Download STAT3 Is Necessary for Proliferation and Survival in Colon Cancer

Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Cancer
Research
Tumor and Stem Cell Biology
STAT3 Is Necessary for Proliferation and Survival in Colon
Cancer–Initiating Cells
Li Lin1,6, Aiguo Liu1,7, Zhengang Peng5, Huey-Jen Lin2,5, Pui-Kai Li3, Chenglong Li3, and Jiayuh Lin1,4
Abstract
STAT3 is constitutively activated in colon cancer but its contributions in cancer-initiating cells have not been
explored. In this study, we characterized STAT3 in aldehyde dehydrogenase (ALDH)-positive (ALDHþ) and
CD133-positive (CD133þ) subpopulations of human colon tumor cells that exhibited more potent tumorinitiating ability than ALDH/CD133 cells in tumor xenograft assays in mice. We found that ALDHþ/CD133þ
cells expressed higher levels of the active phosphorylated form of STAT3 than either ALDH/CD133 or
unfractionated colon cancer cells. STAT3 inhibition by RNA interference–mediated knockdown or smallmolecule inhibitors LLL12 or Stattic blocked downstream target gene expression, cell viability, and tumorsphere-forming capacity in cancer-initiating cells. Similarly, treatment of mouse tumor xenografts with STAT3
short hairpin RNA (shRNA), interleukin 6 shRNA, or LLL12 inhibited tumor growth. Our results establish that
STAT3 is constitutively activated in colon cancer–initiating cells and that these cells are sensitive to STAT3
inhibition. These findings establish a powerful rationale to develop STAT3 inhibitory strategies for treating
advanced colorectal cancers. Cancer Res; 71(23); 1–12. 2011 AACR.
Introduction
Colorectal cancer is a tumor caused by abnormal division of
the cells lining the large intestine. According to the American
Cancer Society, there were an estimated 102,900 new cases and
51,370 deaths due to colorectal cancer in the United States in
2010. As such, there is a need for better treatment approaches
for colorectal cancer. The cellular mechanisms contributing to
colorectal cancer are still not well understood but involve
signaling protein dysregulation which includes the constitutive
activation of STAT3 (1–3). The constitutive activation of
STAT3 is frequently detected in primary human colorectal
carcinoma cells and established human colorectal cancer cell
Authors' Affiliations: 1Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Department of Pediatrics, College
of Medicine; 2Molecular Biology and Cancer Genetics Program, 3Division of
Medicinal Chemistry and Pharmacognosy, College of Pharmacy; 4Experimental Therapeutics Program, 5Medical Technology Division, School of
Allied Medical Professions, The Ohio State University Comprehensive
Cancer Center, The Ohio State University, Columbus, Ohio; and 6Division
of Cardiology, Department of Internal Medicine, 7Department of Pediatrics,
Tongji Hospital, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan, Hubei, PR China
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Jiayuh Lin, Center for Childhood Cancer, The
Research Institute at Nationwide Children's Hospital, Department of Pediatrics, College of Medicine, The Ohio State University, 700 Children's Drive,
Columbus, OH 43205. Phone: 614-722-5086; Fax: 614-722-5895; E-mail:
[email protected]; and Li Lin, Division of Cardiology, Department of Internal
Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of
Science and Technology, 1095 Jiefang Ave.,Wuhan 430030, China; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-10-4660
2011 American Association for Cancer Research.
lines (1–3), and elevated levels of STAT3 phosphorylation were
correlated with the tumor invasion, nodal metastasis, and the
stage (P < 0.05; refs. 1, 3). Constitutive STAT3 activation in
colorectal cancer cells is associated with invasion, survival, and
growth of colorectal cancer cells and colorectal tumor model in
mice in vivo (2, 4–6). These reports indicate that STAT3 is one
of the major oncogenic pathways activated in colorectal cancer
and can serve as an attractive therapeutic target for colorectal
carcinoma. To date, however, whether STAT3 is activated in
colorectal cancer stem cells is unknown.
The concept of the cancer stem cells or cancer-initiating
cells holds that only a minority of cells within a tumor have the
ability to generate a new tumor. Cancer stem cells were
reported to show pluripotency and self-renewal (7). Cancer
stem cells were first identified in leukemias and more recently
in solid tumors. Increasing evidence suggests that the cancer
stem cells concept is also relevant to colorectal cancer (8).
CD133, a transmembrane protein (Prominin-1 or AC133) was
used to isolate stem cells from a host of other normal and
cancerous tissues, including colorectal cancer. However, the
specificity of CD133 alone as a marker for colonic stem cells is
uncertain (9–11).
A promising new marker for cancer stem cells is aldehyde
dehydrogenase 1 (ALDH1). ALDH is a detoxifying enzyme that
oxidizes intracellular aldehydes and thereby confers resistance
to alkylating agents (12). Corti and colleagues (13) showed that
ALDHþ cells isolated from murine brain were capable of selfrenewal and of differentiating into multiple lineages. Further
studies showed that ALDH1 is a specific marker for breast
cancer stem cells (14, 15). ALDH was also investigated as a
specific marker for identifying and isolating normal and malignant human colonic stem cells and as a way to quantify the
number of stem cells over the course of colon cancer
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF1
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
development (16). Xenograft tumors were successfully generated using ALDHþ cells from 7 primary colon cancer cells and
ALDH cells did not generate tumor xenografts (16). When
using ALDH and CD133 together to form tumor xenografts,
ALDHþ/CD133þ cells showed an increased ability to generate
tumor xenografts compared with ALDHþ/CD133 or ALDHþ
alone (16). Taken together, these data suggest that ALDH is a
better marker than CD133 for colorectal cancer stem cells.
However, using both ALDH and CD133 seem to be better than
to enrich the cancer stem cell population using ALDH or CD133
alone.
This study extends that work by using both ALDH and CD133
together as markers for cancer-initiating cells or colorectal
stem cells and examines the STAT3 phosphorylation and interleukin 6 (IL)-6 expression in these cancer-initiating cells. Our
results showed that colorectal cancer–initiating cells, characterized by ALDHþ/CD133þ subpopulation of colorectal cancer
cells expressing higher levels of STAT3 phosphorylation
and IL-6, compared with unseparated and ALDH/CD133
subpopulations. These results suggest that STAT3 is a novel
therapeutic target in colorectal cancer–initiating cells.
Materials and Methods
Colorectal cancer cell lines
Human colorectal cancer cell lines (SW480, HCT116, DLD-1,
and HT29) were purchased from the American Type Culture
Collection (ATCC) and maintained in Dulbecco's Modified
Eagle Medium supplemented with 10% FBS (Invitrogen). These
cancer cell lines have been routinely tested and authenticated
by the ATCC and Asterand, respectively. The known genotype
relative to adenomatous polyposis coli, beta-catenin, and DNA
mismatch repair enzymes (MLH1, MSH2) genes were shown in
Supplementary Table S1. ALDHþ/CD133þ cancer-initiating
cells were grown in a serum-free mammary epithelial basal
medium (MEBM; Clonetics division of Cambrex BioScience),
supplemented with B27 (Invitrogen), 20 ng/mL EGF (BD
Biosciences), 4 mg/mL Gentamycin (Invitrogen), 1 ng/mL
Hydrocortisone (Sigma-Aldrich), 5 mg/mL Insulin, and 100
mmol/L beta-mercaptoethanol (Sigma-Aldrich).
STAT3 inhibitors, LLL12, and Stattic
The laboratory of Dr. Pui-Kai Li's at the Ohio State University
College of Pharmacy synthesized small-molecule LLL12 that
selectively targets STAT3 (17). Stattic, a previously reported
STAT3 inhibitor (18), was purchased from Calbiochem.
