Download Induction of the Stem-like Cell Regulator CD44

Published OnlineFirst September 20, 2012; DOI: 10.1158/0008-5472.CAN-11-3812
Cancer
Research
Tumor and Stem Cell Biology
Induction of the Stem-like Cell Regulator CD44 by Rho
Kinase Inhibition Contributes to the Maintenance
of Colon Cancer–Initiating Cells
Hirokazu Ohata1, Tatsuya Ishiguro1, Yuki Aihara1, Ai Sato1, Hiroaki Sakai1, Shigeki Sekine3,
Hirokazu Taniguchi3, Takayuki Akasu4, Shin Fujita4, Hitoshi Nakagama2, and Koji Okamoto1
Abstract
The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical
characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly
facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem
cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of
cells that express various levels of CD44, whereas CD44high cells were associated with increased sphere-forming
ability, expression of the activating form of b-catenin, and elevated levels of glycolytic genes, CD44/low cells
showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms
of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of cMet. As expected from the predicted hierarchy, CD44high cells differentiated into CD44/low cells. Unexpectedly, a
fraction of CD44/low cells generated CD44high cells, and the ROCK inhibitor or blebbistatin primed the transition
by inducing CD44 expression. We propose that the transition from CD44/low to CD44high state helps to maintain a
CD44high fraction and the tumorigenic diversity in colon cancer. Cancer Res; 72(19); 5101–10. 2012 AACR.
Introduction
Authors' Affiliations: 1Division of Cancer Differentiation and 2Division of
Cancer Development System, National Cancer Center Research Institute;
Departments of 3Pathology and Clinical Laboratories, and 4Gastrointestinal
Oncology, National Cancer Center Hospital, Chuo-ku, Tokyo, Japan
for more than a year without losing the ability to generate
tumors (5). Characterization of the spheroids revealed that a
fraction of the cells that express surface markers such as CD133
(15, 17) or ALDH (16) are attributed to their features as cancerinitiating cells and that extrinsic factors such as IL-4 (15) or
Wnt (12) mediate maintenance of CSC population.
Despite of the progress on the characterization of the colon
spheroid cells, their stable culture in vitro can be maintained
only from a fraction of primary cancers (12, 15), and it will be
instrumental to establish more efficient methods for spheroid
cultivation to clarify the common biochemical nature of colon
cancer–initiating cells.
In this article, we found that inhibitors of Rho-associated
protein kinase (ROCK) or of actomyosin cytokinesis markedly
facilitated the formation of spheroids from primary colon
cancers, and revealed that the CD44high cells in the spheroids
share common characteristics with CSCs. Unexpectedly, a
fraction of CD44/low cells was capable of developing into
CD44high cells via the induction of CD44 expression by the
ROCK inhibitor. We will discuss the potential significance of
the reversible transition between CD44high and CD44/low cells
in light of the plasticity of CSCs and of devising a novel
therapeutic strategy against colon cancer.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Materials and Methods
The emerging picture from recent discoveries revealed that,
in some types of tumors, only a small fraction of cancer cells is
capable of initiating cancer (1–3). These cancer-initiating cells,
or cancer stem cells (CSC), as they are often defined because of
their associated characteristics with stem cells, are one of
the major foci of recent cancer research (1–3). Elucidation of
the biologic and biochemical nature of CSCs will be important
to understand the mechanisms of cancer development and to
devise new strategies for cancer therapy.
It has been reported that CSCs are present in colorectal
cancer (4, 5). Colon CSCs were identified as cells that express
specific surface markers, including CD133, CD44, CD166, and
ALDH1 (4–14).
Several laboratories reported that CSCs from colon cancer
proliferate in vitro as spheroids (5, 12, 14–18). Remarkably, the
spheres can be maintained in conditions of exponential growth
Corresponding Authors: Koji Okamoto, Division of Cancer Differentiation,
National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo
104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-3542-2530; E-mail:
[email protected]; and Hitoshi Nakagama, E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-11-3812
2012 American Association for Cancer Research.
Primary human colon cancer specimens
All human colon cancer samples were resected from
patients with informed consent at the National Cancer Center
Hospital (Chuo-ku, Tokyo, Japan), and all procedures were
conducted under the protocol approved by the ethics committee of the National Cancer Center.
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Ohata et al.
