ORIGINAL PAPER

KLF4 suppresses HDACi induced caspase activationand the SAPK pathway by targeting p57Kip2

Nung Ky Æ Chuan Bian Lim Æ Jinming Li ÆJames P. Tam Æ Mohamed Sabry Hamza ÆYan Zhao

� Springer Science+Business Media, LLC 2009

Abstract Kruppel-like factor 4 (KLF4) belongs to a

family of evolutionarily conserved zinc finger-containing

transcription factors. It has been shown to mediate self

renewal and pluripotency, regulate adipogenesis and play a

critical role in monocyte differentiation. KLF4 is also

highly expressed in squamous cell carcinomas and in 70%

of all primary human breast cancers, suggesting a putative

role for KLF4 as being an oncogene and as an antiapoptotic

factor. However, the mechanism of this regulation remains

unclear. Here, we show that KLF4 is induced during his-

tone deacetylase inhibitor treatment, and regulates the

extrinsic apoptosis pathway by inhibiting caspase cleavage.

In addition, KLF4 binds to the p57Kip2 promoter and

transcriptionally upregulates its expression, which in turn

inhibits the stress activated protein kinase cascade and

c-Jun phosphorylation. Our findings indicate that in cancer

cells that express high levels of KLF4 may be refractory to

HDACi treatment. Results of our study demonstrate an

unexpected antiapoptotic function of KLF4, and suggest an

important cell fate determinant following histone deace-

tylase inhibitor induced apoptosis.

Keywords CDKN1C � HDAC � KLF4 � Kruppel �SAHA � p57Kip2

Introduction

Epigenetic modifications affect a wide range of biological

processes, and thus play key roles in normal development

and tumorigenesis. Among the key chromatin modifying

enzymes that affect the epigenetic state, and thus gene

transcription, include histone acetyltransferases (HATs)

and histone deacetylases (HDACs). HATs and HDACs

have lately garnered attention because of their impact on

tumor development and progression. In several tumors, the

low expression of tumor suppressors or proapoptotic genes

has been directly correlated with the hypoacetylated state

of their respective promoters. The hypoacetylated state is a

consequence of increased expression of HDACs [1, 2].

Lately, several structurally diverse inhibitors of HDACs

(HDACi) have been shown to be potent inducers of growth

arrest and apoptosis in a wide variety of transformed cells.

Of the numerous HDACi that are currently under clinical

investigation as potential treatment strategies for solid and

hematological cancers include vorinostat (SAHA), romi-

depsin (depsipeptide, FK-228), LAQ824/LBH589 and

belinostat (PXD101) [1, 3–5]. Elucidating the apoptotic

signaling pathways induced by HDACi, especially in a

cellular environment replete of p53 function, which is the

scenario in most cancers, will provide valuable insights

into comprehending the biology of epigenetic modifica-

tions on tumor formation and progression. Additionally,

molecular targeted therapies could be devised to target

N. Ky � C. B. Lim � J. Li � J. P. Tam � Y. Zhao (&)

Division of Chemical Biology and Biotechnology, School of

Biological Sciences, College of Science, Nanyang Technological

University, 60 Nanyang Drive, Singapore 637551, Singapore

e-mail: zhaoyan@ntu.edu.sg

M. S. Hamza (&)

Genome Institute of Singapore, Agency for Science, Technology

and Research (A*STAR), 60 Biopolis Street, Genome #02-01,

Singapore 138672, Singapore

e-mail: hamzas@gis.a-star.edu.sg; sabry.hamza@spcorp.com

Present Address:M. S. Hamza

Schering-Plough Technologies Pte Ltd., 8 Biomedical Grove,

#04-01/05, Neuros Building, Singapore 138665, Singapore

123

Apoptosis

DOI 10.1007/s10495-009-0368-0

different types of cancers using the information of signal-

ing pathways affected by HDACi treatment.

In this study, we focused our attention on the Kruppel-

like transcription factor, KLF4, which has not been

explicitly implicated in HDACi induced apoptosis. Krup-

pel-like TFs belong to a family of Sp1-like zinc-finger

proteins, with over twenty members known to date [6–8].

They regulate gene transcription by binding to GC-rich

regulatory elements within promoters and have been

implicated to play a central role in numerous biological

processes, including the regulation of cell growth, prolif-

eration, differentiation and tumorigenesis. The fourth

member of the Kruppel-like TF family, KLF4, has been

shown to mediate self renewal and pluripotency of embry-

onic stem cells [9–11], transcriptionally regulate adipo-

genesis [12], and act as a critical regulator of monocyte

differentiation [13]. Previous studies have also suggested

that KLF4 is deregulated in cancers, with some controversy

of it acting as a tumor suppressor or an oncogene [14, 15].

However, studies conducted by independent laboratories

clearly demonstrate that KLF4 as being a context-depen-

dent oncogene. It induces transformation in RK3E epithelial

cells [16], prevents senescence by activated Ras [15] and is

highly expressed in squamous cell carcinomas and in 70%

of all primary human breast cancers [17, 18].

In this report, we provide a mechanism by which KLF4

plays a significant role in modulating apoptosis induced by

histone deacetylase inhibitor, SAHA. KLF4 expression is

transiently upregulated during SAHA mediated apoptosis.

KLF4 binds to the promoter of CDKN1C (p57Kip2) and

induces its transcription, which in turn modulates the stress

activated protein kinase cascade and phosphorylation of the

transcription factor, c-Jun. In addition, we demonstrate that

the extrinsic apoptosis pathway is also suppressed by KLF4

by inhibiting caspase activation and cleavage. By this

mechanism, KLF4 plays a substantial role in the regulation

of apoptosis induced by histone deacetylase inhibitors, and

may further contribute to tumor formation and malignancy.

