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Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418
Molecular
Cancer
Therapeutics
Cancer Therapeutics Insights
Histone Deacetylase Inhibition Overcomes Drug Resistance
through a miRNA-Dependent Mechanism
Tracy Murray-Stewart1, Christin L. Hanigan1, Patrick M. Woster2, Laurence J. Marton3, and
Robert A. Casero Jr1
Abstract
The treatment of specific tumor cell lines with poly- and oligoamine analogs results in a superinduction
of polyamine catabolism that is associated with cytotoxicity; however, other tumor cells show resistance to
analog treatment. Recent data indicate that some of these analogs also have direct epigenetic effects. We,
therefore, sought to determine the effects of combining specific analogs with an epigenetic targeting agent
in phenotypically resistant human lung cancer cell lines. We show that the histone deacetylase inhibitor
MS-275, when combined with (N1, N11)-bisethylnorspermine (BENSpm) or (N1, N12)-bis(ethyl)-cis-6,7dehydrospermine tetrahydrochloride (PG-11047), synergistically induces the polyamine catabolic enzyme
spermidine/spermine N1-acetyltransferase (SSAT), a major determinant of sensitivity to the antitumor
analogs. Evidence indicates that the mechanism of this synergy includes reactivation of miR-200a, which
targets and destabilizes kelch-like ECH-associated protein 1 (KEAP1) mRNA, resulting in the translocation
and binding of nuclear factor (erythroid-derived 2)-like 2 (NRF2) to the polyamine-responsive element of
the SSAT promoter. This transcriptional stimulation, combined with positive regulation of SSAT mRNA
and protein by the analogs, results in decreased intracellular concentrations of natural polyamines and
growth inhibition. The finding that an epigenetic targeting agent is capable of inducing a rate-limiting step
in polyamine catabolism to overcome resistance to the antitumor analogs represents a completely novel
chemotherapeutic approach. In addition, this is the first demonstration of miRNA-mediated regulation of
the polyamine catabolic pathway. Furthermore, the individual agents used in this study have been
investigated clinically; therefore, translation of these combinations into the clinical setting holds promise.
Mol Cancer Ther; 12(10); 2088–99. 2013 AACR.
Introduction
The naturally occurring polyamines, spermine, spermidine, and putrescine, are essential for cellular growth and
division (1) and, as polycations, they influence cellular
processes such as nucleosome formation, DNA replication, and gene transcription (2–4). Polyamines are typically observed at elevated intracellular concentrations in
proliferating cells, particularly in tumor cells, which readily accumulate polyamine analogs such as (N1, N12)-bis
(ethyl)-cis-6,7-dehydrospermine tetrahydrochloride (PG-
Authors' Affiliations: 1The Sidney Kimmel Comprehensive Cancer Center
at Johns Hopkins University, Baltimore, Maryland; 2Department of Drug
Discovery and Biomedical Sciences at The Medical University of South
Carolina, Charleston, South Carolina; and 3Department of Laboratory
Medicine, University of California, San Francisco, California
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Robert A. Casero, Jr., CRB 1 Room 551, The
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University
School of Medicine, 1650 Orleans Street, Bunting Blaustein Building,
Baltimore, MD 21287. Phone: 410-955-8580; Fax: 410-614-9884; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-13-0418
2013 American Association for Cancer Research.
2088
11047) and (N1, N11)-bisethylnorspermine (BENSpm)
used in current studies (Supplementary Fig. S1). PG11047 is a conformationally restricted version of the antitumor, polyamine mimetic (N1, N12)-bisethylspermine
(BESpm; ref. 5) and has been safely administered in phases
I and II of clinical trials (6). In sensitive cell lines, this class
of analogs rapidly and significantly induces polyamine
catabolism, depletes the natural polyamine pools, increases reactive oxygen species production, and inhibits
growth (7–9). In both in vitro and in human tumor xenograft mouse models of various human cancers, PG-11047
treatment causes significant growth inhibition, resulting
from a dramatic upregulation of polyamine catabolism
and subsequent depletion of the natural polyamines.
However, other cell lines, particularly those derived from
clinically aggressive small-cell lung cancers, show resistance to this induction of polyamine catabolism and
consequently display less growth inhibition following
treatment (5, 7, 10–15).
The mechanism for this superinduction of polyamine
catabolism by the polyamine analogs occurs mainly
through activation of a rate-limiting enzyme, spermidine/spermine N1-acetyltransferase (SSAT). SSAT mRNA
levels are typically expressed at very low levels in the cell,
but can accumulate in the presence of natural polyamines
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Synergistic Polyamine Analog and Epigenetic Therapy
or their analogs (16, 17). We previously discovered that the
nuclear factor (erythroid-derived 2)-like 2 (NRF2 or
NFE2L2) protein plays a role in this regulation. In polyamine analog-sensitive cell lines, NRF2 is constitutively
bound to the polyamine-responsive element (PRE) in the 50
regulatory region of the SSAT gene (18). In the presence of
excess polyamines or their analogs, the NRF2 cofactor
polyamine-modulating factor 1 (PMF1) binds to NRF2,
thereby activating transcription of SSAT (19). However,
in the polyamine analog-resistant H82 cell line used in
the current studies, NRF2 has not been previously detected
in nuclear extracts or at the PRE, consistent with the lack
of SSAT expression observed in these cells either before
or after analog treatment (7, 20). NRF2 function is primarily regulated by kelch-like-ECH-associated protein 1
(KEAP1), which binds to and sequesters NRF2 in the
cytoplasm (21). Inactivation of the KEAP1 protein releases
NRF2, allowing its translocation to the nucleus where it
binds to specific response elements, including the PRE, and
drives gene transcription. Mutations in the KEAP1 gene
that disrupt the KEAP1–NRF2 interaction are frequent in
lung cancers, resulting in the constitutive nuclear localization of NRF2 that is observed in the polyamine analogsensitive cell lines (22).
Histone-modifying enzymes such as histone deacetylases (HDAC) catalyze posttranslational modifications of
specific residues on the N-terminal tails of histone proteins, thereby affecting chromatin structure. The combination of these histone marks at a given promoter, together with DNA methylation, ultimately regulates gene
transcription (23, 24), and tumor cells have been shown
to alter these modifications as a means to evade growth,
repair, and death-control mechanisms (25). These observations, together with the fact that epigenetic changes do
not alter the primary nucleotide sequence of the gene,
suggest the usefulness of strategies reversing these modifications in the treatment of cancer. Several classes of
"epi-drugs" have been developed to target specific modifying enzymes with the goal of restoring the natural
growth-control pathways of tumor cells. Recent studies
have suggested that HDACs play a role in the regulation
of KEAP1, thereby influencing nuclear NRF2 translocation and the transcription of antioxidant response genes,
although the precise mechanism was not determined
(26). A recent study in breast cancer provided evidence
that KEAP1 is negatively regulated by miR-200a, and this
miRNA can be epigenetically activated by HDAC inhibition (27).
