For over 50 years, the fluoropyrimidine class of anticancer agents such as 5-fluorouracil (5-FU) have served as the foundation for the treatment of colon cancer. Chemotherapy-based approaches for the treatment of colon cancer have improved significantly in recent years with 5-FU used in combination with newer agents such as oxaliplatin, irinotecan, cetuximab and bevacizumab. These combinations now demonstrate response rates up to ∼50%.1–3 However, the clinical efficacy of these treatments is hindered due to intrinsic or acquired drug resistance, and novel strategies to overcome resistance are of great clinical importance.
Fluoropyrimidines such as 5-FU and FUdR induce cellular toxicity by inhibiting the enzyme thymidylate synthase (TS) and by incorporating fluoronucleotides into RNA and DNA. The TS enzyme converts dUMP to TMP and serves as the sole de novo source of thymidylate and is essential for DNA synthesis. 5-FU inhibits TS by the formation of a stable ternary complex consisting of the 5-FU metabolite fluorodeoxyuridine monophosphate (FdUMP), the folate cofactor 5,10-methylene tetrahydrofolate, and the TS enzyme leading to thymidylate depletion, cell cycle arrest and apoptosis.1 FUdR is metabolized primarily to FdUMP and has been reported to induce more TS-specific effects when compared to 5-FU. Several clinical studies, conducted by multiple independent laboratories, have demonstrated that elevated TS gene expression is associated with resistance to 5-FU-based therapy in part due to TS overexpression.4–6 Several factors have been shown to contribute to alterations in TS gene expression leading to 5-FU resistance including various polymorphic variations within the TS gene that contribute to differential expression in addition to elevated expression of key regulatory transcription factors such as E2F-1.7–10 Additional studies have shown that TS protein is acutely induced following 5-FU administration due to increased protein stability and translational derepression.11, 12 Despite advances in our understanding of the molecular factors that contribute to the cytotoxicity of 5-FU, ∼50% of patients with advanced colorectal cancer do not respond to current 5-FU-based therapy. Therefore, novel strategies to improve response rates and overcome 5-FU resistance are of great clinical importance.
Histone deacetylase inhibitors (HDACi) have recently emerged as potent and selective anticancer agents. These agents demonstrate pleiotropic anticancer activities by the inhibition of histone deacetylases (HDACs), leading to changes in the acetylation status of both histone and nonhistone proteins including tubulin, Hsp90 as well as multiple transcription factors that can alter protein function.13–15 HDAC inhibition results in modulation of ∼2–10% of genes, promotion of differentiation, inhibition of cell cycle progression, induction of apoptosis and suppression of tumor angiogenesis.16 DNA microarray profiling was used to study the effects of multiple HDACi on gene expression within bladder and breast cancer cell lines. This study identified TS as one of the most heavily downregulated genes following treatment with HDACi.17 Recent reports have extended this initial observation and demonstrated that the HDACi PXD101 has synergistic effects on inhibition of cell growth when combined with 5-FU in colon and gastric cancer cells.18, 19 These authors postulated that HDACi-induced acetylation of Hsp90 destabilized TS protein and that TS mRNA may be downregulated through a transcriptional mechanism.18 Based on these initial observations, we sought to elucidate the mechanistic basis for downregulation of TS mRNA by HDACi within colon cancer cell lines and examine the combinatorial effects of clinically relevant HDACi with fluoropyrimidines on colon cancer cell lines to provide further rationale for the combination of these agents in the clinic.
In this study, we demonstrate that 2 clinically relevant agents of the hydroxamate class of HDACi, vorinostat (suberoylanilide hydroxamic acid, SAHA) and LBH589, cause a potent downregulation of TS gene expression within multiple colon cancer cell lines. Downregulation of TS protein was further demonstrated in vivo in a mouse xenograft model, suggesting that this effect may be achievable by these agents in patient's tumors. We demonstrate that downregulation of TS mRNA by vorinostat and LBH589 occurs through transcriptional repression of the TS gene. Mechanistic studies demonstrate that HDACi abrogate the acute induction of TS following 5-FU treatment and, importantly, enhances the accumulation of dUTP, a key cytotoxic metabolite that is a hallmark of enhanced TS inhibition. Thus, these data provides the first evidence suggesting a direct mechanism-based rationale for the synergy observed between these agents in cell line models. This report further establishes that the combination of HDACi with 5-FU-based regimens represents a viable chemosensitization strategy to overcome TS-mediated resistance.