Isolation of colon cancer–initiating cells
The population with high ALDH enzymatic activity was
isolated by using the ALDEFLUOR Kit (StemCell Technologies)
as previously described (14). Briefly, cells were trypsinized to
single cells and subsequently suspended in ALDEFLUOR assay
buffer containing ALDH substrate and then incubated for 40
minutes at 37 C. In all experiments, the ALDEFLUOR-stained
cells treated with diethylaminobenzaldehyde, a specific ALDH
inhibitor, served as ALDH-negative controls. Anti-human
PE-CD133 antibody were purchased from Miltenyi Biotec.
ALDHþ/CD133þ and ALDH/CD133 subpopulations were
OF2
Cancer Res; 71(23) December 1, 2011
separated from SW480, HCT116, DLD-1, and HT29 colon
cancer cells by a fluorescence-activated cell sorting Wantage
SE (Becton Dickinson) Flow Cytometry. After sorting, ALDHþ/
CD133þ cells were cultured in serum-free stem cell medium
(MEBM) to maintain cancer stem cell characteristics. ALDH/
CD133 and unseperated cells were cultured in regular medium and replaced with stem cell medium (MEBM) for 3 days
before harvesting.
Tissue microarray slides, immunohistochemistry, and
immunofluorescence staining
Human colon cancer tissue microarray slides were obtained
from the Biochain Institute, Inc. and AccuMax ISU ABXIS Co.
containing 109 colon cancer cases. After baking and deparaffinization, the slides were boiled in a pressure cooker
filled with 10 mmol/L sodium citrate (pH 6.0) and then subjected to immunohistochemistry or immunofluorescence
staining. Phospho-STAT3 (Tyr705) antibody (1:25; Signaling
Technology) and or ALDH1 (1:100; BD Pharmingen), CD133
(1:50; Miltenyi Biotec) were used. Alexa Fluor 488–conjugated
anti-rabbit IgG and Alexa Fluor 594–conjugated anti-mouse
IgG (Cell Signaling Technology) and the Histostain-Plus Kits
(Invitrogen) were used in immunofluorescence or immunohistochemistry staining as described by the manufacturer.
Immunostained slides were scored under microscope by using
the criteria of percentage and intensity positive as described
previously by Ginestier (14). Significance of correlation
between phospho-STAT3 and ALDH1 or CD133 was determined, respectively, using 2-sided Pearson x2 test. P < 0.05 was
considered as statistical significance.
Cell viability assay
Colon cancer–initiating cells maintained serum-free MEBM
(3,000 per well in 96-well plates) were incubated with desired
concentrations of compounds in triplicate at 37 C for 72 hours.
MTT viability assay was done according to manufacturer's
protocol (Roche Diagnostics). The absorbance was read at
595 nm.
Western blot analysis
Cells were lysed in cold radioimmunoprecipitation assay
lysis buffer containing protease inhibitors and subjected to
SDS-PAGE. Proteins were transferred on to polyvinylidene
difluoride membrane and were probed with a 1:1,000 dilution
of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The degree of changes in P-STAT3 was determined using
densitometry and normalized to GAPDH.
Reverse transcriptase PCR
Total cell RNA was collected from cells by using RNeasy Kits
(Qiagen). cDNA was generated from 500 ng sample RNA using
Omniscript RT (Qiagen). Two microliter of cDNA was subsequently used for PCR using Taq PCR Master Mix Kit (Qiagen)
according to the manufacturer's instruction. Primer sequences
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 in Colorectal Cancer–Initiating Cells
and source information can be found in Supplementary
Table S2.
Tumorsphere culture
The ALDHþ/CD133þ cells was plated as single cells in ultralow attachment 6-well plates (Corning) and plated at a density of
25,000 viable cells per well. Cells were grown in a serum-free
MEBM as described above in a humidified incubator (5% CO2) at
37 C. At the second day after seeding, the ALDHþ/CD133þ cells
were treated with 2.5 to 5 mmol/L of LLL12 or 5 to 10 mmol/L of
Stattic. Tumorspheres were observed under microscope 15 days
later.
IL-6 ELISA assay
After sorting, ALDHþ/CD133þ and ALDH/CD133 cells
were cultured in 96-well plates at a density of 12,000 viable cells
per well. Twenty-four hours later, the medium was collected
and the IL-6 concentrations were detected by using the Human
IL-6 ELISA Development Kit (Peprotech) as described by the
manufacturer.
Lentiviral infections
Lentivirus short hairpin RNA (shRNA) that specifically targets human STAT3 (19) and control lentivirus that expresses
green fluorescent protein (GFP) were provided by Dr. Antonio
Iavarone at the Columbia University. IL-6 shRNA lentivirus was
purchased from Santa Cruz Biotechnology, Inc. STAT3, IL-6, or
control GFP shRNA lentivirus (CTL shRNA) was introduced
into SW480 and HCT116 colon cancer–initiating cells for 48
hours, followed by selection with puromycin (0.2 mg/mL) for 72
hours. Western blot assay was used to detect the expression of
P-STAT3 and STAT3 in colon cancer–initiating cells. MTT cell
viability and reverse transcriptase PCR (RT-PCR) assay were
conducted and in vivo cancer-initiating cell growth was
determined.
Mouse xenograft tumor model
All animal studies were conducted in accordance with the
principles and standard procedures approved by IACUC at the
Research Institute at Nationwide Children's Hospital. For
tumor-initiation study, the ALDHþ/CD133þ or ALDH/
CD133 cells (1 102, 1 103, or 1 104) from SW480,
HCT116, DLD-1, and HT29 were mixed with 50% Matrigel
(Invitrogen) in a total of 100 mL and were injected subcutaneously into the right flank area of 4- to 5-week-old female
nonobese diabetic/severe combined immunodeficiency
(NOD/SCID) mice, which were purchased from Jackson Laboratory. The tumor incidence ALDHþ/CD133þ and ALDH/
CD133 cells are the numbers of tumor detected/numbers of
mice inoculated and were determined 50 days after the inoculation of cells in mice.
For shRNA lentivirus study, after sorting ALDHþ/CD133þ
HCT116, colon cancer–initiating cells (1 105) were infected
with STAT3, IL-6, or GFP shRNA lentivirus (CTL shRNA) for 48
hours. After 72 hours of selection with puromycin, cells were
mixed with an equal volume of Matrigel and injected subcutaneously into the flanks area of 4- to 5-week-old female NOD/
SCID mice. Tumor growth was determined by measured the
www.aacrjournals.org
length (L) and width (W) of the tumor every week with a caliper
and tumor volume was calculated on the basis of the following
formula: volume ¼ (p/6) LW2.
To detect the effects of STAT3 inhibitor LLL12 in vivo,
ALDHþ/CD133þ SW480 and HCT116 colon cancer-initiating
cells (1 105) were mixed with 50% Matrigel (Invitrogen) in a
total of 100 mL and were injected subcutaneously into the flank
area of female NOD/SCID mice. After 17 and 19 days, SW480
and HCT116 mice were divided into 2 treatment groups
consisting of 5 and 6 mice per group, respectively: (a) dimethyl
sulfoxide (DMSO) control vehicle and (b) 5 mg/kg of LLL12
(dissolved in 10% DMSO, 18% Cremophor EL and 72% sterile
5% dextrose). Tumor growth and body weights of mice were
measured every other day during 14 days period treatments. At
the end of treatments, tumors were harvested from euthanized
mice. A portion of tumor tissues was snap-frozen in liquid
nitrogen and stored in 80 C to examine the expression of
STAT3 phosphorylation by Western blot. The rest of tumors
tissues were dissociated mechanically and enzymatically to
obtain a single-cell suspension. The single-cell suspension was
used for ALDEFLUOR and CD133-PE staining and followed
flow cytometry assay as previously described (20).
