HDAC4 REGULATES HIF1α LYSINE ACETYLATION AND CANCER ...

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1 HDAC4 REGULATES HIF1 LYSINE ACETYLATION AND CANCER CELL RESPONSE TO HYPOXIA Hao Geng, Chris T. Harvey, Janet Pittsenbarger, Qiong Liu, Tomasz M Beer, Changhui Xue, and David Z. Qian From OHSU Knight Cancer Institute Oregon Health & Science University Portland, Oregon 97239 Running head: HDAC4 regulates HIF1 Address correspondence to: David Z. Qian, PhD, 3303 SW Bond Avenue, CH14R, OHSU, Portland, Oregon 97239, Tel: 503-312-5912, Fax: 503-494-6197, Email: [email protected] Keywords: HDAC4, HIF1 Acetylation, Background: HIF1 is a target of anticancer therapy. Results: Lysines within HIF1 N-terminus are targets of HDAC4 deacetylation. HDAC4 inhibition causes the increase of HIF1 protein acetylation and decrease of protein stability, which lead to the reduction of HIF-1 mediated target gene expressions and activities in cancer cells. Conclusion: A novel HIF1 regulatory mechanism by HDAC4. Significance: HIF-1 can be targeted by HDAC4 inhibition. Hypoxia-inducible factor 1 alpha (HIF1) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism and cancer development. In VHL- null kidney cancer cell lines, we previously reported that HIF1 proteins can be acetylated and inhibited by histone deacetylase inhibitors (HDACi) or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1 protein acetylation and stability. Specifically, the HIF1 protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1 protein stability, in contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19 &21) to arginine within the HIF1 N-terminus reduce protein acetylation, but render the mutant HIF1 protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia - target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1 presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1, cancer cell response to hypoxia, and presents a more specific molecular target of inhibition. Hypoxia-inducible factor 1 alpha (HIF1) is an essential part of the HIF-1 transcriptional complex that regulates gene expressions critical to cellular response and adaptation to hypoxia (1,2). HIF-1 is comprised of an alpha () and a beta () subunit. The transcriptional activity of HIF-1 is primarily determined by the availability of HIF1 (3,4). In addition, transcriptional cofactors including histone acetyltransferases (HATs) such as p300 and histone deacetylases (HDACs) have been reported to interact with and influence HIF-1 activity (5-9). However, the relationship between HIF-1 and these cofactors is not fully understood. http://www.jbc.org/cgi/doi/10.1074/jbc.M111.257055 The latest version is at JBC Papers in Press. Published on September 14, 2011 as Manuscript M111.257055 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on April 2, 2018 http://www.jbc.org/ Downloaded from

Transcript of HDAC4 REGULATES HIF1α LYSINE ACETYLATION AND CANCER ...

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HDAC4 REGULATES HIF1 LYSINE ACETYLATION AND CANCER CELL

RESPONSE TO HYPOXIA

Hao Geng, Chris T. Harvey, Janet Pittsenbarger, Qiong Liu, Tomasz M Beer, Changhui Xue, and

David Z. Qian

From OHSU Knight Cancer Institute

Oregon Health & Science University

Portland, Oregon 97239

Running head: HDAC4 regulates HIF1

Address correspondence to: David Z. Qian, PhD, 3303 SW Bond Avenue, CH14R, OHSU,

Portland, Oregon 97239, Tel: 503-312-5912, Fax: 503-494-6197, Email: [email protected]

Keywords: HDAC4, HIF1Acetylation,

Background: HIF1 is a target of anticancer

therapy.

Results: Lysines within HIF1 N-terminus are

targets of HDAC4 deacetylation. HDAC4

inhibition causes the increase of HIF1 protein

acetylation and decrease of protein stability, which

lead to the reduction of HIF-1 mediated target

gene expressions and activities in cancer cells.

Conclusion: A novel HIF1 regulatory

mechanism by HDAC4.

Significance: HIF-1 can be targeted by HDAC4

inhibition.

Hypoxia-inducible factor 1 alpha (HIF1) is

an essential part of the HIF-1 transcriptional

complex that regulates angiogenesis, cellular

metabolism and cancer development. In VHL-

null kidney cancer cell lines, we previously

reported that HIF1 proteins can be acetylated

and inhibited by histone deacetylase inhibitors

(HDACi) or specific siRNA against HDAC4. To

investigate the mechanism and biological

consequence of the inhibition, we have

generated stable HDAC4 knockdown via

shRNA in VHL-positive normal and cancer cell

lines. We report that HDAC4 regulates HIF1

protein acetylation and stability. Specifically,

the HIF1 protein acetylation can be increased

by HDAC4 shRNA and decreased by HDAC4

overexpression. HDAC4 shRNA inhibits HIF1

protein stability, in contrast, HDAC1 or

HDAC3 shRNA has no such inhibitory effect.

Mutations of the first five lysine residues (lysine

10, 11, 12, 19 &21) to arginine within the

HIF1N-terminus reduce protein acetylation,

but render the mutant HIF1 protein resistant

to HDAC4 and HDACi-mediated inhibition.