In the current study, we investigate the use of the class I
histone deacetylase inhibitor (HDACi) MS-275 (reviewed
in ref. 28) in combination with specific antitumor polyamine analogs in human non–small cell lung cancer
(NSCLC) and small-cell lung cancer (SCLC) cell lines that
typically show low sensitivity to the antitumor effects of
the polyamine analogs. Known clinically as entinostat,
MS-275 was selected for the current studies because of its
oral bioavailability, long half-life, and safe administration
in multiple clinical trials. We sought to determine if, in
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phenotypically resistant human lung cancer cell lines,
transcription of the SSAT polyamine catabolic enzyme
could be enhanced using an HDACi to increase NRF2mediated transcriptional activation. In addition, we investigated whether the HDACi alleviated epigenetic histone
modifications contributing to the low levels of basal SSAT
gene expression. Specifically, based on the studies mentioned earlier, we hypothesized and showed that MS-275
could enhance the transcription of SSAT mRNA via activation of a miR-200a–mediated reduction of KEAP1 protein, leading to increased NRF2 translocation and binding
to the PRE of the SSAT gene. This transcriptional stimulation, when combined with the induction of transcription
provided by the analog and followed by the extensive
posttranscriptional effects of the analog on the SSAT
protein, sensitized these cells to the antitumor effects of
the polyamine analogs.
Materials and Methods
Cell lines, culture conditions, and chemicals
The human anaplastic non–small cell lung carcinoma
cell line, Calu-6 [American Tissue Culture Collection
(ATCC), Manassas, VA] was maintained in RPMI1640
medium containing 9% FBS, penicillin, and streptomycin
at 37 C and 5% CO2. The small-cell lung carcinoma line
NCI-H82 (ATCC) was maintained in RPMI1640 containing
9% bovine calf serum. The cell lines were not authenticated
after receipt from the ATCC. The polyamine analog PG11047 was synthesized by Progen Pharmaceuticals, and
BENSpm was synthesized as previously reported (29). A
stock solution (10 mmol/L) of the HDAC inhibitor MS-275
(Alexis Biochemicals) was prepared in dimethyl sulfoxide
(DMSO), with working dilutions in culture medium. Custom primers for PCR were synthesized by Invitrogen,
Sigma, and Integrated DNA Technologies.
Treatment conditions for analyses of gene expression
and nuclear protein
Calu-6 cells were seeded at 7 105 cells per 25-cm2 flask.
At the appropriate time, flasks were aspirated and
refreshed with medium containing increasing concentrations of PG-11047, BENSpm, and/or MS-275. H82 cells
were seeded at 1.67 106 cells/5 mL medium and treated
with the specified combinations. Cells were incubated at
37 C for 24 or 48 hours, as indicated.
RNA extraction, gene expression, and miRNA
expression studies
For gene re-expression studies using reverse transcription-PCR (RT-PCR), total RNA was extracted using TRIzol
reagent (Invitrogen) according to the provided protocol.
RNA was quantified by spectrophotometry, and cDNA
was synthesized using the SuperScript III First Strand Synthesis System (Invitrogen) with oligo-(dT)20 as the primer.
SYBR green-mediated, real-time PCR was conducted using
primer pairs and annealing temperatures as previously
reported for SSAT (30) and GAPDH (31). The primers used
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Murray-Stewart et al.
to quantify KEAP1 gene expression were 50 -CAACCGACAACCAAGACCCC-30 (sense) and 50 -TCAGTGGAGGCGTACATCAC-30 (antisense). NRF2 gene expression
was determined using the primer pair 50 -ACACACGGTCCACAGCTCATC-30 (sense) and 50 -AATGTGGGCAACCTGGGAGTAG-30 (antisense). The optimum annealing temperature for each primer pair was determined on
cDNA using temperature gradients followed by melt
curve analyses and visualization on 2% agarose gels with
GelStar staining (Lonza) and KODAK Digital Science
Image Analysis Software (Rochester, NY). Amplification
conditions consisted of a five-minute denaturation step
at 95 C, followed by 40 cycles of denaturation at 95 C for
30 seconds, annealing at the optimized temperature for
30 seconds, and extension at 72 C for 30 seconds. SYBR
green SuperMix for iQ was purchased from Quanta
BioSciences. Thermocycling was conducted on BioRad
MyiQ and MyiQ2 real-time PCR detection systems, with
data collection facilitated by the iQ5 optical system
software (Hercules, CA). For each of the quantitative
PCR (qPCR) experiments, samples were analyzed in
triplicate, normalized to the GAPDH reference gene, and
the fold-change in expression was determined relative to
cDNA from untreated cells using the 2DDCt algorithm.
For miRNA expression analysis, one microgram of
TRIzol-extracted RNA was converted to cDNA using the
miScript PCR System (SABiosciences). qPCR was conducted using the miR-200a Primer Assay (SABiosciences)
according to the manufacturer’s recommendations with
amplification of U6 snRNA levels as an internal control.
Analyses of SSAT enzyme activity and intracellular
polyamine concentrations
Calu-6 cells were seeded at a density of 2.1 106 cells
per 75-cm2 flask and allowed to attach for two nights, at
which time the medium was aspirated and replaced with
that containing 0, 5, or 10 mmol/L PG-11047 or BENS, with
or without 1 mmol/L MS-275. Following 24 hours of
incubation, cells were trypsinized, counted, and quickfrozen for analysis. H82 cells were seeded and treated at
5 106 cells/15 mL of medium for 24 or 48 hours.
Measurement of SSAT enzyme activity was done as previously reported (7, 32). Concentrations of intracellular
polyamines were determined by pre-column dansylation
followed by reverse-phase, high-pressure liquid chromatography (HPLC), as previously described (33). For each
assay, total cellular protein was measured using the
method of Bradford (34).
Cell proliferation assays
For 96-hour experiments, Calu-6 cells were seeded at
2.8 105 cells per 25-cm2 flask and allowed to attach overnight. Culture medium was replaced with that containing
the appropriate concentration(s) of PG-11047, BENS,
and/or MS-275. NCI-H82 cells were seeded and treated at
7 105 cells per 5 mL of medium. Following incubation for
96 hours, cells were collected and counted using a BioRad
TC-10 automated cell counter (Calu-6) or hemacytometer
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Mol Cancer Ther; 12(10) October 2013
(H82). Viable cells were determined by their ability to
exclude trypan blue. Cells were quick-frozen and acidextracted lysates were used for HPLC analysis of intracellular polyamine pools as described in the previous section.
Analysis of nuclear and total protein expression
Nuclear protein was harvested from Calu-6 and H82
cells treated with PG-11047, BENS, and/or MS-275 using
NE-PER Nuclear and Cytoplasmic Extraction Reagents
according to the manufacturer’s protocol (Pierce Biotechnology). Total protein was isolated from the same cell
treatments by lysing in buffer containing 25 mmol/L
HEPES, pH 7.9, 150 mmol/L NaCl, 0.5 mmol/L EDTA,
0.1% Triton-X, 10% glycerol, 0.1 mg/mL BSA, 1 mmol/L
DTT, and an EDTA-free protease inhibitor cocktail at 4 C
for 20 minutes. Protein was quantified using the BioRad
DC assay with absorbance measured at 750 nm and
converted to protein concentration using interpolation on
a BSA standard curve.