Isolation of cancer cells and cell culture
Tumor samples were minced and enzymatically dissociated
with 1 mg/mL collagenase D (Roche) and 1 mg/mL DNase I
(Roche) for 1 hour at 37 C, and then sequentially filtered
through 100 and 70 mm cell strainers (BD Falcon). After the
lysis of red blood cells with Red Blood Cell Lysis Solution
(Miltenyi Biotec), the filtered cells were grown in STEMPRO
hESC SFM (Invitrogen) supplemented with 8 ng/mL bFGF
(Invitrogen), 20 mmol/L Y-27632 (Wako), and penicillin/streptomycin on ultra-low attachment culture dishes (Corning). For
serial passage, spheroid cells were dissociated into single cells
with Accutase (Invitrogen) once a week and incubated under
the culture conditions described earlier. For differentiation
experiments, spheroid cells were cultivated in the presence of
10% FBS on standard tissue culture dishes (BD Falcon).
Flow cytometry analysis
Dissociated single spheroid cells were filtered, incubated
with 7-AAD (BD Pharmingen) for the exclusion of nonviable
cells, and double-stained with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44 (G44-26; BD Pharmingen) and an allophycocyanin (APC)-conjugated monoclonal
antibody against CD133 (AC133; Miltenyi Biotec). Isotypematched mouse immunoglobulins were used as controls.
Stained cells were then sorted using the FACS Aria II Cell
Sorter (BD Biosciences) under the following conditions: nozzle
tip diameter (100 mm), pressure (20 psi), and threshold rate
(2,000 events/s). Subsequently, the sorted cells were analyzed
using FlowJo ver.7.6 software. Viability of the sorted cells was
examined by Trypan-blue staining.
For analyses of cell-cycle profiles of cells with different levels
of CD44 expression, dissociated cells were double-stained with
PE-conjugated anti-CD44 and APC-conjugated anti-CD133
antibodies, fixed with 70% ethanol, and incubated with 0.1
mg/mL RNase A (Qiagen) and 25 mg/mL propidium iodide
(Sigma), to determine the DNA contents of double-stained cells
from each fraction with different levels of CD44 expression.
In vitro assays for spheroid growth and formation
Accutase-dissociated single cells or fluorescence-activated
cell sorting (FACS)-sorted cells were seeded at a density of
viable 1,000 cells per well on 96-well ultra-low attachment
plates (Corning). Cell growth was quantified by measuring the
amounts of cellular ATP from a pool of spheroid forming and
nonforming cells (CellTiter-Glo Luminescent Cell Viability
Assay; Promega). Spheroid formation was evaluated by counting the number of formed spheres (>20 mm in diameter). For
quantification of the inhibitory effects of CD44 inhibition on
spheroid formation, 10 mg/mL anti-CD44 neutralizing antibodies (IM7; Biolegend and 2C5; R&D Systems) or the control
antibody were added at the beginning of spheroid formation.
Single-cell dilution assays were conducted as described earlier
(12).
Animal experiments
For cell transplantation assays of spheroid cells, the spheroids were dissociated into single cells with Accutase,
suspended in 50 mL medium containing 50% Matrigel (BD
5102
Cancer Res; 72(19) October 1, 2012
Biosciences), and used for subcutaneous injection with a 27G
needle into the flank of NOD/SCID mice (Central Institute for
Experimental Animals, Tokyo, Japan). All mouse procedures
were approved by the Animal Care and Use Committees of the
National Cancer Center and conducted in accordance with
Institutional policies.
Results
ROCK inhibitors markedly improve sphere-forming
efficiency from primary colon cancer cells
To isolate and expand cancer-initiating cells from primary
colon cancer, we dissected and cultivated primary cancer cells
under spheroid culture conditions (5). In an attempt to establish the optimum conditions for the maintenance and growth
of cancer-initiating cells, we examined the effects on spheroid
formation of chemicals that were reported to be effective in
promoting the growth of normal or cancer stem cells (data not
shown). It was previously reported that ROCK inhibitor promotes the survival of embryonic stem cells (19–23). After
extensive screening, Y-27632, a ROCK inhibitor, stood out as
a chemical that greatly facilitated spheroid formation from a
colon cancer specimen (Fig. 1A).
ROCKi-IV, another ROCK inhibitor, was also effective for
spheroid formation (Fig. 1B). The presence of Y-27632 at 10 to
20 mmol/L, a concentration sufficient for maintenance of
embryonic stem cells (21), caused maximum enhancement of
spheroid formation (Fig. 1C and D), and extended in vitro
cultivation revealed that Y-27632 was required for sustained
growth of spheroids (Fig. 1E). There was a striking increase in a
fraction of cells with sub-G1 DNA content in the absence of Y27632 (Fig. 1F) indicating that Y-27632 protects spheroid cells
from apoptotic cell death.
