Published OnlineFirst July 28, 2006; DOI: 10.1158/1541-7786.MCR-06-0029
Suppression of Cell Proliferation and Signaling
Transduction by Connective Tissue Growth
Factor in Non–Small Cell Lung Cancer Cells
Wenwen Chien,1 Dong Yin,1 Dorina Gui,5 Akio Mori,1 Jonathan Mordechai Frank,2
Jonathan Said,5 Donato Kusuanco,3 Alberto Marchevsky,4 Robert McKenna,3
and H. Phillip Koeffler1
1
Division of Hematology/Oncology, 2Department of Neurology, 3Department of Surgery,
and 4Department of Pathology & Laboratory Medicine/Anatomic Pathology,
Cedars-Sinai Research Institute; and 5Department of Pathology, University
of California at Los Angeles School of Medicine, Los Angeles, California
Abstract
Connective tissue growth factor (CTGF) is a secreted
protein that belongs to CCN family. The proteins in this
family are implicated in various biological processes,
such as angiogenesis, adhesion, migration, and
apoptosis. In this study, we explored the roles of CTGF
in lung tumorigenesis. The expression levels of CTGF
in 58 lung cancer samples were reduced by >2 fold in
57% of the samples compared with matched normal
samples using real-time reverse transcription-PCR.
These results were confirmed by immunohistochemical
staining for CTGF in normal lung epithelia and lung
cancer. Cellular proliferation was inhibited in non – small
cell lung cancer (NSCLC) cell lines NCI-H460, NCI-H520,
NCI-H1299, and SK-MES-1 by CTGF overexpression.
Partially purified CTGF suppressed lung cancer cell
growth. The growth inhibition caused by CTGF
overexpression was associated with growth arrest at
G0-G1 and prominent induction of p53 and ADP
ribosylation factor. Most interestingly, overexpression of
CTGF suppressed insulin-like growth factor-I – dependent
Akt phosphorylation and epidermal growth factor –
dependent extracellular signal-regulated kinase 1/2
phosphorylation. In summary, NSCLC cells expressed
decreased levels of CTGF compared with normal lung
cells; this lower expression has an effect on lung cancer
cell proliferation and its cellular response to growth
Received 2/2/06; revised 5/15/06; accepted 5/18/06.
Grant support: NIH, Women’s Cancer Research Institute at Cedars-Sinai
Medical Center, The Cindy & David Horn Trust, The George Harrison Fund, and
Tom Collier Cancer Fund.
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.
Note: H.P. Koeffler holds the Mark Goodson Chair in Oncology Research and is a
member of the Molecular Biology Institute and Jonsson Cancer Center of
University of California at Los Angeles.
Requests for reprints: Dong Yin, Division of Hematology/Oncology, CedarsSinai Medical Center, 8700 Beverly Boulevard, D5022, Los Angeles, CA 90048.
Phone: 310-423-7740; Fax: 310-423-0225. E-mail: [email protected]
Copyright D 2006 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-06-0029
factors. Our data suggest that CTGF may behave as a
secreted tumor suppressor protein in the normal lung,
and its expression is suppressed in many NSCLCs.
(Mol Cancer Res 2006;4(8):591 – 8)
Introduction
Connective tissue growth factor (CTGF; CCN2) is a member
of the CCN protein family, which is composed of cysteine-rich
protein 61 (Cyr61), CTGF, nephroblastoma overexpressed, as
well as WISP-1, WISP-2, and WISP-3 (Wnt-1-induced secreted
proteins; refs. 1-3). The CCN proteins are multifunctional and
are involved in cell adhesion, angiogenesis, cell differentiation,
and tumorigenesis (4-8). CTGF was first identified as a mitogen
from human umbilical vein endothelial cells, and expression of
CTGF is inducible by tumor growth factor-h in fibroblasts (9).