Nuclear proteins (30 mg per lane) were separated on precast 10% Bis–Tris NuPAGE gels with 1 MES running
buffer (Invitrogen) and transferred onto Immun-Blot
PVDF membranes (BioRad). Blots were blocked for one
hour at room temperature in Odyssey blocking buffer (LICOR), followed by overnight incubation at 4 C with
antibodies specific to NRF2 (H-300, Santa Cruz Biotechnology) and b-actin (Sigma). Blots were then incubated
with species-specific, fluorophore-conjugated secondary
antibodies to allow the visualization and quantification of
immunoreactive proteins using the Odyssey infrared
detection system and software (LI-COR).
For total protein Western blots, proteins were separated
on 10% Bis–Tris NuPAGE gels in 1 MOPS running
buffer and immunoblotting was conducted as described
earlier. The KEAP1 antibody (1:500 dilution) was purchased from Santa Cruz Biotechnology and b-actin was
used as a loading control and for normalization.
Quantitative chromatin immunoprecipitation assays
Calu-6 cells were treated with 0 or 5 mmol/L PG-11047
and/or 1 mmol/L MS-275 for 24 hours. H82 cells were
seeded and treated as indicated for 48 hours. Cells were
cross-linked, resuspended in lysis buffer (6 106 cells/mL),
and sonication was conducted using a Branson sonifier
with an output of 2.5 and a duty cycle of 40% for
10 seconds with 20-second rests, 10 times per sample.
The amount of chromatin in sheared samples was approximated using UV spectrophotometry and adjusted to a
concentration of 100 mg of chromatin per 400 mL of lysis
buffer for each immunoprecipitation (IP). For histone acetylation analysis, an antibody to AcH3K9 (Millipore) was
added to the sheared chromatin and incubated with rotation overnight at 4 C. An antibody to pan histone H3
(Abcam) was used for normalization of the histone modification, and the negative control rabbit immunoglobulin
G (IgG) was from DAKO. To analyze NRF2 occupancy,
an NRF2 antibody (C-20, Santa Cruz Biotechnology) was
used, and results were compared relative to input DNA.
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Synergistic Polyamine Analog and Epigenetic Therapy
sary to sustain cell growth and division, any remaining
natural polyamines are diluted through cell division, and
growth is arrested (39, 40). In addition, the superinduction
of polyamine catabolic enzymes by polyamine analogs in
sensitive cell lines can result in the generation of the
reactive oxygen species hydrogen peroxide, resulting in
apoptotic cell death (38).
In the current study, we initially examined the effects
of PG-11047 exposure on polyamine metabolism in the
Calu-6 cells. The Calu-6 NSCLC cell line responds to
PG-11047 with a modest induction of polyamine catabolism and growth inhibition; however, compared with
the superinduction of SSAT detected in other NSCLC
cell lines previously examined (11), Calu-6 cells are
relatively resistant. PG-11047 treatment of Calu-6 cells
induced small increases in the polyamine catabolic
enzyme SSAT at the levels of mRNA (2-fold) and
enzyme activity (10-fold; Fig. 1A and B). This small
induction of catabolism was not sufficient to completely deplete intracellular polyamines; however, it was
accompanied by a modest decrease in the concentrations of the higher natural polyamines spermine and
spermidine, with accumulation of the analog (Fig. 1B).
Overall, these results are more consistent with those
observed in the phenotypically resistant SCLC lines,
where the antitumor polyamine analogs, including PG11047 and BENS, are incapable of superinducing polyamine catabolism and cause only modest growth inhibition (11, 41).
Relative to the superinduction of catabolism often
observed in cells of NSCLC origin, the low level of SSAT
induction achieved with the polyamine analog alone in
Calu-6 cells suggested this would be a good model in
which to investigate the polyamine catabolic effects of
Protein A and protein G Dynabeads were purchased from
Invitrogen.
SYBR green-mediated, quantitative PCR was conducted on the immunoprecipitated DNA to determine
the presence and quantity of AcH3K9 occupancy spanning the proximal promoter region of the SSAT gene.
Multiple primer sets, the sequences of which are available
upon request, were employed that spanned approximately 350 to þ310 base pairs relative to the transcriptional
start site. A primer pair specific to the PRE, located at
1497 of the transcriptional start site of SSAT, was also
used to quantitatively analyze both AcH3K9 and NRF2
chromatin immunoprecipitation (ChIP) products. All
primer pairs were optimized using melt-curve and agarose gel analyses of annealing temperature gradients with
genomic DNA as the template. Fold enrichment of the
modified histone was determined using the 2DDCt algorithm, with treated cells relative to untreated cells and
normalized to the amount of total H3 protein.
Results and Discussion
PG-11047 and MS-275 stimulate enhanced
catabolism of the natural polyamines in Calu-6
NSCLC cells
The original rationale for the use of structural polyamine analogs in cancer therapy was based on the selfregulatory nature of polyamine metabolism (35, 36), and
PG-11047 has exemplified this ability in tumor cell lines of
multiple origins (11, 37, 38). As a polyamine mimetic, PG11047 uses the polyamine transport system for uptake into
dividing cells, stimulating the catabolism and depletion of
natural polyamines (38). As the synthetic molecule is
incapable of fulfilling the functional requirements neces-
10
8
B
0 µmol/L MS-275
1 µmol/L MS-275
1,600
pmol/mgP/min
Fold SSAT mRNA expression
A
6
4
800
400
0
2
5 µmol/L PG-11047
0
0
5
PG-11047 (µmol/L)
P < 0.05
1,200
1 µmol/L MS-275
Spm
Spd
Put
PG-11047
−
−
10.7 ± 1.9
7.3 ± 1.6
1.2 ± 0.2
0
+
−
−
+
6.8 ± 0.4
8.6 ± 0.7
2.9 ± 0.4 5.24 ± 0.4
1.5 ± 1.5
1.1 ± 0.2
18.1 ± 1.1
0
+
+
3.7 ± 0.4
0.7 ± 0.1
0.8 ± 0.1
18.4 ± 2.9
Figure 1. PG-11047 and MS-275 synergistically induce catalytic activity of SSAT with concurrent decreases in intracellular polyamine pools. Calu-6 cells
treated with PG-11047 and/or MS-275 were analyzed for variations in mRNA (A) and protein levels (B) of the polyamine catabolic enzyme SSAT. Values
in A are expressed as average fold-increases in SSAT mRNA expression over untreated cells and represent three independent experiments with
S.E.M. The histogram in B represents the average SSAT enzymatic activity of at least three independent experiments, expressed in picomoles per
milligram of total protein catalyzed per minute, with error bars indicative of S.E.M. Student's paired t test determined a statistically significant P value of
less than 0.05 for the combination treatment over PG-11047 treatment alone with a confidence interval of 95%. Lysates were further analyzed for
intracellular concentrations of the natural polyamines and the polyamine analog (PG-11047); values are expressed as nanomoles of individual polyamine
per milligram of total cellular protein.
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Murray-Stewart et al.