Because Y-27632 was highly effective in establishing
spheroids from the aforementioned case (hereafter referred
as #6), we tested the same conditions for cultivation from
other cases of primary colon cancers. In aggregate, we
successfully cultivated spheroids that could be maintained
in vitro for 1 month in 10 of 16 cases examined (Supplementary Table S1). Continued passage revealed that 5 of 10
spheroids could be further expanded over a period of more
than 6 months (Supplementary Table S1). In all 5 cases, in
which spheroid culture could be sustained, the withdrawal
of Y-27632 resulted in marked reduction of spheroid formation (Fig. 1A; Supplementary Fig. S1A–S1C). ROCKi-IV was
also effective for spheroid formation (#9 and #20; Supplementary Fig. S1D and S1E). As in #6, Y-27632 was required
for continued growth of the spheroids (#20; Supplementary
Fig. S1F), and for the protection of spheroid cells from
apoptotic cell death (#19 and #20; Supplementary Fig. S1G
and S1H). Taken together, the presence of ROCK inhibitor
markedly facilitates the proper maintenance and growth of
spheroids from primary colon cancer.
Spheroids formed in the presence of ROCK inhibitor
express colon CSC markers, differentiate into epitheliallike cells, and are capable of forming tumors in mice
We next examined whether spheroid cells formed in the
presence of Y-27632 share characteristics for CSCs. We
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Regulation of CD44 by ROCK Inhibitor in Colon Cancer–Initiating Cells
A
B
C
E
Relative cell growth
D
mol/L
mol/L
mol/L
Relative cell growth
mol/L
mol/L)
F
3,000
2,000
1,000
4,000
Cell counts
Cell counts
4,000
0
3,000
2,000
1,000
0
Figure 1. ROCK inhibitors improve sphere-forming efficiency from primary colon cancer cells. A and B, bright-phase images of spheroids (#6) in the presence or
absence of 20 mmol/L Y-27632 (A) or 5 mmol/L ROCKi-IV (B). C, bright-phase images of spheroids (#6) in the presence of the indicated concentrations
of Y-27632. D, dose-dependent curves of the cell growth of spheroid cells (#6) by Y-27632 on day 3. Cell growth was quantified by measuring cellular
ATP. E, time course of spheroid cell growth (#6) in the presence or absence of 20 mmol/L Y-27632. F, suppression of cell death of spheroid cells (#6)
by Y-27632. Spheroid cells were stained with propidium iodide, and a fraction of cells with sub-G1 DNA content was measured by flow cytometry analysis.
determined whether spheroid cells were capable of (i)
expressing specific markers, (ii) differentiation, and (iii)
tumor formation in immunocompromised mice, based on
the proposed criteria for CSCs (3). First, spheroid cells
expressed colon CSC–specific markers, CD44 and CD133
(#6 and #20, Fig. 2B; Supplementary Fig. S2B). Second,
spheroid cells were capable of differentiating into epithelial-like cells if grown under differentiating conditions, based
on their morphology (#6 and #20, Fig. 2A; Supplementary Fig.
S2A), reduction of CD44 and CD133, and induction of a
differentiation marker, cytokeratin 20 (CK20; #6 and #20; Fig.
2B and Supplementary Fig. S2B). Third, xenograft experiments using immunocompromised NOD/SCID mice showed
that the injection of spheroid cells (1 103 cells) could form
tumors that were histologically indistinguishable from the
original primary tumor or from its mouse xenograft (#6, #19,
and #20; Fig. 2C and Supplementary Fig. S2C and S2D). Thus,
spheroids included cells that meet the major criteria for
CSCs.
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CD44high cells in spheroids show characteristics of CSCs
To gain insight into whether entire spheroid cells or only a
fraction retain characteristics associated with CSCs, we examined the expression of CD44 and CD133 in spheroid cells by
flow cytometry analysis. Although levels of CD133 expression
did not significantly differ, there were striking differences in
CD44 expression among cells (#6 and #20; Fig. 3A and Supplementary Fig. S3A). To determine whether the difference in
CD44 expression reflects cellular hierarchy among them, we
sorted spheroid cells into CD44/low, CD44med, and CD44high
fractions. As expected, the sorted CD44high cells expressed high
levels of CD44, whereas their expression in CD44/low was not
detectable by Western blot analyses (Fig. 3C). Analyses of the
cell-cycle profile revealed that a significant fraction of
CD44/low cells underwent apoptosis (#6 and #20; Fig. 3B and
Supplementary Fig. S3B). CD44high cells could form spheroids
more effectively than CD44/low cells (Fig. 3D), and were
capable of differentiation based on the induction of CK20 and
downregulation of CD44 and CD133 (#6 and #20; Fig. 3E and
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A
B
Spheroids
C
Differentiated
X
Primary tumor
Supplementary Fig. S3C). In addition, CD44high cells gave rise to
CD44med and CD44/low cells under the conditions of spheroid
culture (#6 and #20; Fig. 3F and Supplementary Fig. S3D),
indicating that there is dynamic transition from CD44high cells
to CD44/low cells in spheroids.