Material and methods
Compounds and reagents
5-Fluorouracil (5-FU), fluorodeoxyuridine (FUdR), Trichostatin A (TSA) and sodium butyrate (NaByr) were purchased from Sigma (St Louis, MO). Vorinostat was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD). LBH589 was obtained from Novartis Pharmaceuticals (East Hanover, NJ). 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) was obtained from Calbiochem (La Jolla, CA). CellTiter96 AQueous One Solution was obtained from Promega (Madison, WI).
The human colon cancer cell lines SW620, HT29 and RKO were all obtained from the American Type Culture Collection (ATCC, Manassas, VA). The HCT116 p53+/+, HCT116 p53−/− and HCT116 p21−/− cell lines were generous gifts from the laboratory of Dr Bert Vogelstein at Johns Hopkins University (Baltimore, MD). HT29 and all HCT116 cell lines were grown in McCoy's 5A. SW620 cells were grown in Leibovitz media and RKO cells were grown in MEM media. All media was supplemented with 10% fetal bovine serum (Lonza, East Rockland, ME) with penicillin/streptomycin, and sodium pyruvate (Invitrogen, Carlsbad, CA). Cells were maintained in a Forma incubator (Thermo, Waltham, MA) at 37°C with 5% CO2. Mycoplasma screening was performed on all cell lines in this study using the MycoALERT Detection kit (Lonza).
Western blot analysis
Cells were harvested and 50 μg of total protein was analyzed by Western blot as described previously.20 Both the TS and dUTPase antibodies were isolated in our lab using methods described previously.21 Blots were probed with the following antibodies overnight at 4°C affinity-purified anti-TS (1:250), affinity-purified anti-dUTPase (1:250), monoclonal anti-β-actin and β-tubulin (1:4,000) (Sigma, St Louis, MO), anti-TK1 (1:500) and anti-UNG (1:1,500) (Abcam, Cambridge, MA). Secondary antibodies antimouse-HRP and anti-rabbit-HRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Western bands were quantified using Scion Image Software (Scion Corporation, Frederick, MD). Relative expression was determined by normalizing pixel intensity for the TS bands versus the respective β-actin bands and compared to untreated controls.
Quantitative polymerase chain reaction
RNA was isolated using the Trizol method (Invitrogen, Carlsbad, CA), resuspended in nuclease-free water and normalized to 100 ng/μl. cDNA was reverse-transcribed using 200 ng total RNA following the M-MLV RT protocol (Invitrogen, Carlsbad, CA). cDNA was then diluted 1:10 and 2 μl of this stock was used as a template using DyNAmo SYBR Green qPCR Kit (New England BioLabs, Beverly, MA) analyzed using real-time quantitative polymerase chain reaction (qPCR). Real-time qPCR was conducted using the ABI 7500 (Applied Biosystems, Foster City, CA) machine. Primers used for analysis were as follows: TS-upper, 5′-GGA GGA GTT GCT GTG GTT TAT CAA G; TS-lower, 5′-AGG CTG TCC AAA AAG TCT CGG G; 18s-upper, 5′-TTG TTG GTT TTC GGA ACT GAG GC; 18s-lower, 5′-GCA TCG TTT ATG GTC GGA ACT ACG; TS pre-mRNA-upper, 5′-GCA CCC TGT CGG TAT TCG; TS pre-mRNA-lower, 5′-CTC GCA GGA TTG AGG TTA GG. Threshold cycle values (CT) were determined in triplicate for each treatment from 4 independently isolated samples. TS expression levels were determined by normalizing against 18s rRNA expression using the 2 method.22 mRNA expression was presented as a percentage of the untreated control set at 100%. The differences in mRNA expression levels among treatment groups were analyzed for statistical significance using a two-tailed unpaired Student's t-test (Graphpad, San Diego, CA).
dUTP determination assay
HT29 cells were seeded at 1 × 106 cells/plate in a 10-cm plate. Twenty-four-hour postseeding cells were treated with specified concentrations of 5-FU, LBH589 and vorinostat both alone and in combination for 48 hr. Following treatment, cells were washed with PBS and harvested in 60% methanol. Cells were centrifuged at 4,000 rpm at 4°C for 5 min. Supernatant was lyophilized by spinning in a Savant SpeedVac (ThermoScientific, Waltham, MA). Lyophilized pellets containing nucleotides were then analyzed for dUTP/TTP content using a radioactive DNA polymerase-based assay developed by Sherman and Fyfe.23 This assay was further modified to detect levels of TTP and dUTP by preincubating cellular extracts with recombinant dUTPase.20, 24 Data was presented as a percentage of dUTP as a fraction of TTP pools (percent dUTP/TTP) within each extract.