Materials and methods

Cell culture and drug treatment

HCT116 and Cal-27 cell lines were obtained from ATCC

(Manassas, VA). Cells were cultured in Dulbecco’s modified

Eagle’s medium, supplemented with 10% fetal bovine serum,

1% penicillin–streptomycin (all from GIBCO� Invitrogen,

Carlsbad, CA) in a humidified 5% CO2 atmosphere at 37�C.

Cells were treated with HDAC inhibitor Suberoylanilide

hydroxamic acid (SAHA, Cayman Chemical, MI) at a final

concentration of 5 lM and dimethyl sulfoxide was used

throughout the experiment as the vehicle control.

Flow cytometry analysis

Cells were harvested after drug treatment and fixed with 70%

ethanol. Fixed cells were treated with RNase (100 lg/ml)

and stained with propidium iodide (50 lg/ml). Subse-

quently, stained cells were analyzed for DNA content by

flow cytometry using FACScalibur (Becton Dickinson,

Franklin Lakes, NJ). Cell cycle fractions were quantifies

using the CellQuest software (BD Biosciences, San Jose,

CA). Further details can be found in [19].

Annexin-V measurements

Direct fluorescence staining of apoptotic cells for flow

cytometric analysis was performed with the Annexin

V-FITC apoptosis detection kit (BD Pharmingen, San Jose,

CA). After the indicated times, cells were harvested and

stained according to the manufacturer’s protocol. Stained

cells were analyzed in a flow cytometer.

Western blotting

Western blotting procedure was followed according to

[19]. Briefly, cells were lysed in appropriate volume of

lysis buffer (Sigma Aldrich, St. Louis, MO). 50 lg of

protein samples were separated by SDS–PAGE and trans-

ferred onto nitrocellulose membrane (Bio-Rad, Hercules,

CA). The membranes were immunoblotted with primary

antibodies against p57Kip2, cleaved caspase 9, 3, 7, 6, 8 and

PARP, Cdc 2, Cyclin D1, D3, Bax, Bik, Puma. Antibodies

were purchased from Cell Signal Technology, Danvers,

MA). Other primary antibodies include, Cdk 2, 4, 6, Cyclin

B1, A, Bcl-2 and actin. Blots were incubated with horse-

radish peroxide-conjugated goat anti-rabbit, goat anti

mouse or rabbit anti-goat. Antibodies were purchased from

Santa Cruz Biotechnology, Santa Cruz, CA.

Real-time quantitative RT–PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen,

Valencia, CA) following the manufacturer’s protocol.

cDNA was synthesized using the reverse transcription

system kit (Promega, Madison, WI) according to the

manufacturer’s protocol. Quantitative real time PCR was

then performed using 7500 Real Time PCR Systems

(Applied Biosystems, Foster City, CA) and SYBR Green

master mix (Applied Biosystems). The expression of genes

was normalized to the housekeeping PPIE gene. Primers

used for qPCR: KLF4 (AGAGTTCCCATCTCAAGGCA

and GTCAGTTCATCTGAGCGGG), CDKN1C (AGCTG

CACTCGGGGATTT and AAGAAATCGGAGATCAGA

GGC).

Apoptosis

123

Chromatin immunoprecipitation assays

ChIP assays were performed on HCT116 cells after SAHA

and DMSO treatments using anti-KLF4 (H-180) X or the

relevant control anti-GST (Z-5) antibody (Santa Cruz Bio-

technology, Santa Cruz, CA) according to the protocol

described in [19, 20]. Briefly, HCT116 cells were seeded

onto 200 mm culture dishes. Upon 80% confluence, cells

were treated with SAHA and dimethyl sulfoxide as a control,

for 8 h. This was followed by 1% formaldehyde treatment

for 10 min at room temp with rotation. The cross-linking was

stopped by the addition of 2.5 M of glycine for 5 min at room

temp with rotation. Cells were then rinsed with cold 19 PBS

and scraped down into tubes. Cells were pelleted by centri-

fugation and washed with cold 19 PBS. Next, cells were

resuspended in 5 ml of IP buffer (150 mM NaCl, 50 mM

Tris–HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1.0% Triton

X-100) and kept on ice for 10 min. Cell were pelleted down

at 4,000 rpm for 5 min and supernatants were discarded. To

nuclear pellet, 300 ll of IP buffer (protease inhibitor added)

was added and left on ice for 30 min. Subsequently, chro-

matin was sonicated to yield chromatin fragments of 500 bp

and fragment size was assessed by agarose gel electropho-

resis. Sonicated chromatin was then pelleted and superna-

tants were transferred to new tubes. To 300 ll extracts,

700 ll of IP buffer and 100 ll of blocked protein

A- sepharose beads (Zymed, San Francisco, CA) was added

and incubated for 30 min, 4�C with rotation. Next, samples

were centrifuged at 1,000 rpm for 2 min, 4�C, and super-

natants were transferred to fresh tubes. Immunoprecipitation

was carried out overnight by rotating the precleared extracts

with anti-KLF4 (H-180) X or the relevant control anti-GST

(Z-5) antibodies (Santa Cruz Biotechnology, Santa Cruz,

CA) and blocked protein A-sepharose beads. Next day, the

immunoprecipitates were washed five times with IP buffer

and centrifuged at 1,500 rpm for 30 s. Afterwards, super-

natants were discarded and 200 ll of 10% Chelex 100 were

added to the tubes. Samples were then incubated for 45 min

at 95�C with occasional vortexing. Samples were cooled

down to room temperature and centrifuged at 800 rpm for

5 min. Supernatants were transferred to new tubes and sub-

jected to amplification using 7500 Real Time PCR system.