Results
LLL12 is a potent agent to inhibit the STAT3
phosphorylation in colorectal cancer cells
STAT3 is frequently activated in many types of human solid
and blood cancers and contributes to progression of those
cancers (21, 22). The STAT3 pathway is also frequently constitutively activated in colorectal cancer and is considered
to play an important role in colorectal cancer carcinogenesis
(1–6). To confirm the important role of STAT3 in colon cancer
cells, the novel STAT3 inhibitor LLL12 (17) was used to target
STAT3 in 3 independent colon cancer cell lines using phosphospecific STAT3 antibody. Our results showed that LLL12
significantly inhibited STAT3 phosphorylation at tyrosine residue 705 (Y705, P-STAT3) in SW480, HCT116, and DLD-1
human colon cancer cell lines (Supplementary Fig. S1). Phosphorylation at Y705 is important to activate STAT3 (23–25).
The inhibition of P-STAT3 by LLL12 is consistent with the
decrease of STAT3 downstream target genes and the induction
of apoptosis, as evidenced by the cleavages of caspase-3
(Supplementary Fig. S1).
ALDHþ/CD133þ cells exhibit more potent tumorinitiating ability than ALDH/CD133 cells in mouse
tumor xenografts
It has been shown that ALDHþ and CD133þ subpopulations
in colorectal cancer cells exhibit colorectal cancer–initiating
cells properties in vitro and in vivo (16). To verify whether
ALDHþ/CD133þ cells contain tumor-initiating ability, we separated ALDHþ/CD133þ and ALDH/CD133 subpopulations
from SW480, HCT116, DLD-1, and HT29 colorectal cancer cells
(Supplementary Fig. S2A). The percentages of ALDHþ/CD133þ
in the 4 colon cancer cell lines were shown in Supplementary
Fig. S2B. Our results showed that although 5 to 8 of 7 to 8 mice
of 102 to 104 ALDHþ/CD133þ cells injected formed tumors,
Cancer Res; 71(23) December 1, 2011
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF3
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
only 1 to 2 of 7 to 8 mice of 103 ALDH/CD133 cells form
tumor, and none of the 102 ALDH/CD133 cells injected
formed tumor (Table 1). The volume of tumor formed by
ALDHþ/CD133þ are also larger than tumors formed by
ALDH/CD133 cells (Supplementary Fig. S3). Taking together, these results suggest that using both ALDHþ and CD133þ
markers can enrich colorectal cancer–initiating cells. These
results increase our confidence to isolate colorectal cancer–
initiating cells using both markers to examine the STAT3
phosphorylation.
there were 64.22% samples which had the similar expression,
much more than the samples with different expression of these
3 proteins (P < 0.05; Table 2). So there was a significant
association between P-STAT3, ALDH1 and CD133. The representative examples of immunohistochemistry/immunofluorescence staining of P-STAT3 and ALDH1/CD133 are shown
in Fig. 1B and C. These results from human colon cancer tissue
further showed that the elevated levels of P-STAT3 is expressed
in colon cancer–initiating cells. These results indicated that
constitutive STAT3 signaling may be a novel therapeutic target
in colorectal cancer–initiating cells.
The ALDHþ/CD133þ subpopulation of colorectal cancer
cells express higher levels of STAT3 phosphorylation
compared with the ALDH/CD133 subpopulation
The STAT3 phosphorylation of ALDHþ/CD133þ and
ALDH/CD133 subpopulations in SW480, HCT116, DLD-1,
and HT29 colorectal cancer cell lines were examined. Interestingly, our results showed that the ALDHþ/CD133þ subpopulation of SW480, HCT116, DLD-1 (Fig. 1A), and HT29 (Supplementary Fig. S4A) colorectal cancer cells expressed higher
levels of P-STAT3 compared with unseparated cells and the
ALDH/CD133 subpopulation. ERK1/2 phosphorylation
(Threonine 202/Tyrosine 204) was not at consistently high
levels in the ALDHþ/CD133þ cells in all 4 colon cancer cell lines
(Fig. 1A; Supplementary Fig. S4A). These results suggested that
STAT3 pathway seems to serve a more important purpose in
colorectal cancer–initiating cells, but ERK may not play a key
role in colorectal cancer–initiating cells, at least in these 4
colorectal cancer cell lines. These results show that colorectal
cancer–initiating cells in the ALDHþ/CD133þ subpopulation
expresses phosphorylated or activated STAT3.
To further investigate the STAT3 activation in clinical colon
cancer samples, P-STAT3 and ALDH1, CD133 protein expression in human colon cancer tissues were also examined using
tissue microarray slides. We observed that there was a significant association (P < 0.01) between staining of P-STAT3 and
staining of ALDH1 (Table 2). In addition, the tumor samples
expressed elevated levels of phosphorylated STAT3 also associated with CD133 (P < 0.01; Table 2). In the 109 cases, there
were 32 samples (29.36%) in which P-STAT3 (Y705), ALDH1,
and CD133 were all positive, and 38 samples (34.86%) with
negative P-STAT3 (Y705), ALDH1, and CD133. Taken together,
STAT3 inhibitors, LLL12 and Stattic, inhibited STAT3
phosphorylation, and STAT3 downstream targets in
ALDHþ/CD133þ cells
To confirm the important role of STAT3 in colon cancer–
initiating cells, we next examined the effect of STAT3 inhibitors, LLL12 and Stattic in SW480, HCT116, DLD-1, and HT29
colorectal cancer–initiating cells. The results showed that
LLL12 and Stattic inhibited P-STAT3 (Y705) but not ERK1/2
phosphorylation (Fig. 2A and B and Supplementary Fig. S4B) in
ALDHþ/CD133þ subpopulation of colorectal cancer cell lines.
In Fig. 2B, Stattic seems to decrease STAT3 expression, which
could explain, in part, the observed decrease in level of
P-STAT3. In addition, STAT3 shRNA also inhibited P-STAT3
in ALDHþ/CD133þ subpopulation of colorectal cancer cell
lines compared with shRNA control (shRNA CTL; Fig. 2C).
The inhibition of STAT3 by LLL12 also downregulates the
expression of many known or putative STAT3-regulated
genes in colorectal cancer–initiating cells such as cyclin
D1 (26), survivin (27), Bcl-2, Bcl-XL (26), Notch1, and Notch3
(28, 29; Fig. 2D-a, Supplementary Fig. S4C). These genes are
related to cancer cell proliferation, survival, and angiogenesis. Moreover, the Notch pathway was reported to be
involved in self-renewal of human cancer stem cells and
tumorigenicity (28, 30). These results indicate that the LLL12
is also potent in terms of inhibiting P-STAT3, downregulating STAT3 downstream genes, and induces apoptosis in
these colorectal cancer–initiating cells. Furthermore, the
expression of STAT3 downstream genes, such as cyclin
D1, survivin, Bcl-XL, Notch1, and Notch3 were also reduced
by STAT3 shRNA (Fig. 2D-b).
Table 1. The tumor-initiating ability (tumor incidence: the numbers of tumor detected/numbers of mice
inoculated) of ALDHþ/CD133þ and ALDH/CD133 cells in NOD/SCID mice for 50 days
Cell numbers inoculated
HCT116
SW480
HT29
DLD-1
OF4
ALDHþ/CD133þ
ALDH/CD133
ALDHþ/CD133þ
ALDH/CD133
ALDHþ/CD133þ
ALDH/CD133
ALDHþ/CD133þ
ALDH/CD133
Cancer Res; 71(23) December 1, 2011
1 104
1 103
1 102
7/7
3/7
7/7
5/7
8/8
6/8
8/8
5/8
6/7
2/7
6/7
2/7
8/8
2/8
7/7
1/7
5/7
0/7
4/7
0/7
5/8
0/8
5/7
0/7
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 in Colorectal Cancer–Initiating Cells
A
Unseparated
ALDH+/
CD133+
ALDH+/
ALDH–/
Unseparated
CD133+
CD133–
ALDH–/
CD133–
Unseparated
ALDH+/
CD133+
ALDH–/
CD133–
P-STAT3
(T705)
STAT3
P-ERK1/2
(T202/Y204)
Figure 1. STAT3 phosphorylation
þ
þ
of ALDH /CD133 subpopulation
of colon cancer cells is higher
than unseparated and the
ALDH/CD133 subpopulations.