It was shown that high Wnt activity is associated with the
colon CSC phenotype (12). To examine whether high levels of
CD44 are associated with the activation of the Wnt pathway,
we examined the activating phosphorylation of b-catenin
(ser552; refs. 24–26) in the CD44high and CD44/low cells. The
fraction of cells with the activating phosphorylation of b-catenin was higher in the CD44high cells than in the CD44/low cells
(Fig. 3G), which was corroborated by Western blot analyses
(Fig. 3H). Thus, b-catenin was activated in the CD44high cells.
Enhancement of aerobic glycolysis is another phenotypic
hallmark associated with normal and CSCs (27–29). We conducted microarray gene expression analyses of the CD44high
and CD44/low cells, and gene set enrichment analyses (30) of
the expression profiles revealed that several pathways associated with glycolysis, that is glucose metabolism and the
pentose phosphate pathway, were upregulated (Supplementary Fig. S3E). These are in agreement with recent reports (31),
and indicate that the glycolytic metabolism is enhanced in the
CD44high cells. Collectively, these results strongly suggest that
cells that express high levels of CD44 are associated with the
known CSC-like characteristics.
Inhibition of CD44 suppresses spheroid formation
To show that high levels of CD44 expression are required for
the CSC-like properties, we inhibited CD44 with the corre-
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X
d
te
tia
ds
oi ren
r
he iffe
Sp D
Figure 2. Spheroids formed in the
presence of ROCK inhibitor
express colon CSC markers,
differentiate into epithelial-like
cells, and are capable of forming
tumors in mice. A, bright-phase
images of spheroids (#6) before and
after differentiation (day 7). B,
Western blot analyses of spheroid
cells (#6) before and after
differentiation with the indicated
antibodies. Asterisk indicates
nonspecific band. C, hematoxylin
and eosin staining of the primary
human colon tumor (left),
subcutaneous xenograft of the
same primary tumor (center), and
subcutaneous xenograft of the
spheroid cells (right). Spheroid cells
were derived from an identical
primary tumor (#6). Scale bars,
100 mm.
sponding shRNAs and determined whether inhibition of CD44
thwarts spheroid formation. Knockdown of CD44 by the
shRNAs was confirmed by quantitative reverse transcription
(qRT)-PCR (#6 and #20; Fig. 4A and Supplementary Fig. S4A)
and Western blot analyses (#6 and #20; Fig. 4B and Supplementary Fig. S4B). In accordance with the elevated levels of
apoptotic cell death in CD44/low cells (Fig. 3B), the inhibition
of CD44 by shRNAs induced apoptotic cell death (data not
shown). Examination of a remaining infectant indicated that
the inhibition of CD44 by shRNAs reduced spheroid formation
(#6 and #20; Fig. 4C and Supplementary Fig. S4C) as well as cell
growth (Fig. 4D and Supplementary Fig. S4D).
In agreement with the inhibition of spheroid formation by
the CD44 shRNAs, spheroid formation and cell growth were
compromised by the inhibition of CD44 by neutralizing antibodies (#6 and #20; Fig. 4E and F and Supplementary Fig. S4E
and S4F). The results from serial dilution assays confirmed that
the anti-CD44 antibody inhibited the clonal growth of #20
spheroid cells (Supplementary Fig. S4G). Combined with the
data presented in Fig. 3, these data indicate that CD44 expression is required to maintain the CSC-like characteristics of
CD44high cells.
ROCK inhibitor induces variant forms of CD44
The functional importance of CD44 presented in Fig. 4
prompted us to examine its expression in spheroids. Of the
5 established spheroids examined, 2 (#6 and #20) expressed
higher levels of CD44 than the others (#9, #17, and #19) in the
presence of Y-27632 (Fig. 5A). Strikingly, CD44 expression was
markedly reduced in the absence of Y-27632 in all the
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Regulation of CD44 by ROCK Inhibitor in Colon Cancer–Initiating Cells
high
cells in spheroids
Figure 3. CD44
show characteristics of CSCs. A, flow
cytometry analyses of spheroid cells
(#6) double-stained with anti-CD44
and anti-CD133 antibodies. B, the
cell-cycle profiles of CD44/low,
CD44med, and CD44high cells. C,
Western blot analyses of FACSsorted CD44high and CD44/low cells
with the indicated antibodies. D,
bright-phase images of spheroids
(day 3) of FACS-sorted CD44high and
CD44/low cells (left). Quantification
of spheroid formation (right). E,
Western blot analyses of FACSsorted CD44high cells (#6) with the
indicated antibodies. CD44high cells
were harvested immediately (day 0)
or cultivated for a week (day 7)
under differentiation conditions.