Primers used : -150 to ?50 of promoter region of p57Kip2

(CTAGCTCGCTCGCTCAGG and CGTGGTGTTGTTGA

AACTGA), negative control primers for normalization

(6 kb upstream of the GADPH promoter, ATGGTTGCC

ACTGGGGATCT and TGCCAAAGCCTAGGGGAAGA).

Luciferase assays

HCT116 cells were transfected with pGL4-p57Kip2 and

luciferase activity was measured in the presence of

endogenous KLF4, overexpression of KLF4 or knockdown

of KLF4 with or without SAHA treatment. Briefly,

HCT116 cells were seeded in each well of a 6-well plate.

Next day, cells were transfected with NC siRNA or KLF4

siRNA using Lipofectamine RNAiMax according to the

manufacturer’s instructions. Twenty-four hours later, these

cells were transfected with 2.5 lg of pGL4-p57Kip2

expression vector (obtained from SwitchGear Genomics,

Menlo Park, CA) and 0.2 lg of CMV b-galactosidase as a

control for transfection efficiencies using Lipofectamine

2000 (Invitrogen, Carlsbad, CA) according to the manu-

facturer’s instructions. pGL4-p57Kip2 was constructed

using primers situated on coordinates 2,863,456 and

2,864,550 on chr11 (hg17)). Cells were transfected in the

same manner with 2.0 lg of empty vector (pCDNA3.1) or

KLF4 overexpressing vector (kind gift from Dr Zhi Yi

Chen, Dept. of Medicine, Boston University) using

FuGENE 6 transfection reagent (Roche, Indianapolis, IN)

following manufacturer’s instructions. Twenty-four hours

post-transfection, cells were treated with 5 lM SAHA or

DMSO for 24 h, and cell extracts were analyzed for

luciferase activity by using a TD-20/20 luminometer

(Turner Designs, Sunnyvale, CA). Luciferase activity was

normalized using b-galactosidase levels.

siRNA transfection

HCT116 cells were transfected with siRNA using Lipo-

fectamine RNAimax according to the manufacturers pro-

tocol. HCT116 cells were transfected with non-targeting

control siRNA (NC), siRNA KLF4 or siRNA p57Kip2 when

cells reached 80% confluency. After 24 h, cells were split

1:3, and treated with SAHA, TSA or DMSO the next day.

Final siRNA concentration was 100 nM and transfection

was performed using Lipofectamine RNAimax (Invitrogen,

Carlsbad, CA) according to the manufacturer’s protocol.

Target sequences used for siRNA against KLF4 and

p57Kip2 are CCUUCAACCUGGCGGACAUTT and

GGCCUCGGCUGGGACCGUUTT, respectively.

Results

Histone deacetylase inhibitors (HDACi) induce caspase

cleavage, p57Kip2 and apoptosis

When HCT116 cells were treated with the HDACi, SAHA,

there was a substantial increase in apoptosis when com-

pared with the DMSO control treated cells (Fig. 1a). A

similar increase in apoptosis was observed for the tongue

squamous carcinoma cell line, Cal-27 (Fig. 1b) treated with

SAHA, and in HCT116 cells treated with another HDACi,

TSA (Fig. 1c). In addition, upon HDACi treatment, the

protein levels of cleaved caspases and PARP increased in a

Apoptosis

123

subG1 3.4% subG1 10.9%

subG1 3.1% subG1 10.1%

subG1 28.2%

subG1 3.4% subG1 10.9%

subG1 3.1% subG1 10.1%

subG1 28.2%

0

10

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80

90

Untreated DMSO SAHA (5µM)

24h48h

subG1-32.2%

subG1-5.9%

subG1-60.3%

subG1-6.9% subG1-7.3%

subG1-3.2%

Unt

rea t

edD

MS

OS

AH

A

subG1-32.2%

subG1-5.9%

subG1-60.3%

subG1-6.9% subG1-7.3%

subG1-3.2%

Unt

rea t

edD

MS

OS

AH

A

SAHA 24h SAHA 48h

subG1-4.7% subG1-9.8%

Unt

reat

edE

tOH

TS

A

subG1-4.5% subG1-6.8%

subG1-38.4% subG1-82.5%

24h 48h

subG1-4.7% subG1-9.8%

0

10

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30

40

50

60

70

80

90

Untreated 95% EtOH TSA (400nM)

24h

48h

A B

C

Fig. 1 Histone deacetylase inhibitors induce apoptosis. a HCT116

cells were mock, DMSO or 5 lM SAHA treated for 24 and 48 h.

FACS analysis was performed to measure percentage of apoptosis. bCal-27 cells were untreated or treated with DMSO or 5 lM SAHA for

24 and 48 h. Percentage of apoptotic cells was measured using FACS

analysis. c Graph showing percentage of apoptotic cells in EtOH,

TSA or mock treated cells after 24 and 48 h treatment

Apoptosis

123

time-dependent manner in HCT116 cells (Fig. 2a) and cal-

27 cells (Fig. 2b) when compared to cells treated with

DMSO. This response was also observed in HCT116 cells

treated with TSA (Fig. 2c). Western blot analysis for

p57Kip2 protein expression levels also increased upon

HDACi treatment in a time-dependent manner confirming

our microarray results (data not shown).

