Inhibition of CK2a Down-Regulates Hedgehog/Gli
Signaling Leading to a Reduction of a Stem-Like Side
Population in Human Lung Cancer Cells
Shulin Zhang1,2,3, Yucheng Wang4, Jian-Hua Mao5, David Hsieh1, Il-Jin Kim1, Li-Min Hu6, Zhidong Xu1,
Hao Long1*, David M. Jablons1, Liang You1*
1Thoracic Oncology Laboratory, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco,
California, United States of America, 2Lung Cancer Institute, Sun Yat-sen University, Guangzhou, People’s Republic of China, 3Department of Surgical Oncology, The
Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, People’s Republic of China, 4Department of Surgery, Helen Diller Family Comprehensive Cancer
Center, University of California San Francisco, San Francisco, California, United States of America, 5Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
California, United States of America, 6Department of Obstertrics and Gynecology, University of California San Francisco, San Francisco, California, United States of America
Protein kinase CK2 is frequently elevated in a variety of human cancers. The Hedgehog (Hh) signaling pathway has been
implicated in stem cell maintenance, and its aberrant activation has been indicated in several types of cancer, including lung
cancer. In this study, we show that CK2 is positively involved in Hh/Gli signaling in lung cancer cell lines A549 and H1299.
First, we found a correlation between CK2a and Gli1 mRNA levels in 100 primary lung cancer tissues. Down-regulation of
Gli1 expression and transcriptional activity were demonstrated after the silencing of CK2a in lung cancer cells. In addition,
CK2a siRNA down-regulated the expression of Hh target genes. Furthermore, two small-molecule CK2a inhibitors led to a
dose-dependent inhibition of Gli1 expression and transcriptional activity in lung cancer cells. Reversely, forced over-
expression of CK2a resulted in an increase both in Gli1 expression and transcriptional activity in A549 cells. Finally, the
inhibition of Hh/Gli by CK2a siRNA led to a reduction of a cancer stem cell-like side population that shows higher ABCG2
expression level. Thus, we report that the inhibition of CK2a down-regulates Hh/Gli signaling and subsequently reduces
stem-like side population in human lung cancer cells.
Citation: Zhang S, Wang Y, Mao J-H, Hsieh D, Kim I-J, et al. (2012) Inhibition of CK2a Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-
Like Side Population in Human Lung Cancer Cells. PLoS ONE 7(6): e38996. doi:10.1371/journal.pone.0038996
Editor: Alan P. Fields, Mayo Clinic College of Medicine, United States of America
Received January 16, 2012; Accepted May 14, 2012; Published June 29, 2012
Copyright: ? 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The present work was supported by National Institutes of Health grant R01 CA140654-01A1 (LY) and Natural Science Foundation of Guangdong
Province, P.R.C. 2009 (9451008901003072). We are also grateful for support from the Kazan, McClain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman
Foundation, Inc; the Estate of Robert Griffiths; the Jeffrey and Karen Peterson Family Foundation; Paul and Michelle Zygielbaum; the Estate of Norman Mancini;
and the Barbara Isackson Lung Cancer Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (HL); Liang.You@ucsfmedctr.org (LY)
Protein kinase CK2 (formerly known as casein kinase II) is a
highly conserved serine/threonine kinase that phosphorylates
more than 300 proteins . CK2 has a heterotetrameric structure
consisting of two catalytic subunits (42-kDa a or 38-kDa a’) and
the regulatory subunit (28-kDa b), forming the configurations
a2b2, aa’b2 and a’2b2. CK2 is a multifunctional protein kinase
, that has been shown to be involved in nearly every aspect of
cell proliferation and survival [3,4,5]. The level of CK2a
expression is tightly regulated in normal cells , and increased
CK2a level and activity has been consistently observed in a variety
of human cancers [7,8,9]. For instance, the high level and/or
nuclear localization of CK2a is a marker of poor prognosis for
patients with acute myeloid leukemia, chronic lymphocytic
leukemia, prostate cancer and gastric cancer [10,11,12,13]. CK2
also affects several cell signaling pathways, including PI3K, NFkB
and Wnt [6,14,15].
The Hedgehog (Hh) family of secreted proteins, which consists
of Sonic, Indian and Desert Hedgehog, plays important roles in
mammalian development and in stem cell maintenance [16,17].
Activation of the Hh pathway is initiated at the cell surface by the
Hh ligand binding to its receptor Patched (Ptc), resulting in
derepression of the effector protein, a G-protein-coupled receptor,
Smoothened (Smo) . Ultimately, Smo activates the Gli family
of transcription factors and target genes. There are three Gli
proteins in humans: Gli1 serves to activate Hh target genes, Gli2
acts both as activator and repressor of Hh target genes, while Gli3
acts as a repressor of Hh target genes [19,20]. Deregulation of
Hh/Gli signaling is implicated as an initiating or maintaining
factor in the progression of various cancers, including basal cell
carcinomas, medulloblastomas, leukemia, lung, gastrointestinal,
lung, ovarian, breast and prostate cancers [19,21]. For instance,
the Gli1 gene is amplified in human glioma and activated in basal
cell carcinoma [22,23,24]. Transgenic over-expression of Gli1 in
mice leads to the development of basal cell carcinoma . Gli1
activation has been demonstrated in non-small cell lung cancer
(NSCLC) cells and tissues .