Functionally, in VHL-positive cancer cell lines,

stable inhibition of HDAC4 decreases both the

HIF-1 transcriptional activity and a subset of

HIF-1 hypoxia - target gene expression. On the

cellular level, HDAC4 inhibition reduces the

hypoxia-related increase of glycolysis and

resistance to docetaxel chemotherapy. Taken

together, the novel biological relationship

between HDAC4 and HIF1 presented here

suggests a potential role for the deacetylase

enzyme in regulating HIF-1, cancer cell

response to hypoxia, and presents a more

specific molecular target of inhibition.

Hypoxia-inducible factor 1 alpha (HIF1) is an

essential part of the HIF-1 transcriptional complex

that regulates gene expressions critical to cellular

response and adaptation to hypoxia (1,2). HIF-1 is

comprised of an alpha () and a beta () subunit.

The transcriptional activity of HIF-1 is primarily

determined by the availability of HIF1 (3,4). In

addition, transcriptional cofactors including

histone acetyltransferases (HATs) such as p300

and histone deacetylases (HDACs) have been

reported to interact with and influence HIF-1

activity (5-9). However, the relationship between

HIF-1 and these cofactors is not fully understood.

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.257055The latest version is at JBC Papers in Press. Published on September 14, 2011 as Manuscript M111.257055

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Biologically, the HIF1 protein is constantly

synthesized, but rapidly degraded by oxygen under

non-hypoxic conditions. The newly synthesized

HIF1 protein is post-translationally modified by

hydroxylations at proline (P) residues 402 and 564

via oxygen-dependent prolyl hydroxylases (PHDs)

(1). The von Hippel-Lindau (VHL) protein then

binds hydroxylated HIF1, and recruits an E3

ubiquitin ligase complex that targets HIF1 for

26S proteasome mediated degradation (10). There

are also oxygen-independent mechanisms in

HIF1 protein regulation. Hsp90 is a key

molecular chaperon in maintaining HIF1 stability

(11). In contrast, RACK1, Hsp70 and CHIP

promote HIF1 degradation (12,13).

Besides hydroxylation, the HIF1 protein can

also be post-translationally modified by reversible

lysine (K) acetylation (14-16), which can be

pharmacologically modulated by HDAC inhibitors

(HDACi) (16-18). Treating cells with HDACi also

reduces HIF1 on the protein level under

normoxic, hypoxic and hypoxia mimic conditions

(16,19-22). The protein inhibition is dependent on

the 26S proteasomal degradation system, but can

be VHL-independent (16,19). Two of the

acetylation sites within HIF1 protein have been

identified at residues K532 and K674 (14,15).

Functionally, the acetylation at K532 can lead to

VHL-dependent HIF1 protein degradation (14).

In contrast, the acetylation at K674 is required for

HIF-1 transcriptional activity (15).

Currently, the mechanism and the functional

consequence of HIF1 acetylation / deacetylation

at different lysine residues are unclear. While the

identity of HDAC isozymes deacetylating K532 is

unknown (14), the K674 deacetylation is mediated

by a class III HDAC, Sirt1 (15). Previously, we

have shown that the inhibition of class II HDAC

isozymes HDAC4 and HDAC6 via siRNA inhibits

HIF1 protein in VHL-null kidney cancer cell

lines. The HDAC6 siRNA-mediated HIF1

inhibition is thought to be related with the

acetylation of Hsp90, which disrupts the Hsp90

chaperone function toward its client proteins

including HIF1 (16,19). However, the

mechanism for HIF1 inhibition via HDAC4

siRNA is unclear. HDAC4 siRNA does not

increase Hsp90 acetylation, nor disrupts the

interaction between HIF1 and Hsp90/Hsp70

(16). On the other hand, the inhibition of HDAC4,

not HDAC6, increases HIF1 protein acetylation

(16). These results suggest that HDAC4 can

regulate HIF1 protein acetylation and stability.

In this study, we first recapitulated the HIF1

protein acetylation and inhibition by stable

HDAC4 shRNA knockdown in VHL-positive

prostate and liver cancer cell lines. We then

identified that the HIF1 N-terminus lysine

residues are targets of HDAC4, and associated

with HIF1 sensitivity toward HDAC inhibition.

Further, we observed that stable HDAC4

knockdown attenuates cancer cell response and

adaptation to hypoxia in terms of HIF-1 mediated

gene transcriptional upregulation, glycolysis and

chemoresistance.

Cell lines and reagents - Hep3Bc1 is a gift

from Dr. Gregg Semenza at Johns Hopkins

University. It was engineered by stable

transfection of plasmids for hypoxia/HIF-1

response element (HRE)-driving firefly luciferase

reporter (p2.1) and constitutive renilla luciferase

reporter (pSV-renilla) in human liver cancer cell

line Hep3B (23). The prostate cancer C42B cell

line was a gift from Dr. John Isaacs at Johns

Hopkins University. The Hek293 cells were kindly

provided by Dr. Zhengfeng Zhou’s lab at OHSU.