Inhibition of caspase cleavage blocks HDACi induced

apoptosis

To confirm our findings that SAHA induces caspase

cleavage resulting in apoptosis, we used the caspase-3

specific inhibitor, Z-DEVD-fmk to determine if SAHA-

mediated apoptosis is reduced. Z-DEVD-fmk on this own

did not affect apoptosis and was similar to that of the DMSO

control. A combination of SAHA and Z-DEVD-fmk sig-

nificantly inhibited apoptosis ([50%) when compared with

SAHA only treated cells (Fig. 3a). In addition, the levels of

caspase 3 and PARP cleavage were significantly reduced

(Fig. 3b). Albeit these experiments show a reduction of

HDACi induced caspase cleavage and apoptosis, the

inability of Z-DEVD-fmk to completely rescue cells sug-

gests an additional mechanism by which HDAC inhibitors

induce apoptosis.

0

5

10

15

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30

35

40

45

50

DMSO SAHA Z-DEVD-fmk Z-DEVD-fmk +SAHA

% o

f ce

lls in

Su

b-G

1

Cleaved caspase 3

Cleaved PARP

Cleaved caspase 6

Cleaved caspase 7

Cleaved caspase 9

Actin

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDaD

MS

O

SA

HA

Z-D

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mk

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+ S

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Cleaved PARP

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Cleaved caspase 7

Cleaved caspase 9

Actin

35 kDa

19 kDa

A

B

17 kDa

20 kDa

18 kDa

89 kDa

45 kDaD

MS

O

SA

HA

Z-D

EV

D-f

mk

Z-D

EV

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mk

+ S

AH

A

Fig. 3 Z-DEVD-fmk attenuates HDACi mediated apoptosis. aHCT116 cells were treated with DMSO, SAHA, Z-DEVD-fmk

(caspase 3 inhibitor) or Z-DEVD-fmk ? SAHA for 24 h and percent

of cells undergoing apoptosis was measured using FACS analysis. bWestern blot analysis for cleaved caspases after 24 h of indicated

drug treatment. Actin was used as a loading control. All experiments

were performed in biological triplicates

Cleaved PARP

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 6

p57kip2

SAHA (5uM)

Actin

Cleaved caspase 7

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

57 kDa

HCT116 Cell Line

Cleaved PARP

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 6

p57kip2

Actin

Cleaved caspase 7

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

57 kDa

HCT116 Cell LineA

Cleaved caspase 7

Cleaved PARP

Actin

Cleaved caspase 319 kDa17 kDa

20 kDa

89 kDa

45 kDa

Cleaved caspase 7

Cleaved PARP

Actin

Cleaved caspase 319 kDa17 kDa

20 kDa

89 kDa

45 kDa

SAHA 24hrs B

Cleaved PARP

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 6

p57kip2

Actin

Cleaved caspase 7

57 kDa

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

Cleaved PARP

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 6

p57kip2

Actin

Cleaved caspase 7

57 kDa

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

C

20 84 12

TSA 24hrs 20 84 12

24hrs 20 84 12

Fig. 2 HDACis induce p57Kip2 expression and activates caspase

cleavage. a HCT116 cells were treated with SAHA and protein was

extracted at the indicated timepoints. Western blots were performed

for cleaved caspases, PARP and p57Kip2. Actin was used as a loading

control. b Cal-27 were treated with 5 lM SAHA and whole cell

lysates were extracted at the indicated timepoints. Western blots were

performed for cleaved caspases. Actin was used as the loading

control. c Western blots for cleaved caspases and p57Kip2 from whole

cell lysates harvested from HCT116 cells treated with 400 nM TSA at

the indicated timepoints

Apoptosis

123

Induction of KLF4 suppresses HDACi induced

apoptosis and caspase cleavage and increases p57kip2

protein levels

Upon SAHA treatment, we observed a substantial increase

in caspase cleavage for both HCT116 and Cal-27 cell lines

(Fig. 2a, b) and in HCT116 cells treated with TSA (Fig. 2c).

In addition, we observed the transcript levels of KLF4 to be

significantly increased upon SAHA treatment in our

microarray data. We did not observe other members of the

Kruppel-like transcription factors to be affected (data not

shown). To confirm our findings, we validated our micro-

array results using qPCR, and confirmed KLF4 expression

levels increased upon SAHA treatment (Fig. 4a). To ana-

lyze further the role of KLF4 in HDACi induced apoptosis,

we used short interfering RNAs (siRNA) to selectively

deplete KLF4 expression. The mRNA levels of KLF4 were

measured by qPCR after SAHA and TSA treatment, and a

knockdown efficiency of 50% was achieved (Fig. 4b, c).

When HCT116 cells depleted of KLF4 expression were

treated with SAHA, the levels of apoptosis were higher

when compared to HCT116 cells transfected with a non-

targeting siRNA (Fig. 4d). To recapitulate these findings

and confirm our results, we used the MitoLight apoptosis

Detection Kit and Annexin V staining (Fig. 4e, f). To

demonstrate that this phenotype was not peculiar to the

HCT116 cell line, we carried out similar experiments on

Cal-27 and the breast cancer cell line, MCF-7 (Fig. 4g,

and data not shown). We observed a similar result when

KLF4 depleted HCT116 cells were treated with TSA

(Fig. 4h).