Direct evidence that Hh/Gli signaling plays an important role
in cancer stem cells (CSCs) derives from a series of studies in
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different tumor types . Recent studies have indicated that
ATP-binding cassette transporter member 2 of G family protein
(ABCG2) is a direct transcriptional target of Hh/Gli signaling
. A subpopulation, named side population (SP) with higher
ABCG2 expression level in human cancer cells including lung
cancer A549 cells, showed series of CSCs’ characteristics
[28,29,30]. Several lines of recent evidence suggest that hedgehog
signaling regulates stem-like side population in human lung cancer
cells. For instance, the side population of lung cancer cell line
H460 preferentially expresses ABCG2 and SMO, a critical
mediator of the hedgehog signaling. Cyclopamine, a natural
hedgehog pathway inhibitor, greatly inhibits cell-cycle progression
and cell proliferation of H460 cell line . Cyclopamine also
reduced side population in human cancer cells . Furthermore,
Hedgehog pathway inhibitor GDC-0449, a FDA approved drug
for treatment of metastatic basal cell carcinoma, effectively
reduced cell growth in human lung cancer cell lines. The effect
is mediated by the inhibition of stem-like side population .
To date, there is no evidence for the relationship between CK2
and Hh/Gli signaling in mammalian cells. To investigate whether
CK2 is involved in the Hh pathway in human lung cancer cells, we
tested the activity of Gli1 after CK2 inhibition.
CK2a and Gli1 Genes are Activated and Correlated in
Both CK2a and Gli1 genes have been shown to be over-
expressed in a variety of cancers, including lung cancer [26,33].
Through the use of semi-quantitative RT-PCR (Figure 1A) and
Western blot analysis (Figure 1B), we examined the CK2a gene
and protein in eight of NSCLC cell lines. Data showed that CK2a
is expressed in all these cancer cells. Among them, at least five cell
lines (A549, A427, H1299, H358 and H838) showed relatively
higher expression of CK2a both at the mRNA and protein levels.
CK2a expression was previously shown to be minimal in normal
lung cells . Gli1 gene and protein expressions were broadly
detected in all cell lines except H358. Interestingly, there appeared
to be a correlation between the expression of CK2a and Gli1 in
these cell lines. We performed real-time RT-PCR of CK2a and
Gli1 in 100 primary NSCLC samples. A mild correlation between
CK2a and Gli1 mRNA levels was found in these tissues (r=0.37,
P,0.05) (Figure 1C). For subsequent experimental studies, A549
was chosen because the status of cancer-related pathways in A549
cells has been well characterized. H1299 was also chosen because
of its relatively higher expression of CK2a and Gli1 genes.
CK2a Knockdown Inhibits Hh/Gli Signaling Through
To investigate whether CK2 suppression have an effect on the
Hh pathway, we silenced CK2 expression using CK2 subunit-
specific siRNAs. Forty-eight
efficiency of RNA interference was monitored by semi-quantita-
tive RT-PCR (Figure S1). The corresponding mRNA levels of
the three subunits decreased, and the knockdown of a and a9
was confirmed by co-transfection of their siRNAs. The expres-
sion of the indicated Hh pathway components was also
determined (Figure S2). Overall, the RT-PCR results showed
that Ptc1 and Gli1 mRNA levels in A549 and H1299 cells were
consistently down-regulated after CK2a or CK2b knockdown,
whereas minimal changes were observed in other Hh pathway
components. By real-time RT-PCR, we confirmed that the
silencing of CK2a significantly inhibited Gli1 expression both in
A549 and H1299 cell lines (Figure 2A). Silencing of CK2b also
resulted in a significant decrease of Gli1 in both cell lines
(Figure 2A). In addition, Gli1 expression was minimal in the
normal lung control. At the protein level, silencing of CK2a led
to a 71% (A549) and a 73% (H1299) decrease of Gli1, while
silencing of CK2b led to a 67% (A549) and a 35% (H1299)
decrease of Gli1. Treatment with CK2a9-targeting siRNA
produced no obvious difference, rather than decrease of Gli1
in A549, and produced a 60% increase in H1299, both at the
mRNA and protein levels (Figure 2B). Furthermore, we
performed immunofluorescence staining with a monoclonal
anti-Gli1 antibody, since nuclear localization of Gli1 reflects
activity of the Hh pathway . Nuclear Gli1 proteins were
dramatically decreased in the presence of CK2a siRNA
(Figure 2C). Moreover, silencing of CK2a resulted in a
significant decrease (45% at 25 mM and 60% at 50 mM,
P,0.01) in the Gli1-boosted Gli reporter activity, compared
with the non-targeting siRNA (control) (Figure 2D).
Small-Molecule CK2a Inhibitors Down-Regulate Gli1
Expression and Transcriptional Activity
To further validate the role played by CK2a in the Hh pathway,
we used two small-molecule CK2a inhibitors: TBB (4,5,6,7-
tetrabromobenzotriazole), a well-known inhibitor of CK2a ,
and CX-4945 (5-(3-chlorophenylamino)benzo[c] [2,6]naphthyri-
dine-8-carboxylic acid), a first-in-class, selective, oral inhibitor of
CK2a under investigation in Phase 1 clinical trials .
Cells were treated with various concentrations of TBB (10, 30,
50 mM) or CX-4945 (1, 3, 10 mM), or with the vehicle DMSO for
48 hours. Treatments with TBB or CX-4945 led to a dose-
dependent decrease of Gli1 mRNA and protein levels both in
A549 and H1299 cell lines (Figure 2E, 2F and S3). The
quantitative mRNA levels were detected by real-time RT-PCR.
As shown in Figure 2E, Gli1 expression in A549 decreased
noticeably at the dosage levels of 1 mM CX-4945 and 10 mM
TBB, and decreased significantly at 10 mM CX-4945 and 50 mM
TBB (50% and 60%, P,0.05). At the protein level, these decreases
reached to 76% (10 mM CX-4945) and 89% (50 mM TBB) in
A549, and 81% (10 mM CX-4945) and 93% (50 mM TBB) in
H1299, respectively (Figure 2F). Immunofluorescence staining
with a monoclonal anti-Gli1 antibody on cultured cells showed a
dramatically lower green florescence in the presence of 30 mM
TBB (Figure 2G). We then demonstrated that the small molecules
inhibited Gli reporter activity in A549 cell line, where a significant
decrease (55%, P,0.01) was detected in the presence of 10 mM
CX-4945 or 10 mM TBB (Figure 2H). These results are consistent
with what we found in siRNA studies.
