CD44 Expression Denotes A Subpopulation of Gastric Cancer Cells in Which Hedgehog Signaling Promotes Chemotherapy Resistance
CD44 Expression Denotes A Subpopulation of Gastric Cancer Cells in Which Hedgehog Signaling Promotes Chemotherapy Resistance
CD44 Expression Denotes A Subpopulation of Gastric Cancer Cells in Which Hedgehog Signaling Promotes Chemotherapy Resistance
Cancer
Cancer Therapy: Preclinical Research
Abstract
Purpose: Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including
chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key
developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of
HH signaling in CD44(þ) gastric cancer cells.
(ab38686) and Gli-1 (ab49314) from Abcam; anti-CD133 Afterward, the slices were counterstained by hematoxylin.
(CA1217) from Applications, Inc.; and anti–b-actin from Antibodies used were anti-PCNA (Santa Cruz Biotechnol-
Sigma. ogy) and CD44 (eBioscience, San Diego, CA). CD44 stain
was predominantly localized to the outer cell membrane.
FACS and magnetics cell sorting CD44 scores (0–300) were calculated by multiplying the
For FACS, cells were dissociated using Accutase and staining intensity (0, 1, 2, or 3) by the staining extent (0%–
resuspended in PBS containing 0.5% BSA. The cells were 100%).
stained with FITC-conjugated CD44 (BD555478) or isotype For detection of apoptosis, terminal deoxynucleotidyl
control antibody (BD555742) from BD Biosciences on ice transferase–mediated dUTP nick end labeling (TUNEL)
for 30 minutes. Cells were then washed with PBS and immunofluorescence was performed. Paraffin-embedded
analyzed on a BD FACSCalibur (BD Biosciences) using Cell sections were deparaffinized, and sections were treated with
Quest software. the DeadEnd Fluorometric TUNEL system (Promega) to
CD44-positive cells were sorted by a magnetic-activated detect apoptosis following the manufacturer’s instruction.
cell sorting (MACS) system (Miltenyi Biotech). After Subsequently, sections were stained with DAPI (0.2 mg/mL)
collecting spheroids, cells were washed with PBS, disso- for 3 minutes. Images were analyzed using Molecular
3976 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
C D
AGS MKN-45 N87 CFPAC-1 PANC-1 AGS MKN-45 N87
Monolayer
Monolayer
Monolayer
Monolayer
Monolayer
Monolayer
Monolayer
Monolayer
Spheroid
Spheroid
Spheroid
Spheroid
Spheroid
Spheroid
Spheroid
Spheroid
Shh Sox2
Ptch1 Oct-4
Smo Nanog
Gli1 β-Actin
β-Actin
Figure 1. Gastric cancer cells grown as spheroids show upregulation of CD44 and Hedgehog pathway proteins. A, photos of gastric cancer cell lines
AGS, MKN-45, and N87 and pancreatic cancer cell lines CFPAC-1 and PANC-1 grown as spheroids. B, immunofluorescence photos of gastric cancer
cells in regular media (monolayer) or in spheroid formation media at specified time points. Cells stained for CD44 (green), Gli1 (red), and DAPI (blue).
C, Western blot showing expression of Hedgehog pathway proteins Shh, Ptch1, Smo, and Gli1 in cell lines grown as monolayer cells versus as
spheroids. D, Western blot showing expression of self-renewal proteins Sox2, Oct-4, and Nanog in cell lines grown as monolayer cells versus as
spheroid.
associated with sustained increases in levels of CD44, We next examined the effects of Hedgehog pathway
Hedgehog pathway proteins, and some self-renewal inhibition on CD44 expression and spheroid formation.
proteins. The Hedgehog pathway was inhibited either genetically
3978 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
using Smo shRNA or pharmacologically using the drug invasion, colony formation, and anchorage-independent
vismodegib. Effective knockdown of Smo by shRNA in growth in gastric cancer monolayer cells and spheroid cells.