CTGF mediates wound healing, implantation, and fibrosis (10);
hammerhead ribozymes targeting CTGF mRNA inhibit tumor
growth factor-h – mediated cell proliferation (11). Northern blot
analysis shows increase in CTGF mRNA in pancreatic cancer
tissue compared with normal control, but in situ hybridization
indicates that CTGF mRNA localizes to fibroblasts and not in
the pancreatic cancer cells (12). Furthermore, survival rates for
pancreatic cancer patients were directly correlated to their
CTGF expression levels (12). Low levels of CTGF were
associated with increased metastasis and poor prognosis in
breast cancer patients (13, 14). Similarly, the metastatic breast
cancer cell line, MDA-MB-231, expresses lower levels of
CTGF than the less invasive MCF-7 cells (13). Forced
expression of CTGF in MCF-7 cells induces apoptosis (15).
In the progression of colorectal cancer, patients with high
CTGF expression in their tumors have higher survival rates
compared with patients with low CTGF expression in their
tumors (16). Transfection of colorectal cancer cell lines with
antisense CTGF increases their invasiveness both in vitro and
in vivo (16).
Non – small cell lung cancer (NSCLC) represents f80% of
the total lung cancer cases, and the subtypes include
adenocarcinoma, adenosquamous, squamous, and large cell
carcinoma (17). A recent study showed that lower CTGF
expression is associated with advanced stage and mortality in
lung adenocarcinoma (18). Overexpression of CTGF in lung
adenocarcinoma cell lines decreases invasive and metastatic
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592 Chien et al.
activities, perhaps mediated by a protein called collapsing
response mediator protein 1 (18). In the current study, we found
that expression levels of CTGF in lung cancer samples were
reduced compared with matched normal samples. Force
expression of CTGF in NSCLC cell lines NCI-H460, NCIH520, NCI-H1299, and SK-MES-1 suppressed their proliferation. A similar effect was noted when partially purified CTGF
was added to the cells. Overexpression of CTGF induced G1
cell cycle arrest concurrently with induction of p53 and p14
[ADP ribosylation factor (ARF)]. Kinase activation by the
growth factors, insulin-like growth factor (IGF)-1 and epidermal growth factor (EGF), was suppressed by CTGF overexpression. In summary, blunted expression of CTGF may play
a role in lung tumorigenesis, and the mechanism may be
associated with lack of suppression of downstream signals of
IGF-1 and EGF.
Results
Differential Gene Expression of CTGF in Lung Cancers
To determine the CTGF expression profile in lung tumors,
total RNA was extracted from lung tumors and matched normal
lung tissues from the same patients. Complementary DNA was
prepared, and quantitative real-time PCR was used to measure
the CTGF mRNA expressions. The relative CTGF expression
levels were calculated in a panel of 58 tumor/normal pairs
(Fig. 1A). Of the 58 pairs, 16 pairs (28%) showed <2-fold
change in CTGF expressions, 9 pairs (16%) had increased
CTGF expression in the tumors, and 33 pairs (57%) had >2-fold
decreased CTGF expression in the tumors. To determine
whether the CTGF mRNA expression levels corresponded to
CTGF protein expressions, immunohistochemical staining for
CTGF was evaluated in normal epithelia and lung tumors.
Strong positive staining was found in bronchiolar normal
epithelia (Fig. 1B). In contrast, only very weak CTGF staining
occurred in the tumor cells (Fig. 1B). These results suggested
that low expression of CTGF conferred a growth advantage to
lung cancer cells.