F, flow cytometry analyses of sorted
CD44high cells (#6) cultivated in
spheroid conditions for the indicated
periods. G, immunofluorescence
staining of dissociated spheroid
cells. Left, immunostaining with the
indicated antibody. Right, a fraction
of the spheroid cells that were
positive for staining with the antiser552 phosphorylated b-catenin
antibody. H, Western blot
analyses of FACS-sorted CD44high
and CD44/low cells with the
indicated antibodies.
A
B
C
D
E
F
G
spheroids, whereas CD133 expression was not significantly
affected (Fig. 5A). The withdrawal of Y-27632 caused rapid
reduction of CD44 within 3 days (#6 and #20; Fig. 5B and
Supplementary Fig. S5A).
In addition to the standard form (CD44s), there are several
variant isoforms of CD44 (CD44v) as a result of alternative
splicing (32). The molecular weight of the major polypeptide
detected with the anti-pan-CD44 antibody (170 kD) indicated that CD44v was a major form induced by Y-27632 (#6
and #20; Supplementary Fig. S5B and S5C, left columns).
Western blot analyses with variant-specific antibodies
revealed that CD44v9 and CD44v6 were induced by Y27632 (Supplementary Fig. S5B and S5C, middle and right
columns). The molecular weight of the major band detected
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H
with the anti-pan-CD44 antibody approximately matched
that of CD44v9 (Supplementary Fig. S5B and S5C), although
a longer exposure revealed that the anti-pan-CD44 antibody
detected the polypeptide that roughly corresponds to the
size of CD44v6 (data not shown). Thus, several variant forms
of CD44 were induced by Y-27632.
To determine whether CD44 expression can be reinitiated
after readdition of the ROCK inhibitor, dissociated spheroid
cells were cultivated in the absence of Y-27632 for 3 days, and
then reincubated with the inhibitor. The readdition of Y-27632
resumed CD44 expression, and 10 to 20 mmol/L Y-27632 was
sufficient for maximum induction of CD44 (#6; Fig. 5C). Timecourse analyses of CD44 induction by Y-27632 showed
that CD44 was induced for 3 days after incubation (#6 and
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B
R
A
D
N
R
C
F
R
N
E
Figure 4. Inhibition of CD44 suppresses spheroid formation. A, qRT-PCR
analyses of CD44 expression in spheroid cells (#6) after infection with
lentiviruses expressing the indicated CD44 shRNAs. B, Western blot
analyses of CD44 expression in the spheroid cells presented in A. C,
spheroid formation after shRNA-mediated inhibition of CD44. Extent of
spheroid formation was measured by counting the number of spheroids.
D, cell growth of spheroid cells after shRNA-mediated inhibition of CD44.
Cell growth was quantified by measuring cellular ATP. E, spheroid
formation after treatment with the indicated antibodies. F, cell growth of
spheroid cells after treatment with the indicated antibodies. C and D,
3
1 10 cells were plated per well for 3 days, and average values from
3 independent experiments are shown. , P < 0.05; , P < 0.01;
, P < 0.001.
#20; Fig. 5D and Supplementary Fig. S5D). ROCKi-IV as well as
Y-27632 was capable of inducing CD44 (#6 and #20; Supplementary Fig. S5E). The kinetics of the induction of CD44 were
overall in line with those of spheroid formation (Fig. 1E and
data not shown). Thus, given the functional role of CD44
expression in spheroid formation, these data strongly suggest
that the induction of CD44v contributes to the formation of
spheroids by ROCK inhibitors.
ROCK inhibitor primes the retrograde transition from
CD44/low to CD44high cells
In accordance with the induction of CD44 after the readdition of ROCK inhibitor, the incubation of FACS-sorted
CD44/low cells with Y-27632 caused an increase of CD44med
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and CD44high cells (#6 and #20; Fig. 5E and Supplementary
Fig. S5F). The induction of CD44-positive cells by Y-27632 was
supported by Western blot analyses (#6 and #20; Fig. 5F and
Supplementary Fig. S5G). These results suggest that the ROCK
inhibitor, by inducing CD44, induced reverse transition from
CD44/low to CD44high cells in spheroids.
qRT-PCR analyses indicated that there was no significant
increase of CD44 mRNA over the same time course, indicating
that CD44 was posttranscriptionally induced (#6 and #20; Fig.