A, ALDHþ/CD133þ and
ALDH/CD133 subpopulations
were separated from SW480,
HCT116, and DLD-1 colon cancer
cells by flow cytometry.
Phosphorylation of STAT3 (Y705),
ERK 1/2 (T202/Y204), phosphoindependent STAT3, and GAPDH
were detected by Western blot. B,
representative examples of the
expression of P-STAT3, ALDH1,
and CD133 were shown by
immunohistochemistry (IHC) using
colon cancer tissue microarray
slides. Negative/weak staining of
P-STAT3 (Y705)/ALDH1/CD133 (a)
and positive staining of P-STAT3
(Y705)/ALDH1/CD133 (b and c)
tumor tissues were shown. The
spots for P-STAT3 (Y705), ALDH1
and CD133 were from the matched
tissues section from the same
patient. The negative controls are
stained with no antibody. C,colon
cancer tissue microarray slides
were double-stained with P-STAT3
(Y705) and ALDH1 using
immunofluorescence (IF). ALDH1
high expression tumor cells
(cytoplasm, green) also expressed
phosphylated-STAT3 in nuclei
(red). Scale bar: 10 mm.
GAPDH
HCT116
SW480
Fold
0.699
changes 0.819
B
1
1
0.026
1.150
P-STAT3
ALDH1
1
1
0.594
0.922
CD133
0.207
0.954
1
1
0.044
1.091
P-STAT3
STAT3
Negative control
a
b
c
C
LLL12 and Stattic inhibit cell viability and tumorsphereforming capacity of ALDHþ/CD133þ subpopulation of
colorectal cancer cells
We next examined the inhibitory effects of LLL12, Stattic,
and STAT3 shRNA on cell viability in colorectal cancer–initiating cells. Our results showed that LLL12, Stattic, and STAT3
shRNA could inhibit cell viability of the ALDHþ/CD133þ cells
from SW480, HCT116, DLD-1 (Fig. 3A–C), and HT29 (Supplementary Fig. S4D) colorectal cancer cells, further supporting
www.aacrjournals.org
0.363
1.056
DLD-1
the idea that colorectal cancer–initiating cells are sensitive to
the inhibition of STAT3. LLL12 and Stattic also inhibited the
cell viability of ALDH/CD133 subpopulation (Supplementary Fig. S5A and B) and the bulk tumor cells (Supplementary
Fig. 5C and D). These results may be explained by at least 2
possibilities: Some (HCT116) or low (SW480 and DLD-1) levels
of P-STAT3 are expressed in ALDH/CD133 cells; Stattic and
LLL12 may also inhibit both P-STAT3 (Tyr705) and unphosphorylated STAT3 or nonnuclear function of STAT3 (31). The
Cancer Res; 71(23) December 1, 2011
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF5
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
Table 2. The association of P-STAT3 (Y705) with the expression of ALDH1 and CD133 in colon carcinoma
Colon
carcinoma
Total numbers
of cancer tissue Positive, n (%) Negative, n (%)
x2
Association with P-STAT3
Pa
Both positive, n (%) Both negative, n (%)
P-STAT3
ALDH1
CD133
ALDH1/CD133
109
109
109
109
60 (55.05)
54 (48.54)
45 (41.28)
35 (32.11)
49 (44.95)
55 (50.45)
64 (59.6)
45 (41.28)
45 (41.28)
40 (36.70)
32 (29.36)
40 (36.70)
44 (40.37)
38 (34.86)
34.606 <0.01
35.473 <0.01
10.771 <0.05
a
The correlation of P-STAT3 (Y705) with ALDH1or/and CD133 (both positive or negative) was assessed by x2 test. P < 0.05 is
considered as statistically significant. Colon cancer tissues from a total numbers of 109 cancer patients were examined.
inhibition of unphosphorylated STAT3 or nonnuclear function
of STAT3 in ALDH/CD133 cells might contribute to the
reduction of cell viability.
Furthermore, we examined the efficacy of LLL12 and Stattic
in inhibiting colorectal cancer–initiating cells to survive and
proliferate in anchorage-independent conditions and ability to
form tumorspheres. Our results showed that LLL12 and Stattic
can inhibit tumorsphere-forming capacity in the ALDHþ/
CD133þ cells of SW480, HCT116, DLD-1 (Fig. 3D), and HT29
(Supplementary Fig. S4E) colorectal cancer cells. The effects of
LLL12 are more potent than Stattic. These results indicate that
LLL12 is an excellent drug candidate for targeting colorectal
cancer–initiating cells for inhibiting phosphorylated or activated STAT3 in human colorectal cell lines.
STAT3 inhibitors, LLL12 and Stattic, depleted ALDHþ/
CD133þ subpopulation and the expression of ALDH1,
CD133 in colon cancer cells
Colon cancer stem cells are resistant to current chemotherapy and radiation regimens available (8). To examine whether
STAT3 inhibition might eliminate the ALDHþ/CD133þ subpopulation, we treated cancer cells with 5 mmol/L of LLL12, 10
mmol/L of Stattic, 10 mmol/L of doxorubicin, and 10 mmol/L of
5-Fu for 24 hours, and sorted for the percentage of ALDHþ/
CD133þ subpopulation by flow cytometry. Our results showed
that LLL12 could decrease the ALDHþ/CD133þ subpopulation
in HCT116 and SW480 colon cancer cells (Fig. 4A; Supplementary Fig. S6A), suggesting that this subpopulation of colon
cancer–initiating cells is sensitive to STAT3-mediated inhibition. We found that 10 mmol/L of Stattic also decreased the
percentage of ALDHþ/CD133þ subpopulation (Fig. 4A; Supplementary Fig. S6A). However, 10 mmol/L of doxorubicin or
5-Fu increased the percentage of ALDHþ/CD133þ colorectal
cancer–initiating cells (P < 0.05; Fig. 4A), which might indicate
that colon cancer–initiating cells are resistant to chemotherapy. We also detected the effects of Dox or 5-Fu (10 mmol/L) in
STAT3 activation in the bulk tumor cell population (Supplementary Fig. S6B). The results showed different responses to
both drugs on P-STAT3 in 2 independent colon cancer cell lines
(SW480 and HCT116).
To further investigate the role of IL-6/STAT3 pathway in
ALDHþ/CD133þ colorectal cancer–initiating cells, ALDHþ/
CD133þ and ALDH/CD133 colon cancer cells were collected
OF6
Cancer Res; 71(23) December 1, 2011
after sorting. ELISA assay showed that ALDHþ/CD133þ cells
secreted higher levels of IL-6 than ALDH/CD133 cells (Fig.
4B). Interestingly, the expression of IL-6, IL-6R, GP130, and IL-8
were higher in ALDHþ/CD133þ cells than ALDH/CD133
cells (Fig. 4C-a) as detected by RT-PCR assay. We also examined the ALDH1 and CD133 expression after the LLL12 treatment. We found that the expression of ALDH1 and CD133 was
lower after treatment with LLL12 (Fig. 4C-b). In addition,
LLL12 inhibited the expression of IL-6, GP130, and IL-8 (Fig.