The Hek293T cells were purchased from ATCC.

All cell lines were maintained as previously

described (23,24). Cell culture hypoxic condition

was defined as 1% oxygen, 5% CO2 and 94%

nitrogen. Cycloheximide (CHX), cobalt chloride

(CoCl2), HDACi-SAHA, and MG132 were

purchased from Sigma and Cayman.

Plasmids and transient transfection – Plasmids

encoding for 3×Flag-HIF1, p2.1, and constitutive

pSV-renilla were gifts of Dr. Gregg Semenza at

Johns Hopkins University. Plasmids encoding HA-

HIF1, Flag-HDAC1, and Flag-HDAC4 were

purchased from Addgene (Cambridge, MA).

Transfection was performed using Fugene6

(Roche). All transfections were equalized with

empty vector (Ev) to ensure the same DNA

amount.

Stable shRNA knockdown cell lines – The

pLKO.1-puro vector-based lentiviral transduction

particles containing HDAC1, HDAC3, HDAC4

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and HIF1 shRNA or scramble control were

purchased from Sigma.

Immunoprecipitation (IP) and antibodies – IP

was performed by incubating whole cell lysates

with the primary antibody at 4 degree followed by

incubation with protein A/G plus-agarose beads

(Santa Cruz). For Flag- and HA- based IP, anti-

Flag M2 affinity gel and anti-HA agarose were

purchased from Sigma. The beads were

extensively washed with lysis buffer, and the

associated proteins were eluted and analyzed by

western blots. Key antibodies used for IP and

western blots include anti-HIF1 (R&D Systems),

anti-acetyl-lysine (Ace-K) (Cell Signaling), anti-

Flag (Sigma), anti-HA (Santa Cruz) and anti-

HDAC4 (Cell Signaling).

HIF1 protein half-life measurement using

cycloheximide (CHX) - cells were seeded in 10-cm

culture dishes. HIF1 proteins were induced by

CoCl2 (150 M) for 6 hours. Then, protein

synthesis inhibitor - CHX was added into the

media, and proteins were harvested at the

indicated time points. Western blotting for HIF1

and -tubulin were scanned by LI-COR Odyssey

Fluorescence Scanner, and quantified using LI-

COR odyssey Infrared software.

Site-directed mutagenesis – We used the wild

type 3xFlag HIF1 plasmid (HIF1-wt) as the

template and two-step PCR to generate the mutant

HIF1 (HIF1-mut), which contains the lysine to

arginine mutation at residues K10, K11, K12, K19

and K21. The resulted HIF1-mut plasmid was

sequenced to confirm to mutations.

Real time quantitative RT-PCR – qRT-PCR

was performed as previously described by us (24),

the ΔΔCt method was used to calculate mRNA

transcripts level.

Reporter gene assay - Reporter assays using

Hep3Bc1 were done as described (23). For

transient assays in Hek293, C42B cell lines, cells

were co-transfected by 1.0 g of plasmid p2.1, and

0.1 g of plasmid pSV-renilla. For assays in

Hek293 cells, 20 ng of HIF1-wt or –mut

plasmids were also transfected. 24 hours later,

cells were incubated in normal or hypoxic

incubators overnight and the firefly and renilla

luciferase activities for each sample were

measured as described by the dual-luciferase

activity kit (Promega) (23).

Lactate measurement - Cells were added with

fresh media, cultured under normal and hypoxic

condition for 48 hours. The lactate concentrations

in media were measured with a kit (Biovision),

and all values were adjusted with the total cell

number, and normalized to the value of sh-Scr cell

lines under the normoxic condition.

In vitro deacetylation assay – Hek293T cells

were transfected with plasmid expressing HA-

HIF1 for 48 hours. Then, total HA-HIF1

proteins (including the acetylated and the

unacetylated) were IP-purified using anti-HA

antibodies. Simultaneously, Flag-HDAC1 or

Flag-HDAC4 was overexpressed in HEK293T

cells and IP-purified using anti-Flag M2 agarose.

The agarose beads containing the immunocomplex

of either HDAC4 or HDAC1 were mixed with the

agarose containing HA-HIF1 in deacetylation

buffer (Upstate-Millipore) as described (25,26). At

various time points, the deacetylation was

terminated by adding western blot loading buffer.

The total HA-HIF1 and its acetylation can be

visualized by western blots using anti-HA and

anti-Acetyl-K antibodies.

Statistical analysis - Differences between the

means of unpaired samples were evaluated by the

Student's t-test. P values < 0.05 were considered to

be statistically significant.