Forced Over-Expression of the CK2a Gene Leads to Gli1
Up-Regulation and Transcriptional Activation
To confirm whether the CK2 gene positively affects the
transcriptional activity of Gli1, we transfected A549 cells with
either a pcDNA3.1-CK2a or control pcDNA3.1-LacZ plasmid. As
expected, the over-expression of CK2a was attributed to the
activation of Gli1 in the A549 cell line. The over-expression of
CK2a was detected by RT-PCR (Figure 3A) and Western blot
(Figure 3B). The elevated Gli1 mRNA level was also detected by
RT-PCR (Figure 3A). By Western blot analysis, we showed the
elevated Gli1 protein level (2.35 folds) in cells with ectopic over-
expression of CK2a (Figure 3B). Moreover, the reporter assay also
showed a significant (.2 folds, P,0.01) increase of Gli1
transcriptional activity (Figure 3C). These findings suggest that
the over-expression of CK2a gene leads to an up-regulation of
Gli1 gene expression and transcriptional activity.
CK2a Regulates Gli1 Activity
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Figure 1. Over-Expression and Correlation of CK2a and Gli1 Genes in NSCLC. (A) RT-PCR. (B) Western blot. The CK2a gene was activated in
the eight NSCLC cell lines examined (A549, A427, H460, H1299, H1650, H358, H838 and H322), and the Gli1 gene was expressed in all the cell lines
except H358. (C) Linear correlation curve of CK2a and Gli1 mRNA levels. A mild correlation (r=0.37, P,0.05) was shown in linear correlation analysis
by SPSS. Primary NCSCL tissues from patients undergoing resection were collected at the time of surgery and immediately snap-frozen in liquid
nitrogen. These tissue samples were kept at –170uC in a liquid nitrogen freezer before use.
CK2a Regulates Gli1 Activity
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CK2a Regulates Gli1 Activity
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The Silencing of CK2a Promotes Gli1 Degradation
We further carried out a time-course experiment to examine
Gli1 half-time. A549 cells were transfected with CK2a or control
siRNA, and Gli1 protein levels were detected at the time points of
0, 1 and 2 hours after treatment with the protein inhibitor,
cycloheximide. In the CK2a knockdown group, the Gli1 protein
level reduced to mimimum at 2 hours after treatment with
cycloheximide (when compared with that at 0 hour), which
suggested that the half life of Gli1 after CK2a siRNA knockdown
is ,2 hours. These data indicate that CK2a knockdown results in
degradation of Gli1 (Figure 3D). This, in turn, suggests that CK2a
regulates Gli1 activity by preventing its degradation.
CK2a Knockdown Down-Regulates Hh Downstream
Genes and Reduces SP Profile via ABCG2 in A549
Hh reportedly controls the proliferation of several cell types
through various molecular mechanisms and targets downstream
genes, including Ptc, Cyclin family members, and self-induction of
Gli1 [20,38]. We analyzed mRNA levels of four (Gli1, Ptc1, Ptc2
and Cyclin E1) of these genes by using real-time RT-PCR
(Figure 4A). The expression of the four Hh target genes decreased
significantly (P,0.05) after CK2a knockdown, which suggests a
depressed transcriptional activity of the Hh pathway.
It has been demonstrated that ABCG2 is the primary
contributor to the SP phenotype in several cancer cell lines
including A549 . The SP phenotype has been characterized in
a series of studies of cancer stem cells derived from prostate, breast,
colon,glioma, bladder, ovary,
[29,39,40,41,42]. Recent studies show that ABCG2 is a direct
target of the Hh pathway which is involved in stem cell
maintenance . We first examined ABCG2 expression after
CK2a knockdown and found that the ABCG2 level decreased
dramatically in CK2a-silenced A549 cells (Figure 4B). Consistent
with other findings, relatively higher percentage (26.8%) of A549
cells were classified as SP cells in our study. In the presence of
verapamil, an ABC transporter inhibitor, the proportion of SP
cells dropped to 9.4%. After treatment with CK2a siRNA, the
proportion dropped to 15.4% (without verapamil) or 3.4% (with
verapamil) (Figure 4C and 4D). We then sorted SP and non-SP
cells in this system. In further analysis by semi-quantitative RT-
PCR, the sorted SP cells showed higher expression of ABCG2
than did non-SP cells, but no difference of ABCG2 expression in
SP or non-SP cells was shown between the CK2a siRNA and
control (Figure S4). In brief, we showed a 42.5% reduction of SP
proportion after CK2a knockdown.
Our results suggest that CK2 is a positive regulator in Hh/Gli1
signaling in human lung cancer. This is supported by several lines
of evidence. First of all, the correlation of CK2 and Gli1
expressions were noticed in several lung cancer cell lines. We
further demonstrated a linear correlation in 100 primary NSCLC
tissues. Secondly, the inhibition of CK2a by siRNA or small-
molecular inhibitors resulted in down-regulation of Gli1 expres-
sion and transcriptional activity. Thirdly, forced over-expression of
CK2a resulted in increased Gli1 expression and transcriptional
activity. Finally, CK2a knockdown led to a reduction of side
population via down-regulating ABCG2, a direct target of Hh/Gli
To date, there is no evidence for the correlation between CK2
and Hh/Gli signaling in human cancer cells, although CK2 was
suggested as a positive regulator of the Hh signal transduction
pathway and two serine residues in Smo were phosphorylated by
CK2 in Drosophila . However, this mechanism on Smo
phosphorylation by CK2 does not appear to apply to humans,
as these two Smo residues are not conserved in human Smo when
aligned with Clustal W  (Figure S5). Furthermore, several
studies have suggested that mammalian Smo and Drosophila Smo
are regulated by fundamentally distinct mechanisms [45,46].