4C-b). However, the expression of IL-6R was not changed
consistently in all 4 cell lines. Our data also observed that
IL-6 (25–50 ng/mL) induced the expression of IL-8 in SW480
and HCT116 colon cancer cells, which could be blocked by
LLL12 (Supplementary Fig. S6C). The results confirm that the
IL-6/STAT3 pathway plays a central role in the maintenance of
the ALDHþ/CD133þ subpopulation in colon cancer cells.
IL-6 shRNA decreased STAT3 phosphorylation of
colorectal cancer–initiating cells in vitro and inhibited
cancer-initiating cell growth in vivo
To determine whether the increased levels of phosphorylated or activated STAT3 in ALDHþ/CD133þ cells is dependent
on upstream ligand, IL6, we treated ALDHþ/CD133þ cells with
lentiviral IL-6 shRNA versus control lentivirus without encoding IL-6 shRNA. Our results in Fig. 4D-a shows that lentiviral IL6 shRNA, but not control lentivirus, downregulated the phosphorylated STAT3. These results provide evidence that elevated levels of phosphorylated STAT3 in ALDHþ/CD133þ cells is
IL-6–dependent and the inhibition of IL-6 downregulates
phosphorylated STAT3.
To further show the tumor dependence on STAT3 and its
upstream activation (IL-6), we used shRNA that specifically
knock down STAT3 and its upstream signaling protein, IL-6.
Our results in Fig. 4D-b shows that STAT3 and IL-6 shRNA
significantly suppressed colon cancer stem cell tumor growth
compared with lentivirus GFP (as a control). These data
supported tumor dependence on STAT3 and its upstream
activation by IL-6.
LLL12 suppresses tumor growth of colorectal cancer–
initiating cells in mouse tumor model in vivo
We have shown that LLL12 inhibited P-STAT3, cell viability,
and tumorsphere growth in colorectal cancer–initiating cells
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 in Colorectal Cancer–Initiating Cells
A
DMSO
ALDH+/CD133+
LLL12
DMSO 5 µmol/L
LLL12
5 µmol/L
DMSO
LLL12
5 µmol/L
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
SW480
Figure 2. STAT3 inhibitors LLL12,
Stattic, and STAT3 shRNA inhibited
STAT3 phosphorylation and STAT3
downstream genes expression in
þ
þ
ALDH /CD133 colon cancer–
initiating cells. The ALDHþ/CD133þ
subpopulation was separated from
SW480, HCT116, and DLD-1 colon
cancer cells by flow cytometry, and
cultured in serum-free stem cell
medium (MEBM) to maintain cancer
stem cell characteristics. A and B,
ALDHþ/CD133þ colon cancer–
initiating cells were treated with
DMSO, LLL12 (5 mmol/L) or Stattic
(10–20 mmol/L) for 24 to 48 hours. C,
STAT3 or control GFP shRNA
lentivirus (CTL shRNA) was
introduced into colon cancer–
initiating cells for 48 hours, followed
by selection with puromycin for 72
hours. Phosphorylation of STAT3
and ERK1/2, STAT3 protein, and
GAPDH in colon cancer–initiating
cells were detected by Western blot
as described in Materials and
Methods. D, ALDHþ/CD133þ
subpopulation of SW480, HCT116,
and DLD-1 colon cancer cells
were treated with DMSO, LLL12
(5 mmol/L), CTL, or STAT3 shRNA.
Reverse-transcriptase PCR reveals
decreased expression of STAT3
downstream target genes in LLL12
or STAT3 shRNA treated cells as
compared with control.
Fold
changes
B
1
1
DMSO
0.043
1.073
Stattic
10 µmol/L
DMSO
DLD-1
1
0.043
1
1.022
Stattic
10 µmol/L
DMSO
P-STAT3
STAT3
Stattic
20 µmol/L
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
SW480
1
0.554
1
0.725
Fold
changes
C
CTL
shRNA
1
1
STAT3
shRNA
HCT166
0.603
0.572
CTL
shRNA
DLD-1
1
1
0.012
0.375
P-STAT3
STAT3
STAT3
shRNA
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
SW480
HCT166
Fold
changes
1
1
0.054
0.293
1
1
P-STAT3
STAT3
0.112
0.447
D
a
SW480
ALDH+/CD133+
5 µmol/L
DMSO LLL12
HCT116
ALDH+/CD133+
5 µmol/L
DMSO LLL12
expressing elevated levels of STAT3 phosphorylation in vitro.
We further tested LLL12 against colorectal cancer–initiating
cells isolated from the SW480 and HCT116 colon cancer cells in
NOD/SCID mice xenograft model in vivo. The results showed
that LLL12 significantly suppresses (P < 0.01) the tumor growth
(Fig. 5A-a) and tumor mass (Fig. 5A-b). The average reduction
in tumor weight is 49.67% to 61.89% in LLL12-treated mice
www.aacrjournals.org
HCT166
1
0.024
1
0.825
b
DLD-1
ALDH+/CD133+
5 µmol/L
DMSO LLL12
SW480
HCT116
ALDH+/CD133+ ALDH+/CD133+
CTL STAT3
shRNA shRNA
CTL STAT3
shRNA shRNA
Cyclin D1
Cyclin D1
Survivin
Survivin
Bcl-2
Bcl-2
Bcl-XL
Bcl-XL
Notch1
Notch1
Notch3
Notch3
GAPDH
GAPDH
compared with DMSO vehicle in xenograft mouse model
(Fig. 5B). LLL12 also inhibited STAT3 but not ERK1/2 phosphorylation of SW480 and HCT116 colon cancer–initiating
cells (Fig. 5C). We also used flow cytometry to determine the
percentage of ALDH1þ/CD133þ subpopulation in the tumors
treated with vehicle or LLL12. Our results in Fig. 5D-a, b
showed that LLL12 reduced the percentage of ALDHþ/CD133þ
Cancer Res; 71(23) December 1, 2011
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF7
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
Fold changes in cell viability
A
HCT116
SW480
120
ALDH+
100
80
ALDH+
100
80
80
60
60
60
40
40
20
20
*
*
*
0
*
ALDH+
LLL12
120
DLD-1
120
ALDH+
100
100
100
80
80
80
*
60
*
*
*
100
D
Stattic
HCT116
ALDH+
80
*
120
DLD-1
ALDH+
100
80
80
60
60
40
20
20
0
*
STAT3 shRNA
Stattic
5 µmol/L
CTL
Stattic
10 µmol/L
ALDH+
40
*
20
0
0
DMSO
120
100
40
CTL
DMSO 5 µmol/L 10 µmol/L 20 µmol/L
Stattic
SW480
60
*
0
DMSO 5 µmol/L 10 µmol/L 20 µmol/L
Stattic
120
20
*
0
DMSO 5 µmol/L 10 µmol/L 20 µmol/L
C
*
40
20
0
ALDH+
60
*
40
40
*
DMSO 0.5 µmol/L 1 µmol/L 2.5 µmol/L
HCT116
SW480
20
*
0
DMSO 1 µmol/L 2.5 µmol/L 5 µmol/L
LLL12
120
60
*
20
0
B
ALDH+
40
*
LLL12
Fold changes in cell viability
120
100
DMSO 1 µmol/L 2.5 µmol/L 5 µmol/L
Fold changes in cell viability
DLD-1
120
STAT3 shRNA
Stattic
20 µmol/L
CTL
LLL12
2.5 µmol/L
STAT3 shRNA
Figure 3. LLL12 (A), Stattic (B), and
STAT3 shRNA (C) inhibited cell
viability of SW480, HCT116, and
DLD-1 colon cancer–initiating cells.