RESULTS

HDAC4 regulates HIF1 protein acetylation

and stability – Since most types of cancer cell

have functional VHL, to determine the effect of

HDAC4 inhibition on HIF1 in a VHL-positive

background, we transduced a human prostate

cancer cell line C42B and a variant of human liver

cancer cell line Hep3Bc1 with pseudo-lentivirus

containing shRNA against HDAC4 (sh-HDAC4)

or scramble control (sh-Scr). We incubated the

cells under normal oxygen or hypoxic condition

for 6 hours, and we observed that the HIF1

protein level was significantly reduced in hypoxic

cells of sh-HDAC4 compared to the sh-Scr (Figure

1A). Similar inhibitory results were observed

when we used cobalt chloride (CoCl2) as a

hypoxic mimic (Figure 1B). To measure the

HIF1 acetylation, we treated cells with CoCl2

and the proteasome inhibitor MG132. As

previously seen in the VHL-null cell lines (16), the

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inhibited HIF1 protein due to HDAC4

knockdown can be rescued by MG132 (Figure 1C

IB: HIF1), and there was no significant change in

HIF1 interaction with Hsp90/Hsp70 (data not

shown). Significantly, the HIF1 acetylation level

was higher in the HDAC4 knockdown cells

(Figure 1C IB: Ace-K) than the scramble controls.

To measure the kinetics of HIF1 protein

degradation, we treated sh-HDAC4 and sh-Scr

cells with CoCl2 to stabilize HIF1 protein. Then,

we added cycloheximide (CHX) to arrest protein

synthesis. The degradation rate of existing HIF1

protein was obtained by measuring HIF1 and

tubulin in cell lysates harvested at 0, 15, 30, 60, 90

and 120 minutes after the CHX addition. We

observed that HIF1 protein degraded faster in sh-

HDAC4 cells than in the sh-Scr cells (Figure 1D),

which was confirmed by densitometry of multiple

western blot results (Figure 1E). These data

confirm our ongoing hypothesis that HDAC4

shRNA knockdown causes an increase of HIF1

acetylation and a decrease of HIF1 stability.

Previously we reported that HDAC6 inhibition

can also reduce HIF1 protein level, however,

such inhibition was not associated with HIF1

protein acetylation (16). To further investigate the

differential regulation of HIF1 by HDAC4 and

other HDAC isozymes, similar stable shRNA

knockdown experiments were performed in

Hek293 cells with shRNA against HIF1,

HDAC1, HDAC3, and HDAC4. HDAC4 shRNA

robustly inhibited HIF1 proteins under hypoxic

condition; in contrast, neither HDAC1 nor

HDAC3 shRNA had a significant effect on the

HIF1 protein level (Figure 2A). Previously we

reported that HDAC4 interacts with HIF1 (16).

To further examine the relationship between

HIF1 acetylation level and HDAC4, we

transiently co-overexpressed a hemagglutinin

(HA)-tagged HIF1with either an empty vector or

a Flag-HDAC4 plasmid in Hek293T cells. Co-IP

experiments showed that Flag-HDAC4 interacted

with HA-HIF1 (Figure 2B). Importantly,

HDAC4 overexpression reduced the HA-HIF1

protein basal acetylation (Figure 2B). In addition,

we performed an in vitro deacetylation assay (25)

using overexpressed and IP-purified Flag-HDAC1,

Flag-HDAC4 and HA-HIF1. In the course of 2

hours of deacetylation reaction, we observed that

the level of HA-HIF1 acetylation gradually

decreased by the presence of the HDAC4, but not

by HDAC1 (Figure 2C). These data further

suggest that the HIF1 acetylation level and

protein stability can be regulated by HDAC4.

HDAC4 regulates HIF1 protein stability via

HIF1 N-terminal lysines – To identify the lysine

residues that are deacetylated by HDAC4, we

ectopically overexpressed the full-length Flag-

HIF1 in Hek293 cells with shRNA against

HDAC4 (sh-HDAC4) or scramble control (sh-

Scr). The over expressed Flag-HIF1 proteins

were anti-Flag IP-purified, resolved on SDS-

PAGE gel, digested by trypsin, and subjected to

liquid chromatography – tandem mass

spectrometry analysis (LC-MS/MS) at OHSU

Proteomic Core Facility. MS/MS analysis of the

trypsin-digested Flag-HIF1 peptides did not

reveal a significant change in lysine acetylation

between the sh-Scr and sh-HDAC4 cells. In

repeated experiments, however, trypsin digestion

(cleaves at the un-acetylated lysine) did not

generate a peptide, containing the first 30 amino

acids of HIF1, that was suitable for LC-MS/MS

analysis. There is a cluster of 5 lysine residues

(K10, K11, K12, K19, and K21) within the HIF1

N-terminal amino acids 1-30. We speculated that

this can be a region regulated by HDAC4

deacetylation. Thus we took an alternative

approach. We used the site-directed mutagenesis

to replace all five lysines to arginines in the full-

length Flag-HIF1 wild type (HIF1-wt)

construct. We observed that the acetylation level

of the resulted HIF1 mutant (HIF1-mut) was

significantly lower than HIF1-wt (Figure 3A).