Growth Inhibition of NSCLC Cells by CTGF Overexpression
To assess the effect of CTGF expression on lung cancer cell
proliferation, four lung cancer cell lines (NCI-H1299 and NCIH460: large cell lung cancer; NCI-H520 and SK-MES-1:
squamous cell carcinoma) were transfected with either CTGFpcDNA3.1 expression vector or empty vector pcDNA3.1. The
effect of CTGF on clonogenic growth of these cells was
evaluated. The empty vector – transfected cells (V) formed
numerous colonies, whereas the number of colonies decreased
after plating the cells transfected with CTGF-pcDNA3.1
(Fig. 2). G418-resistant clones were then screened and selected
for CTGF expression by both real-time PCR and Western blot
analysis. Two stable clones overexpressing CTGF were selected
for each of two cell lines (NCI-H460, 4F1 and 4F2; NCI-H520,
5F1 and 5F2; Fig. 3A). These sublines were used for further
study. Compared with 4V and 5V control cells, the two stable
clones (4F1 and 4F2 and 5F1 and 5F2, respectively) expressing
prominent levels of CTGF showed reduction in their growth
rate as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Fig. 3B).
FIGURE 1.
Relative expression of CTGF in paired
human lung tumors and normal lung tissues and CTGF
immnohistochemical staining
of these tissues. A. Quantitative real-time PCR was done
on 58 pairs of matched
NSCLC RNA samples. The
CTGF expression levels were
normalized to that of h-actin.
Data were calculated from
triplicates. Each bar is the
log2 value of the ratio of CTGF
expression levels between
lung tumors (T ) and matched
normal tissues (N ) from the
same patients. Less than
2-fold change: the ratio between tumor and normal is <2.
Because Log2 2 = 1, bar value
>1 represents >2-fold increase (T > N, 16%), whereas
bar value < 1 represents >2fold decrease (T < N, 57%). B.
Immunohistochemical staining
of CTGF in lung tissues.
Strong CTGF staining occurred in normal bronchiolar
epithelia (black arrows ),
whereas no CTGF staining
appeared in the squamous
carcinoma cells (similar findings were seen with adenocarcinoma of the lung, data
not shown). All photos were
taken at 400.
Mol Cancer Res 2006;4(8). August 2006
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Published OnlineFirst July 28, 2006; DOI: 10.1158/1541-7786.MCR-06-0029
The Role of CTGF in NSCLC
Fig. 4B, right). No difference in growth (MTT assay) of NCIH460 and MCF-7 cells occurred when they were grown in
control conditioned medium either with or without heparin
(Fig. 4C).
CTGF Induced Growth Arrest and Expression of Genes
Related to Growth Suppression
To explore the mechanisms by which CTGF caused growth
inhibition, we examined cell cycle profiles. The G0-G1 phase
increased in CTGF-overexpressing cells compared with the
control cells (4F1 versus 4V; 5F1 versus 5V). In contrast, the
proportion of cells in S phase decreased in CTGF-overexpressing cells (Table 1). Expression of cyclin D1 is important
in the promotion of cell cycle progression from G1 to S phase
(22). We found that moderately high cyclin D1 was expressed
in control cells (4V and 5V; Fig. 5); whereas in CTGFoverexpressing cells, cyclin D1 levels diminished (4F and 5F;
FIGURE 2. Effect on clonogenic growth by the forced expression of
CTGF in NSCLC cell lines. Cells were transfected with either a CTGF
expression vector or empty vector (V) and grown under G418 selection.
Colony formation was stained with crystal violet after 2 weeks and
photographed. Results from one experiment. Similar findings were
observed in an identical duplicate experiment. The staining was dissolved,
and absorbance at 590 nm was measured and displayed in bar graph
form. The number of cells showed a linear relationship to absorbance.
NCI-H460 and NCI-H1299: large cell lung cancer cell lines; NCI-H520 and
SK-MES-1: squamous cell carcinoma cell lines.
Biologically active CTGF is both secreted and expressed
on the cell membrane (19, 20). Conditioned medium prepared
from 4F1 cells had prominent expression of CTGF compared
with control 4V cells (Fig. 4A, left). After 48 hours of
incubation, the CTGF-enriched conditioned medium from 4F1
cells (CTGF) inhibited cell growth compared with control
conditioned medium from 4V cells (V) in both lung cancer
NCI-H460 and breast cancer MCF-7 cells (Fig. 4A, right).
