Download KLF4 inhibition of lung cancer cell invasion by suppression of

Research Paper
Research paper
Cancer Biology & Therapy 9:7, 507-513; April 1, 2010; © 2010 Landes Bioscience
KLF4 inhibition of lung cancer cell invasion
by suppression of SPARC expression
Yanbin Zhou,1,2 Wayne L. Hofstetter,2 Yong He,2 Wenxian Hu,2 Abujiang Pataer,2 Li Wang,2 Ji Wang,2 Yihong Zhou,3 Liping Yu,4
Bingliang Fang2 and Stephen G. Swisher2,*
1
Department of Pulmonary Medicine; First Affiliated Hospital of Sun Yat-sen University; Guangzhou, China; 2Department of Thoracic and Cardiovascular Surgery;
the University of Texas M.D. Anderson Cancer Center; Houston, TX USA; 3Department of Neurological Surgery and Biological Chemistry; University of California Irvine;
Irvine, CA USA; 4Ziren Research LLC; Irvine, CA USA
Key words: KLF4, tumor invasion, lung, carcinoma, SPARC
Krüppel-like factor 4 (KLF4) functions as a tumor suppressor in some cancers, but its molecular mechanism is not clear.
Our recent study also showed that the expression of KLF4 is dramatically reduced in primary lung cancer tissues. To
investigate the possible role of KLF4 in lung cancer, we stably transfected KLF4 into cells from lung cancer cell lines
H322 and A549 to determine the cells’ invasion ability. Our results showed that ectopic expression of KLF4 extensively
suppressed lung cancer cell invasion in Matrigel. This effect was independent of KLF4-mediated p21 upregulation
because ectopic expression of p21 had minimal effect on cell invasion. Our analysis of the expression of 12 genes
associated with cell invasion in parental, vector-transfected and KLF4-transfected cells showed that ectopic expression of
KLF4 resulted in extensively repressed expression of secreted protein acidic and rich in cysteine (SPARC), an extracellular
matrix protein that plays a role in tumor development and metastasis. Knockdown of SPARC expression in H322 and A549
cells led to suppression of cell invasion, comparable to that observed in KLF4-transfected cells. Moreover, retrovirusmediated restoration of SPARC expression in KLF4-transfected cells abrogated KLF4-induced anti-invasion activity.
Together, our results indicate that KLF4 inhibits lung cancer cell invasion by suppressing SPARC gene expression.
Introduction
Secreted protein acidic and rich in cysteine (SPARC, also
named osteonectin or BM40) is an extracellular matrix protein that binds selectively to collagen fibrils and minerals and
plays an important role in bone calcification.1 A growing body
of evidence has shown that SPARC also plays a role in tumor
development and metastasis. Overexpression of SPARC has
been associated with the progression of, and poor outcome in,
various cancers, including melanoma, 2 glioma,3,4 cancers of the
breast,5,6 prostate,7 liver,8 pancreas,9 bladder,10 head and neck11,12
and lung.13 Molecular characterization has revealed that SPARC
can suppress E-cadherin expression, induce mesenchymal transition, and promote tumor cell invasion and metastasis.14-16 It
can also promote cell survival by activating AKT, focal adhesion
kinase and/or integrin-linked kinase.17,18 In addition, SPARCderived peptides may play a role in angiogenesis.19 Suppression
of SPARC expression by a SPARC antisense expression vector
suppressed in vitro adhesive and invasive capacities of melanoma cells and completely abolished their in vivo tumorigenicity.20 These observations collectively indicated that SPARC
plays a critical role in invasive/metastatic phenotype in various
tumors. However, controversial results associated with both
the overexpression and underexpression of SPARC have been
reported in colorectal cancer.21,22 SPARC has also been found
to induce apoptosis in ovarian cancer23 but to inhibit metastasis
in some breast cancer cells.24 Thus, the role of SPARC in tumor
progression and invasion may be dependent on tissue type or
cell context. Nevertheless, little is known about the regulation
of SPARC expression in normal and tumor tissues.