Flow cytometric analysis
HT29 and HCT116 cells were seeded in 6-well dishes at 3 × 105 cells/well. Cells were treated with indicated concentrations of 5-FU, FUdR, LBH589 and vorinostat either alone or in combination for 24 hr as indicated. Both floating and adherent cells were harvested and fixed in 100% ethanol and stained with propidium iodide. DNA content was analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA) and Cellquest Pro software (BD Biosciences, San Jose, CA).
Growth inhibition assay and drug combination analysis
HT29 and HCT116 cells were seeded in 96-well plates at 3 × 103 cells/well in 100 μl of growth media. Cells were treated with the indicated concentrations of 5-FU, LBH589 or vorinostat for 96 hr either alone or in combination as indicated. Following treatment, 10 μl of CellTiter96 AQueous One Solution (Promega) was added to each well and incubated for 4 hr at 37°C with 5% CO2. Absorbance was measured using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA) at 490 nm. Absorbance of treated cells was compared to untreated controls set at 100%. Fraction affected (FA) was calculated from % growth inhibition using the following equation: (100 − % growth inhibition)/100. Combination index (CI) values for drug combinations were calculated to determine the degree of synergy according to the methods of Chou–Talalay using Calcusyn software (Biosoft, Ferguson, MO).25 CI values were interpreted as follows: <1, synergism; 1–1.2, additive; >1.2, antagonism.
In vivo studies
All animal studies were conducted under the approval of the USC Animal Care and Use Committee. For the generation of colon cancer xenografts, 3 × 106 HCT116 cells in a volume of 300 μl of sterile 1× PBS were injected subcutaneously into athymic male BABL/C nu/nu mice (Charles River Laboratories, Wilmington, MA). Following tumor growth to ∼100 mm3, mice were treated with 100 mg/kg/day of vorinostat in 10% DMSO/45% PEG400 or with 10 mg/kg/day LBH589 dissolved in a saline solution with 5%DMSO/1%Tween for 3 days. Mice were sacrificed 6 hr following treatment on the third day, and tumors were excised and snap-frozen in liquid nitrogen or fixed in 10% formalin. Formalin-fixed tumor specimens were paraffin-embedded and subsequently analyzed by immunohistochemistry (IHC) (QualTek Laboratories, Newtown, PA). IHC for TS was conducted as described previously using the TS polyclonal antibody.26 Slides were examined under a microscope at 200× magnification. For Western blot analysis of tumor samples, snap-frozen tumor specimens were processed by grinding, using a motorized pestle in a microfuge tube in 300 μl PBS + protease inhibitor cocktail III (Calbiochem, La Jolla, CA). The lysate was sonicated and centrifuged at 17,000 g for 15 min at 4°C. Cell lysates were then analyzed by Western blot as described above.