MCF-7 cells were used as control because recombinant CTGF
protein has been shown to cause growth inhibition in MCF-7
cells (15). Heparin affinity chromatography has been used in
CTGF purification (21). Therefore, we used heparin agarose
beads to deplete CTGF from the CTGF-conditioned medium
and investigated the effect on cell growth using the CTGFdepleted conditioned medium. After heparin binding, CTGF
expression was negligible in the conditioned medium
(nonbinding; Fig. 4B, left), and the growth-inhibitory effect
of the conditioned medium was lost (heparin nonbinding;
FIGURE 3. Suppression of lung cancer cell proliferation by overexpression of CTGF. A. Immunoblot analysis of CTGF expression. Two
CTGF stably transfected clones from NCI-H460 (4F1 and 4F2) and NCIH520 (5F1 and 5F2) cells were probed for CTGF expression. Glyceraldehyde-3-phosphate dehydrogenase was used as loading control. V,
empty vector – transfected clones (4V and 5V). B. MTT assay. Growth
rates were compared between the CTGF and control transfected cell lines.
Viable cells were measured by MTT activity every 24 hours from day 0
(D0 ) to day 4 (D4 ). MTT activity measured at day 0 was set at 100%.
Points, mean of two independent experiments done in triplicates; bars, SE.
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594 Chien et al.
Fig. 5). Both NCI-H460 and NCI-H520 cells express the wildtype p53 tumor suppressor protein. We examined the effect of
CTGF on p53 levels. Significantly increased p53 expression
occurred in CTGF-overexpressing cells compared with control
cells (Fig. 5). One important upstream regulator of p53 levels is
ARF (23), which was also markedly induced in CTGFoverexpressing cells (Fig. 5). ARF can be activated by E2F1
(24), which was also increased in CTGF-overexpressing cells
(Fig. 5).
CTGF Suppression of IGF-1-Dependent Akt Phosphorylation and EGF-Dependent Extracellular Signal-Regulated
Kinase 1/2 Phosphorylation
Various signaling pathways inducible by growth factors are
keys to regulation of tumorigenesis (25). We investigated the
effect of CTGF on IGF-1- and EGF-dependent kinase
activation. Phosphorylation of Akt was inducible by addition
of IGF-I to 5V cells (Fig. 6A, compare lane 2 with lane 1); in
contrast, the induction was suppressed in CTGF-overexpressing
5F1 cells (Fig. 6A, compare lane 5 with lane 2). Similar results
were found in 4F1 and 4F2 cells compared with 4V cells
(data not shown). Because Akt is a downstream effector of
phosphatidylinositol 3-kinase, we also used a phosphatidylinositol 3-kinase inhibitor, Wortmannin, to test whether it blocks
Akt activation in these cells. Specific inhibition of Akt
phosphorylation was mediated by Wortmannin (Fig. 6A,
lanes 3 and 6). When exposed to EGF, phosphorylation of
extracellular signal-regulated kinase 1/2 (ERK1/2) was found
in 5V cells (Fig. 5, compare lane 2 with lane 1), and the
level of phosphorylation was decreased in the CTGFoverexpressing cells 5F1 (Fig. 6B, compare lane 4 with
lane 5). When the ERK1/2 inhibitor, PD98059, was added to
the cells, it specifically blocked the ERK1/2 activation (Fig. 6B,
lanes 3 and 6).
FIGURE 4. Inhibitory activity of the CTGF
conditioned medium on lung cancer cell growth.
A. Cells were incubated with conditioned
medium prepared from 4V and 4F1 cells for
48 hours, and cell numbers were determined
by MTT assay. Left, expression of CTGF in
conditioned medium was analyzed by immunoblots. B. Cells were incubated with either
CTGF-enriched or CTGF-depleted conditioned
medium (heparin nonbinding) for 48 hours, and
cell numbers were determined by MTT assay.