As seen with SPARC, altered expression of Krüppel-like factor 4 (KLF4) has been reported in various cancers, and downregulation of KLF4 has been associated with cancer development,
progression and metastasis.25,26 KLF4, a SP1-like zinc finger
transcriptional factor, 27 has been reported to play an important
role in stem cells.28 Our recent study showed that KLF4 may
function as a tumor-suppressive gene in lung cancer because
expression of KLF4 is downregulated in a substantial number of
primary lung cancers and because ectopic expression of KLF4
suppressed lung cancer cell proliferation and clonogenic formation in vitro. Moreover, enforced expression of KLF4 in lung
cancer cells by ex vivo transfection or by adenovector-mediated
gene transfer suppressed tumor growth in vivo.29 However, the
molecule mechanisms underlying KLF4’s tumor-suppressive
function in lung cancer remain to be determined.
To further explore the possible role of KLF4 in lung cancer,
we analyzed lung cancer cell invasion with or without ectopic
expression of KLF4. Our results showed that ectopic expression of KLF4 extensively suppressed lung cancer invasion and
that this anti-invasion effect was not caused by upregulation
*Correspondence to: Stephen G. Swisher; Email: [email protected]
Submitted: 11/03/09; Revised: 12/30/09; Accepted: 01/04/10
Previously published online: www.landesbioscience.com/journals/cbt/article/11106
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Cancer Biology & Therapy
507
Figure 1. Ectopic expression of KLF4 suppressed lung cancer cell invasion. The cell-invasion assay was performed with use of Matrigel, as described
in the Materials and Methods section. Parental, vector-transfected, and KLF4-transfected H322 and H549 cells were used in the study. (A) Representative areas of invaded cells. (B) Number of invaded cells/field in three transwells. Values represent mean + SD number of cells/field. The experiment was
repeated at least twice with similar results.
of p21, a cell cycle regulator whose expression is regulated by
KLF4,30 because ectopic expression of p21 had no effect on lung
cancer invasion. Analysis of several genes involved in cell invasion revealed that ectopic expression of KLF4 led to a drastic
suppression of SPARC gene expression, suggesting that KLF4
suppresses lung cancer cell invasion by suppressing SPARC
expression.
Results
Enforced expression of KLF4-suppressed lung cell invasion.
We recently found that ectopic expression of KLF4 resulted in
marked inhibition of lung cancer cell growth and clonogenic
formation and that knockdown of KLF4 promoted cell growth
in immortalized human bronchial epithelial cells.29 To further
explore the biologic function of the KLF4 gene in lung cancer cells, we determined the extent of lung cancer cell invasion after retrovirus-mediated KLF4 gene transfer. H322 and
A549 cells were infected with retrovirus expressing KLF4 or a
control vector and selected with geneticin. The parental, KLF4transfected, or control vector-transfected H322 and A549 cells
were then analyzed for their ability to invade a Matrigel-coated
membrane. The results showed that ectopic expression of KLF4
in H322 and A549 cells, compared with that of parental and
control vector-transformed cells, significantly suppressed cell
invasion (p < 0.01) (Fig. 1). This suppression of cell invasion
is unlikely caused by KLF4-mediated cell growth inhibition
because KLF4 stably transfected cells had similar growth rate
as parental cells when tested at 24–72 h after cell seeding,
although those cells had dramatically reduced clonogenic formation ability when compared with parental cells at a relatively
long-term cell culture (9 d). This result indicated that KLF4 is
critical in lung cancer cell invasion.
508
KLF4-mediated anti-invasion activity is independent of
p21 upregulation. KLF4 is known to activate p21(WAF1/Cip1)
through a specific Sp1-like cis-element in the p21(WAF1/Cip1)
proximal promoter.31 Our recent studies also revealed that ectopic
expression of KLF4 in lung cancer cells upregulated p21 expression.29 To test whether KLF4-mediated anti-invasion activity is
associated with upregulation of p21 and consequent cell cycle
arrest at G1 phase, we established H322 and A549 cells stably
overexpressing p21 and evaluated their in vitro invasion ability.
