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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 Mol Cancer Ther; 12(10) October 2013 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 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 www.aacrjournals.org 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 Mol Cancer Ther; 12(10) October 2013 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 2089 Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418 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 2090 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. 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 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. www.aacrjournals.org Mol Cancer Ther; 12(10) October 2013 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 2091 Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418 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. 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 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 www.aacrjournals.org 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 Mol Cancer Ther; 12(10) October 2013 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 2093 Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418 Murray-Stewart et al. 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 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 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). www.aacrjournals.org Mol Cancer Ther; 12(10) October 2013 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 2095 Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418 Murray-Stewart et al. 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 2096 Mol Cancer Ther; 12(10) October 2013 PG-11047 BENSpm 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 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 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 2097 Published OnlineFirst August 13, 2013; DOI: 10.1158/1535-7163.MCT-13-0418 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 Downloaded from mct.aacrjournals.org on June 18, 2017. © 2013 American Association for Cancer Research. 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. Updated version Supplementary Material Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-13-0418 Access the most recent supplemental material at: http://mct.aacrjournals.org/content/suppl/2013/08/15/1535-7163.MCT-13-0418.DC1 Cited articles This article cites 52 articles, 22 of which you can access for free at: http://mct.aacrjournals.org/content/12/10/2088.full#ref-list-1 E-mail alerts Sign up to receive free email-alerts related to this article or journal. 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