Downregulation of TS protein by HDACi in colon cancer cell lines
To investigate whether downregulation of TS in colon cancer cells was a common downstream event following exposure to various HDACi, we treated HCT116 cells with the short-chain fatty acid sodium butyrate (NaByr) or the hydroxamates TSA, vorinostat and LBH589 for 24 hr. As exhibited in Figure 1a, TS protein was significantly downregulated by each of the HDACi tested, suggesting that TS downregulation is a common outcome following HDACi treatment in HCT116 cells (Fig. 1a). To determine the influence of cellular background on TS downregulation by HDACi, a panel of colon cancer cell lines was treated for 24 hr with the clinically relevant HDACi vorinostat and LBH589. The cell lines selected for this analysis were selected based on differences in p53 status, p21 status and mutation of HDAC isozymes. The HDACi vorinostat and LBH589 were chosen for use in subsequent experiments, as both are currently undergoing extensive clinical evaluation. Vorinostat is approved for the treatment of cutaneous T-cell lymphoma, and both vorinostat and LBH589 are currently in clinical trials for a variety of malignancies including hematological and solid tumors. Analysis of protein expression by Western blot and subsequent densitometric analysis demonstrated a significant dose-dependent decrease in TS protein expression by both HDACi in all cell lines examined (Figs. 1b and 1c). This downregulation was determined to be independent of p53 status as demonstrated by the use of the HCT116 p53+/+ and HCT116 p53−/− cells as well as SW620 and HT29 cells that harbor the gain of function p53 R273H mutation (Figs. 1b and 1c).27 Downregulation of TS by HDACi was independent of p21 status, a well-established inducible marker of HDAC inhibition as demonstrated by the use of the HCT116 p21−/− cell line. TS downregulation was also independent of the HDAC2 enzyme as demonstrated by use of the RKO cell line that expresses a mutant nonfunctional HDAC2 enzyme (Figs. 1b and 1c).28 These results indicate that downregulation of TS protein by HDACi occurs within multiple cell lines with various genotypes, suggesting that suppression of TS is a common response to these agents in colon cancer cells. To extend this observation, we tested the ability of HDACi to downregulate TS protein in vivo. Results from these experiments confirm that downregulation of TS was achievable following administration of HDACi in vivo in nude mice bearing HCT116 xenografts (Supporting Information Fig. 1).
Characterization of HCT116 and HT29 colon cancer cell lines
HCT116 and HT29 cells were selected for subsequent analysis based on their relative sensitivities to 5-FU. Growth inhibition analysis indicated that the HCT116 had an IC50(96h) of ∼2.5 μM, whereas HT29 cells were significantly more resistant with an IC50(96h) of 10.5 μM (Fig. 2a). Both cell lines were subsequently evaluated for their sensitivity to FUdR and the HT29 were 2-fold more resistant than the HCT116 cells with IC50(96h) of 2 and 0.98 μM, respectively (Fig. 2a). Both cell lines were analyzed for key enzymes, which are reported to confer resistance to 5-FU including TS, thymidine kinase (TK1), deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG). Interestingly, despite being more resistant to both 5-FU and FUdR, HT29 cells have lower expression of TS and dUTPase when compared to HCT116 cells (Fig. 2b). Expression of the thymidine salvage enzyme TK1 and the base excision repair enzyme UDG appeared similar between both cell lines (Fig. 2b). HCT116 and HT29 cells were subsequently evaluated to determine their sensitivities to both vorinostat and LBH589. Both cell lines appeared to demonstrate similar sensitivities to vorinostat with IC50(96h) of 0.8 and 1.1 μM for the HCT116 and HT29 cells, respectively. HT29 cells appeared modestly less sensitive to LBH589 with an IC50(96h) of 10 nM compared to that obtained in the HCT116 cells of 6 nM (Fig. 2a). To further investigate the toxic effects of these agents in the HCT116 and HT29 cells, analysis of apoptosis was performed using the APO-Direct BrdU detection kit (BD Biosciences, San Jose, CA) following treatment with FUdR, 5-FU, vorinostat and LBH589. Consistent with the growth inhibition analysis, HT29 cells were significantly more resistant to the induction of apoptosis following treatment with both 5-FU and FUdR than the HCT116 (Fig. 2c). Specifically, 2 μM FUdR resulted in a modest increase in 2% cells staining positive for BrdU in HT29 cells, whereas 1 μM FUdR in HCT116 cells resulted in 6% of cells positive for BrdU. HT29 cells demonstrated no significant increase in cells positive for apoptosis following 10μM 5-FU treatment. In contrast, HCT116 cells treated with 2.5 μM 5-FU resulted in 13.2% of cells staining positive for apoptosis (Fig. 2c). Despite both the HT29 and HCT116 cells displaying similar IC50(96h) for both HDACi, HCT116 cells were substantially more susceptible to the rapid onset of apoptosis following both vorinostat and LBH589 treatment with both HDACi inducing apoptosis in 29% of cells compared to HT29 cells with 10 and 12% of cells apoptotic following vorinostat and LBH589 treatment, respectively (Fig. 2c). Based on their differing sensitivities to the fluoropyrimidines, the HCT116 and HT29 cells were utilized for further analysis.