Western blot analysis revealed that H322 and A549 cells transfected with KLF4 and p21 had equivalent levels of p21 expression (Fig. 2A). Nevertheless, an in vitro Matrigel cell-invasion
assay showed that ectopic expression of p21 in H322 and A549
cells, compared with that in parental and empty vector-transfected cells, had no obvious effect on cell invasion (p > 0.05)
(Fig. 2B and C). These results indicated that KLF4-mediated
anti-invasion activity was not associated with the upregulation
of p21 expression in lung cancer.
Ectopic expression of KLF4 leads to extensive downregulation of SPARC. To further investigate the mechanisms
underlying KLF4-induced anti-invasion activity, we analyzed
the expression of various genes that were reported to play roles
in cancer cell invasion,32,33 including MMP1, MMP9, SPARC,
TERT, PKLR, IL3RA, IGF1, VEGFA, RHOC, TGFB1, PLAU
and VCAM1.28-34 For this purpose, we isolated total RNA and
determined mRNA levels in these genes in parental, vectortransfected, and KLF4-transfected H322 and A549 cells by
using real-time PCR. The housekeeping gene ACTB (also know
as β-actin) was used as the internal reference gene to control
mRNA quantity. As expected, cells transfected with KLF4 had
higher levels of KLF4 mRNA than did parental or vector control cells. For most genes tested, ectopic expression of KLF4
did not lead to extensive changes at mRNA levels. Of interest,
Cancer Biology & Therapy
Volume 9 Issue 7
a substantial reduction in mRNA
levels was observed for SPARC in
both KLF4-transfected H322 and
A549 cells, compared with levels
in parental and vector-transfected
cells (Table 1). This result was confirmed by protein analysis. SPARC
expression was easily detectable
in parental and vector-transfected
cells but was completely abolished
in KLF4-transfected cells (Fig. 3).
These results indicated that expression of SPARC is regulated by KLF4
in lung cancer cells.
Knockdown of SPARC inhibited cell invasion in vitro. SPARC,
also known as osteonectin, is an
extracellular matrix protein that
plays an important role in the calcification of bone.1 It is reported to
promote cancer cell invasion and
migration in glioma and prostate
cancer.4,34 SPARC overexpression
is often associated with the most
aggressive and highly metastatic
tumors.35 To investigate whether
downregulation of SPARC alters
cell invasion activity in human
lung cancer, we inhibited SPARC
expression in H322 and A549 cells
Figure 2. Effects of KLF4 and p21 expression on cancer cell invasion. (A) Western blots of KLF4 and p21
by using SPARC-specific siRNA.
expression in H322 and A549 cells transfected with either KLF4 or p21. Parental and vector-transfected
Transfection with SPARC siRNA
cells were used as controls. β-actin was used as a loading control. (B and C) Cell-invasion assay
markedly inhibited the expresperformed in cells listed in (A): (B) Representative areas of invaded cells. (C) Number of invaded cells/
field in three transwells. Values represent mean + SD number of cells/field. The experiment was
sion of SPARC protein in both
repeated at least twice with similar results.
H322 and A549 cells compared
with the expression seen when
control siRNA and mock-treated
Table 1. mRNA levels some invasion-related genes determined by
cells were used (Fig. 4A). Cell viability in H322 and A549
real-time PCR
cells was determined after treatment with SPARC siRNA
H322
A549
or control siRNA, and results showed that knockdown of
SPARC did not affect cell viability compared with viability in
Genes
Parental Vector
KLF4
Parental
Vector
KLF4
parental- and control siRNA-treated cells (data not shown).