5G and Supplementary Fig. S5H).
Blebbistatin induces CD44 and promotes spheroid
growth
It was reported that the enhanced survival of embryonic
stem cells by ROCK inhibitors is mediated via the interference of the cytokinesis pathway, and that survival of
dissociated embryonic stem cells was greatly enhanced by
Blebbistatin, an inhibitor of myosin hyperactivation, as well
as by ROCK inhibitor (20). Treatment of spheroid cells with
blebbistatin, as well as with Y-27632, induced CD44 expression (Fig. 5H and Supplementary Fig. S5I) and promoted cell
growth of spheroid cells (Fig. 5I and Supplementary Fig.
S5H). Thus, it is likely that the ROCK/myosin pathway
mediates the CD44-dependent enhancement of spheroid
cell growth as well as cell survival of dissociated embryonic
stem cells.
Expression of CD44 variants is associated with the
activating phosphorylation of c-Met
To determine the type of CD44 variant isoforms expressed in
spheroid cells (Supplementary Fig. S5B and S5C), we conducted RT-PCR analyses using isoform-specific primers (Fig.
6A; ref. 33). Overall profiles of CD44 isoforms were similar
between those from the spheroids and HT29 cells (Fig. 6B and
C), which were reported to express a variety of CD44 variants
including CD44v6 and CD44v9. Of note, CD44v6 mediates the
activation of the hepatocyte growth factor (HGF)/c-Met signaling (34). Indeed, CD44 induction by Y-27632 was associated
with activating phosphorylation of c-Met (Fig. 6D), suggesting
that the induction of CD44v6 augments the HGF/c-Met
signaling.
CD44/low cells are capable of forming CD44-positive
tumors in mice
Finally, we examined the capacity of CD44high and
CD44/low cells to generate tumors in immunocompromised
mice. Both types of cells were capable of forming tumors,
although the frequency of tumor formation by CD44/low
cells was less than that by CD44high cells (Supplementary
Fig. S6A). Hematoxylin and eosin staining showed that
tumors derived from CD44high cells were histologically indistinguishable from the original primary tumor (#6; Figs. 2C
and 7A). CD44/low cells also formed similar adenocarcinoma, although cribriform and more PAS-positive vacuoles
were found in some parts of the tumor (Fig. 7A and data not
shown).
Remarkably, some adenocarcinoma cells from CD44/low
cells as well as from CD44high cells were CD44-positive (Fig. 7B),
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Regulation of CD44 by ROCK Inhibitor in Colon Cancer–Initiating Cells
B
A
Figure 5. ROCK inhibitor primes
/low
retrograde transition from CD44
to CD44high cells. A, Western blot
analyses of 5 spheroid cells in the
presence or absence of 20 mmol/L
Y-27632. The indicated spheroids
were grown under standard spheroid
conditions or in the absence of
Y-27632 for 7 days. Western blot
analyses were conducted with the
indicated antibodies. B, Western blot
analyses of spheroid cells (#6) after
withdrawal of Y-27632 for the
indicated periods. C and D,
dissociated spheroid cells (#6) were
incubated in the absence of Y-27632
for 3 days and recultivated with the
indicated concentration of Y-27632
for 7 days (C) or with 20 mmol/L Y27632 for the indicated periods (D).
Western blot analyses were
conducted as described in A. E, flow
cytometry analyses of FACS-sorted
CD44/low cells (#6) that were grown
under the spheroid conditions for the
indicated periods. F, Western blot
analyses of sorted CD44/low cells
(#6) harvested immediately (day 0) or
grown under the spheroid conditions
for 8 days. G, qRT-PCR analyses of
CD44 and CD133 of the spheroid
cells (#6) described in D. GAPDH
expression was used as a control. H,
Western blot analyses of spheroid
cells incubated with the indicated
concentration of Y-27632 or
blebbistatin for 3 days. I, cell growth
of spheroid cells (#6) grown under
conditions described in H. Cell
growth was quantified by measuring
cellular ATP.
D
C
E
mol/L)
G
F
I
H
mol/L)
mol/L)
suggesting that CD44/low cells generated CD44-positive
tumors. Supporting this, flow cytometry analyses showed
that a fraction of the CD44/low cell-derived tumors as well
as CD44high cell-derived ones expressed CD44 (Fig. 7C).