When KLF4 depleted cells were analyzed by Western

blot, there was a significant increase in caspase cleavage

and PARP upon SAHA treatment when compared to con-

trols (Fig. 5a). In addition, expression levels for p57Kip2

were reduced in KLF4 depleted cells. We confirmed our

findings using the Cal-27 cell line (Fig. 5b) and obtained

similar results. These finding were recapitulated using TSA

in KLF4 HCT116 depleted cells (Fig. 5c). Concomitantly,

Western blots to determine protein levels of cyclins,

cyclin-dependent kinases or intrinsic apoptosis factors in

cells after SAHA treatment (Fig. 5d–f) could not account

for the increased levels of apoptosis in KLF4 depleted

cells. Levels of p53 and p21 were also unaffected by KLF4

status (data not shown). Inhibition of KLF4 consequently

increased apoptosis when treated with SAHA or TSA for

cell lines studied. Knockdown of KLF4 did not increase

apoptosis when cells were left untreated or treated with the

vehicle, DMSO. When KLF4 was exogenously expressed,

apoptosis was reduced in cells treated with SAHA (Fig. 6).

These experiments confirm that KLF4 modulates the

apoptotic response through the extrinsic apoptosis pathway

and strongly suggest an association between KLF4, caspase

cleavage and p57Kip2 expression.

p57Kip2 is a direct target of KLF4 during HDACi

induced apoptosis

Examination of our gene expression data identified several

transcripts that were significantly upregulated during

HDACi induced apoptosis. We observed the expression

levels of the cyclin dependent kinase inhibitor, p57Kip2

(CDKN1C), to strongly correlate with the transcript levels

of KLF4 and the depletion of KLF4 diminished the levels

of p57Kip2 during HDACi induced apoptosis (microarray

analysis, Fig. 5a–c). Based on a previously published study

[21], inhibitors of class I/II HDACs, but not of class III

HDACs significantly induces p57Kip2 in several cell lines,

but the mechanism of this induction was unknown. We

analyzed the promoter region of p57Kip2 to determine

whether it contains putative Sp1 binding sites, suitable for

KLF4 binding. Our analysis showed numerous putative

binding sites very close to the TSS of p57Kip2. We per-

formed chromatin immunoprecipitation (ChIP) in HCT116

cells subject to SAHA treatment: KLF4 antibodies were

used to pull down chromatin fractions bound to KLF4, and

PCR was performed using two set of primers flanking

Fig. 4 Inhibition of KLF4 expression augments HDACi induced

apoptosis. a HCT116 cells were treated with SAHA and RNA was

extracted at the indicated timepoints. mRNA expression levels for

KLF4 was normalized against actin. b Relative mRNA expression

levels of KLF4 in HCT116 cells transfected with NC or KLF4 siRNA

at the indicated timepoints after 5 lM SAHA treatment. Expression

levels were normalized to Actin and were performed using qPCR.

Error bars denote one SD. c HCT116 cells were transfected with non-

targeting siRNA (NC siRNA) or KLF4-specific siRNA for 24 h and

then treated with 400 nM TSA. RNA was extracted at the indicated

timepoints after TSA treatment and mRNA levels of KLF4 was

measured by qPCR (normalized to Actin expression). d HCT116 cells

were transfected with non-targeting (NC) or KLF4 siRNA. Twenty-

four hours post transfection, cells were treated with SAHA for 24 and

48 h and FACS analysis was performed to measure percent of

apoptosis. e and f HCT116 cells were transfected with non-targeting

siRNA (NC siRNA) or KLF4-specific siRNA for 24 h and then

treated with SAHA (5 lM) for an additional 24 and 48 h. Cells were

harvested for the analysis of apoptotic cells by FACS using the (e)

Annexin V-FITC apoptosis kit following the manufacture’s protocol

or (f) MitoLight mitochondrial apoptosis Detection Kit from Chem-

icon. The lower right (LR) quadrant of the FACS histograms indicates

the percentage of early apoptotic cells (Annexin V-FITC stained

cells) and the upper right (UR) quadrant indicates the percentage of

late apoptotic cells (Annexin V-FITC ? propidium iodide stained

cells). For the MitoLight mitochondrial apoptosis detection, the upper

right quadrant indicates healthy cells (RED) fluorescence, lower right

quadrant indicates apoptotic cells (GREEN) fluorescence. g Percent-

age of apoptotic Cal-27 cells after 24 and 48 h of 5 lM SAHA

treatment (comparison between cells transfected with NC or KLF4

siRNA. h FACS analysis of NC and KLF4 siRNA transfected

HCT116 cells treated with TSA after 24 and 48 h

c

Apoptosis

123

segments very close to the TSS of p57Kip2. PCR showed

that treatment with SAHA results in increased binding of

KLF4 to the Sp1-binding region located around the TSS of

p57Kip2 compared to untreated controls (Fig. 7a). This

suggests that in response to SAHA treatment, KLF4

binds to the p57Kip2 promoter. To determine if KLF4

regulates the transcription of p57Kip2, we cloned the

500 bp region upstream and around the TSS of p57Kip2 into

0102030405060708090

NC siRNA KLF4 siRNA

%of

cells

i nS

ub-G

1

SAHA-24h

SAHA-48h

subG1-35.3%

subG1-41.4%

subG1-62.9%

subG1-82.9%

SAHA-24h

NC

siR

NA

KLF

4si

RN

A

SAHA-48h

subG1-35.3%

subG1-41.4%

subG1-62.9%

subG1-82.9%

SAHA-24h

NC

siR

NA

KLF

4si

RN

A

SAHA-48hTSA-48h

subG1-49.4% subG1-75.9%

subG1-59.0% subG1-91.1%

NC

siR

NA

KL F

4si

RN

A

TSA-24h TSA-48h

subG1-49.4% subG1-75.9%

subG1-59.0% subG1-91.1%

NC

siR

NA

KL F

4si

RN

A

TSA-24h

0102030405060708090

100

NC siRNA KLF4 siRNA

%o

f cel

lsin

Su

b- G

1 TSA-24h

TSA-48h

SAHA (5uM)