HDACi interact synergistically with TS inhibitors to inhibit cell growth
To investigate potential synergistic interactions between HDACi and fluoropyrimidines in our selected colon cancer models, we investigated the effects of vorinostat and LBH589 in combination with 5-FU on colon cancer cell growth inhibition. HT29 and HCT116 cells were treated with increasing concentrations of 5-FU in combination with increasing concentrations of either vorinostat or LBH589 over a period of 96 hr. The combined drug effect was analyzed using the MTS growth inhibition assay and the methods of Chou and Talalay as outlined in the Material and Methods. To analyze the combination effect of the fluoropyrimidines/HDACi combinations, combination indices (CI) were calculated at each concentration with CI < 1 demonstrating synergy while CI > 1 demonstrating antagonism.25 The data is presented as CI and FA to indicate the level of growth inhibition at each combination index. Potentiation of cell growth inhibition was demonstrated at all concentrations tested in HCT116 cells treated with 5-FU in combination with vorinostat, while significant growth inhibition was only observed at the lower FA values in HT29 cells (Figs. 3a and 3b). CI analysis demonstrated synergistic interactions between both 5-FU and vorinostat at multiple concentrations in HCT116 cells and at lower concentrations in HT29 cells (Figs. 3a and 3b). The combination of LBH589 with 5-FU in HCT116 cells also resulted in enhanced growth inhibition at all combinations tested with CI analysis determining all these to be additive and synergistic interactions. LBH589 in combination with 5-FU also resulted in the potentiation of growth inhibition with modest but significant effects in HT29 cells (Fig. 3b). Data presented here is consistent with previous studies that have demonstrated promising synergistic interactions between TS inhibitors and HDACi in colon and gastric cancer cell lines.18, 19
HDACi repress TS gene transcription
Previous studies have demonstrated that TS mRNA is downregulated by HDACi; however, the mechanistic basis for this downregulation remains unknown. To investigate the mechanistic basis for TS mRNA downregulation, both HCT116 and HT29 cells were treated with increasing concentrations of vorinostat or LBH589 for 24 hr and TS steady-state mRNA levels were determined using quantitative real-time RT-PCR (qPCR) using a primer set spanning an exon–exon boundary. Both vorinostat and LBH589 induced a dose-dependent downregulation of steady-state TS mRNA after 24 hr drug treatment in HCT116 and HT29 cell lines (Figs. 4a and 4b). Specifically, treatment with 10 μM vorinostat and 50 nM LBH589 downregulated TS mRNA to less than 10% of the untreated control at 24 hr (Figs. 4a and 4b). Notably, the downregulation observed with 2 and 10 μM vorinostat and 25 and 50 nM LBH589 persisted for 48 hr (data not shown).
HDACi are known to induce changes in histone acetylation and transcription factor activity, resulting in modulation of gene expression. Based on the observation that TS mRNA is downregulated in colon cancer cells, we postulated that this event may be due to transcriptional regulation or alternatively through TS mRNA destabilization. To address these possibilities, we measured the effect of HDACi treatment on TS pre-mRNA, which provides a close estimate of the transcription rate of the gene of interest.29 To measure newly synthesized TS pre-mRNA transcripts, we used qPCR employing primers that spanned an exon–intron boundary of this gene. As a positive control for TS pre-mRNA downregulation in our experiments, we used the transcriptional inhibitor, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), that inhibits RNA pol II activity and will validate our analysis of pre-mRNA expression. As shown in Fig. 4c, treatment of the cells with the transcriptional inhibitor DRB as well as vorinostat and LBH589 all resulted in a potent downregulation of TS pre-mRNA with ∼75% downregulation observed after a 2-hr exposure. At 24-hr post-DRB treatment, mature TS mRNA downregulation persisted with only 25% of control levels remaining, confirming its effect as a transcriptional inhibitor and demonstrating that our observations with the TS pre-mRNA extend to the mature mRNA. Treatment with vorinostat and LBH589, however, reduced mature TS mRNA to almost undetectable levels (Fig. 4c). These data indicate that treatment with HDACi results in the downregulation of newly synthesized pre-mRNA through potent transcriptional repression of the TS gene.