Comparison of CD44 expression between CD44/low cellderived tumor cells and the original FACS-sorted cells
indicated that approximately 10% of the CD44/low cellderived tumor cells became CD44-positive (Supplementary
Fig. S6B). It is unlikely that CD44 expression in tumors from
CD44/low cells was attributed to cells other than tumors,
because CD44 staining was almost exclusively detected in
the tubular structure of adenocarcinoma (Fig. 7B). Western
blot analyses further confirmed the expression of CD44 in
tumors derived from CD44/low cells (Fig. 7D). Judging
from levels of human Topo I expression, levels of CD44
www.aacrjournals.org
expression in CD44high cell-derived tumors were approximately 3-fold higher than those in CD44/low cell-derived
ones (Fig. 7D).
We also conducted transplantation assays using spheroid
cells that were cultivated either in the presence or absence or Y27632 (Fig. 5). The generated tumor from these cells showed
similar CD44 staining (Supplementary Fig. S6C). Taken together, our data indicate that CD44-negative cells are capable of
forming CD44-positive tumor.
Discussion
In this report, we showed that the addition of ROCK inhibitors markedly facilitated the continuous growth of cells with
properties for colon CSCs in vitro. Characterization of the
cultivated cells showed that ROCK inhibitors were capable of
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Ohata et al.
A
C
Exon
Primer
B
D
sustaining spheroids that contained cells that met the major
criteria for CSCs. In the formed spheroids, CD44high cells
shared characteristics for CSCs, and were capable of generating their differentiated progeny, CD44/low cells, as well as
CD44high cells themselves.
Recently, it was reported that CD44v contributes to
the ROS resistance of gastrointestinal cancer stem-like cells
(35), and associates with enhanced aerobic glycolysis (31).
CD44 expression in spheroid cells is associated with the
enhanced glycolytic pathways (Supplementary Fig. S3E),
which may be attributed to CD44v expressed in the presence of ROCK inhibitor (Figs. 6A–C and Supplementary Fig.
S5B and S5C).
Of particular interest among the CD44 variants is CD44v6,
because it was reported that CD44v6 mediates the activation
of HGF/c-Met pathway (34). Indeed, our data indicate that
CD44 expression is associated with c-Met activation (Fig.
6D). Considering that HGF restores the colon CSC phenotype (12), CSC-like characteristics associated with CD44
expression may be induced via the activation of the HGF/
c-Met pathway.
What is the mechanism for the induction of CD44 by
ROCK inhibitors? It was reported that the functions of
ROCK center on the regulation of cytoskeletons via the
phosphorylation of its targets (36). Considering that CD44
induction by the ROCK inhibitor is posttranscriptional (Fig.
5D and G and Supplementary Fig. S5D and S5I), alterations
in the cytoskeleton caused by ROCK inhibitor may lead to
stabilization or enhanced translation of CD44v protein.
5108
Cancer Res; 72(19) October 1, 2012
Figure 6. Expression of CD44
variants is associated with the
activating phosphorylation of cMet in spheroid cells. A, schematic
presentation of the position of
primers for exon-specific RT-PCR
analyses. The forward primer is
located at the constant exon 5
(C13) or each variant exon (v2-v10)
of CD44, and the reverse primer is
located at the constant exon 15. B,
RT-PCR analyses of CD44 variants.
C13 and the exon 15 primer are
used to detect the variants from the
spheroid cells and HT29 cells. RTPCR analyses were conducted as
described before (33). C, RT-PCR
analyses with isoform specific
primers. Expression of each CD44
variants was detected with the
primers shown in A. HepG2 cells,
which do not express CD44, were
used as negative controls (33). D,
induction of the activating
phosphorylation of c-Met in the
presence of Y-27632. The spheroid
cells in the presence or absence of
Y-27632 for 3 days were used for
Western blot analyses with the
indicated antibodies.
Notably, ROCK inhibitors facilitate the in vitro growth of
embryonic stem cells by inhibiting dissociation-induced apoptosis (21), and the inhibition of the cell death is mediated via
the blockage of ROCK/myosin hyperactivation (20). Similarly,
the inhibition of dissociation-induced apoptosis may also be
important for the establishment of colon cancer spheroids, and
indeed the inhibitor was used to block cell death of dissociated
organoids that are derived from colon adenocarcinoma (18).
ROCK inhibitors and blebbistatin may contribute to CD44mediated spheroid formation through the inhibition of actomyosin hyperactivation.
ROCK inhibitor may also regulate other pathways independent of CD44 induction to facilitate the formation of spheroids.