Synergistic induction of SSAT activity in SCLC cells
To confirm this sensitization by MS-275 to the effects of
polyamine analogs, we used a cell line of small-cell lung
cancer origin, NCI-H82. Well characterized as phenotypically resistant to the induction of SSAT activity, NCI-H82
SCLC cells have extremely low basal levels of SSAT
message and activity and have historically displayed little
response to the antitumor polyamine analogs (7, 45). In
fact, induction of SSAT to a level sufficient to deplete
polyamine pools to growth inhibitory levels has never
been obtained from the endogenous gene in these cells
(45). We examined the effects of combining MS-275 with
PG-11047 in this cell line, as well as the combination of MS275 and BENSpm–an extensively studied polyamine analog known to be one of the most potent inducers of
polyamine catabolism–and found that both combinations
were capable of synergistically inducing SSAT activity.
Treatment with MS-275 alone did not induce transcription
of SSAT in H82 cells as it did in the Calu-6 cells, nor did it
have an effect on SSAT activity (Fig. 2A). However, an
additive induction of SSAT mRNA and a significant
synergistic induction of SSAT enzyme activity was
detected after 48 hours of cotreatment with PG-11047
(Fig. 2A and B). Increasing the concentration of PG-11047
to 10 mmol/L had no additional effect on either mRNA or
supplementing PG-11047 treatment with MS-275. Singleagent treatment with the HDAC inhibitor MS-275 affected polyamine catabolism in Calu-6 cells, as detected by
an induction of SSAT at the level of transcription. After
24 hours, MS-275 induced the expression of SSAT mRNA
by approximately 5-fold that of untreated cells, and
adding PG-11047 enhanced this expression to approximately 7-fold (Fig. 1A). Because of the substantial posttranslational regulation of the SSAT protein by this class
of polyamine analogs (42–44), the combination of PG11047 and MS-275 produced a synergistic increase in
SSAT activity (80-fold) that exceeded the sum of the
activities determined with either agent alone (Fig. 1B).
This synergy was reflected in the corresponding decreases in intracellular polyamine pools, where MS-275
alone had a minor effect and adding it to the PG-11047
treatment enhanced spermine and spermidine depletion
beyond that seen with either agent alone. PG-11047
competes with the natural polyamines for transport
into the cell and was accumulated in equal intracellular
amounts in all treatment groups. That the SSAT gene can
be transcriptionally regulated by a HDAC inhibitor has
not been previously reported and provides a completely
novel strategy for therapeutic exploitation of polyamine
catabolism.
Fold SSAT mRNA expression
A
10
8
0 µmol/L MS-275
0.25 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
6
*
pmol/mgP/min
80
2
0
PG-11047
C
0 µmol/L MS-275
0.25 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
60
40
BENSpm
1,800
* *
20
1,500
1,200
0 µmol/L MS-275
0.25 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
*
*
900
*
600
300
0
0
0
5
PG-11047 (µmol/L)
2092
* *
pmol/mgP/min
100
*
4
no combo
B
* *
Mol Cancer Ther; 12(10) October 2013
0
10
BENSpm (µmol/L)
Figure 2. MS-275 and either PG11047 or BENSpm synergistically
induce the catalytic activity of
SSAT in NCI-H82 SCLC cells. Cells
were treated for 48 hours with PG11047 or BENS and increasing
concentrations of MS-275, alone
or in combination. Real-time,
reverse-transcriptase PCR results
of the SSAT gene are shown in A.
Values are expressed as average
fold-increases in mRNA
expression over untreated cells,
relative to GAPDH, and represent
three independent experiments
with S.E.M. The data in histograms
B and C are derived from the same
experiments and represent the
average SSAT enzymatic activities
following 48-hour exposures to
MS-275 and either PG-11047 (B)
or BENSpm (C). The values
corresponding to treatment with
MS-275 alone are, therefore, the
same in B and C but are plotted
on different y-axis scales to show
the significance of each
combination treatment. Values are
expressed as picomoles per
milligram of total protein catalyzed
per minute, with error bars
indicative of range. , P < 0.05
relative to cells treated with
polyamine analogs without
MS-275.
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Synergistic Polyamine Analog and Epigenetic Therapy
Effects of polyamine analog and MS-275
combination treatments on cell proliferation
The ultimate goal of therapeutic induction of polyamine
catabolism is tumor-specific growth inhibition. As the
H82 cell line displayed the greatest difference in SSAT
activity between the HDACi/BENSpm cotreatment and
either of the single-agent treatments, we used this cell
line and treatment strategy to evaluate the effects of
cotreatment in terms of intracellular polyamine pools and
growth inhibition.
Cotreating H82 cells with MS-275 and BENSpm over 96
hours revealed a dose-dependent decrease in growth rate,
resulting in complete inhibition of growth (N1/N0 ¼ 1) in
the presence of 0.25 mmol/L MS-275 and 5 mmol/L or
more BENSpm (Fig. 3, top). It should be noted that the H82
cells displayed greater sensitivity to the cytotoxic effects of
MS-275, and, thus, concentrations were scaled back
accordingly. Intracellular polyamine pool analysis correlated with growth inhibition and revealed decreasing
concentrations of spermine, spermidine, and putrescine
when supplementing BENSpm treatment with increasing
concentrations of MS-275 (Table 1). H82 cells maintain
much higher basal levels of all three natural polyamines
than do the other cell lines examined, reflecting the low
endogenous polyamine catabolic enzyme levels. Replacing BENSpm with PG-11047 produced similar dosedependent results, but did not culminate in cytostatic
levels over 96 hours, corresponding to the lower level of
SSAT induction and maintenance of higher natural polyamine pools (Fig. 3, bottom).
Alterations in chromatin acetylation following
HDACi/polyamine analog cotreatment are not
responsible for the observed synergy
As polycations, the polyamines are protonated at
physiologic pH and electrostatically interact with negatively charged molecules, including nucleic acids and
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8
No combination
0.05 µmol/L MS-275
0.1 µmol/L MS-275
0.25 µmol/L MS-275
7
6
N1/N0
5
4
3
2
1
0
0.1
1
10
BENSpm (µ
µmol/L)
8
No combination
0.05 µmol/L MS-275
0.1 µmol/L MS-275
0.25 µmol/L MS-275
7
6
5
N1/N0
activity level (data not shown). Most impressively,
cotreatment with BENSpm and MS-275 caused an accumulation of SSAT mRNA over 48 hours that resulted in a
dramatic, synergistic increase in catabolic activity in a
dose-dependent manner (Fig. 2A and C). This is the first
demonstration of significant levels of SSAT activity from
the endogenous SSAT gene in this cell line. Likewise,
similar results were obtained using a second SCLC cell
line, NCI-H69 (data not shown).
It should be noted that PG-11047 and BENSpm display
similar abilities to induce SSAT transcription, both alone
and in combination with MS-275 over the 48-hour period.
It is likely that the well-studied, posttranslational regulatory abilities of BENSpm on the SSAT protein, for example, enzyme stabilization, are more effective than those
of PG-11047, thereby accounting for the more dramatic
increase in enzyme activity (42, 46). These results also
suggest that the main contribution of MS-275 in the
observed SSAT induction is at the level of enhanced
transcription.