0

50

100

150

200

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350

400

0 2 4 8 12 24h

KLF

4m

RN

Aex

pres

s ion

(%)

0

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400

0 2 4 8 12 24h

KLF

4m

RN

Aex

pres

s ion

(%)

400nM TSA

0

20

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120

140

160

180

200

0 6 12 24h

KLF

4m

RN

Aex

pres

sion

(%) NC siRNA

KLF4 siRNA

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180

200

0 6 12 24h

KLF

4m

RN

Aex

pres

sion

(%) NC siRNA

KLF4 siRNA

400nM TSA

0

50

100

150

200

250

300

0 6 12 24hrs

KL

F4

mR

NA

exp

ress

i on

leve

l(%

)

NC siRNA

KLF4 siRNA

5uM SAHA

0

50

100

150

200

250

300

0 6 12 24hrs

KL

F4

mR

NA

exp

ress

i on

lev e

l(%

)

NC siRNA

KLF4 siRNA

5uM SAHA

UR 9.0%

LR 26.5%

UR 49.9%

LR 12.4%

5uM SAHA-24hrs 5uM SAHA-48hrsN

CsiR

NA

UR 13.9%

LR 36.5%

UR 57.4%

LR 22.1%

KL

F4

siR

NA

SAHA (5uM)-24h SAHA (5uM)-48h

NC

siR

NA

KL

F4

siR

NA

subG1-43.6%

subG1-61.6%

subG1-84.1%

subG1-91.3%

0102030405060708090

100

NC siRNA KLF4 siRNA

%o

fcel

lsin

Su

b-G

1

SAHA-24h

SAHA-48h

A B C

D

G H

E F

Apoptosis

123

pGL4-luciferase. There was a strong induction of luciferase

activity upon SAHA treatment versus that of the DMSO

control (Fig. 7b). To further substantiate our findings we

exogenously expressed KLF4 together with the p57Kip2

promoter construct. An increase in luciferase activity in

KLF4 over-expressing cells was observed when compared

to cells transfected with an empty vector (Fig. 7c). Exog-

enously expressed KLF4 had no effect on promoter activity

in the absence of SAHA (Fig. 7c), suggesting an activating

mechanism is needed for KLF4 protein to bind to the

p57Kip2 promoter. Altogether, these experiments show

that upon SAHA treatment, KLF4 binds to the promoter of

p57Kip2 and transcriptionally activates it expression.

p57Kip2 suppresses activation of the MAPkinase

pathway and apoptosis during HDACi induced

apoptosis

To investigate the significance of p57Kip2 in cells under-

going HDACi induced apoptosis, we depleted p57Kip2 in

NC siRNA KLF4 siRNA

cleaved caspase 9

cleaved caspase 3

cleaved PARP

cleaved caspase 7

cleaved caspase 6

p57

Actin

57 kDa

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

cleaved caspase 843 kDa41 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24

NC siRNA KLF4 siRNA

cleaved caspase 9

cleaved caspase 3

cleaved PARP

cleaved caspase 7

cleaved caspase 6

p57

Actin

57 kDa

35 kDa

19 kDa17 kDa

20 kDa

18 kDa

89 kDa

45 kDa

cleaved caspase 843 kDa41 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 7

Actin

SAHA 0 24 0 24h

NC siRNA KLF4 siRNA

35 kDa

19 kDa17 kDa

20 kDa

34 kDa

57 kDa p57

Cleaved caspase 9

Cleaved caspase 3

Cleaved caspase 7

Actin

SAHA 0 24 0 24h

NC siRNA KLF4 siRNA

35 kDa

19 kDa17 kDa

20 kDa

34 kDa

57 kDa p57

Actin

SAHA 0 24 0 24h

NC siRNA KLF4 siRNA

35 kDa

19 kDa17 kDa

20 kDa

34 kDa

57 kDa p57

Actin

19 kDa17 kDa

35 kDa

57 kDa

45 kDa

TSA 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Cleaved caspase 9

Cleaved caspase 3

p57kip2

Actin

19 kDa17 kDa

35 kDa

57 kDa

45 kDa

TSA 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Cleaved caspase 9

Cleaved caspase 3

p57kip2

NC siRNA KLF4 siRNA

Bcl-2

Actin45 kDa

Bax

Bik

PUMA

28 kDa

20 kDa

20 kDa

23 kDa

18 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24

NC siRNA KLF4 siRNA

Bcl-2

Actin45 kDa

Bax

Bik

PUMA

28 kDa

20 kDa

20 kDa

23 kDa

18 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24

cyclin A

Actin45 kDa

cyclin D1

67 kDa

36 kDa

cyclin D331 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

cyclin B160 kDa

cyclin A

Actin45 kDa

cyclin D1

67 kDa

36 kDa

cyclin D331 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

cyclin B160 kDaActin45 kDa

Cdk2

Cdc2

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Cdk434 kDa

34 kDa

34 kDa

Actin45 kDa

Cdk2

Cdc2

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Cdk434 kDa

34 kDa

34 kDa

A B

C D

E F

Fig. 5 Inhibition of KLF4 expression reduces p57Kip2 expression

and enhances caspase cleavage upon HDACi treatment. Western blot

analysis of cells transfected with NC or KLF4 siRNA and treated

either 5 lM SAHA or 400 nM TSA for the indicated timepoints. Cells

were harvested and westerns were performed to measure for cleaved

caspases and p57Kip2 in cells treated with a SAHA in HCT116 cells, b

SAHA in Cal-27 cells and c TSA in HCT116 cells. Western blot

analysis of HCT116 cells transfected with NC or KLF4 siRNA to

measure protein levels in the d intrinsic apoptosis pathway e Cyclins

and f cyclin-dependent kinases. Actin was used as a loading control.