Left, Western blot analysis confirmed CTGF
depletion from the conditioned medium after
binding to heparin agarose. C. Cells were
incubated with conditioned medium prepared
from control 4V cells for 48 hours either with or
without heparin, and cell numbers were determined by MTT assay. Proliferation data are
expressed as MTT activity. Columns, mean of
triplicate wells in two separate experiments;
bars, SE. NCI-H460, lung cancer cells; MCF-7,
breast cancer cells.
Mol Cancer Res 2006;4(8). August 2006
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The Role of CTGF in NSCLC
Table 1. Percentage of Cells in Different Phases of the Cell
Cycle Analyzed by Flow Cytometry
DNA content (%)
G0-G1
S
G2-M
NCI-H460
NCI-H520
4V
4F1
5V
5F1
48
40
12
56
32
12
52
40
8
63
30
7
NOTE: Cells were methanol fixed and stained with propidium iodide for flow
cytometry. Results are mean F SE of triplicate experiments.
Discussion
Several studies have shown the antigrowth activity of
CTGF in cancer cells; however, the molecular mechanism by
which CTGF exerts its effect is not well understood. The
survival rates of colorectal and pancreatic cancer patients
directly correspond to their CTGF expression levels (12, 16).
In addition, breast cancer patients with a poor prognosis and
mortality had significantly lower CTFG levels compared with
their normal breast tissues (13). Forced expression of CTGF in
MCF-7 breast cancer cells increased cell apoptosis (15). In
addition, patients with lung adenocarcinoma with low CTGF
expression more often had an advanced stage and a poorer
prognosis (18).
In the current study, we initially examined the expression
levels of CTGF in 58 pairs of matched lung tumor and normal
samples. By quantitative real-time PCR, 57% of the samples
expressed lower levels of CTGF in tumors compared with their
matched normal tissues. Immunohistochemistry identified
prominent CTGF protein localization in the normal bronchiolar
epithelia but not in tumor cells. A similar decreased CTGF
expression has been shown between normal breast tissue and
tumor. CTGF was expressed highly in normal breast epithelia,
endothelia, and stromal cells but weakly in breast cancer cells
(14). This is similar to pancreatic cancer tissue, in which weak
CTGF staining was found in tumor cells surrounded by
fibroblasts staining strongly for CTGF (12). The growth
disadvantage conferred by CTGF expression probably occurs
via similar mechanisms in lung and breast and maybe in
pancreas.
We evaluated the effect of CTGF on NSCLC cell growth by
forced expression of CTGF. Both MTT and colony formation
assays showed reduction in growth rates after CTGF overexpression in four NSCLC cell lines, including NSCLC line
(NCI-H1299), large cell lung cancer line (NCI-H460), and two
squamous cell carcinoma lines (NCI-H520 and SK-MES-1).
This showed that CTGF is antiproliferative in these lung cancer
cells. Inhibition of cell proliferation in vitro, however, may not
translate into decreased tumorigenesis in vivo. CTGF may
modulate expression of extracellular matrix components and
promote angiogenesis and tumorigenesis. Expression of CTGF
in prostate stromal fibroblasts promoted microvessel density
(26); in MDA-MB-231 cells, hypoxia-stimulated CTGF
induced neovascularization by modulating expression levels
of matrix metalloproteinases and their inhibitors (27). Future
studies will examine how CTGF-overexpressing lung cancer
cells behave in vivo and what effect CTGF will have on the
synthesis and degradation of extracellular matrix.