4
3
2
1
0
0.1
1
10
PG-11047 (µmol/L)
Figure 3. Effects of combination treatments on growth inhibition and
intracellular polyamine content in NCI-H82 cells. Cells were incubated
with increasing concentrations of BENSpm, PG-11047, and/or MS-275
for 96 hours. Effects on proliferation were determined by trypan blue
exclusion assay of two independent experiments, with each containing at
least duplicate determinations of each treatment condition. Data is
presented as averages of final numbers of live cells remaining after
treatment, divided by initial seeding density (N1/N0), where a value of one
represents cytostasis, and values less than one indicate cytotoxicity.
Error bars indicate range.
certain proteins (47). The analogs, therefore, have the
potential to displace the natural polyamines from their
functional sites, affecting chromatin organization and
gene expression (5). Considering the observed transcriptional changes in the SSAT gene induced by the current
studies and the fact that MS-275, as well as the natural
polyamines and their analogs, are capable of altering
chromatin structure, we investigated the changes in an
acetylated histone H3 modification known to contribute
to an active state of transcription.
Due to the low level of basal SSAT gene expression in
the cell lines studied, we sought to determine if the
increase in histone acetylation induced by MS-275 correlated with changes in local chromatin architecture at
the SSAT promoter following treatment with the
HDACi, either alone or in combination with the polyamine analogs. In response to MS-275, quantitative ChIP
of H82 cells revealed an increase in acetylated H3K9, a
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Table 1. Intracellular polyamine concentrations following 96-hour treatments with the indicated
combinations
Putrescine (nmol/mg protein)
mmol/L MS-275
BENSpm (mmol/L)
0
0.1
1
5
10
0
0.05
0.1
7.51 0.34
8.77 0.06
8.79 0.96
2.56 0.21
0.61 0.87
7.00 0.95
8.28 0.38
10.02 0.61
1.98 0.01
0.90 0.91
7.53 1.43
8.96 0.76
9.45 1.15
1.99 0.06
0.00
0
0.05
0.1
21.84 1.07
22.47 1.48
21.32 0.82
4.16 0.04
2.57 0.28
16.04 0.42
21.09 2.80
14.68 2.04
3.44 0.01
2.55 0.31
17.15 1.16
19.08 1.20
13.92 3.05
3.24 0.02
1.88 0.27
0
0.05
0.1
34.87 0.41
30.58 0.61
23.46 1.25
7.57 0.34
5.28 0.07
26.22 1.87
30.69 1.63
16.03 2.67
5.60 0.15
5.58 0.08
25.73 0.05
27.77 3.14
15.99 3.37
5.00 1.08
4.12 0.38
0
0.05
0.1
0.25
0.00
1.09 0.02
17.52 4.09
42.85 1.89
37.86 1.54
0.00
1.06 0.18
12.73 2.33
33.49 2.48
38.29 1.34
0.00
0.86 0.22
12.53 0.65
33.27 1.04
30.70 1.03
0.00
0.66 0.15
13.07 1.22
33.17 7.59
26.46 2.59
Spermidine (nmol/mg protein)
mmol/L MS-275
BENSpm (mmol/L)
0
0.1
1
5
10
Spermine (nmol/mg protein)
mmol/L MS-275
BENSpm (mmol/L)
0
0.1
1
5
10
BENSpm (nmol/mg protein)
mmol/L MS-275
BENSpm (mmol/L)
0
0.1
1
5
10
0.25
7.08 9.63 9.05 1.38 0.55 1.00
1.08
1.29
0.07
0.55
0.25
15.99 17.40 12.06 2.87 1.35 3.44
3.03
3.25
0.02
0.52
0.25
27.53 23.86 13.81 4.41 3.07 6.28
4.77
3.73
1.94
0.85
NOTE: Data is presented as nanomoles of individual polyamine per milligram of total cellular protein and represent dual determinations
of two independent experiments, range.
chromatin mark associated with transcriptionally active
chromatin, in the region of the SSAT promoter corresponding to 225–124 nucleotides 50 of the transcriptional
start site. However, the addition of PG-11047 or BENSpm
to the treatment lessened these increases to a level
comparable with that detected in untreated cells (Supplementary Fig. S2), suggesting that the mechanism
through which the MS-275-polyamine analog combination is inducing SSAT transcription is not dependent on
its ability to increase histone acetylation. Although occupancy by AcH3K9 was also increased at multiple sites
spanning the SSAT promoter when Calu-6 cells were
treated with MS-275, cotreatment with the analog
returned this enrichment to a near-basal level (Supplementary Fig. S2).
2094
Mol Cancer Ther; 12(10) October 2013
It is clear that adding either of the polyamine analogs to
the HDAC inhibitor treatment affects the histone-modifying abilities of the HDAC inhibitor. One possibility is
that the enhanced depletion of natural polyamines following the synergistic induction of SSAT activity by the
combination HDACi-analog treatment further facilitates
binding of the analog to chromatin, resulting in an altered
accessibility of the chromatin to the modifying enzyme. In
addition, it is possible that the increased abundance of
SSAT protein competes with the histone acetyltransferases for their substrate, acetyl-CoA. Regardless of the
mechanism, it does not appear that histone hyperacetylation at the interrogated sites makes a significant contribution to the increase in transcription that is observed
when combining MS-275 with the polyamine analogs.
Molecular Cancer Therapeutics
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Synergistic Polyamine Analog and Epigenetic Therapy
Changes in NRF2 occupancy at the PRE of SSAT
To confirm that the increased NRF2 in the nucleus of
the MS-275-treated cells is indeed playing a role in the
enhanced transcription of SSAT, we determined the
occupancy of NRF2 at the PRE locus of the SSAT gene
by ChIP analysis (Fig. 4D). NRF2 was not detected at the
PRE of untreated H82 cells. This is consistent with results
from previous studies using electrophoretic mobility
shift assay (EMSA) and DNase I protection analyses
(18). Treating with either of the polyamine analogs
resulted in the detection of low levels of NRF2 at the
PRE, whereas MS-275-treatment substantially increased
the abundance of NRF2 bound to the PRE, consistent with
the increase in global nuclear NRF2 protein observed.
Adding a polyamine analog to the MS-275 treatment had
no additional effect. This is not surprising, as the polyamine analog itself is not known to increase NRF2 binding, but increases the binding of the PMF1 cofactor to
NRF2, further activating transcription. These results
show that treatment with the HDACi MS-275 induces
the translocation of NRF2 into the nucleus, where it binds
Nuclear translocation of NRF2 is enhanced following
HDACi and polyamine analog treatment
One of the ways in which the natural polyamines and
their analogs exert their transcriptional regulatory effects
on polyamine catabolism is through the binding of NRF2
to the PRE of the SSAT gene. Unlike in many of the NSCLC
cell lines that are extremely sensitive to the polyamine
analogs due to constitutive occupancy of the PRE by
NRF2, H82 cells have historically shown little NRF2 presence in the nucleus or at the PRE, even in the presence of
polyamine analog treatment (18). We, therefore, analyzed
the levels of NRF2 mRNA, total cellular protein, and
nuclear protein in these cells. Treatment with MS-275
and/or the polyamine analogs had no effect on NRF2
mRNA expression level or the abundance of total NRF2
protein in the cell (Fig. 4A and B). However, treatment
with MS-275 increased the abundance of NRF2 protein
that was localized in the nucleus, and this increase was
further enhanced by the addition of the polyamine analogs to the treatment, in an MS-275–dose-dependent manner (Fig. 4C).