Recently, Chen et al. demonstrated that mammalian Smo is
activated through multi-site phosphorylation by CK1a and GRK2
and proposed two-step mechanism for Smo phosphorylation in
mammalian cells .
To investigate the potential mechanism through which CK2
positively regulates human Gli1 expression and transcriptional
activity, we performed protein degradation assay of Gli1 after
treatment with CK2a siRNA. Our results indicated that CK2
silencing reduces the half-life of human Gil1 protein in A549 cells.
Furthermore, both CK2 siRNA and small molecule inhibitors
down-regulated Gli1-boosted transcriptional activity. Moreover,
forced over-expression of CK2a resulted in Gli1 transcriptional
activity. In addition, we found two predicted CK2 phosphoryla-
tion sites in human Gli1 by using Scansite with medium stringency
 (Figure S6). Our data suggest that CK2 may regulate Gli1 in
human cancer cells in a similar manner with that in Drosophila. For
instance, Jia et al. reported that CK2 directly phosphorylates Ci in
Drosophila, and then prevents its ubiquitination and degradation
. On the other hand, it has been proposed that glycogen
synthase kinase-3 beta negatively regulates Gli1 transcription
factors cooperating with other kinases such as PKA and CK1s in
lung cancer A549 cells . Thus, two steps are probably involved
in the positive regulation of Gli1 by CK2. In the first step,
inhibition of CK2 promotes Gli1 degradation, followed by
Figure 2. CK2a Inhibition Down-Regulates Gli1 Expression and Transcriptional Activity. (A) Quantitative Gli1 mRNA levels after treatment
with CK2 subunit-specific siRNA detected by real-time RT-PCR. Silencing of CK2a significantly reduced Gli1 mRNA levels both in A549 and H1299 cell
lines (by 50% and 45%, respectively). Silencing of CK2b also resulted in a significant decrease of Gli1 in both cell lines. Minimal Gli1 mRNA level was
noticed in the normal lung (NL). * P,0.05, ** P,0.01, Student’s t-test. (B) Protein levels detected by Western blot. Silencing of CK2a led to 71% (A549)
and 73% (H1299) reduction of Gli1, while silencing of CK2b led to 67% (A549) and 35% (H1299) decrease of Gli1. Treatment by CK2a’-targeting siRNA
produced no obvious difference, rather than decrease of Gli1 in A549, and produced a 60% increase in H1299, both in mRNA and protein levels. (C)
Localization of Gli1 detected by green fluorescence. The Gli1 protein mainly localizes in the nuclear compartment of the cells, and shows a great
reduction after CK2a knockdown. Scale bar=30 mm. (D) The transcriptional activity of Hh pathway in A549 detected by Gli reporter assay. Silencing of
CK2a resulted in a significant decrease (more than 40% at 25 mM and 60% at 50 mM) of the transcriptional activity, compared with the control siRNA.
* P,0.05, ** P,0.01, Student’s t-test. (E) Quantitative Gli1 mRNA levels after treatment with TBB and CX-4945 by real-time RT-PCR. Gli1 expression in
A549 decreased noticeably at the dosage levels of 1 mM CX-4945 and 10 mM TBB, significantly at 10 mM CX-4945 and 50 mM TBB (50% and 60%).
* P,0.05, ** P,0.01, Student’s t-test. (F) Protein levels detected by Western blot. In protein level, these decreases rose to 76% (10 mM CX-4945) and
89% (50 mM TBB) in A549, and 81% (10 mM CX-4945) and 93% (50 mM TBB) in H1299, respectively. (G) Protein expression of Gli1 detected by green
fluorescence. Cells were treat with 30 mM TBB or vehicle DMSO, immunoflorescence staining with anti-Gli1 mAb on cultured cells showed a distinctly
lower green florescence in the presence of 30 mM TBB. (Scale bar=30 mm). (H) The transcriptional activity of Hh pathway in A549 detected by Gli
reporter assay. A 50% decrease was detected in the presence of 10 mM CX-4945 or 10 mM TBB. * P,0.05, ** P,0.01, Student’s t-test.
CK2a Regulates Gli1 Activity
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reduced accumulation of Gli1 in nuclear compartment. In the
second step, as the key transcription factor of the Hh pathway, the
reduced Gli1 protein subsequently suppresses the transcription of
the Hh target genes. This in turn, forms a feedback loop to further
decrease the expression level of Gli1, which is acting as a target
gene of the pathway (Figure 4E). Although the mechanism of CK2
regulation in human cancers remains largely unknown, there is
evidence that CK2 is essential for Wnt/beta-catenin signaling
[50,51]. For instance, it was implicated that CK2 may bind and
phosphorylate b-catenin and promotes its degradation . Taken
together, these results suggest that CK2a may stabilize of human
Gli1 protein through direct phosphorylation. Further studies are
needed to elucidate the precise mechanisms.