This differential protein acetylation level was also

associated with differential protein stability in the

context of HDAC4. Co-overexpression of HDAC4

with either the HIF1-wt or HIF1-mut showed

that HDAC4 decreased the protein acetylation of

HIF1-wt, but not the HIF1-mut (Figure 3B). On

the other hand, HDAC4 overexpression increased

the protein level of HIF1-wt, but not the HIF1-

mut (Figure 3B). When we overexpressed the

HIF1-wt and –mut constructs in Hek293 cells

with stable sh-Scr or sh-HDAC4, under both

normal and hypoxic mimic conditions, we

observed that HIF1-wt (high in acetylation) was

inhibited by HDAC4 shRNA knockdown

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compared to sh-Scr (Figure 3C). This was

consistent with the results in Figure 2A. In

contrast, HIF1-mut (low in acetylation) was

resistant to the HDAC4 shRNA knockdown

(Figure 3C). Further, we overexpressed the wild-

type or mutant Flag-HIF1 construct in Hek293T

cells, and treated cells with solvent or HDACi-

SAHA. Under both normal and hypoxic mimic

conditions, the HIF1-wt was sensitive to HDACi;

in contrast, the HIF1-mut was resistant to

HDACi-induced protein degradation (Figure 3D).

These data show that the acetylation and stability

of HIF1 can be regulated by HDAC4 via N-

terminal lysines.

HDAC4 regulates HIF-1 protein activity via

HIF1 N-terminal lysines – Using a hypoxia/HIF-

1 response element (HRE)-driving luciferase

reporter (p2.1) assay (23), we also compared the

transcriptional activity of HIF1-wt and HIF1-

mut under non-hypoxic condition, where the

endogenous HIF1 protein is inhibited by oxygen

and does not confound the reporter assay result.

We overexpressed either the HIF1-wt or HIF1-

mut in Hek293 cells along with a firefly luciferase

reporter gene plasmid (p2.1) under the control of

HRE and a constitutive renilla luciferase gene.

The firefly activity can be robustly induced by

HIF1 or hypoxia stimulation. The renilla activity

can be used as a control for equal transfection and

cell density(23). We observed that the HIF1-mut

had a higher transcriptional activity than HIF1-

wt (Figure 4A). Co-overexpression with HDAC4

significantly enhanced the HIF1-wt activity, but

had no effect on HIF1-mut (Figure 4A). This is

consistent with the HIF1 protein level regulation

by HDAC4 in Figure 3B, in which HIF1-wt

protein level was lower than HIF1-mut, but can

be upregulated by HDAC4 overexpression. We

also tested the transcriptional activity of HIF1-wt

and HIF1-mut in HDAC4 shRNA knockdown

conditions by performing reporter gene assay in

Hek293 cells with sh-Scr or sh-HDAC4. Similar to

the protein results in Figure 3C, we observed that

the HIF1-wt transcriptional activity was

significantly reduced in the presence of HDAC4

shRNA knockdown (Figure 4B). In contract, the

HIF1-mut had a higher activity than HIF1-wt,

and was not significantly affected by HDAC4

shRNA (Figure 4B).

The effect of HDAC4 knockdown on HIF-1

activity in cancer cells - To measure the effect of

HDAC4 shRNA knockdown on HIF-1

transcriptional activities in cancer cell lines, we

performed the luciferase reporter assay using

Hep3Bc1 sh-Scr and sh-HDAC4 cells. Previously,

Hep3Bc1 cells had been stably co-transfected with

the HRE-firefly luciferase reporter gene plasmid

(p2.1) and the constitutive renilla luciferase gene

(23). We observed that the sh-Scr cells exhibited

very robust firefly reporter activity due to hypoxia,

in contrast, the sh-HDAC4 cells did not respond to

hypoxia stimulation (Figure 5A). To confirm this,

we transiently co-transfected the HRE-firefly and

constitutive renilla plasmids into C42B cells

containing either stable sh-HDAC4 or sh-Scr, and

performed similar experiments under normal and

hypoxic conditions. Similar to the results in

Hep3Bc1 cell lines, hypoxia upregulated firefly

activity in sh-Scr cells. HDAC4 shRNA

significantly inhibited this upregulation (Figure

5B). Based on these results, we measured the

effect of HDAC4 shRNA on endogenous HIF-1

target gene upregulation in hypoxia by qRT-PCR.

We found that hypoxia significantly upregulated a

subset of HIF-1 target genes including VEGFa,

LDHA, and Glut1 in sh-Scr cell lines, however,

the upregulation was significantly inhibited in the

sh-HDAC4 cells (Figure 5C & 5D).

The effect of HDAC4 knockdown on cellular

response to hypoxia - Based on the reduced

glycolytic gene (LDHA and Glut1) upregulation

by HDAC4 shRNA (Figure 5C), we hypothesized

that HDAC4 shRNA will also affect hypoxia-

induced glycolysis. We measured glycolysis levels

based on the lactate production in the sh-Scr and

sh-HDAC4 cancer cells growing under normal or

hypoxic condition. Indeed, we found that the

lactate levels were significantly increased in the

hypoxic sh-Scr cells, and HDAC4 shRNA

significantly reduced the hypoxia induced lactate

production (Figure 6A). Next, in cell proliferation

assays, we observed that HDAC4 shRNA did not

change the growth rate of the cancer cell lines

under normal oxygen and short-term (48 hours)

hypoxic conditions (Figure 6B). When cells were

cultured in long-term hypoxia (96 hours), C42B

cells didn’t survive regardless the HDAC4 status

(data not shown), but the Hep3Bc1 sh-HDAC4

cells grew significantly slower than the sh-Scr

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cells (Figure 6B). Hypoxia and HIF-1 are

considered as key factors in causing resistance

toward cytotoxic therapies in solid cancer (27,28).