Combining HDACi with fluoropyrimidines enhances cell cycle arrest
It has been reported that exposure of various cancer cell lines to HDACi induces differential cell cycle arrest, either a G1 or G2/M depending on the cell line tested.30, 31 In contrast, treatment of cancer cells with fluoropyrimidines is characterized by an increased number of cells in the G1 and S phases of the cell cycle that is dependent on inhibition of TS.32, 33 As fluoropyrimidines target actively replicating cells, it is critical to understand how combining agents with different cell cycle effects influence each other. To better understand the cell cycle influence of combining HDACi with fluoropyrimidines, we analyzed the cell cycle distribution of HCT116 and HT29 cells following treatment with 5-FU or FUdR alone and in combination with HDACi for 24 hr by using flow cytometric analysis.
Treatment of either cell line with the fluoropyrimidines for 24 hr resulted in the reduction of cells in the G2/M phase of the cell cycle and the increase of G1/S fractions (Fig. 5a). The cell cycle effects of 2 μM vorinostat and 50 nM LBH589 treatment were different, depending on the cell line. In HCT116 cells, a G2/M arrest was observed as indicated by decreased cell numbers in the G1 and S phases of the cell cycle and a two-fold or greater increase in the number of cells in G2/M (Figs. 5a and 5b). In contrast, HT29 cells treated with vorinostat exhibited a G1 arrest along with a decreased cell population in the S and G2/M phases of the cell cycle, whereas LBH589 induced a reduction of cells in G1 and an increase in cells in Sub-G1 (Fig. 5b). Interestingly, LBH589 also resulted in the detection of a small population of cells with DNA content >2N, indicative of mitotic catastrophe in HCT116 cells. Interestingly, the addition of vorinostat to the fluoropyrimidines did not alter the fluoropyrimidine-mediated G1/S arrest and reduction of the G2/M fraction. In both cell lines, when vorinostat was combined with either 5-FU or FUdR, no decrease in the S-phase fraction was observed, but rather there was an overall decrease in the G2/M fraction and modest increase in cell death (Fig. 5a). HCT116 cells treated with LBH589 in combination with either 5-FU or FUdR still retained a significant percentage of cells in G1 unlike and small increases in Sub-G1 were observed. In HT29 cells, a significant percentage of cells were retained in S-phase with the combination than with LBH589 treatment alone (Fig. 5b). Overall, these results would suggest that although the HDACi induce different modes of cell cycle arrests in the 2 colon cancer cell lines, the combination of HDACi and the fluoropyrimidines are effective at inducing cell cycle arrest.
HDACi inhibit fluoropyrimidine-mediated induction of TS protein
A well-characterized resistance mechanism to effective treatment with fluoropyrimidines is the acute induction of TS following treatment.34 The mechanisms leading to 5-FU-mediated TS upregulation include increased protein stability following formation of the ternary complex, and translational derepression.11, 12 This model proposes that, under normal cellular conditions, TS protein binds and sequesters its own transcript resulting in translational repression. However, following 5-FU treatment, the formation of the inhibitory ternary complex causes the TS enzyme to become stabilized and resistant to proteasome-induced degradation as well as causing dissociation of the TS mRNA from the TS protein. This release of the TS mRNA results in translational derepression leading to elevated translation and increased TS protein expression, which can abrogate the effects of TS inhibitors.
To determine if HDACi could abrogate the fluoropyrimidine-mediated acute upregulation of TS, HCT116 and HT29 cells were treated with 5-FU and FUdR alone and in combination with vorinostat or LBH589 and subsequently analyzed by Western blot analysis. Treatment with either HDACi in both cell lines reduced TS expression to nearly undetectable levels. Treatment with 10 μM 5-FU or 2 μM FUdR for 24 hr in either cell line led to the formation of the inhibited ternary complex at 38.5 kDa and continued expression of the free active enzyme at 36 kDa (Fig. 6a). Quantification of total TS protein demonstrated that 5-FU and FUdR treatment led to an ∼1.5- to 2-fold increase in TS protein in both cell lines (Fig. 6a). Interestingly, coincubation of either 5-FU or FUdR with 2 μM vorinostat or 50 nM LBH589 inhibited the acute induction of TS. Expression of free TS protein following coincubation was significantly less than that of the control in both the HCT116 cells and HT29 cells. Importantly, combination treatment resulted in the majority of TS protein detected in the form of the ternary complex relative to uninhibited active TS when compared to fluoropyrimidine treatment alone (Fig. 6a).