þ
þ
The ALDH /CD133 subpopulation of colon cancer cells was
seeded in 96-well plates (3,000 cells
per well) in triplicates in a serumfree MEBM. The following day,
cancer-initiating cells were treated
with LLL12, Stattic, CTL, or STAT3
shRNA. MTT assay was used to
determine cell viability. D, Stattic
and LLL12 inhibited tumorsphere
formation of ALDHþ/CD133þ
subpopulation of SW480, HCT116,
and DLD-1 colon cancer cells. The
ALDHþ/CD133þ cancer-initiating
cells were plated as single cells and
treated with Stattic (5–20 mmol/L) or
LLL12 (2.5 and 5 mmol/L) at the
second day. Tumorspheres were
observed under microscope 15
days posttreatments.
LLL12
5 µmol/L
SW480
HCT116
DLD-1
subpopulation in tumor. In addition, the body weights of mice
have no obvious reduction in both DMSO vehicle- and LLL12treated mice (Supplementary Fig. S7). These results showed
that LLL12 is potent in suppressing tumor growth from the
colorectal cancer–initiating cells in vivo.
Discussion
STAT3 is frequently activated in many types of human
solid and blood cancers, including colon cancer (1–3, 22).
OF8
Cancer Res; 71(23) December 1, 2011
Blocking signaling to STAT3 inhibits cancer cell growth,
showing that STAT3 is crucial to the survival and growth of
tumor cells (21, 22, 32) and is an attractive therapeutic target
for cancer. At the present time, the main effort to target
constitutive STAT3 signaling is only focused on the bulk of
cancer cells. No published literatures have been reported
about whether STAT3 is activated in colon cancer–initiating
cells, and no approach has been initiated to explore the
STAT3 as a possible therapeutic target in colon cancer–
initiating cells. In this article, we took a pilot study to explore
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 in Colorectal Cancer–Initiating Cells
C
a
*
*
*
SW480
20
*
ALDH+/
CD133+
SW480
ALDH–/
CD133–
3
2
*
1
0
ALDH+/
CD133+
ALDH–/
CD133–
DLD-1
HCT116
ALDH+/ ALDH–/ ALDH+/ ALDH–/
CD133+ CD133– CD133+ CD133–
*
10
3
2.5
2
1.5
1
0.5
0
b
ALDH+/ ALDH–/
CD133+ CD133–
LLL12
Stattic Doxorubicin 5-FU
5 µmol/L 10 µmol/L 10 µmol/L 10 µmol/L
DLD-1
*
ALDH+/
CD133+
ALDH–/
CD133–
SW480
ALDH+/CD133+
5 µmol/L
DMSO
LLL12
ALDH1
*
*
0
DMSO
HCT116
4
*
30
Stattic Doxorubicin 5-FU
DMSO LLL12
5 µmol/L 10 µmol/L 10 µmol/L 10 µmol/L
3
2.5
2
1.5
1
0.5
0
HCT116
40
Relative IL-6 level
ALDH+/CD133+ %
*
Relative IL-6 level
Relative IL-6 level
B
SW480
14
12
10
8
6
4
2
0
Relative IL-6 level
ALDH+/CD133+ %
A
2.5
2
1.5
1
0.5
0
HCT116
HT29
*
ALDH+/
CD133+
ALDH–/
CD133–
DLD-1
HT29
ALDH+/CD133+ ALDH+/CD133+
5 µmol/L
5 µmol/L
DMSO
DMSO
LLL12
LLL12
ALDH+/CD133+
5 µmol/L
DMSO
LLL12
ALDH
CD133
CD133
IL-6
IL-6
IL-6R
IL-6R
GP130
GP130
IL-8
IL-8
GAPDH
GAPDH
D
1
1
0.052
0.035
a
CTL
shRN
1
1
IL-6
shRN
0.165
0.204
CTL
shRN
1
1
0.059
0.140
IL-6
shRN
ALDH1
CD133
b
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
SW480
Fold
changes
1
1
0.024
0.988
HCT116
1
1
0.075
0.889
P-STAT3
STAT3
HCT116ALDH+/CD133+
Tumor volume (mm3)
Fold
changes
700
600
500
400
300
200
100
0
GFP shRNA
STAT3 shRNA
IL-6 shRNA
*
*
Day 0
Day 14
Day 21
Day 28
þ
þ
Figure 4. A, LLL12 (5 mmol/L) and Stattic (10 mmol/L) decreased the percentage of ALDH /CD133 subpopulation. However, 10 mmol/L doxorubicin or 5-Fu
increased the percentage of ALDHþ/CD133þ colorectal cancer–initiating cells ( , P < 0.05). B, ELISA assay showed that ALDHþ/CD133þ subpopulation
secreted more IL-6 than ALDH/CD133 subpopulation of colon cancer cells. C, the expression of ALDH1, CD133, IL-6, IL-6R, GP130, IL-8 was
detected by RT-PCR. (a) ALDHþ/CD133þ and ALDH/CD133 colon cancer cells were collected after sorting. The expression of IL-6, IL-6R, GP130, IL-8 were
higher in ALDHþ/CD133þ cells than ALDH/CD133 cells; (b) the expression of ALDH1, CD133, IL-6, GP130, and IL-8 were lower after treated with LLL12 in
ALDHþ/CD133þ colon cancers. D, after sorting, ALDHþ/CD133þ HCT116 and/or SW480 colon cancer–initiating cells were infected with CTL, IL-6,
and/or STAT3 shRNA. (a) IL-6 shRNA decreased STAT3 phosphorylation of SW480 and HCT116 colorectal cancer–initiating cells in vitro; (b) IL-6 and
STAT3 shRNA inhibited ALDHþ/CD133þ HCT116 cancer-initiating cell growth in vivo.
the STAT3 in colon cancer–initiating cells characterized
by ALDHþ/CD133þ subpopulation. Our study confirmed
that ALDHþ/CD133þ colon cancer cells exhibited more
potent tumor-initiating ability than ALDH/CD133 cells
www.aacrjournals.org
in mouse tumor xenografts. We showed that elevated PSTAT3 is detected in colon cancer–initiating cells cell lines
and in human colon cancer tissues derived from cancer
patients. These results suggest that activated STAT3 is
Cancer Res; 71(23) December 1, 2011
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF9
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
SW480ALDH+/CD133+
a
1,600
Tumor volume (mm 3)
1,400
HCT116ALDH+/CD133+
800
Vehicle
5 mg/kg LLL12
3
Tumor volume (mm )
A
1,200
1,000
800
600
400
200
Vehicle
5 mg/kg LLL12
700
600
500
400
300
200
100
0
17
19
21
23 25
Days
27
29
0
30
19
21
23
25 27
Days
29
31
33
b
Vehicle
Vehicle
LLL12
5 mg/kg
LLL12
5 mg/kg
HCT116ALDH+/CD133+
SW480ALDH+/CD133+
C
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
SW480ALDH+/CD133+
0.6
Tumor weight (g)
Tumor weight (g)
B
*
Vehicle
Vehicle
0.4
LLL12
5 mg/kg
*
0.3
0.2
0.1
0
LLL12
5 mg/kg
HCT116ALDH+/CD133+
0.5
Vehicle
LLL12
5 mg/kg
Vehicle 5LLL12
mg/kg
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
SW480ALDH+/CD133+
Fold 1
changes 1
0.368
0.721
D
P-STAT3
STAT3
P-STAT3
(Y705)
STAT3
P-ERK1/2
(T202/Y204)
GAPDH
HCT116ALDH+/CD133+
1
1
Figure 5. LLL12 suppressed tumor
growth in mouse xenografts with
SW480 or HCT116 colon cancer
stem cells. The mice were given
daily intraperitoneal dosages of 5
mg/kg LLL12 or DMSO vehicle.