All experiments were performed in biological triplicates

Apoptosis

123

HCT116 cells using siRNA-mediated knockdown. We

observed an increase in apoptosis in cells depleted of

p57Kip2 compared to cells treated with a non-targeting

control siRNA (Fig. 8a). This result suggests KLF4 mod-

ulates HDACi induced apoptosis via p57Kip2. To determine

whether the increase in caspase cleavage observed in cells

depleted of KLF4 expression could be recapitulated in cells

depleted of p57Kip2, we performed Western blot analysis

for caspase cleavage. No differences in caspase cleavage

were observed in cells depleted of p57Kip2 treated with

SAHA to those of control cells (data not shown), sug-

gesting an alternative pathway of apoptosis induction via

p57Kip2. In order to establish a significance for p57Kip2

during HDACi induced apoptosis, we considered whether

p57Kip2 had a role in modulating apoptosis via the stress-

activated protein kinase pathway. Based on a previously

published study [22], p57Kip2 physically interacts with and

inhibits c-Jun NH2-terminal kinase/stress-activated protein

0

10

20

30

40

50

60

NC vector KLF4 Induced

%o

fcel

lsin

Su

b-G

1

SAHA-24h

SAHA-48h

SAHA (5uM)-24h SAHA (5uM)-48h

NC

vect

or

KL

F4

SAHA (5uM)-24h SAHA (5uM)-48h

KL

F4

OE

subG1-14.6%

subG1-51.9%

subG1-38.8%

subG1-17.9%

Fig. 6 KLF4 protects cells

from histone deacteylase

inhibitor induced apoptosis.

HCT116 cells were transfected

with empty vector or vector

expressing KLF4 for 24 h and

then treated with SAHA for an

additional 24–48 h. Cells were

harvested for the analysis of

apoptotic cells by FACS.

Experiments were performed in

biological triplicates

Apoptosis

123

kinase (JNK/SAPK) and over-expression of p57KIP2 sup-

pressed UV- and MEKK1-induced apoptotic cell death. To

establish if HDAC inhibitors modulate the SAPK/JNK

pathway through p57Kip2, we depleted HCT116 cells of

p57Kip2 using siRNA mediated knockdown and performed

Western blot analysis to detect changes in phosphorylation

levels for SAPK/JNK and c-Jun. We observed differences

in phosphorylation levels for JNK and c-Jun, with higher

phosphorylation levels seen in cells depleted of p57Kip2

upon SAHA treatment compared to control NC siRNA

treated cells (Fig. 8c). This result was recapitulated in

HCT116 cells depleted of KLF4 (Fig. 8b), strongly sug-

gesting that KLF4 modulates JNK/SAPK activation via

p57Kip2. Results were verified using the Cal-27 and MCF-7

cell lines (data not shown).

Discussion

KLF4 suppresses HDACi induced apoptosis

Previous studies have implicated KLF4 to play a central

role in numerous biological processes, including regulation

of cell growth, proliferation, differentiation and tumori-

genesis. However, the underlying molecular mechanism for

KLF4 in cancer pathogenesis remains largely unclear, with

some controversy of it acting as a tumor suppressor or an

oncogene [14, 15]. The apparent contradiction suggests

KLF4’s role in these pathways as being context dependent.

The evidence that KLF4 induces transformation [16],

prevents senescence [15], is highly expressed in carcino-

mas and breast cancers [17, 18] and our preliminary data

suggesting KLF4 being induced by histone deacetylase

inhibitors prompted us to investigate if KLF4 functions as

an antiapoptotic factor and whether, it serves, in vivo, as an

essential factor for tumor formation and progression.

Our subsequent findings derived from experiments per-

formed in cells where KLF4 was depleted consistently

resulted in an increase in apoptosis when treated with

histone deacetylase inhibitors. Despite the increase in cell

death, most of the key mediators controlling the intrinsic

apoptosis pathways were unaffected. Instead the increased

sensitivity seen was associated with increased levels of

caspase cleavage, suggesting activation of the extrinsic

apoptosis pathway. Indeed, when caspase cleavage was

blocked using a specific inhibitor, the reduction of

0

10

20

30

DMSO SAHA

Arb

itra

ry U

nit

s (

R.L

.U)

0

5

10

15

20

O/E N

EG + D

MSO

O/E N

EG + S

AHA

O/E K

LF4 + D

MSO

O/E K

LF4 + S

AHA

Arb

itar

y U

nit

s (R

LU

)

0

1

2

3

4

5

Cdkn1C Cdkn1C

DMSO

SAHA

Fo

ld E

nri

chm

ent

ove

r Ig

G

A

B C

Fig. 7 KLF4 binds to the promoter of p57Kip2 upon HDACitreatment. a ChIP assay was performed on HCT116 cells after 6 h

of DMSO or SAHA treatment. Sheared chromatin was immunopre-

cipitated using control IgG or KLF4 antibody. qPCR was performed

on enriched DNA using two primer pairs around the p57Kip2

promoter. Fold enrichment was calculated over IgG and normalized

using primers 6 kb upstream of the GAPDH promoter. b HCT116

cells were transfected with pGL4-p57Kip2 and CMV-Bgal vector for

24 h. Post transfection, cells were treated with DMSO or SAHA and

luciferase activity was measured and normalized against b-galacto-

sidase levels. c HCT116 cells were transfected with pGL4-p57Kip2

and CMV-Bgal together with empty vector or vector expressing

KLF4. Twenty-four hours post transfection, cells were treated with

DMSO or SAHA, and luciferase activity was measure and normalized

to Bgal levels. All experiments were performed in biological

triplicates and error bars indicate one SD

Apoptosis

123

KLF4 Caspase cleavage

Apoptosis

p57Kip2

Jnk

P-Jnk

KLF4 Caspase cleavage

Apoptosis

p57Kip2

Jnk

P-Jnk

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Phospho-c-Jun (Ser63)