all three gastric cancer cell lines was confirmed by Western The three gastric cancer cell lines demonstrated 5.3- to 6.8-
blot analysis (Fig. 2A). Smo shRNA treatment of gastric fold more migration (Fig. 4A) and 6.5- to 13.6-fold more
cancer cells reduced expression of CD44 as measured by invasion (Supplementary Fig. S4A and S4B) for spheroid
immunocytochemistry and also reduced spheroid forma- cells compared with monolayer cells. Smo inhibition using
tion from 20 to 30 colonies per visual field to 6 to 10 shRNA or vismodegib reduced the migration of spheroid
colonies (Fig. 2B). Vismodegib treatment also reduced cells by 50.2% to 65.6% and reduced the invasion of
CD44 expression and reduced the number of spheroid spheroid cells by 57.4% to 66.3%. We also measured colony
colonies from 25 to 35 to 10 to 15 per visual field (Fig. formation ability for monolayer and spheroid cells and
2B). Spheroid cells were next dissociated and grown in a found spheroid cells formed 3.8- to 4.6-fold more colonies
single-cell assay under spheroid formation conditions. than monolayer cells (Fig. 4B). As seen with migration and
Hedgehog pathway inhibition reduced the diameter of invasion, Smo shRNA and vismodegib reduced colony
colonies in this single-cell assay after 10 days by 70.3% to formation of spheroid cells by 68.6% to 72.3% and
78.4% for Smo shRNA and 66.9% to 70.8% for vismodegib 38.7% to 76.5%, respectively. Gastric cancer cells grown as
Scramble shRNA
Scramble shRNA
Scramble shRNA
Smo shRNA
Smo shRNA
Smo shRNA
Smo
β-Actin
DMSO
30 µm
Vismodegib
shRNA
Smo-
Number of spheroids
per visual field
30
per visual field
30
20 20
10 10
0 0
75
C 75 1 day 1 day
Spheroid diameter (μmol/L)
Spheroid diameter (μmol/L)
4 days 4 days
7 days 7 days
10 days 10 days
50 50
25 25
0 0
DMSO
DMSO
DMSO
Smo.shRNA
Scr.shRNA
Smo-shRNA
Scr.shRNA
Smo-shRNA
Figure 2. Hedgehog pathway inhibition with Smo shRNA or vismodegib blocks spheroid formation. A, Western blot demonstrating knockdown of Smo in
gastric cancer cell lines AGS, MKN-45, and N87 following transduction with Smo shRNA (Smo.shRNA) lentivirus compared with scrambled shRNA control
(Scr.shRNA) lentivirus. B, immunofluorescence photos for CD44 (green) and nuclei (blue) of gastric cancer cell lines treated with Smo.shRNA or Scr.shRNA
and grown in spheroid formation conditions or treated with vismodegib (10 mmol/L) or carrier (DMSO) and grown in spheroid formation conditions.
C, single-cell assay of spheroid cells showing diameter of spheroids at selected time points following treatment with Smo.shRNA versus Scr.shRNA or
vismodegib (Vis, 10 mmol/L) versus carrier (DMSO). Bars, SD.
3980 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
CD44
DAPI
Shh
CD44
Ptch1
Smo
Merge
Gli1
30 μm
β-Actin
Vis
5-FU
75
Cisplatin
Cell viability (%)
Vis + 5-FU
50 Vis + cisplatin
25
0
CD44(–) CD44(+) CD44(–) CD44(+) CD44(–) CD44(+)
Cisplatin/Smo shRNA
50
25
0
Monolayer Spheroid Monolayer Spheroid Monolayer Spheroid
Figure 3. CD44(þ) gastric cancer cells demonstrate chemotherapy resistance, which can be reversed with Hedgehog pathway inhibition. A,
immunofluorescence images of CD44 (green) and nuclei (blue) for CD44(þ) and CD44() cells following magnetic bead sorting of AGS, MKN-45, and N87
gastric cancer cell lines. B, Western blot showing expression of Hedgehog pathway proteins Shh, Ptch1, Smo, and Gli1 in CD44(þ) and CD44() cells.
Proliferation assay for CD44(þ) and CD44() cells (C) and spheroid cells and monolayer cells (D) following treatment with 5-fluorouracil (5-FU) or cisplatin
chemotherapy and vismodegib (Vis, 10 mmol/L) compared with DMSO or Smo shRNA compared with Scramble shRNA. Bars, SD.