Tumor volume (A-a), tumor mass
(A-b), and tumor weight (B) were
reduced in all LLL12-treated mice
compared with DMSO vehicle
group. C, one representative
sample from tumor tissues
generated from SW480 or HCT116
colon cancer–initiating cells
showing STAT3 but not ERK1/2
phosphorylation were also inhibited
by LLL12 treatment. D, at the end of
treatments, tumors tissues were
dissociated to obtain a single-cell
suspension and analyzed by flow
cytometry. LLL12 reduced the
þ
þ
percentage of ALDH /CD133
subpopulation in tumor.
0.301 P-STAT3
0.610 STAT3
b
Vehicle
18
16
14
12
10
8
6
4
2
0
*
Vehicle
LLL12
LLL12
5 mg/kg
CD133
ALDH+/CD133+ %
a
ALDH activity
indeed a novel therapeutic target in colon cancer–initiating
cells.
To explore the role thatSTAT3 plays in colon cancer–
initiating cells, we examined the effects of STAT3 inhibition.
Two small molecular STAT3 inhibitors, LLL12 (17) and
Stattic (18) were employed. LLL12 is a novel and more
potent derivative of LLL3, a previously reported STAT3
inhibitor from our laboratories (33, 34). Our results showed
that LLL12 was potent inhibitor to inhibit P-STAT3; STAT3
OF10
Cancer Res; 71(23) December 1, 2011
downstream targets expression and induces apoptosis in
nonseparated colon cancer cells. Importantly, STAT3 inhibitors, LLL12 and Stattic inhibited P-STAT3, STAT3 downstream gene expression, cell viability, and the formation of
tumorspheres in ALDHþ/CD133þ subpopulation of colon
cancer–initiating cells. In addition, STAT3 shRNA was used
to inhibit the STAT3 expression and activity. Our results
showed that STAT3 shRNA also inhibited STAT3 phosphorylation, cell viability, and STAT3 downstream genes
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 in Colorectal Cancer–Initiating Cells
expression in ALDHþ/CD133þ subpopulation of colon cancer–initiating cells.
We compared the expression of IL-6/STAT3 signal pathway, such as IL-6, IL-6R, GP130, and IL-8 between ALDHþ/
CD133þ and ALDH/CD133 subpopulations. IL-6 has been
shown to activate STAT3 (35) and may play a role in colon
cancer oncogenesis (36–38). Interestingly, our results showed
that ALDHþ/CD133þ cells expressed higher levels of IL-6,
GP130, and IL-8 and secreted higher levels of IL-6 than those
in ALDH/CD133 cells. In addition, introduction of the IL-6
shRNA in ALDHþ/CD133þ cells downregulated the expression of STAT3 phosphorylation. These results provide evidence that STAT3 activation in ALDHþ/CD133þ cells is IL-6
dependent. The expression of IL-6 and IL-8 could be reduced
by STAT3 inhibitor, LLL12. It was speculated that STAT3 may
regulate the expression of IL-6 (28). Our data showed that IL-6
is downregulated by STAT3 inhibitor, LLL12, supporting that
IL-6 may be regulated by STAT3. Furthermore, it has been
reported that activated STAT3 could selectively bind to IL-8
promoter and induce IL-8 transcription (39). Our data
showed that IL-6 induced the expression of IL-8, which could
be blocked by LLL12. These results suggest that IL-8 may be a
downstream target of IL-6/STAT3 in colon cancer cells.
Recent studies have suggested a role for interleukins, such
as IL-6 and IL-8, in breast cancer stem cells (15), which imply
that inflammatory microenvironment is important in promoting the oncogenesis. Ginestier and colleagues reported
that blockade of the IL-8 receptor CXCR1 selectively depletes
human breast cancer stem cells (40). Our data suggested that
IL-6/STAT3/IL-8 activation in colon cancer–initiating cells
might play an important role in the development of colon
cancer.
We found that the expression of ALDH1 and CD133 was
reduced after treatment with LLL12, and this may be due to
the inhibition of their expression. It may also be an effect of
LLL12 on cellular heterogeneity, whereby it decreases the
proportion of ALDHþ/CD133þ cells in the tumor cell population which was shown by our in vitro and in vivo data. In
addition, our data showed that STAT3 inhibitors, but not
other cancer therapeutic drugs such as doxorubicin and 5Fu, eliminated ALDHþ/CD133þ subpopulation of colon cancer–initiating cells in colon cancer cell lines. These results
suggested that colon cancer–initiating cells, which are more
resistant to conventional drugs, might be sensitive to STAT3
inhibitors.
Furthermore, our results showed that STAT3, IL-6 shRNA
and LLL12 exhibited suppressive activity on the tumor growth
of human colon cancer–initiating cells derived from bulk colon
cancer cells. These results suggest that constitutive active
IL-6/STAT3 in these cancer-initiating cells enhanced proliferation and survival, as well as tumor growth in mice, whereas
STAT3 blockade by STAT3, IL-6 shRNA and LLL12, suppressed
tumor-initiating cell growth in vivo.
In summary, this study is the first report to show that
IL-6/STAT3 is activated in colon cancer–initiating cells.
Targeting IL-6/STAT3 may be able to eliminate the colon
cancer–initiating cells and provides a promising approach to
treat advanced colon cancer. Our study also showed that
LLL12 is a potent STAT3 inhibitor for cancer-initiating cells
and is a promising drug candidate to target constitutive
STAT3 signaling in colon cancer–initiating cells. Most
recently, 2 literatures reported that IL-6/STAT3 pathway
may be activated in glioblastoma stem cells (41, 42). In
addition, targeting STAT3 by 2 small molecules, STA-21 and
S31-201 or IL-6 shRNAs, respectively, can inhibit cell viability
of these glioblastoma stem cells (41, 42). Furthermore, we
also observed that high levels of STAT3 phosphorylation are
detected in breast cancer–initiating cells compared with
unseparated and nonbreast cancer–initiating cells (data not
shown). These results are consistent with our observation in
colon cancer–initiating cells that activated STAT3 seems to
play an important role in cancer-initiating cells. It is also of
interest to investigate whether STAT3 is also activated in
cancer-initiating cells in other types of human cancer. If
STAT3 is constitutively activated in other types of cancerinitiating cells or cancer stem cells, it may open a new
therapeutic opportunity to target STAT3 in cancer-initiating
cells of those cancer types.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Cynthia McAllister and Dave Dunaway at the Flow
Cytometry Core of Nationwide Children's Hospital.
Grant Support
This research was partly supported by start-up fund from the Department of
Pediatrics to J. Lin and supported by National Natural Science Foundation of
China (81001005) to L. Lin.
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 December 29, 2010; revised July 15, 2011; accepted August 19, 2011;
published OnlineFirst September 7, 2011.
References
1.
2.
Ma X, Wang S, Ye Y, Du R, Cui Z, Somsouk M. Constitutive activation of
Stat3 signaling pathway in human colorectal carcinoma. World J
Gastroenterol 2004;10:1569–73.
Corvinus FM, Orth C, Moriggl R, Tsareva SA, Wagner S, Pfitzner EB,
et al. Persistent STAT3 activation in colon cancer is associated with
enhanced cell proliferation and tumor growth. Neoplasia 2005;7:
545–55.
www.aacrjournals.org
3.
4.
Kusaba T, Nakayama T, Yamazumi K, Yakata Y, Yoshizaki A,
Nagayasu T, et al. Expression of p-STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors. J Clin Pathol 2005;58:833–8.
Lin Q, Lai R, Chirieac LR, Li C, Thomazy VA, Grammatikakis I, et al.
Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and
cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and
Cancer Res; 71(23) December 1, 2011
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
OF11
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
Lin et al.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
OF12
cell cycle arrest of colon carcinoma cells. Am J Pathol 2005;167:
969–80.