KLF4
1.79
0.35
5.47
0.66
0.26
4.08
Nevertheless, in vitro cell invasion analysis performed on
SPARC
0.95
0.99
0.02
0.53
0.37
0.00
Matrigel showed that knockdown of SPARC extensively supMMP-1
0.76
0.34
0.5
0.11
0.12
0.10
pressed cell invasion in both H322 and A549 cells, compared
MMP-9
0.11
0.04
0.05
0.06
0.08
0.02
with cells treated with control siRNA or phosphate-buffered
TERT
0.27
0.15
1.63
1.87
0.52
2.23
saline (PBS) (parental cells) (Fig. 4B). These results, like
PKLR
0.07
0.03
0.11
0.18
0.06
0.18
those observed in glioma and prostate cancer, suggested that
IL3RA
0.08
0.03
0.17
0.43
0.23
0.58
SPARC plays an important role in cell migration and invasion
in lung cancer cells.
IGF1
0.09
0.01
0.01
0.09
0.03
0.01
Enforced expression of SPARC restores KLF4-mediated
VEGFA
46.7
21.4
58.1
41.3
28.3
31.2
inhibition of cell invasion. To further determine the role of
RHOC
19.6
8.45
25.5
28.9
20.1
36.6
SPARC in KLF4-mediated anti-invasion activity, we introTGFB1
5.1
1.4
2.12
13.3
8.50
13.5
duced SPARC cDNA via retrovirus-mediated gene transfer to
PLAU
54.6
58.1
26.6
22.3
14.8
7.26
H322 and A549 cells stably transfected with KLF4. A retrovirus
VCAM1
0.05
0.04
0.1
0.13
0.02
0.09
expressing the GAPDH gene was used as a vector control. The
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Cancer Biology & Therapy
509
Discussion
Figure 3. Effects of KLF4 on SPARC protein expressions. SPARC
expression was determined by western blot analysis on parental,
vector-transfected and KLF4-transfected H322 and A549 cells. Enforced
expression of KLF4 in H322 and A549 led to extensive downregulation
of SPARC expression. β-actin was used as a loading control.
Figure 4. Effects of SPARC siRNA in cell invasion. (A) Western blots of
SPARC expression at 48 h after treatment with siRNA. Parental and
KLF4-transfected cells were used as controls. (B) Effect of SPARC siRNA
on lung cancer cell invasion. Values represent mean + SD number of
cells/field in three transwells. The experiment was repeated at least
twice with similar results. *p < 0.01 among the groups.
SPARC retroviral vector effectively restored SPARC expression in KLF4-transfected H322 and A549 cells (Fig. 5A). The
in vitro Matrigel cell-invasion assay showed that restoration
of SPARC expression in KLF4-transfected H322 and A549
cells restored the cells’ invasion ability, similar to the results
seen in parental H322 and A549 cells (Fig. 5B). These results
indicated that KLF4-induced suppression of SPARC expression is critical for KLF4-mediated inhibition of lung cancer
cell invasion.
510
Our results demonstrated that ectopic expression of KLF4 inhibited lung cancer cell invasion in vitro and that KLF4-mediated
anti-invasion activity is caused by inhibition of SPARC gene
expression. The role of the SPARC gene in cancer cell invasion
and cancer metastasis has been studied by several groups, and
accumulating evidence has shown that SPARC promotes cancer
cell invasion and metastasis in glioma, melanoma and prostate
cancer.4,14,34 Although the role of SPARC in lung cancer invasion
and metastasis was not well characterized previously, SPARC had
been extensively induced by treating bronchial epithelial cells
with cigarette smoke condensate.36 High levels of SPARC expression had been observed in the stroma of patients with non-small
cell lung cancer13,36 and had been associated with nodal metastasis and poor prognosis13 suggesting that SPARC may play a role
in lung cancer cell invasion and metastasis. Our results showed
that downregulation of SPARC, either by siRNA or by ectopic
expression of KLF4, suppressed lung cancer cell invasion, providing direct evidence of the role of SPARC in lung cancer cell
invasion.