Monolayer Monolayer
Spheroid/ Spheroid/
Scramble shRNA DMSO
Spheroid/ Spheroid/
Smo shRNA vismodegib
Monolayer Monolayer/DMSO
Spheroid/Scramble shRNA Spheroid/DMSO
Spheroid/Smo shRNA Spheroid/Vis 10 μmol/L
75
Migrated cells
75
50 50
25 25
0 0
AGS MKN-45 N87 AGS MKN-45 N87
Monolayer Monolayer
B Spheroid/Scramble shRNA Spheroid/DMSO
Spheroid/Smo shRNA Spheroid/Vis 10 μmol/L
40 40
Colonies per visual field
30 30
20 20
10 10
0 0
AGS MKN-45 N87 AGS MKN-45 N87
C AGS MKN-45 N87
40
Monolayer
Monolayer
30
Colonies per
Spheroid/Scramble shRNA
visual field
Figure 4. Gastric cancer spheroid cells demonstrate increased migration, colony formation, and anchorage-independent growth, which can be attenuated
by Hedgehog pathway inhibition. A, photos and graphs of migration assay of gastric cancer monolayer cells and spheroid cells treated with Smo shRNA
compared with control (Scramble shRNA) or vismodegib (vis; 10 mmol/L) compared with DMSO. B, colony formation assay of gastric cancer monolayer
cells and spheroid cells treated with Smo shRNA compared with control (Scramble shRNA) or vismodegib (10 mmol/L) compared with DMSO.
C, photos and graph of soft agar assay of gastric cancer monolayer cells and spheroid cells treated with Smo shRNA compared with control
(Scramble shRNA). Bars, SD.
3982 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
0
1 2 3 6 7 8 9 10 13 14 15 16 17
75
(% of Control)
shRNA shRNA
50
PBS
25
0
Scramble Scramble Smo shRNA Smo shRNA
Cisplatin
C 30 **
50 μm
Number of apoptotic cells
20
*
PBS
10
*
Cisplatin
0
Scramble Scramble Smo shRNA Smo shRNA
shRNA + shRNA + + PBS + cisplatin
PBS cisplatin
D 50 μm
100
*
CD44-positive cells
PBS
(% of Control)
75
50
*
Cisplatin
25
**
0
Scramble Scramble Smo shRNA Smo shRNA 20 μm
shRNA + shRNA + + PBS + cisplatin
PBS cisplatin
Figure 5. Cisplatin chemotherapy combined with Hedgehog pathway inhibition with Smo shRNA synergistically blocks growth of MKN-45 gastric cancer
xenografts. A, proliferation of MKN-45 cells transduced with Smo shRNA or control (Scramble shRNA), and tumor growth curves for MKN-45 xenografts
treatment with Scramble shRNA or Smo.shRNA and PBS or cisplatin. Immunohistochemical analysis of tumors for proliferation using PCNA IHC (B), apoptosis
using TUNEL immunofluorescence (C), and CD44 expression using CD44 IHC (D). Bars, SD. , P < 0.05 compared with control; , P < 0.05 compared with all
other groups.
increased from 4.7 to 10.9 cells per field with single-agent because only the subset of gastric and gastroesophageal
therapy to 20.9 cells per field with combined therapy junction cancers with high levels of CD44-expressing cells
(Supplementary Fig. S5C), and CD44-positive cells were would benefit from vismodegib. We thus performed CD44
reduced to only 23% of control tumors (Supplementary IHC on 97 available tumor samples from this study and
Fig. S5D). scored the CD44 levels (see Materials and Methods). Exam-
In a randomized phase II study of chemotherapy with or ples of tumors with low, intermediate, and high CD44
without vismodegib, 124 patients with advanced gastric or scores are shown in Fig. 6B. The gender and tumor char-
gastroesophageal junction adenocarcinoma were random- acteristics, radiologic response, follow-up, and survival of
ized to receive 5-fluorouracil, oxaliplatin, leucovorin (FOL- these patients are listed in Supplementary Table S1. The
FOX) chemotherapy with placebo or with vismodegib median CD44 score was 15.0 (SD, 63.6) and the range was 0
(Fig. 6A). There was no difference in response rate, progres- to 270. Two patients in the chemotherapy plus vismodegib
sion-free survival, or overall survival with the addition of group had a complete response, and these 2 patients had a
vismodegib to chemotherapy. We hypothesized that the median CD44 score of 187.5 compared with 26.4, 31.5, and
lack of benefit of vismodegib in this clinical trial was 42.0 for those with a partial response, stable disease, and
Advanced gastric
or GE junction Randomize
adenocarcinoma
1.0 1.0
Overall survival
0.0 0.0
0 10 20 30 40 50 0 10 20 30 40 50
Months Months
Figure 6. CD44 may be a response biomarker in patients with advanced gastric and gastroesophageal cancer treated with chemotherapy with or without
vismodegib. A, schema of the clinical trial. B, CD44 IHC of patient tumor samples showing low, intermediate, and high CD44 expression. C, Kaplan–
Meier overall survival curves for patients receiving chemotherapy alone and patients receiving chemotherapy plus vismodegib stratified by low
and high CD44 score.