Fold NRF2 mRNA expression
3
B
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
2
1
0
Fold nuclear NRF2 protein
8
6
3
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
2
1
0
No combo
C
Fold total NRF2 protein
A
PG-11047
No combo
BENSpm
PG-11047
BENSpm
D
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
−
−
−
+
−
−
−
+
−
−
−
+
+
−
+
−
+
+
5 µmol/L PG-11047
10 µmol/L BENSpm
1 µmol/L MS-275
NRF2
4
WCE
IgG
2
0
No combo
PG-11047
BENSpm
Figure 4. Treatment with MS-275 increases NRF2 nuclear localization and occupancy at the PRE of the SSAT gene. NRF2 mRNA (A) and total protein
expression (B) were detected in H82 cells after 48-hour cotreatments. The histogram in B is derived from quantitative Western blots using whole-cell
lysates based on b-actin normalization. C, nuclear localization of the NRF2 protein was determined by quantitative Western blotting of nuclear
extracts, using b-actin for normalization. Western blots were conducted for three independent experiments; histograms represent the average with S.E.M. D,
occupancy by NRF2 at the PRE of SSAT was determined by ChIP after 48 hours of treatment in H82 cells. Data is presented from a representative
experiment repeated three times with similar results. WCE, whole-cell extract (input DNA).
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to the PRE of the SSAT gene and makes it available for
activation by the polyamine analogs in a manner consistent with the constitutive NRF2 binding observed in
sensitive cell lines resulting from mutant KEAP1 proteins
(18, 22).
Another recent study provided evidence that KEAP1
can be negatively regulated by the miR-200a miRNA (27).
Members of the miR-200 family of miRNAs play a critical
role in maintaining the epithelial phenotype and are often
downregulated in cancer (48). In addition, these miRNAs
are frequently the subject of aberrant epigenetic silencing
(49, 50). Eades and colleagues determined that treatment
of breast cancer cells with an HDACi restored the expression of miR-200a, which downregulated KEAP1 mRNA by
binding to its 30 -UTR, ultimately activating NRF2-dependent antioxidant response pathways (27). To determine if
this same mechanism could be responsible for the activation of the NRF2/SSAT pathway observed in our
experiments, we determined the expression levels of
miR-200a following treatment with MS-275 and/or the
polyamine analogs. PCR amplification of miRNA cDNA
using miR-200a-specific primers revealed a very low level
of basal miR-200a that was unchanged with analog exposure. Treatment with MS-275 resulted in an obvious dosedependent increase in miR-200a expression that was not
affected by cotreatment with the polyamine analog
(Fig. 5C). These results suggest that the epigenetic activation of miR-200a by the HDACi likely contributes to the
mechanism responsible for the observed decrease in
KEAP1 mRNA and protein that enables NRF2 nuclear
translocation.
Overall, the results presented here show that the catabolic enzyme SSAT can be transcriptionally regulated by
HDACi treatment reduces KEAP1 expression in
association with the activation of miR-200a
Under normal, unstressed conditions, the adapter protein KEAP1 sequesters NRF2 in the cytoplasm. A recent
study using an in vivo cerebral ischemia mouse model
reported that various HDAC inhibitors, including MS275, could reduce KEAP1 mRNA and protein levels,
thereby enhancing NRF2 translocation to the nucleus
(26). We, therefore, analyzed the mRNA and protein levels
of KEAP1 in H82 cells following 48-hour exposures to MS275 and/or the polyamine analogs. MS-275 treatment
significantly decreased KEAP1 mRNA levels in a dosedependent manner, and adding either of the polyamine
analogs to the treatment had no effect (Fig. 5A). Likewise,
the level of KEAP1 protein in the cell decreased with
increasing concentrations of MS-275, regardless of the
presence of the polyamine analog (Fig. 5B). Therefore,
the decrease in KEAP1 protein resulting from MS-275
treatment releases NRF2 from its cytoplasmic sequestration, allowing it to translocate to the nucleus where it can
bind to specific gene promoter region response elements
and stimulate transcription.
A
B
1.5
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
1.0
*
*
*
0.5
*
*
*
Fold KEAP1 protein
Fold KEAP1 mRNA expression
1.5
0.0
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
1.0
*
0.5
*
*
*
0.0
No combo
PG-11047
Fold miR-200a expression
C
No combo
BENSpm
8
0 µmol/L MS-275
0.5 µmol/L MS-275
1 µmol/L MS-275
6
*
*
*
*
4
*
PG-11047
BENSpm
Figure 5. Treatment with MS-275
decreases expression of KEAP1
and increases miR-200a
expression in H82 cells. KEAP1
mRNA (A) and protein (B)
expression levels were determined
by qRT-PCR and quantitative
Western blot, respectively,
in H82 cells following 48-hour
cotreatments. Expression levels
of miR-200a (C) relative to U6
snRNA were determined after
48-hour cotreatments as
indicated. , P < 0.05 indicates a
statistically significant change
relative to cells treated with the
respective polyamine analog
alone.
*
2
0
No combo
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Mol Cancer Ther; 12(10) October 2013
PG-11047
BENSpm
Molecular Cancer Therapeutics
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Synergistic Polyamine Analog and Epigenetic Therapy
KEAP1 ORF
MRE
AAAA
KEAP1 NRF2
Mature miR-200a
Nucleus
H3
Ac
Ac
H3
H3
NRF2
PRE
SSAT transcription
PMF1
pri-miR-200a transcription
Cellular
sensitization
Ac
MS-275
BENSpm or PG-11047
HDACs
Figure 6. Schematic representation of the transcriptional stimulation of SSAT by MS-275 and polyamine analog cotreatment that results in the sensitization
of resistant cells to the growth-inhibitory effects of polyamine analogs. Before treatment, NRF2 is absent from the PRE. MS-275 inhibits deacetylation of the
miR200a promoter region, enhancing transcription. Mature miR-200a binds the MRE in the 30 UTR of KEAP1 mRNA, destabilizing it. The consequent
decrease in KEAP1 protein releases NRF2, which translocates to the nucleus and binds the PRE. Polyamine analogs increase expression of the NRF2 cofactor
PMF1, which binds NRF2 and further enhances transcription. Subsequent posttranscriptional and -translational regulation of the SSAT mRNA and protein by
the analogs further induce SSAT catabolic activity resulting in polyamine depletion and growth inhibition. MRE, miRNA response element.
an HDACi; in solid tumor cell lines that are typically
resistant to the induction of polyamine catabolism,
cotreating with MS-275 and specific polyamine analogs
sensitizes these cells to the antitumor effects of the analogs, as detected by synergistic increases in SSAT activity
that further deplete intracellular polyamine concentrations and enhance growth inhibition. We show that these
increases in SSAT transcription are not dependent upon
histone acetylation changes at the SSAT gene promoter,
but rather are the result of an increase in NRF2 nuclear
localization resulting from a decrease in KEAP1 protein
mediated by the epigenetic activation of miR-200a (Fig. 6).