We therefore tested the effect of HDAC4 shRNA

on prostate cancer cell response to docetaxel,

which is a microtubule targeting agent and

standard chemotherapy for late-stage metastatic

prostate cancer patients. We treated C42B sh-Scr

and sh-HDAC4 cells with increasing doses of

docetaxel for 48 hours under normal or hypoxic

condition. Cell viability at each drug concentration

was calculated and adjusted to the solvent control.

We observed that HDAC4 shRNA did not

significantly modify docetaxel efficacy in

normoxic conditions, but significantly inhibited

the hypoxia-induced resistance (Figure 6C).

Since the HIF1 protein plays a pathological

role in human diseases including cancer,

understanding novel mechanisms regulating the

HIF1 and HIF-1 activity is important for

developing more effective and targeted therapies.

In the current study, we determined that a member

of HDACs family – HDAC4 plays an important

role in regulating HIF1 protein N-terminal lysine

acetylation level, stability and HIF-1 activity.

These results provide a mechanistic explanation to

the well-observed phenomena that HDACi can

induce HIF1 protein acetylation, reduce HIF1

protein level, and HIF-1 target gene expression in

cancer cell lines, and angiogenesis in preclinical

mouse xenograft models (16,19,29).

A likely scenario is that the N-terminal lysine

acetylation level of HIF1 protein is in part

regulated by HDAC4. The inhibition of HDAC4

by either HDACi or shRNA causes an

inappropriate increase of HIF1 protein

acetylation (hyperacetylation), which in turn

disrupts the protein stability and leads to the

reduction in HIF1 protein level and HIF-1

activity. In the previous study, we have shown that

the HIF1 protein inhibition due to HDAC4

inhibition requires proteasomal degradation

system, but is independent of VHL, and does not

impact the HIF1 interaction with Hsp70/90 (16).

Currently, the exact molecular detail connecting

the protein acetylation and degradation is

unknown, and warrants further additional studies.

The HIF1 N-terminal sequence is also highly

similar to the HIF2 N-terminus, both are critical

for mediating protein-protein and protein-DNA

interaction. However, we did not observe a

significant HIF2 inhibition in sh-HDAC4 cells.

Based on the novel information regarding the

HIF1 N-terminal lysines and HDAC4 presented

here, we will, in additional studies, investigate in

details whether the N-terminal lysine acetylation

plays a role in HIF1 interaction with DNA and

the additional degradation machinery such as

RACK1, CHIP, and the ubiquitin.

In the current study, the first five lysine

residues in the HIF1 N-terminus collectively

play a pivotal role in HIF1 protein acetylation

and stability in the context of HDAC4. We have

not been able to identify the exact location (s) of

acetylation. Since the single lysine mutants were

not significantly different from the HIF1-wt

(data not shown), it is likely that a combination of

these five lysines (if not all five) is involved.

Based on the results in this and other studies, it

is apparent that multiple lysine residues within the

HIF1 protein can be substrates for multiple

HATs and HDACs. Up to date, HIF1 can be

deacetylated at least at two lysine residues, K532

and K674 by different HDACs (14,15). The K532

deacetylation is likely to be carried out by the

class I and/or II HDACs (14). The K674

deacetylation requires a class III HDAC-Sirt1

(15). In our study, using HIF1 containing point

mutation at K532 or K674 did not indicate a clear

regulation of HDAC4 toward these two lysine

residues (data not shown). It is unclear whether

these regulations occur to HIF1 ubiquitously or

only at specific physiological conditions. Taken

together, these suggest that multiple HDACs are

involved in HIF1 regulation. We speculate that

these specific associations between HDAC

isozymes and deacetylation reflect the biological

complexity and redundancy on HIF-1 regulation.

Further, the acetylation and deacetylation at

different lysine residues within HIF1 can lead to

different biological consequences. Acetylations at

the N-terminus (shown in this study) and at the

oxygen dependent degradation (ODD) domain

(K532) can promote HIF1 protein degradation

and inhibition of downstream HIF-1 activity (14).

In contrast, the acetylation at K674 promotes the

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transcriptional activity of HIF-1 by recruiting the

transcriptional co-factor p300 (15). Recently, it

has been shown that PCAF can act as HIF1

acetylase at K674 (15). Although our primary goal

is to identify the deacetylase of HIF1, we did

observe that endogenous PCAF are well expressed

in all the cell lines, and PACF overexpression

increased HIF1-wt and HIF1-mut acetylation

level. Whether the HIF1 N-terminal lysines are

substrates of PCAF warrants more careful

investigation in additional studies.