3984 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
progressive disease, respectively (P ¼ 0.001). In the che- the gastric CSC marker CD44 as well as increased levels of
motherapy only group, patients with no progression of Hedgehog pathway proteins and certain self-renewal pro-
disease had significantly lower CD44 scores than patients teins. Inhibition of Hedgehog signaling using shRNA
with progression of disease, and patients who were alive at targeting Smo or pharmacologic Smo inhibition blocked
the end of the study period had significantly lower CD44 spheroid formation. CD44(þ) spheroid cells were highly
scores than those who had died by the end of the study resistant in vitro to 5-fluorouracil or cisplatin chemother-
period (Table 1). Conversely in the chemotherapy plus apy, and this chemotherapy resistance was reversed with
vismodegib group, patients with no progression of disease Hedgehog pathway inhibition. Cells grown as spheroids
tended to have higher CD44 scores, and patients who were had increased migration, invasion, colony formation, and
alive at the end of the study period had significantly higher anchorage-independent growth in soft agar, and all these
CD44 scores. Separate Kaplan–Meier curves were generat- phenotypes were attenuated following Hedgehog path-
ed for actuarial overall survival of patients receiving way inhibition. Hedgehog inhibition in tumor xenografts
chemotherapy alone and chemotherapy with vismodegib. acted synergistically with chemotherapy to block tumor
In the chemotherapy only group, estimated overall sur- growth, and histologic examination of treated tumors
vival was not significantly different in those with high found synergistic increases in tumor cell apoptosis and
CD44 score
Status N (%) (mean SD) P
All patients
No progression 27 (27.8) 35.7 56.6 0.473
Definite progression 70 (72.2) 46.1 66.3
Alive 24 (26.4) 44.4 56.8 0.804
Dead 67 (73.6) 40.7 64.4
Chemotherapy only
No progression 15 (29.4) 20.7 29.9 0.011
Definite progression 36 (70.6) 60.3 76.6
Alive 12 (25.0) 24.2 28.7 0.041
Dead 36 (75.0) 55.4 73.8
Chemotherapy plus vismodegib
No progression 12 (26.1) 54.6 75.8 0.233
Definite progression 34 (73.9) 31.2 50.1
Alive 12 (27.9) 64.6 70.9 0.032
Dead 31 (72.1) 23.6 47.0
genetically engineered mouse models of tumors com- locally advanced basal cell carcinoma, vismodegib led to
bined with fluorescent markers to genetically trace CSCs a partial response in 22% of patients and a complete
have been performed. Chen and colleagues demonstrated response in 21% of patients (31). One lesson learned in
that a quiescent subset of endogenous glioma cells was our study is that for any given cancer, the Hedgehog
responsible for tumor regrowth after chemotherapy (21). pathway may promote chemotherapy resistance in only
Schepers and colleagues found that for intestinal adeno- a subset of patients and that a biomarker may be needed
mas, the crypt stem cell marker Lgr5 marks a subpopu- to identify this subset. The clinical trial described in this
lation of 5% to 10% of adenoma cells that can generate study of chemotherapy with or without vismodegib for
additional Lgr5(þ) cells as well as all other adenoma cell patients with advanced gastric or gastroesophageal cancer
types (22). There are certainly subsets of cancer cells was a negative study in that vismodegib did not increase
within heterogeneous solid tumors with varied proper- progression-free or overall survival for the group as a
ties. In this study, we demonstrate one such subpopula- whole. However, the minority of patients with tumor
tion of CD44(þ) cells, which have the CSC phenotypes of having high CD44 expression seemed to benefit from the
spheroid formation, formation of colonies from single addition of vismodegib. This association between high
cells, and chemotherapy resistance. However, given the CD44 expression and response to vismodegib needs to be
3986 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research
Hedgehog Signaling in CD44(þ) Gastric Cancer Cells
beneficial in a minority of patients with gastric cancer Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): H.-J. Lee, S.S. Yoon
whose tumors express high levels of CD44. Study supervision: S.S. Yoon
Conception and design of clinical trial portion of the study: D.J. Cohen
Disclosure of Potential Conflicts of Interest
Y. Y. Janjigian reports receiving commercial research grants from Amgen, Acknowledgments
Boehringer Ingelheim, and Genentech and is a consultant/advisory board The authors thank Sanghoon Oh at the Molecular Cytology Core Facility
member for Eli Lilly. No potential conflicts of interest were disclosed by the of Memorial Sloan-Kettering Cancer Center for image acquisition and
other authors. analysis.
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