Although CTGF is antiproliferative in our lung cancer cells,
a previous study showed that CTGF-transfected lung adenocarcinoma A549 cells lost their invasive ability but continued to
have a comparable growth rate as control cells (18). Reason for
these differences may relate to variability in the cell lines. This
is shown from experiments with another CCN family protein,
Cyr61 (8). Forced expression of Cyr61 had no effect on the
growth rates of NCI-H125 (adenosquamous), NCI-H157
(squamous), and NCI-H1299 but inhibited proliferation of
NCI-H460 (large cell) and NCI-H520 (squamous) carcinoma
lung cancer cells (8). This growth inhibition by Cyr61 in
NCSLC was p53 dependent. The cell growth of the NSCLC
cell lines (NCI-H157, NCI-H125, and NCI-H1299) expressing
mutant p53 was not suppressed by Cyr61 (28). Likewise, all
four lung cancer cell lines in our study (NCI-H1299, NCIH460, NCI-H520, and SK-MES-1) expressed wild-type p53.
Future studies will explore if growth inhibition by CTGF is
mediated in part by p53. Nevertheless, we found significant
increase of p53 expression in NCI-H460 and NCI-H520 cells
overexpressing CTGF compared with control cells. Upregulated p53 can activate p21, inhibit cyclin-dependent kinase
2 kinase activity, and activate Rb tumor suppressor gene to
promote cell cycle arrest (29). In NSCLC cells overexpressing
Cyr61, p53 is induced by h-catenin/TCF4 complex activation
and c-myc expression (8, 28).
The levels of p53 can be regulated by ARF, which is an
antagonist of MDM2 by binding to this protein and preventing
p53 degradation (23, 30, 31). In fibroblasts, ARF working
through p53 induces cell cycle arrest at both G1 and G2 phases
FIGURE 5. Growth arrest and induction of genes related to growth
suppression by CTGF. Western blot analysis. Cell lysates were prepared
and probed for the expression of cyclin D1, p53, ARF, and E2F1.
Glyceraldehyde-3-phosphate dehydrogenase was used as loading control.
Blots are representative of two separate experiments. 4F1 and 5F1 are
CTGF-overexpressing NSCLC sublines of NCI-H460 and NCI-H520, and
4V and 5V are their control cell lines.
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FIGURE 6.
CTGF suppressed IGF-I- and EGF-induced kinase
phosphorylation of target proteins. 5V and 5F1, control and CTGFoverexpressing sublines of NCI-H520 cells, were serum starved overnight
before addition of growth factor. Cell lysates were extracted and analyzed
with immunoblotting. A. Phosphorylation of Akt was analyzed by Western
blot analysis. The phosphatidylinositol 3-kinase inhibitor Wortmannin
(100 nmol/L) was added to cells 1 hour before treatment with IGF-I
(100 ng/mL) for an additional 15 minutes. B. ERK1/2 activation was
analyzed by Western blot analysis. Cells were pretreated with the inhibitor
for ERK1/2 [PD98059 (50 Amol/L)] for 1 hour before stimulation with EGF
(25 ng/mL) for an additional 15 minutes. Representative Western blots.
Similar results were observed with 4V and 4F1 (data not shown).
of the cell cycle (32, 33). ARF is frequently inactivated or
not expressed (19-55%) in NSCLC (34-36). Our results show
that forced expression of CTGF in NCI-H460 and NCI-H520
cells increases ARF expression levels. The cell cycle analysis
showed growth arrest at the G1 phase of the cell cycle and
down-regulation of cyclin D1, which could be partially caused
by ARF activation in these cells. Studies have shown that ARF
is not activated by DNA-damaging agents (24), rather by myc,
ras, and E2F1 (24, 37, 38). Unregulated E2F1 activity induces
ARF protein expression (37), which in turn stabilizes p53 and
thus leads to cell cycle arrest and prevents uncontrolled cell
proliferation (39, 40). We found increased levels of E2F1 in our
NSCLC cells overexpressing CTGF. Possibly, the growth arrest
in these cells occurred by CTGF-inducing expression of p53
through E2F1 and ARF.
