovery
Hua-Jung Li, Ferenc Reinhardt, Harvey R. Herschman, et al.
Signaling
2
Stem Cell Niche via Prostaglandin E
Cancer-Stimulated Mesenchymal Stem Cells Create a Carcinoma
Updated version
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Published OnlineFirst July 3, 2012; DOI: 10.1158/2159-8290.CD-12-0101