In the absence of Y-27632, some spheroids (#6 and #20) express
residual amounts of CD44, the levels of which are roughly
equivalent to those from other spheroids in the presence of the
inhibitor (#9, #17, and #19; ref. Fig. 5A). Because the capacity to
form spheroids was lower in the former than the latter (Supplementary Fig. S1C), the CD44 pathway and other unknown
pathways may synergize to facilitate the formation of
spheroids.
The retrograde transition from CD44/low to CD44high cells
by ROCK inhibitor may lead to the formation of a dynamic
equilibrium between these cells. Of note, similar dynamic
transitions between CSC and non-CSC states were also
observed in melanoma cells (37, 38) and in breast cancer cells
(39, 40). It was proposed that, through mathematical analyses,
stochastic transition between cancer stem-like cells and nonstem-like cells forms a dynamic equilibrium in cancer (39), and
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Published OnlineFirst September 20, 2012; DOI: 10.1158/0008-5472.CAN-11-3812
Regulation of CD44 by ROCK Inhibitor in Colon Cancer–Initiating Cells
C
A
/low
cells are
Figure 7. CD44
capable of forming CD44-positive
tumors in mice. A, hematoxylin and
eosin staining of xenografts derived
from CD44high and CD44/low cells
(#6). B, immunostaining with
anti-CD44 antibody of tumors from
CD44high and CD44/low cells (#6). C,
measurement of CD44 expression in
tumors derived from CD44high
and CD44/low cells (#6) by flow
cytometry. The tumor cells were
double-stained with anti-CD44 and
anti-CD133 antibodies (right) or with
control IgG (left). D, Western blot
analyses of CD44 expression in the
tumors described in C.
B
D
Xenograft
such equilibrium may be influenced by extracellular factors
(12). Thus, the phenotypic plasticity of CSC-like cells we
observed may be a widespread feature of solid cancer cells.
In devising a therapeutic strategy against colon cancer–
initiating cells, tumors with high activity for retrograde transition may pose a major problem, because elimination of both
CD44high and CD44/low cells may be required for effective
therapy; CD44/low cells, if not simultaneously neutralized, will
revert back to CD44high cells with cancer-initiating ability. The
combination of killing cancer-initiating cells and blocking
retrograde differentiation may be considered an effective
therapy in the future.
Disclosure of Potential Conflicts of Interest
Y. Aihara is employed (other than primary affiliation; e.g., consulting) in
Sysmex Corporation as a Researcher. No potential conflicts of interest were
disclosed by the other authors.
Authors' Contributions
Conception and design: H. Ohata, H. Nakagama, K. Okamoto
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): H. Ohata, T. Ishiguro, Y. Aihara, A. Sato, H. Sakai, S.
Sekine, T. Akasu, S. Fujita
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): H. Ohata, H. Nakagama, K. Okamoto
Writing, review, and/or revision of the manuscript: H. Ohata, H. Nakagama,
K. Okamoto
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Taniguchi, H. Nakagama
Study supervision: H. Nakagama, K. Okamoto
Acknowledgments
The authors thank the laboratory members for critical reading of the
manuscript, Ibuki Kobayashi, and Naoaki Uchiya for technical assistance,
Yasuhide Yamada for clinical suggestion, and Hideyuki Saya for anti-CD44v9
antibody. SCADS inhibitor kit, which includes Y-27632, was a gift from the
Screening Committee of Anticancer Drugs supported by Grant-in-Aid for
Scientific Research on Priority Area "Cancer" from MEXT, Japan.
Grant Support
This study was supported by a Grant-in-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the MHLW (K. Okamoto); the
Program for Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NiBio; K. Okamoto); Grant-in-Aid
for Scientific Research in Innovate Areas from MEXT (K. Okamoto); Grant-in-Aid
for Scientific Research (C) from Japan Society for the Promotion of Science (JSPS;
K. Okamoto); Grant-in-Aid for Cancer Research from Foundation for Promotion
of Cancer Research (K. Okamoto); Research Resident Fellowship from the
Foundation for Promotion of Cancer Research (FPCR, Japan; T. Ishiguro).
Grant-in-Aid for Young Scientist (B) from JSPS (H. Ohata and T. Ishiguro).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
Received November 30, 2011; revised June 19, 2012; accepted July 14, 2012;
published OnlineFirst September 20, 2012.
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Published OnlineFirst September 20, 2012; DOI: 10.1158/0008-5472.CAN-11-3812
Induction of the Stem-like Cell Regulator CD44 by Rho Kinase
Inhibition Contributes to the Maintenance of Colon Cancer−
Initiating Cells
Hirokazu Ohata, Tatsuya Ishiguro, Yuki Aihara, et al.
Cancer Res 2012;72:5101-5110. Published OnlineFirst September 20, 2012.
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