Despite many reports about the association between SPARC
gene expression and cancer progression, little is known about
the regulation of SPARC expression. Aberrant SPARC promoter
methylation has been reported in lung cancer and colon cancer.37,38 In contrast, c-Jun has been reported to induce SPARC
in MCF7 breast cancer cells39 but to suppress SPARC expression
in rat embryo fibroblasts,40 suggesting that transcriptional factors involved in SPARC regulation might be species- or cell typespecific. Our study showed that SPARC expression in lung cancer
cells was extensively suppressed by ectopic expression of KLF4,
which was found to be downregulated in lung cancer tissues,29
thus, indicating that KLF4 is a negative regulator of SPARC in
lung cancer.
Moreover, the apparent inhibitory effect of KLF4 on lung cancer cell invasion led us to test its effect on invasion-related genes.
Among the 12 genes tested, SPARC was the only one that was
extensively suppressed by KLF4 in both H322 and A549 cells.
Knockdown of SPARC by siRNA also inhibited lung cancer
cell invasion, as observed in KLF4 overexpression. Furthermore,
restoration of SPARC expression in KLF4-transfected cells abrogated KLF4-mediated anti-invasion activity. Thus, SPARC is one
of the major downstream molecules of KLF4-induced anti-invasion activity in lung cancer cells.
KLF4 is a zinc-finger transcription factor of the SP/KLF
family of transcriptional factors, which includes three domains
of Kruppel-like zinc fingers. All of the members of the SP/KLF
family of transcriptional factors can recognize and specifically
bind to GC-rich sequences41 thereby regulating various target
genes involved in differentiation, proliferation and apoptosis.
Evidence has shown that KLF4 competes with SP1 in promoter
binding and suppresses the expression of SP1-regulated genes
such as cyclin D1 and ornithine decarboxylase.42,43 Whether
KLF4 regulates SPARC through a similar mechanism remains to
be determined, however, the SP1 consensus sequence was identified in the SPARC promoter.39,44 The expression of KLF4 itself is
Cancer Biology & Therapy
Volume 9 Issue 7
Figure 5. Enforced expression of SPARC in KLF4-transfected cells. (A) Western blots of SPARC expression. β-actin was used as a loading control.
(B) Number of invaded cells/field in three transwells. Values represent mean + SD number of cells/field. The experiment was repeated at least twice
with similar results. *p < 0.01 among the groups.
frequently lost in various human cancer types, such as gastric,25
colorectal,45 esophageal squamous cell,46 intestinal,47 prostate,48
bladder49 and lung cancers;29 of interest, SPARC was reportedly overexpressed in many of these cancers. Whether increased
expression of SPARC in these cancers is caused by loss of KLF4
function is not clear. Nevertheless, our results showed that at
least in some lung cancer cells, SPARC expression is negatively
regulated by KLF4 and that KLF4 may execute its anti-invasion
function by suppressing SPARC expression.
Materials and Methods
Human lung cancer cell lines. Lung cancer cell lines A549 and
H322 are maintained in our laboratory. They are routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and
antibiotics (penicillin G at 100 units/mL and streptomycin at
100 µg/mL) and incubated at 37°C under regular culture conditions of 100% humidity, 95% air and 5% CO2.
Retrovirus-mediated gene transfection. Plasmids containing
coding sequences for KLF4, SPARC and P21 were obtained either
from OriGene Technologies, Inc., (Rockville, MD) or from the
American Type Culture Collection (Manassas, VA). The coding
sequences were cloned to pBabe-based retrovirus plasmids with
either puromycin or neomycin as a selection marker for mammalian cells. (Detailed cloning procedures and plasmid maps are
available upon request). The plasmids were then transfected to
293/Phoenix cells by using FuGENE HD reagent (Roche Applied
Science, Indianapolis, IN) according to the manufacturer’s instructions. Retroviral vectors were collected from a culture medium of
293/Phoenix cells at 48–72 h after transfection and used to infect
lung cancer cells. After selection with geneticin or puromycin
(Invitrogen Corporation, Carlsbad, CA) for 2 w, the positive clones
were further studied.