CTGF is a secreted protein that can mediate its activity
through binding to integrins (4), bone morphogenetic protein
(41), and low-density lipoprotein receptor – related proteins
(42, 43). Conditioned medium from CTGF-transfected cells
inhibited cell proliferation of the NSCLC NCI-H460 cells as
well as the breast cancer MCF-7 cells; these latter cells have
been shown to have enhanced apoptosis in the presence of
recombinant CTGF (15). After the conditioned medium was
incubated with heparin-coated beads, the inhibitory effect was
reversed, whereas control conditioned medium showed no
effect on cell proliferation either with or without heparin.
Nevertheless, the heparin affinity beads might be expected to
deplete not only CTGF but also other secretory heparin-binding
protein, such as Cyr61, basic fibroblast growth factor, and
vascular endothelial growth factor (44-46). One of these may
also slow the growth of NSCLC cells. In addition, CTGF, like
all other CCN proteins, has multiple modules: module I
contains the IGF-binding protein domain; module II, the von
Willebrand factor type C repeat; module III, the thrombospondin type 1 repeat; and module IV, the cystein knot. The
multimodules provide the opportunity for the CCN proteins to
interact with multiple proteins, which of modules is essential
for the inhibitory function of CTGF in lung cancer cells
requires further studies.
As noted above, CTGF protein contains an IGF-binding
protein domain. In our study, IGF-I-dependent Akt phosphorylation and EGF-dependent ERK1/2 phosphorylation were
suppressed by CTGF overexpression in NSCLC. Previous
studies in inflammatory breast cancer showed that WISP3
(CCN6) was able to modulate IGF-I receptor signaling by
interaction with IGF-1 (47). A similar mechanism may be
feasible for CTGF, although studies have suggested that CTGF
has a relatively low affinity toward IGF-1 (48). The Akt and
the p53 pathways form a feedback loop. Akt activation leads
to MDM2 phosphorylation resulting in inactivation of p53,
whereas p53 activation suppresses Akt activity (49). CTGF
expression may sway this cross-talk toward the p53 pathway,
which concurrently will suppress the Akt pathway.
In summary, we showed that CTGF is able to suppress
NSCLC cell growth. CTGF blunts cell growth probably by
activation of E2F1, ARF, and p53 and by suppression of cyclin
D1. Decreased expression of CTGF may play a role in lung
tumorigenesis by allowing IGF-1 and EGF to have greater
progrowth activity.
Materials and Methods
RNA Extraction, cDNA Preparation, and Real-time PCR
Lung tissues of matched tumor and normal samples were
obtained from lung cancer patients after informed consent.
RNA was extracted using Trizol reagent (Invitrogen, Carlsbad,
CA) and cleanup with RNeasy column (Qiagen, Valencia, CA).
The first strand cDNA was synthesized using the SuperScript
reverse transcription-PCR kit (Invitrogen). Quantitative realtime PCR was done using Taqman PCR Core Reagent kit (PE
Biosystems, Foster City, CA) as described previously. Amplification of h-actin was used as control for cDNA normalization.
The oligonucleotide primers (first two sequences) and probe
(third sequence) were 5¶-AGGTATGGCAGAGGTGCAAG,
5¶-ATGTCTTCATGCTGGTGCAG, and 5¶-TGCGAAGCTGACCTGGAAGAGAACA (CTGF) and 5¶-GATCATTGCTCCTCCTGAGC, 5¶-ACTCCTGCTTGCTGATCCAC, and
5¶-CTCGCTGTCCACCTTCCAGCAGAT (h-actin).
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The Role of CTGF in NSCLC
Immunohistochemistry
Immunohistochemical staining for CTGF was done with
polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz,
CA). Heat-induced epitope retrieval was done with a pressure
cooker and Tris buffer (pH 9.0) for 2 minutes. Localization was
done with DAKO Envision (DAKO, Carpinteria, CA) conjugated to horseradish peroxidase followed by the diaminobenzidine
reaction. Negative controls consisted of substitution of the
primary antiserum with normal rabbit serum at the same dilution.