The Hh pathway transcription factor Gli1 regulates the
expression of the ABC transporter protein ABCG2 by directly
binding to the ABCG2 promoter , ABCG2 is a molecular
determinant of the SP phenotype and expressed at higher levels in
SP cells, compared with non-SP cells [28,31]. The SP cells are
reportedly enriched with cancer stem cells, as they shows stem-cell
properties (highly tumurigenic and chemo-resistant). Previous
studies have detected SP phenotype with higher ABCG2
expression level and implicated ABCG2 as a CSC marker in lung
Figure 3. Over-Expression of CK2a Gene Leads to Gli1 Up-Regulation and Transcriptional Activation, and Loss of CK2a Function
Promotes Gli1 Degradation. (A)A549 cells were transfected either with a pcDNA3.1-CK2a or control pcDNA3.1-LacZ plasmid vectors, the over-
expressed CK2a and up-regulated Gli1was detected by real-time RT-PCR. * P,0.05, ** P,0.01, Student’s t-test. (B)Western blot. The protein level of
Gli1 was up-regulated in this system with a two-fold increase. (C)Moreover, the reporter assay also showed a significant (more than 2-fold) increase of
the Gli1 transcriptional activity. * P,0.05, ** P,0.01, Student’s t-test. (D)In a protein degradation analysis, A549 cells showed reduced Gli1 protein at
the time point of 2 hours after treated with CK2a siRNA.
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cancer A549 cells . For instance, SP cells in A549 showed
more tumurigenic potential . In our study, the target gene of
Hh/Gli signaling ABCG2 was down-regulated after CK2a
inhibition, where ABCG2-driven side population was reduced
subsequently. Thus, we report that CK2 participates in CSCs
maintenance by regulating Hh/Gli signaling.
Hh pathway may play key roles in the maintenance of CSCs,
however the druggable targets in Hh pathway is very limited. CK2
provide an additional target for the inhibition of Hh/Gli signaling.
CK2 inhibitors have not been extensively developed as therapeutic
agents, partially due to that the ATP-binding pocket of CK2 is not
as druggable as some other protein kinases [50,53,54]. To date,
only one small-molecule CK2 inhibitor has been entered to
clinical trials as a potential anticancer drug. CX-4945, a highly
selective CK2 small molecule inhibitor, is a promising first-in-class
oral therapeutic agent targeting multiple human cancers. CX-
4945 shows a favorable safety profile in Phase I clinical trials .
In addition, CIGB-300 (a synthetic peptide-based drug targeting
the CK2 phosphoaceptor domain) has proved to be safe and of
clinical benefit in Phase I cervical cancer trials .
In summary, we report that CK2 is a positive regulator in the
Hh/Gli signaling pathway, and the inhibition of CK2a down-
regulates Hh/Gli signaling in human lung cancer cells. Given the
emerging importance of Hh/Gli signaling in tumor initiation and
progression, our findings provide an important evidence for the
potential benefits of CK2 inhibitors.
Materials and Methods
Cell Culture and Small Molecule Treatment
Human NSCLC cell lines (A549, A427, H460, H1299, H1650,
H358, H838 and H322) were obtained from American Type
Culture Collections (Manassas, VA). Cells were routinely main-
tained in RPMI-1640 supplemented with 10% heat-inactivated
fetal bovine serum, penicillin (100 mg/ml) and streptomycin
(100 mg/ml). All cells were routinely cultivated at 37uC in a
humid incubator with 5% CO2. Treatment with CX-4945
(Synkinase, San Diego, CA) and TBB (Sigma, St. Louis, MO)
dissolved in DMSO was administered at several dosages (1, 3 and
10 mM of CX4945; 10, 30, 50 mM of TBB). Cells were grown in
medium for 48 hours after treatment.
SiRNA and Plasmid DNA Transfection
CK2a, CK2a9 and CK2b-specific siRNAs (ON-TARGET plus
SMARTpool) and control RNA were purchased from Thermo
Scientific (Waltham, MA). In brief, cells were seeded in a 6-well
plate as 105cells/well one day before transfection, with a target of
30–50% confluency at the time of transfection. Cells were
transfected with 50 nmol/L of siRNA using Lipofectamine
RNAiMAX (Invitrogen, Carlsbad, CA) according to the manu-
facturer’s protocol. Adequate inhibition of the siRNA-mediated
knockdown was confirmed by RT-PCR. The pcDNA3.1-CK2a or
control pcDNA3.1-LacZ plasmid vectors were then transfected
into the A549 cells (0.5 mg/ml in 24-well plate) using Lipofecta-
mine 2000 transfection reagent (Invitrogen), according to the
manufacturer’s protocol. Cells were harvested for RT-PCR and
Western blot or used in reporter assays at 48 hours post-
RNA Isolation, cDNA Synthesis and Semi-quantitative RT-
Isolation of RNA was performed using RNeasy Mini kit
(Qiagen, Valencia, CA). Human Lung Total RNA was purchased
from Applied Biosystems (Foster City, CA). Five-hundred ng of
total RNA was converted into 20 ml cDNA using iScript cDNA
Synthesis Kits (Bio-Rad, Hercules, CA,) according to the
manufacturer’s recommendations. PCR bands were visualized
under UV light and photographed.
A total of 2 ml of the reverse transcription reaction were used as
template for real-time detection of Gli1 expression using TaqMan
Technology on an Applied Biosystems 7000 sequence detection
system (Applied Biosystems, Foster City, CA). Gene expression was
endogenous control gene b-glucuronidase (GUSB) using the primer
and probe sequences commercially (Applied Biosystems).
Western Blot Analysis and Immunofluorescence Staining
Whole protein was extracted by M-PER Mammalian Protein
Extraction Reagent (Thermo) from cell lines added with
Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego,
CA) and Complete Protease Inhibitor Cocktails (Roche, Lewes,
UK) according to manufactures’ protocols. The proteins were
separated on 4–15% gradient SDS–polyacrylamide gels and
transferred to Immobilon-P membranes (Millipore, Bellerica,
MA). The following primary antibodies were used: anti-CK2a
(Millipore), anti-Gli1 (Cell Signaling, Beverly, MA), anti-ABCG2
(Millipore), and anti-GAPDH (Trevigen, Gaithersburg, MD).