Phospho-SAPK/JNK (Thr183/Tyr185)

Actin

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA p57 siRNA

Phospho-SAPK/JNK (Thr183/Tyr185)

Phospho-c-Jun (Ser63)

Actin

p5757 kDa

54 kDa46 kDa

54 kDa46 kDa

48 kDa

48 kDa

45 kDa

45 kDa

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA KLF4 siRNA

Phospho-c-Jun (Ser63)

Phospho-SAPK/JNK (Thr183/Tyr185)

Actin

SAHA (5uM) 0 6 12 24 0 6 12 24h

NC siRNA p57 siRNA

Phospho-SAPK/JNK (Thr183/Tyr185)

Phospho-c-Jun (Ser63)

Actin

p5757 kDa

54 kDa46 kDa

54 kDa46 kDa

48 kDa

48 kDa

45 kDa

45 kDa

subG1-46.1% subG1-67.9%

subG1-57.4% subG1-76.3%

SAHA-24h SAHA-48h

NC

siR

NA

p57

siR

NA

subG1-46.1% subG1-67.9%

subG1-57.4% subG1-76.3%

SAHA-24h SAHA-48h

NC

siR

NA

p57

siR

NA

A

B

C

D

Fig. 8 p57Kip2 inhibits

phosphorylation of SAPkinases

and apoptosis after HDACitreatment. HCT116 cells were

transfected with non targeting

siRNA (NC siRNA), p57Kip2 or

KLF4 specific siRNA for 24 h

and then treated with 5 lM

SAHA for the indicated

timepoints. a Percent of

apoptosis after 24 and 48 h of

SAHA treatment in NC and

p57Kip2 siRNA treated cells. bWestern blots for phospho-

SAPK/JNK and phospho-c-Jun

and c Western blots for p57Kip2,

p-SAPK/JNK and p-c-Jun.

Western blots were performed

on lysates extracted from cells

transfected with NC siRNA,

p57Kip2 or KLF4 specific siRNA

and treated with SAHA at the

indicated timepoints. All

experiments were performed in

triplicate. d Model for the role

of KLF4 in histone deacetylase

inhibitor induced apoptosis

Apoptosis

123

apoptosis observed was dramatically reduced, but total

abrogation of apoptosis was not observed. To explain this,

we analyzed our gene expression data and observed a

stalwart relationship between KLF4 expression and the

cyclin dependent kinase inhibitor, p57Kip2 upon HDACi

treatment. We hypothesized that p57Kip2 was a direct target

of KLF4, given that previous reports have suggested Sp1

transcription factors up-regulating p57Kip2 expression dur-

ing HDACi treatment. Analyzing the promoter of p57Kip2,

we found numerous Sp1 binding elements capable of

binding KLF4. Using chromatin immunoprecipitation and

luciferase assays we were able to experimentally prove that

p57Kip2 was indeed a direct target and depends on KLF4

for its expression. To account for the importance of p57Kip2

expression during HDACi induced apoptosis, we studied

the effect of depleting p57Kip2. We observed an increase in

apoptosis, suggesting KLF4 acts as an anti-apoptotic factor

via p57Kip2 expression. Further experimental evidence

strongly suggested that the anti-apoptotic effect of p57Kip2

expression was through the inhibition of activation of the

stress-activated protein kinase pathway. This is in accor-

dance with previous work which suggests that p57Kip2

associates with SAPK/JNK and inhibits it activation. The

model we propose is cartooned in Fig. 8d. On the basis of

our studies we predict that cells that express high levels of

KLF4 show an intrinsically lower sensitivity to apoptosis

induced by histone deacetylase inhibitors.

KLF4 expression has been shown to be highly expressed

in several type of cancers [17, 18], leading us to hypoth-

esize from our observations that KLF4 might play a role in

tumor formation and progression. In an environment of

nutrient deprivation, i.e. within tumors, KLF4 might con-

tribute to tumor cell survival by inhibiting the stress-kinase

and extrinsic apoptosis pathway, and subsequently show a

remarkable tolerance to nutrient deprivation. Equally pro-

vocative is the notion that inhibitors of KLF4 and p57Kip2

could be used in a therapeutic setting to augment tumor

sensitivity to chemotherapeutic drugs. This could be of

significant interest to scientist and clinical researchers in

the field, since cancer cells that express high levels of

KLF4 may be refractory to HDACi treatment. Another

noteworthy observation gained from our study illustrates

that histone deacetylase are not only capable of reactivating

tumor suppressor and proapoptotic genes, but also induce

antiapoptotic genes capable of protecting cells against

HDACi mediated apoptosis. Particular care should be taken

in treating patients with histone deacetylase inhibitors as a

potential serious outcome would be selecting drug resistant

cells with substantially more malignancy.

Acknowledgments This work was supported by the Academic

Research Fund (AcRF), Tier 1 (RG78/07), Ministry of Education,

Singapore, to ZY.

Author contributions N.K. carried out most of the experiments;

C.B.L. assisted with some experiments; M.S.H. and Z.Y. conceived

and designed the project; M.S.H., N.K. and Z.Y. wrote the article. All

authors participated in data analysis. All authors read and edited the

article.

Conflict of interest statement The authors declare no competing

financial interests.

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