Xiong H, Zhang Z, Tian X, Sun D, Liang Q, Zhang Y, et al. Inhibition of
JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and
reduces tumor cell invasion in colorectal cancer cells. Neoplasia
2008;10:287–97.
€tz A, Kovacic
Tsareva S, Moriggl R, Corvinus F, Wiederanders B, Schu
B, et al. Signal transducer and activator of transcription 3 activation
promotes invasive growth of colon carcinomas through matrix metalloproteinase induction. Neoplasia 2007;9:279–91.
Boman BM, Wicha MS. Cancer stem cells: a step toward the cure.
J Clin Oncol 2008;26:2795–9.
Boman BM, Huang E. Human colon cancer stem cells: a new paradigm
in gastrointestinal oncology. J Clin Oncol 2008;26:2828–38.
Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C,
et al. Identification and expansion of human colon-cancer-initiating
cells. Nature 2007;445:111–5.
O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell
capable of initiating tumour growth in immunodeficient mice. Nature
2007;445:106–10.
Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T,
et al. CD133 expression is not restricted to stem cells, and both
CD133þ and CD133- metastatic colon cancer cells initiate tumors.
J Clin Invest 2008;118:2111–20.
Magni M, Shammah S, Schiro R, Mellado W, Dalla-Favera R, Gianni
AM. Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer. Blood 1996;87:1097–103.
Corti S, Locatelli F, Papadimitriou D, Donadoni C, Salani S, Del Bo R,
et al. Identification of a primitive brain-derived neural stem cell population based on aldehyde dehydrogenase activity. Stem Cells
2006;24:975–85.
Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown
M, et al. ALDH1 is a marker of normal and malignant human mammary
stem cells and a opredictor of poor clinical outcome. Cell Stem Cell
2007;1:555–67.
Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti
P, et al. Breast cancer cell lines contain functional cancer stem cells
with metastatic capacity and a distinct molecular signature. Cancer
Res 2009;69:1302–13.
Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H,
et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant
human colonic stem cells (SC) and tracks SC overpopulation during
colon tumorigenesis. Cancer Res 2009;69:3382–9.
Lin L, Hutzen B, Li P, Ball S, Zuo M, DeAngelis S, et al. A novel small
molecule, LLL12, inhibits STAT3 phosphorylation and activities and
exhibits potent growth-suppresive activity in human cancer cells.
Neoplasia 2010;12:39–50.
Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule
inhibitor of STAT3 activation and dimerization. Chem Biol 2006;13:
1235–42.
Carro MS, Lim WK, Alvarez MJ, Bollo RJ, Zhao X, Snyder EY, et al. The
transcriptional network for mesenchymal transformation of brain
tumours. Nature 2010;463:318–25.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.
Prospective identification of tumorigenic breast cancer cells. Proc Natl
Acad Sci U S A 2003;100:3983–8.
Buettner R, Mora LB, Jove R. Activated STAT signaling in human
tumors provides novel molecular targets for therapeutic intervention.
Clin Cancer Res 2002;8:945–54.
Turkson J, Jove R. STAT proteins: novel molecular targets for cancer
drug discovery. Oncogene 2000;19:6613–26.
Kaptein A, Paillard V, Saunders M. Dominant negative stat3 mutant
inhibits interleukin-6-induced Jak-STAT signal transduction. J Biol
Chem 1996;271:5961–4.
Cancer Res; 71(23) December 1, 2011
24. Schaefer T, Sanders L, Park O, Nathans D. Functional differences between STAT 3 and STAT 3. Mol Cell Biol 1997;17:5307–
16.
25. Faruqi T, Gomez D, Bustelo X, Bar-Sagi D, Reich N. Rac1 mediates
STAT3 activation by autocrine IL-6. Proc Natl Acad Sci U S A
2001:9014–9.
26. Bromberg J, Wrzeszczynska M, Devgan G, Zhao Y, Pestell R, Albanese
C, et al. Stat3 as an oncogene. Cell 1999;98:295–303.
27. Gritsko T, Williams A, Turkson J, Kaneko S, Bowman T, Huang M, et al.
Persistent activation of stat3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells.
Clin Cancer Res 2006;12:11–9.
28. Grivennikov S, Karin M. Autocrine IL-6 signaling: a key event in
tumorigenesis? Cancer Cell 2008;13:7–9.
29. Studebaker AW, Storci G, Werbeck JL, Sansone P, Sasser AK,
Tavolari S, et al. Fibroblasts isolated from common sites of breast
cancer metastasis enhance cancer cell growth rates and invasiveness in an interleukin-6-dependent manner. Cancer Res 2008;68:
9087–95.
30. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM,
Wicha MS. Role of notch signaling in cell-fate determination of
human mammary stem/progenitor cells. Breast Cancer Res 2004;6:
R605–15.
31. Gough DJ, Corlett A, Schlessinger K, Wegrzyn J, Larner AC, DE L.
Mitochondrial STAT3 supports Ras-dependent oncogenic transformation. Science 2009;324:1713–6.
32. Ling X, Arlinghaus RB. Knockdown of STAT3 expression by RNA
interference inhibits the induction of breast tumors in immunocompetent mice. Cancer Res 2005;65:2532–6.
33. Fuh B, Sobo M, Cen L, Josiah D, Hutzen B, Cisek K, et al. LLL-3 inhibits
STAT3 activity, suppresses glioblastoma cell growth and prolongs
survival in a mouse glioblastoma model. Br J Cancer 2009;100:
106–12.
34. Bhasin D, Cisek K, Pandharkar T, Regan N, Li C, Pandit B, et al. Design,
synthesis, and studies of small molecule STAT3 inhibitors. Bioorg Med
Chem Lett 2008;18:391–5.
35. Zhong Z, Wen Z, Darnell JE Jr. Stat3: a STAT family member activated
by tyrosine phosphorylation in response to epidermal growth factor
and interleukin-6. Science 1994;264:95–8.
36. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S,
et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells
and development of colitis-associated cancer. Cancer Cell 2009;
15:103–13.
37. Bollrath J, Phesse T, von Burstin V, Putoczki T, Bennecke M, Bateman
T, et al. gp130-mediated Stat3 activation in enterocytes regulates cell
survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell 2009;15:79–80.
38. Becker C, Fantini MC, Wirtz S, Nikolaev A, Lehr HA, Galle PR, et al. IL-6
signaling promotes tumor growth in colorectal cancer. Cell Cycle
2005;4:217–20.
39. Gharavi NM, Alva JA, Mouillesseaux KP, Lai C, Yeh M, Yeung W, et al.
Role of the Jak/STAT pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis
in vivo. J Biol Chem 2007;282:1460–8.
40. Ginestier C, Liu S, Diebel ME, Korkaya H, Luo M, Brown M, et al.
CXCR1 blockade selectively targets human breast cancer
stem cells in vitro and in xenografts. J Clin Invest 2010;120:
485–97.
41. Sherry MM, Reeves A, Wu JK, Cochran BH. STAT3 is required for
proliferation and maintenance of multipotency in glioblastoma stem
cells. Stem Cells 2009;27:2383–92.
42. Wang H, Lathia JD, Wu Q, Wang J, Li Z, Heddleston JM, et al. Targeting
interleukin 6 signaling suppresses glioma stem cell survival and tumor
growth. Stem Cells 2009;27:2393–404.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.
Published OnlineFirst September 7, 2011; DOI: 10.1158/0008-5472.CAN-10-4660
STAT3 Is Necessary for Proliferation and Survival in Colon
Cancer−Initiating Cells
Li Lin, Aiguo Liu, Zhengang Peng, et al.
Cancer Res Published OnlineFirst September 7, 2011.
Updated version
Supplementary
Material
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-10-4660
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2011/11/23/0008-5472.CAN-10-4660.DC1
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 2011 American Association for Cancer
Research.