After being incubated with appropriate secondary antibodies, the
antigen-antibody complexes were detected by using an ECL
blotting analysis system (Amersham Pharmacia Biotech, Piscat-
away, NJ). Immunofluorescence staining was carried out. Exam-
ination was done using laser scanning confocal microscopy
(LSM510, Carl Zeiss, Oakland, CA). Digital images of single
confocal slices were prepared using Adobe Photoshop 6.0.
Protein Degradation Assay
The CK2a- and control siRNA-transtected A549 cells were
exposed to 50 mg/ml cycloheximide and harvested at the time
points of 0 and 6 hours. Total cellular proteins were extracted and
were analyzed by western blot analysis.
Luciferase Reporter Assays
To measure Gli-mediated Hh transcriptional activity, the
luciferase reporter constructs, 86 wild-type Gli binding site (86
GliwtLuc) or 86mutant Gli binding site (86GlimutLuc) plasmids
 and a human Gli1 expression vector (pcDNA3.1-Gli1) were
co-transfected into A549 cells in 24-well plate. The Renilla
Figure 4. CK2a Knockdown Down-Regulates Hh Signal Pathway Transduction and Reduces SP Proportion in A549. (A) Hh
downstream genes expression detected by real-time RT-PCR. The mRNA level of the four Hh target genes (Gli1, Ptc1, Ptc2 and Cyclin E1) decreased
significantly after CK2a knockdown. * P,0.05, ** P,0.01, Student’s t-test. (B) The expression of ABCG2 decreased in CK2a-silenced A549 cells. (C) The
proportion of SP cells dropped from 26.8% to 15.4%, and from 9.4% to 3.4% in presence of 50 mM verapamil, respectively. Cells were labeled with the
Hoechst 33342 and then analyzed by flow cytometry. (right) Results when the cells were treated with 50 mM verapamil during the labeling procedure.
The SP is outlined and shown as a percentage of the total cell population. These experiments were repeated at three times with similar results. (D) Bar
chart for (C). (E) Model of Hh/Gli1 signaling regulated by CK2. Details are described in the text.
CK2a Regulates Gli1 Activity
PLoS ONE | www.plosone.org8June 2012 | Volume 7 | Issue 6 | e38996
luciferase pRL-TK plasmid (Promega, Madison, WI), whose
expression is driven by the housekeeping thymidine kinase gene
promoter, was co-transfected to normalize for transfection
efficiency. All transfection experiments were performed using the
Lipofectamine2000 (Invitrogen) in accordance with the manufac-
turer’s instructions. After 24 h cells were lysed and luciferase
assays were performed as described previously . Results are
expressed as fold induction, which is the ratio of luciferase activity
induced in Gli-transfected cells relative to basal luciferase activity
in control transfected A549 cells. All experiments were performed
in triplicate; means and standard errors were calculated using
Flow Cytometry Analysis and Sorting
Identification of SP cells was performed as described previously
by Goodell et al. . The A549 cells were incubated with 5 mg/
ml Hoechst 33342 dye (Invitrogen) for 90 min with and without
50 mM verapamil. Cell samples were analyzed and sorted using a
Moflo MLS cell sorter (Beckman-Coulter, Hialeah, FL) with UV
capabilities and SUMMIT software for data acquisition and
analysis. An argon laser was used to excite the Hoechst dye.
Fluorescence emission was collected with a 405/30 nm bandpass
filter for Hoechst blue and a 670/40 nm bandpass filter for
Hoechst red. Dead cells were excluded by propidium iodide
fluorescence at 670/30 nm. The SP and non-SP cells were sorted
for further test of their ABCG2 gene expression.
Data were expressed as mean 6 standard deviation (SD) from
three independent experiments. All of the statistical analyses were
performed using the SPSS 13.0 for Windows software system
(SPSS Inc, Chicago, IL). Student’s t-test was used to compare the
differences among groups. Pearson product correlation tests (in the
form of a correlation matrix) were used to analyze the mRNA level
of CK2 and Gli1. A significant difference was declared if the P
value from a two-tailed test was less than 0.05 (* P,0.05,
Supplementary materials and methods are shown in Text S1.
by siRNA. Forty-eight hours after transfection, the
efficiency of RNA interference was monitored by semi-
quantitative RT-PCR. The corresponding mRNA levels of the
three subunits decreased, and the knockdown of a and a9 was
confirmed by co-transfection.
Silencing of CK2 substrates genes expression
components, detected by semi-quantitative RT-PCR.
The expression of the indicated Hh pathway
The results showed that Ptc and Gli1 gene expression in A549
and H1299 was consistently down-regulated after CK2a and b
knockdown, whereas no obvious changes were revealed in other
HH pathway components.
dose-dependent decrease of Gli1 mRNA level both in
A549 and H1299, which was detected by semi-quantita-
Treatments with TBB or CX4945 led to a
of ABCG2 than non-SP cells in semi-quantitative RT-
PCR, however, no difference of ABCG2 expression in SP
or non-SP cells was shown between the CK2a siRNA and
The sorted SP cells showed higher expression
with peptides indentified by Proteomics analysis. Hh
membrane receptor Smo in human (NCBI Reference Sequence:
EAL24102.1) and Drosophila melanogaster (NCBI Reference
Sequence: NP_523443.1) was aligned with Clustal W software.
The two serine phosphorylation sites of CK2 (indicated in box) in
Drosophila do not exist in humans, indicating that the regulation
of Hh by CK2 is a Smo phosphorylation-independent reaction.
Multiple sequence alignment of Smoothened
human Gli1 with CK2. Two CK2 phosphorylation sites
(indicated in box) in Gli1 (NCBI Reference Sequence:
AAM13391.1) were predicted using Scansite 2.0 with
The phosphorylation prediction results of
Supplementary materials and methods.
We thank Pamela Derish and Li Tai Fang of the UCSF Department of
Surgery for editorial assistance with the manuscript and correlation
analysis. We also thank Tara Rambaldo of USCF Laboratory for Cell
Analysis for cell sorting.