Absent from the PRE of untreated H82 cells, NRF2 binds
to the PRE of the SSAT gene in cells treated with MS-275
and poises it for cofactor activation following induction by
the polyamine analogs. The resulting increased SSAT
mRNA then undergoes posttranscriptional regulation by
the polyamine analog as well as SSAT enzyme stabilization, ultimately resulting in enhanced catabolic activity
sufficient to deplete intracellular polyamines and arrest
growth.
Both of the polyamine analogs used in the current study
have been clinically evaluated and were well tolerated by
patients (6, 51, 52). The best responders of these studies
achieved stable disease; however, current knowledge
suggests that the dosing in those trials might not have
been optimal and that combining low doses of the analogs
with other agents may enhance their therapeutic efficacy.
The results provided here represent the first demonstration of synergy between an HDACi and a polyamine
analog and provide the first evidence of the effect of
www.aacrjournals.org
miRNA regulation on the polyamine catabolic pathway.
The sensitization of tumor cells to the effects of a polyamine analog through the use of an epigenetic therapy
targeting polyamine catabolism is a completely novel
chemotherapeutic approach. Furthermore, as most epigenetically related clinical studies have focused on hematologic tumors, the current data are derived from solid
tumor models, using agents already evaluated as single
agents and found to be well tolerated in the clinic. The
results of these studies, therefore, hold great potential for
rapid and effective translation into the clinic.
Disclosure of Potential Conflicts of Interest
L.J. Marton and R.A. Casero have ownership interest in a patent. No
potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: T. Murray-Stewart, C.L. Hanigan, P.M. Woster,
L.J. Marton, R.A. Casero
Development of methodology: C.L. Hanigan, R.A. Casero
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): T. Murray-Stewart
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Murray-Stewart, R.A. Casero
Writing, review, and/or revision of the manuscript: T. Murray-Stewart,
C.L. Hanigan, P.M. Woster, L.J. Marton, R.A. Casero
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Murray-Stewart
Study supervision: R.A. Casero
Grant Support
This work was financially supported by the National Institutes of
Health (CA51085 and CA98454 to R.A. Casero; CA149095 to P.M. Woster)
and the Samuel Waxman Cancer Research Foundation (to R.A. Casero).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
Mol Cancer Ther; 12(10) October 2013
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2097
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Murray-Stewart et al.
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received May 23, 2013; revised July 12, 2013; accepted July 31, 2013;
published OnlineFirst August 13, 2013.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
2098
Nowotarski SL, Woster PM, Casero RA Jr. Polyamines and cancer:
implications for chemotherapy and chemoprevention. Expert Rev Mol
Med 2013;15:e3.
Snyder RD. Polyamine depletion is associated with altered chromatin
structure in HeLa cells. Biochem J 1989;260:697–704.
Balasundaram D, Tyagi AK. Polyamine–DNA nexus: structural ramifications and biological implications. Mol Cell Biochem 1991;100:129–40.
Feuerstein BG, Pattabiraman N, Marton LJ. Molecular mechanics of
the interactions of spermine with DNA: DNA bending as a result of
ligand binding. Nucleic Acids Res 1990;18:1271–82.
Reddy VK, Valasinas A, Sarkar A, Basu HS, Marton LJ, Frydman B.
Conformationally restricted analogues of 1N,12N-bisethylspermine:
synthesis and growth inhibitory effects on human tumor cell lines.
J Med Chem 1998;41:4723–32.
Smith MA, Maris JM, Lock R, Kolb EA, Gorlick R, Keir ST, et al. Initial
testing (stage 1) of the polyamine analog PG11047 by the pediatric
preclinical testing program. Pediatr Blood Cancer 2011;57:268–74.
Casero RA Jr, Celano P, Ervin SJ, Porter CW, Bergeron RJ, Libby PR.
Differential induction of spermidine/spermine N1-acetyltransferase in
human lung cancer cells by the bis(ethyl)polyamine analogues. Cancer
Res 1989;49:3829–33.
Devereux W, Wang Y, Stewart TM, Hacker A, Smith R, Frydman B, et al.
Induction of the PAOh1/SMO polyamine oxidase by polyamine analogues in human lung carcinoma cells. Cancer Chemother Pharmacol
2003;52:383–90.
Pledgie A, Huang Y, Hacker A, Zhang Z, Woster PM, Davidson NE,
et al. Spermine oxidase SMO(PAOh1), not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine
analogue-treated human breast cancer cell lines. J Biol Chem
2005;280:39843–51.
Dredge K, Kink JA, Johnson RM, Bytheway I, Marton LJ. The polyamine analog PG11047 potentiates the antitumor activity of cisplatin
and bevacizumab in preclinical models of lung and prostate cancer.
Cancer Chemother Pharmacol 2009;65:191–5.
Hacker A, Marton LJ, Sobolewski M, Casero RA Jr. In vitro and in vivo
effects of the conformationally restricted polyamine analogue CGC11047 on small cell and non-small cell lung cancer cells. Cancer
Chemother Pharmacol 2008;63:45–53.
Holst CM, Frydman B, Marton LJ, Oredsson SM. Differential polyamine
analogue effects in four human breast cancer cell lines. Toxicology
2006;223:71–81.
Kuo WL, Das D, Ziyad S, Bhattacharya S, Gibb WJ, Heiser LM, et al. A
systems analysis of the chemosensitivity of breast cancer cells to the
polyamine analogue PG-11047. BMC Med 2009;7:77.
Ignatenko NA, Yerushalmi HF, Pandey R, Kachel KL, Stringer DE,
Marton LJ, et al. Gene expression analysis of HCT116 colon tumorderived cells treated with the polyamine analog PG-11047. Cancer
Genomics Proteomics 2009;6:161–75.
Cirenajwis H, Smiljanic S, Honeth G, Hegardt C, Marton LJ, Oredsson
SM. Reduction of the putative CD44þCD24- breast cancer stem cell
population by targeting the polyamine metabolic pathway with
PG11047. Anticancer Drugs 2010;21:897–906.
Fogel-Petrovic M, Shappell NW, Bergeron RJ, Porter CW. Polyamine
and polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME-3M human melanoma cells. J Biol Chem
1993;268:19118–25.
Xiao L, Casero RA Jr. Differential transcription of the human spermidine/spermine N1-acetyltransferase (SSAT) gene in human lung carcinoma cells. Biochem J 1996;313(Pt 2):691–6.
Wang Y, Xiao L, Thiagalingam A, Nelkin BD, Casero RA Jr. The
identification of a cis-element and a trans-acting factor involved in
the response to polyamines and polyamine analogues in the regulation
of the human spermidine/spermine N1-acetyltransferase gene transcription. J Biol Chem 1998;273:34623–30.
Mol Cancer Ther; 12(10) October 2013
19. Wang Y, Devereux W, Stewart TM, Casero RA Jr. Cloning and
characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene. J Biol Chem 1999;274:
22095–101.