In the current study, not all HIF-1 target genes

are downregulated by HDAC4 inhibition. This

suggests that i) the downregulation of HIF1

protein by HDAC4 shRNA is not the only

mechanism responsible for the downregulation of

HIF-1 target gene expression, ii) HDAC4 is only

required for a subset of HIF1 target genes, or iii)

HDAC4 directly regulates a subset of HIF-1 target

genes either independently or in collaboration with

HIF-1. HDAC4 is known to regulate the activity

of transcriptional factors and co-factors via

participating in the formation of transcriptional

complexes including HIF-1 (30-33). Therefore, we

speculate that only a subset of HIF-1 target genes

require HDAC4 as a HIF-1 co-factor to stabilize

HIF1 and to form an effective transcriptional

complex. In HDAC4 knockdown cell lines, the

upregulation of these genes in hypoxia is inhibited.

In addition, the regulation of glycolysis and

cytotoxic stress by HDAC4 in hypoxic cells

suggests a novel role for HDAC4 in cellular

adaptation toward hypoxia. Genetic studies have

shown that HDAC4 regulates cellular proliferation

and differentiation, and is an important

determinant for the development of muscle, bone

and heart (30-32,34). Because the proper

development of these tissues depends on the

availability of oxygen (35), the regulatory role of

HDAC4 in tissue development can potentially

includes the participation in responding and

adapting to the change of oxygen concentrations.

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This work is supported by NIH/NCI grant R01CA149253 01 to D.Z.Q. D.Z.Q. is also a recipient of a

new investigator award from Department of Defense (DOD) Prostate Cancer Research Program.

Abbreviations used: HIF1, hypoxia-inducible factor 1 alpha; HIF1-wt, HIF1 wild type; HIF1-

mut, HIF1-mutant (K to R mutation at K10, 11, 12, 19, 21), HDAC, histone deacetylase; HDAC4,

histone deacetylase isozyme 4; HDACi, histone deacetylase inhibitors; O2, oxygen; HRE, HIF-1 response

element; aa, amino acid; K, lysine; R, arginine; CoCl2, cobalt chloride, IP, immunoprecipitation; IB:

immunoblotting; SAHA, Suberoylanilide Hydroxamic Acid; Glut1, glucose transporter 1; LDHA, Lactate

dehydrogenase A; VEGFa, Vascular endothelial growth factor A; VHL, von Hippel-Lindau; PCAF,

P300/CBP-associated factor.

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Figure 1: HDAC4 regulates HIF1 protein acetylation and stability in cancer cells. (A) Hep3Bc1 cells

with stable sh-Scr (-) or sh-HDAC4 (+) were cultured under normal or hypoxic condition for 6 hours. (B)

C42B sh-Scr and sh-HDAC4 cells were cultured with or without CoCl2 for 6 hours. The HIF1, HDAC4

and tubulin protein levels were measured using whole cell lysates by western blots. (C) Hep3Bc1 sh-Scr

and sh-HDAC4 cells were co-treated with proteasome inhibitor MG132 and CoCl2 for 6 hours. HIF1

was IP-purified (+) from whole cell lysates, and immuno blotted (IB) for HIF1 and its acetylation (ace-

K). (D)Hep3Bc1 sh-Scr and sh-HDAC4 cells were treated with CoCl2 for 6 hours, followed by protein

synthesis inhibitor cycloheximide (CHX) treatment for the indicated times. HIF1 and tubulin protein

levels were measured by western blots. (E) The decay of HIF1 protein level in (D) was quantified by

florescence densitometry. The HIF1 protein band intensity was divided by the tubulin band intensity in

each sample and normalized to the time 0 of CHX treatment. Lines represent mean and standard deviation

of 3 experiments. * P < 0.01, sh-Scr vs. sh-HDAC4 at the indicated time points.

Figure 2: HDAC4 specifically regulates HIF1 proteins. (A) Hek293 cells were transduced with

lentivirus containing shRNA against scramble sequence (sh-Scr), HIF1 (sh-HIF1), HDAC1 (sh-

HDAC1), HDAC3 (sh-HDAC3) or HDAC4 (sh-HDAC4). The indicated cells were cultured in either

normal or hypoxic condition for 6 hours before western blot analysis. (B) An equal amount of HA-HIF1

plasmid was co-overexpressed with either empty vector (-) or Flag-HDAC4 (+). 48 hours after

transfection, whole cell lysates were used for IP with anti-HA antibodies. The level of HA-HIF1 protein

and its acetylation were measured by western blots using anti-HA and anti-acetyl-lysine (Ace-K)

antibodies, respectively. The Flag-HDAC4 was measured by the anti-Flag antibody. (C) The HA-HIF1

plasmid or Flag-HDAC4 or Flag-HDAC1 plasmid was overexpressed in HEK293T cells. 48 hours after

transfection, the HA-HIF1, Flag-HDAC4 and Flag-HDAC1 were IP-purified using anti-HA and anti-

Flag antibodies, respectively. The purified HA-HIF1 was mixed with purified HDAC4 or HDAC1 in

deacetylase buffer for the indicated time in an in vitro deacetylation assay. The resulted proteins were

analyzed by western blotted using anti-HA or anti-ace-K antibodies.