Conceived and designed the experiments: SZ YW JHM LY. Performed the
experiments: SZ IJK DH. Analyzed the data: SZ YW DH JHM IJK ZX
HL DMJ LY. Contributed reagents/materials/analysis tools: YW IJK
LMH. Wrote the paper: SZ LY.
1. Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase
CK2? FASEB J 17: 349–368.
2. Raaf J, Bischoff N, Klopffleisch K, Brunstein E, Olsen BB, et al. (2011)
Interaction between CK2alpha and CK2beta, the subunits of protein kinase
CK2: thermodynamic contributions of key residues on the CK2alpha surface.
Biochemistry 50: 512–522.
3. Guo C, Yu S, Davis AT, Ahmed K (1999) Nuclear matrix targeting of the
protein kinase CK2 signal as a common downstream response to androgen or
growth factor stimulation of prostate cancer cells. Cancer Res 59: 1146–1151.
4. Buchou T, Vernet M, Blond O, Jensen HH, Pointu H, et al. (2003) Disruption of
the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-
autonomous defect and early embryonic lethality. Mol Cell Biol 23: 908–915.
5. Ahmad KA, Wang G, Unger G, Slaton J, Ahmed K (2008) Protein kinase CK2–
a key suppressor of apoptosis. Adv Enzyme Regul 48: 179–187.
6. Dominguez I, Sonenshein GE, Seldin DC (2009) Protein kinase CK2 in health
and disease: CK2 and its role in Wnt and NF-kappaB signaling: linking
development and cancer. Cell Mol Life Sci 66: 1850–1857.
7. Trembley JH, Wang G, Unger G, Slaton J, Ahmed K (2009) Protein kinase CK2
in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 66:
8. Piazza FA, Ruzzene M, Gurrieri C, Montini B, Bonanni L, et al. (2006) Multiple
myeloma cell survival relies on high activity of protein kinase CK2. Blood 108:
9. Wang G, Ahmad KA, Harris NH, Ahmed K (2008) Impact of protein kinase
CK2 on inhibitor of apoptosis proteins in prostate cancer cells. Mol Cell
Biochem 316: 91–97.
CK2a Regulates Gli1 Activity
PLoS ONE | www.plosone.org9 June 2012 | Volume 7 | Issue 6 | e38996
10. Martins LR, Lucio P, Silva MC, Anderes KL, Gameiro P, et al. (2010) Targeting Download full-text
CK2 overexpression and hyperactivation as a novel therapeutic tool in chronic
lymphocytic leukemia. Blood 116: 2724–2731.
11. Laramas M, Pasquier D, Filhol O, Ringeisen F, Descotes JL, et al. (2007)
Nuclear localization of protein kinase CK2 catalytic subunit (CK2alpha) is
associated with poor prognostic factors in human prostate cancer. Eur J Cancer
12. Kim JS, Eom JI, Cheong JW, Choi AJ, Lee JK, et al. (2007) Protein kinase
CK2alpha as an unfavorable prognostic marker and novel therapeutic target in
acute myeloid leukemia. Clin Cancer Res 13: 1019–1028.
13. Lin KY, Fang CL, Chen Y, Li CF, Chen SH, et al. (2010) Overexpression of
nuclear protein kinase CK2 Beta subunit and prognosis in human gastric
carcinoma. Ann Surg Oncol 17: 1695–1702.
14. Duncan JS, Litchfield DW (2008) Too much of a good thing: the role of protein
kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2.
Biochim Biophys Acta 1784: 33–47.
15. Guerra B (2006) Protein kinase CK2 subunits are positive regulators of AKT
kinase. Int J Oncol 28: 685–693.
16. Beachy PA, Karhadkar SS, Berman DM (2004) Tissue repair and stem cell
renewal in carcinogenesis. Nature 432: 324–331.
17. Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development:
paradigms and principles. Genes Dev 15: 3059–3087.
18. Ingham PW, Nakano Y, Seger C (2011) Mechanisms and functions of Hedgehog
signalling across the metazoa. Nat Rev Genet 12: 393–406.
19. Ng JM, Curran T (2011) The Hedgehog’s tale: developing strategies for
targeting cancer. Nat Rev Cancer 11: 493–501.
20. Takebe N, Harris PJ, Warren RQ, Ivy SP (2011) Targeting cancer stem cells by
inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol 8: 97–
21. Ruiz i Altaba A (2008) Therapeutic inhibition of Hedgehog-GLI signaling in
cancer: epithelial, stromal, or stem cell targets? Cancer Cell 14: 281–283.
22. Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, et al. (1987)
Identification of an amplified, highly expressed gene in a human glioma. Science
23. Dahmane N, Lee J, Robins P, Heller P, Ruiz i Altaba A (1997) Activation of the
transcription factor Gli1 and the Sonic hedgehog signalling pathway in skin
tumours. Nature 389: 876–881.
24. Epstein EH (2008) Basal cell carcinomas: attack of the hedgehog. Nat Rev
Cancer 8: 743–754.
25. Nilsson M, Unden AB, Krause D, Malmqwist U, Raza K, et al. (2000) Induction
of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1.
Proc Natl Acad Sci U S A 97: 3438–3443.
26. Yuan Z, Goetz JA, Singh S, Ogden SK, Petty WJ, et al. (2007) Frequent
requirement of hedgehog signaling in non-small cell lung carcinoma. Oncogene
27. Singh RR, Kunkalla K, Qu C, Schlette E, Neelapu SS, et al. (2011) ABCG2 is a
direct transcriptional target of hedgehog signaling and involved in stroma-
induced drug tolerance in diffuse large B-cell lymphoma. Oncogene 30(49):
28. Scharenberg CW, Harkey MA, Torok-Storb B (2002) The ABCG2 transporter
is an efficient Hoechst 33342 efflux pump and is preferentially expressed by
immature human hematopoietic progenitors. Blood 99: 507–512.
29. Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer
cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67:
30. Shi Y, Fu X, Hua Y, Han Y, Lu Y, et al. (2012) The Side Population in Human
Lung Cancer Cell Line NCI-H460 Is Enriched in Stem-Like Cancer Cells. PLoS
One 7: e33358.
31. Balbuena J, Pachon G, Lopez-Torrents G, Aran JM, Castresana JS, et al. (2011)
ABCG2 is required to control the sonic hedgehog pathway in side population
cells with stem-like properties. Cytometry A 79A: 672–683.
32. Tian F, Mysliwietz J, Ellwart J, Gamarra F, Huber RM, et al. (2012) Effects of
the Hedgehog pathway inhibitor GDC-0449 on lung cancer cell lines are
mediated by side populations. Clin Exp Med 12: 25–30.
33. Scaglioni PP, Yung TM, Cai LF, Erdjument-Bromage H, Kaufman AJ, et al.
(2006) A CK2-dependent mechanism for degradation of the PML tumor
suppressor. Cell 126: 269–283.
34. Hung MS, Lin YC, Mao JH, Kim IJ, Xu Z, et al. (2010) Functional
polymorphism of the CK2alpha intronless gene plays oncogenic roles in lung
cancer. PLoS One 5: e11418.
35. Niemann C, Unden AB, Lyle S, Zouboulis ChC, Toftgard R, et al. (2003) Indian
hedgehog and beta-catenin signaling: role in the sebaceous lineage of normal
and neoplastic mammalian epidermis. Proc Natl Acad Sci U S A 100 Suppl 1:
36. Sarno S, Reddy H, Meggio F, Ruzzene M, Davies SP, et al. (2001) Selectivity of
4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein
kinase CK2 (‘casein kinase-2’). FEBS Lett 496: 44–48.
37. Pierre F, Chua PC, O’Brien SE, Siddiqui-Jain A, Bourbon P, et al. (2011)
Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-
carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2
for the treatment of cancer. J Med Chem 54: 635–654.
38. Duman-Scheel M, Weng L, Xin S, Du W (2002) Hedgehog regulates cell growth
and proliferation by inducing Cyclin D and Cyclin E. Nature 417: 299–304.
39. Kondo T, Setoguchi T, Taga T (2004) Persistence of a small subpopulation of
cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A 101:
40. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, et al.
(2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas
ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65:
41. Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, et al. (2006)
Characterization of a side population of cancer cells from human gastrointestinal
system. Stem Cells 24: 506–513.
42. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and
functional properties of murine hematopoietic stem cells that are replicating in
vivo. J Exp Med 183: 1797–1806.
43. Jia H, Liu Y, Xia R, Tong C, Yue T, et al. (2010) Casein kinase 2 promotes
Hedgehog signaling by regulating both smoothened and Cubitus interruptus.
J Biol Chem 285: 37218–37226.
44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007)
Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
45. Varjosalo M, Li SP, Taipale J (2006) Divergence of hedgehog signal
transduction mechanism between Drosophila and mammals. Dev Cell 10:
46. Huangfu D, Anderson KV (2006) Signaling from Smo to Ci/Gli: conservation
and divergence of Hedgehog pathways from Drosophila to vertebrates.
Development 133: 3–14.
47. Chen Y, Sasai N, Ma G, Yue T, Jia J, et al. (2011) Sonic Hedgehog dependent
phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation
and activation of smoothened. PLoS Biol 9: e1001083.
48. Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: Proteome-wide
prediction of cell signaling interactions using short sequence motifs. Nucleic
Acids Res 31: 3635–3641.
49. Mizuarai S, Kawagishi A, Kotani H (2009) Inhibition of p70S6K2 down-
regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines. Mol
Cancer 8: 44.
50. Sarno S, Pinna LA (2008) Protein kinase CK2 as a druggable target. Mol Biosyst
51. Gao Y, Wang HY (2006) Casein kinase 2 Is activated and essential for Wnt/
beta-catenin signaling. J Biol Chem 281: 18394–18400.
52. Song DH, Sussman DJ, Seldin DC (2000) Endogenous protein kinase CK2
participates in Wnt signaling in mammary epithelial cells. J Biol Chem 275:
53. Cozza G, Meggio F, Moro S (2011) The dark side of protein kinase CK2
inhibition. Curr Med Chem 18: 2867–2884.
54. Cozza G, Bortolato A, Moro S (2010) How druggable is protein kinase CK2?
Med Res Rev 30: 419–462.
55. Pierre F, Chua PC, O’Brien SE, Siddiqui-Jain A, Bourbon P, et al. (2011) Pre-
clinical characterization of CX-4945, a potent and selective small molecule
inhibitor of CK2 for the treatment of cancer. Mol Cell Biochem 356: 37–43.
56. Perea SE, Baladron I, Garcia Y, Perera Y, Lopez A, et al. (2011) CIGB-300, a
synthetic peptide-based drug that targets the CK2 phosphoaceptor domain.
Translational and clinical research. Mol Cell Biochem 356: 45–50.
57. Sasaki H, Hui C, Nakafuku M, Kondoh H (1997) A binding site for Gli proteins
is essential for HNF-3beta floor plate enhancer activity in transgenics and can
respond to Shh in vitro. Development 124: 1313–1322.
58. Dhoot GK, Gustafsson MK, Ai X, Sun W, Standiford DM, et al. (2001)
Regulation of Wnt signaling and embryo patterning by an extracellular sulfatase.
Science 293: 1663–1666.
CK2a Regulates Gli1 Activity
PLoS ONE | www.plosone.org10 June 2012 | Volume 7 | Issue 6 | e38996