20. Casero RA Jr, Mank AR, Xiao L, Smith J, Bergeron RJ, Celano P.
Steady-state messenger RNA and activity correlates with sensitivity to
N1,N12-bis(ethyl)spermine in human cell lines representing the major
forms of lung cancer. Cancer Res 1992;52:5359–63.
21. Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, et al.
Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain.
Genes Dev 1999;13:76–86.
22. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, et al.
Dysfunctional KEAP1–NRF2 interaction in non-small-cell lung cancer.
PLoS Med 2006;3:e420.
23. Lachner M, O'Sullivan RJ, Jenuwein T. An epigenetic road map for
histone lysine methylation. J Cell Sci 2003;116:2117–24.
24. Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:
1074–80.
25. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer - a mechanism
for early oncogenic pathway addiction? Nat Rev Cancer 2006;6:
107–16.
26. Wang B, Zhu X, Kim Y, Li J, Huang S, Saleem S, et al. Histone
deacetylase inhibition activates transcription factor Nrf2 and protects
against cerebral ischemic damage. Free Radic Biol Med 2012;52:
928–36.
27. Eades G, Yang M, Yao Y, Zhang Y, Zhou Q. miR-200a regulates Nrf2
activation by targeting Keap1 mRNA in breast cancer cells. J Biol
Chem 2011;286:40725–33.
28. Piekarz RL, Bates SE. Epigenetic modifiers: basic understanding and
clinical development. Clin Cancer Res 2009;15:3918–26.
29. Bergeron RJ, Neims AH, McManis JS, Hawthorne TR, Vinson JR,
Bortell R, et al. Synthetic polyamine analogues as antineoplastics.
J Med Chem 1988;31:1183–90.
30. Babbar N, Hacker A, Huang Y, Casero RA Jr. Tumor necrosis factor
alpha induces spermidine/spermine N1-acetyltransferase through
nuclear factor kappaB in non-small cell lung cancer cells. J Biol Chem
2006;281:24182–92.
31. Murray-Stewart T, Wang Y, Goodwin A, Hacker A, Meeker A, Casero
RA Jr. Nuclear localization of human spermine oxidase isoforms–
possible implications in drug response and disease etiology. FEBS
J 2008;275:2795–806.
32. Wang Y, Murray-Stewart T, Devereux W, Hacker A, Frydman B,
Woster PM, et al. Properties of purified recombinant human polyamine oxidase, PAOh1/SMO. Biochem Biophys Res Commun 2003;
304:605–11.
33. Kabra PM, Lee HK, Lubich WP, Marton LJ. Solid-phase extraction and
determination of dansyl derivatives of unconjugated and acetylated
polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue.
J Chromatogr 1986;380:19–32.
34. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:248–54.
35. Porter CW, Sufrin JR. Interference with polyamine biosynthesis and/or
function by analogs of polyamines or methionine as a potential anticancer chemotherapeutic strategy. Anticancer Res 1986;6:525–42.
36. Porter CW, Bergeron RJ. Enzyme regulation as an approach to interference with polyamine biosynthesis–an alternative to enzyme inhibition. Adv Enzyme Regul 1988;27:57–79.
37. Mitchell JL, Thane TK, Sequeira JM, Marton LJ, Thokala R. Antizyme
and antizyme inhibitor activities influence cellular responses to polyamine analogs. Amino Acids 2007;33:291–7.
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418
Synergistic Polyamine Analog and Epigenetic Therapy
38. Casero RA Jr, Marton LJ. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug
Discov 2007;6:373–90.
39. Seiler N, Atanassov CL, Raul F. Polyamine metabolism as target for
cancer chemoprevention (review). Int J Oncol 1998;13:993–1006.
40. Seiler N. Pharmacological aspects of cytotoxic polyamine analogs and
derivatives for cancer therapy. Pharmacol Ther 2005;107:99–119.
41. Lima e Silva R, Kachi S, Akiyama H, Shen J, Hatara MC, Aslam S, et al.
Trans-scleral delivery of polyamine analogs for ocular neovascularization. Exp Eye Res 2006;83:1260–7.
42. Coleman CS, Huang H, Pegg AE. Role of the carboxyl terminal MATEE
sequence of spermidine/spermine N1-acetyltransferase in the activity
and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine.
Biochemistry 1995;34:13423–30.
43. Casero RA, Pegg AE. Polyamine catabolism and disease. Biochem J
2009;421:323–38.
44. Libby PR, Bergeron RJ, Porter CW. Structure-function correlations of
polyamine analog-induced increases in spermidine/spermine acetyltransferase activity. Biochem Pharmacol 1989;38:1435–42.
45. Murray-Stewart T, Applegren NB, Devereux W, Hacker A, Smith R,
Wang Y, et al. Spermidine/spermine N1-acetyltransferase (SSAT)
activity in human small-cell lung carcinoma cells following transfection
with a genomic SSAT construct. Biochem J 2003;373:629–34.
46. McCloskey DE, Pegg AE. Properties of the spermidine/spermine N1acetyltransferase mutant L156F that decreases cellular sensitivity to
www.aacrjournals.org
47.
48.
49.
50.
51.
52.
the polyamine analogue N1, N11-bis(ethyl)norspermine. J Biol Chem
2003;278:13881–7.
Feuerstein BG, Basu HS, Marton LJ. Theoretical and experimental
characterization of polyamine/DNA interactions. Adv Exp Med Biol
1988;250:517–23.
Eades G, Yao Y, Yang M, Zhang Y, Chumsri S, Zhou Q. miR-200a
regulates SIRT1 expression and epithelial to mesenchymal transition
(EMT)-like transformation in mammary epithelial cells. J Biol Chem
2011;286:25992–6002.
Tellez CS, Juri DE, Do K, Bernauer AM, Thomas CL, Damiani LA, et al.
EMT and stem cell-like properties associated with miR-205 and miR200 epigenetic silencing are early manifestations during carcinogeninduced transformation of human lung epithelial cells. Cancer Res
2011;71:3087–97.
Vrba L, Jensen TJ, Garbe JC, Heimark RL, Cress AE, Dickinson S, et al.
Role for DNA methylation in the regulation of miR-200c and miR-141
expression in normal and cancer cells. PLoS ONE 2010;5:e8697.
Hahm HA, Ettinger DS, Bowling K, Hoker B, Chen TL, Zabelina Y, et al.
Phase I study of N(1),N(11)-diethylnorspermine in patients with nonsmall cell lung cancer. Clin Cancer Res 2002;8:684–90.
Wolff AC, Armstrong DK, Fetting JH, Carducci MK, Riley CD, Bender
JF, et al. A Phase II study of the polyamine analog N1,N11-diethylnorspermine (DENSpm) daily for five days every 21 days in patients
with previously treated metastatic breast cancer. Clin Cancer Res
2003;9:5922–8.
Mol Cancer Ther; 12(10) October 2013
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2099
Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418
Histone Deacetylase Inhibition Overcomes Drug Resistance
through a miRNA-Dependent Mechanism
Tracy Murray-Stewart, Christin L. Hanigan, Patrick M. Woster, et al.
Mol Cancer Ther 2013;12:2088-2099. Published OnlineFirst August 13, 2013.
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