Cell Culture and Transfections
Large cell lung cancer cell lines (NCI-H1299 and NCIH460) and squamous cell lung carcinoma cell lines (NCI-H520
and SK-MES-1) were maintained in DMEM supplemented with
10% FCS. Cells were transfected with either 5 Ag CTGF
expression vector or empty vector pcDNA3.1 using LipofectAMINE 2000 (Invitrogen). Forty-eight hours after transfection,
cells were replated in complete medium containing 600 Ag/mL
G418, and stable clones were selected after 2 weeks. Several
G418-resistant stable clones after CTGF transfection were
screened for CTGF expression with real-time PCR and Western
blot analysis. Two clones with the most prominent CTGF
expression were used for this study. A G418-resistant clone was
also selected from an empty vector pcDNA3.1 transfection for
each cell line and they were designated 4V for the one selected
from NCI-H460 and 5V for the clone from NCI-H520 cells.
Colony Formation Assay
Cells were seeded at 1 105 per well in 12-well plates.
Triplicates of each cell line were plated. Cells were passaged
under G418 selection for 2 to 3 weeks, fixed and stained with
0.1% crystal violet in 50% methanol, and photographed with an
inverted phase-contrast microscope. The dye was then dissolved in 0.2% (v/v) Triton X-100, and absorbance was
measured at 590 nm. The number of cells showed a linear
relationship to absorbance.
MTT Assay
Cells were seeded at 1 104 per well in 96-well plates.
Triplicates of each cell line were plated. MTT reagents were
added to each well, and absorbance was measured according to
the manufacturer’s instructions (Sigma, St. Louis, MO).
Western Blot Analysis
Total cell lysates were prepared with lysis buffer [50 mmol/L
Tris (pH 7.6), 5 mmol/L EDTA, 300 mmol/L NaCl, 0.1% NP40,
0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT,
protease inhibitor mixture (Roche Molecular Biochemicals,
Pleasanton, CA)] and resolved by SDS-PAGE followed by
blotting. Membranes were probed with anti – glyceraldehyde-3phosphate dehydrogenase (PharMingen, San Diego, CA) as
loading control. Antibodies against CTGF, p53, ARF, and E2F1
were purchased from Santa Cruz Biotechnology. Signals were
detected by West Pico reagents (Pierce, Rockford, CA).
Conditioned Medium Preparation
CTGF-pcDNA3.1 (4F1) and pcDNA3.1 (4V) transfected
cells were grown to 80% confluency and replaced with serum-
free medium for another 48 hours. The conditioned media were
centrifuged at 3,000 g for 10 minutes, and the supernatants
were aliquoted and stored at 80jC. Heparin agarose beads
(Sigma) were prewashed, equilibrated with serum-free medium,
and incubated with conditioned medium at 4jC overnight to
remove CTGF from the conditioned media.
Growth Factor Treatment
Cells were plated in serum-free medium for 24 hours
followed by treatment with either IGF-I (100 ng/mL; Sigma) or
EGF (25 ng/mL; Sigma) for 15 minutes and then harvested for
Western blot analysis. For inhibitor studies, cells were
pretreated with either PD98059 (50 Amol/L) or Wortmannin
(100 nmol/L) before addition of growth factors. Antibodies
used for these studies included phosphorylated Akt, ERK1/2,
as well as Akt and ERK1/2 (Santa Cruz Biotechnology).
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Published OnlineFirst July 28, 2006; DOI: 10.1158/1541-7786.MCR-06-0029
Suppression of Cell Proliferation and Signaling Transduction
by Connective Tissue Growth Factor in Non −Small Cell Lung
Cancer Cells
Wenwen Chien, Dong Yin, Dorina Gui, et al.
Mol Cancer Res 2006;4:591-598. Published OnlineFirst July 28, 2006.
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