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Cell-invasion assay. The Matrigel-based in vitro cell-invasion
assay was performed by using a Falcon cell culture insert with reconstituted basement membrane matrix (Matrigel, ­Becton-Dickinson
Biosciences, NJ). Cells suspended in DMEM containing 1%
FBS were seeded onto the upper sides of insert filters coated
with Matrigel (60 µg/insert) at a density of 1 x 105 cells/insert.
The lower compartment of the Falcon 24-well plates contained
600 µL of DMEM medium with 10% FBS and 5 µg/mL fibronectin. After 24–48 h at 37°C under the regular culture condition, the
noninvading cells on the upper side of the chamber membranes
were removed with a cotton swab. The lower surfaces of the culture
inserts were fixed with 5% glutaraldehyde and stained with 5%
Giemsa. The number of invading cells on the opposite side of the
chamber membranes was counted on each of three transwells.
Cell-viability assay. Cell viability was determined by using
the sulforhodamine B assay, as described previously.50 In brief,
3 x 103 cells were seeded in each well of 96-well flat-bottom plates
and incubated for 24 h. Next, SPARC siRNA and control siRNA
were transfected, as described below. At 72 h after incubation,
70 µL of 0.4% sulforhodamine B (w/v) in 1% acetic acid solution was added to each well and incubated at room temperature for
an additional 60 min. The culture medium was removed, bound
sulforhodamine B was solubilized with 100–200 µL of 10 mM
unbuffered Tris-base solution (pH, 10.5), and absorbance was read
on a multidetection microplate reader (Synergy HT, BIO-TEK
Instruments, Inc., Winooski, VT) at 570 nm. Relative cell viability was determined by setting the viability of the control cells at
100% and comparing the viability of the treated cells with that of
the controls. Each experiment was performed in quadruplicate and
repeated at least twice.
Gene knockdown with siRNA. The siRNA for human SPARC
coding sequence (GenBank accession No. NM_003118) was
obtained from Dharmacon Research, Inc., (Chicago, IL). The
forward and reverse RNA strands were CGA UGU UGU CAA
Cancer Biology & Therapy
511
GGA UGG U dtdt and GCU ACA ACA GUU CCU ACC A
dtdt, respectively. A control siRNA for random sequence UGA
GAC CGA AGU UUU GGG U dtdt was also obtained from
Dharmacon Research, Inc. For siRNA transfection, 10 µL of
Lipofectamine 2000 (Invitrogen, San Diego) was mixed with
500 µL of Opti-MEM (Invitrogen, Carlbad) and then mixed with
200 pmol siRNA diluted in 500 µL of Opti-MEM. After incubation at room temperature for 20 min, the mixture was added to
60-mm plates with 5 mL of fresh medium. The cells were incubated at 37°C under regular conditions for indicated times before
analysis.
Real-time absolute quantitative reverse transcriptasepolymerase chain reaction (real-time AqRT-PCR). Total RNA
was extracted from cells using Trizol reagent (Invitrogen). 1 µg of
RNA from each sample was reverse transcribed in a 20 µL reaction volume with use of the Taqman reverse transcription reagents
(Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The cDNAs were diluted and quantified
for expression of above given gene using real-time PCR (SYBR
Green I) performed by Ziren Research LLC (Irvine, CA), with
use of a single standard for getting the absolute ratio of each target and reference gene expressions, the procedure was performed
as previously described.33,51 The primer sequences for KLF4,
SPARC, MMP1, MMP9, TERT, PKLR, IL3RA, VEGFA, RHOC,
TGFB1, IGF1, VCAM1, PLAU, ADAMTS1 and ACTB (β-actin
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Acknowledgements
We thank Tamara Locke for editorial review. This work is
supported by National Cancer Institute grants: R01CA092487 (B. Fang), RO1CA-124951 (B. Fang), Lung SPORE
Developmental Award 5P50CA-070907-100007 (S. Swisher),
National Institutes of Health Core Grant 3P30CA-01667232S3, Homer Flower Gene Therapy Research Fund, Charles
Rogers Gene Therapy Fund, Flora & Stuart Mason Lung
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