Figure 3: HDAC4 regulates HIF1 protein via HIF1 N-terminal lysines. (A) Flag-HIF1-wt or Flag-

HIF1-mut was overexpressed in Hek293T cells for 48 hours, then HIF1 proteins were IP-purified by

anti-Flag M2 agarose, and analyzed by western blot using anti-HIF1 and anti-ace-K antibodies. (B)

Flag-HIF1-wt and Flag-HIF1-mut were co-overexpressed with Ev (-) or HDAC4 (+) in Hek293T cells.

To control transfection efficiency and specific regulation by HDAC4, all cells were also co-transfected

with a green fluorescent protein (GFP) plasmid. 48 hours after transfection, whole cell lysates (WCL)

were harvested and were analyzed either by western blot using anti-Flag, HDAC4, GFP and tubulin

antibodies or IP-purified by anti-Flag M2 agarose and probed for acetylation. (C) Flag-tagged HIF1-wt

and –mut were overexpressed in Hek293 sh-Scr cells (-) and sh-HDAC4 (+) cells for 48 hours. CoCl2 was

added 4 hours prior to protein harvest to mimic hypoxia. WCL were analyzed by western blots. (D) Flag-

tagged HIF1-wt and –mut were overexpressed in Hek293T cells for 48 hours. 4 hours prior to protein

harvest, CoCl2 was added to mimic hypoxia, and cells were treated with either vehicle (-) or 5 M of

HDACi-SAHA (+). WCL were analyzed by western blot.

Figure 4: HDAC4 regulates HIF-1 transcriptional activity via HIF1 N-terminal lysines. (A) HDAC4,

HIF1-wt, HIF1-mut, or empty vector (Ev) was overexpressed in Hek293 cells as a single agent or in

combination along with HRE-firefly and constitutive renilla luciferase. (B) HIF1-wt, HIF1-mut or Ev

was overexpressed in Hek293 sh-Scr and sh-HDAC4 cells along with HRE-firefly and constitutive renilla

luciferase plasmids. 48 hours later, dual-luciferase activities were measured, and expressed as values

relative to the Ev. *, and ** P < 0.01. All bars represent mean and standard deviation of three

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experiments.

Figure 5: HDAC4 regulates HIF-1 transcriptional activity and target gene expression in cancer cells.

(A) Hep3Bc1 cells containing stable sh-Scr or sh-HDAC4 were cultured under normal and hypoxic

incubators overnight. The ratio of firefly/renilla of all samples was normalized to the value of sh-Scr

under normal condition, and expressed as relative luciferase (luc). * P < 0.001. (B) C42B sh-Scr and sh-

HDAC4 cells were transiently co-transfected with HRE-firefly (p2.1) and constitutive-renilla plasmids.

48 hours after transfection, the transfected cells were cultured under normal or hypoxic condition

overnight. The luciferase activity was measured and calculated as above. ** P < 0.01. (C&D) The

indicated cells were cultured in normal or hypoxia for 24 hours. Selective HIF-1 target gene expressions

were measured by realtime qRT-PCR, and the fold change was expressed as relative values to sh-Scr at

normal condition. # P < 0.01, & P < 0.05. All bars represent mean and standard deviation of three

experiments.

Figure 6: HDAC4 regulates cancer cell response to hypoxia. (A) Lactate levels (indicative of

glycolysis) were measured in sh-Scr and sh-HDAC4 cells in normoxia (n) and hypoxia (h), normalized

with total cell numbers, and expressed as relative fold change to the value of sh-Scr in normoxia. * P <

0.05. (B) Hep3Bc1 sh-Scr and sh-HADC4 cells were cultured under normoxia (n) and hypoxia (h) for 5

days. The viable cells were quantitated at day 1, 3 and 5 of the experiments, and normalized to day 1.

Lines represent mean and standard deviation of three experiments. # P < 0.05, sh-Scr (h) vs. sh-HDAC4

(h) on day 5. (D) C42B sh-Scr and sh-HDAC4 cells were treated with the indicated dose of docetaxel for

48 hours under the normal (n) and hypoxic (h) conditions. For each condition, the viable cells were

counted and compared to the solvent control as % of viability. ** P < 0.05, sh-Scr (h) vs. sh-HDAC4 (h)

at 20 nM and 30 nM of docetaxel. All experiments represent mean and standard deviation of three

experiments.

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Xue and David Z. QianHao Geng, Chris T. Harvey, Janet Pittsenbarger, Qiong Liu, Tomasz M. Beer, Changhui

lysine acetylation and cancer cell response to hypoxiaαHDAC4 regulates HIF1

published online September 14, 2011J. Biol. Chem. 

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