To determine if the downregulation of TS mRNA expression also correlates with the inhibition of fluoropyrimidine-mediated induction of TS protein following coincubation with HDACi, we examined TS mRNA expression following single agent and combination treatment after 24 hr. Both HDACi reduced TS mRNA to less than 10% of untreated controls (Fig. 6b). A statistically significant 2-fold upregulation of TS mRNA was observed following treatment with 10 μM 5-FU in both cells lines and with 2 μM FUdR in HCT116 cells (Fig. 6b). In contrast, vorinostat combined with either 5-FU or FUdR resulted in the abrogation of TS mRNA induction and further downregulated TS mRNA to less than 10% of untreated controls in both cell lines (Fig. 6b). When 5-FU or FUdR was combined with LBH589, the downregulation of TS mRNA was even more pronounced to less than 5% of control levels. These data demonstrate that combining the fluoropyrimidines with HDACi does not interfere with TS mRNA downregulation and suggests that this contributes to the mechanistic basis for the abrogation of acute TS induction during coincubation of HDACi with either 5-FU or FUdR.
HDACi enhances the accumulation of the cytotoxic nucleotide intermediate dUTP
To determine if the drug interaction observed between fluoropyrimidines and HDACi is mechanistically linked to TS downregulation, we analyzed alterations in the downstream cytotoxic events following TS inhibition. Well-established events following TS inhibition include the severe depletion of TTP pools, and in some cases, a simultaneous increase in dUTP pool levels.35 The accumulation of dUTP is cytotoxic due to aberrant misincorporation of uracil into DNA followed by iterative uracil–DNA repair and reinsertion of uracil that results in enhanced DNA strand breaks.20, 36
To determine if HDACi could enhance the accumulation of dUTP, HT29 cells were treated with 5-FU alone or in combination with vorinostat or LBH589 for 48 hr, and nucleotides were isolated from cells and measured using a primer/template-based radioactive assay that measures the amounts of dUTP and TTP in the cell.23 In HT29 cells, dUTP pools were undetectable in both the untreated control and cells treated for 48 hr with 2 μM vorinostat (Fig. 7a). Consistent with previous reports, HT29 cells treated with 0.5 μM 5-FU single agent demonstrated a significant accumulation of intracellular dUTP pools (Figs. 7a and 7b). Interestingly, the addition of 2 μM vorinostat caused a significant increase in the total percent accumulation of dUTP within the cell from 33% dUTP accumulation to 56% (Fig. 7a). Interestingly, treatment of HT29 cells with LBH589 alone resulted in the accumulation of 21% dUTP, which was further increased to 61% when LBH589 was combined with 5-FU (Fig. 7b).
Another possible explanation for the increase in dUTP pool accumulation in cells treated with the combination of vorinostat and fluoropyrimidines is the downregulation of the enzyme dUTPase, the sole regulator of intracellular dUTP pools in the cell. To account for this alternative possibility, we performed Western blot analysis using extracts from HT29 cells treated with either HDACi, 5-FU and combinations to determine the effect of HDACi treatment on dUTPase protein expression. Treatment of HT29 cells with vorinostat alone resulted in a modest decrease in dUTPase protein levels at 48 hr with TS levels reduced to almost undetectable levels (Fig. 7a). Treatment with both 5-FU alone and 5-FU in combination with vorinostat resulted in the detection of the TS ternary complex and induced a small increase in dUTPase expression. It would be anticipated that this modest increase in dUTPase protein expression with the combination would prevent accumulation of dUTP; however, exposure of cells to the Vor/5-FU combination demonstrates an increase in dUTP pools indicative of enhanced TS inhibition and pathway blockade. In addition, analysis of TS expression with the 5-FU/LBH589 combination at 48 hr indicated that TS was downregulated to undetectable levels, either as ternary complex or as free unbound enzyme. The depletion of free TS enzyme observed with the LBH589 combination would enhance the metabolic blockade and contribute to the accumulation of the TS substrate dUMP and subsequently dUTP. Overall, these data suggests that downregulation of TS by HDACi leads to enhanced accumulation of dUTP pools despite modest increases in dUTPase expression. These data provide the first direct mechanistic evidence suggesting that the downregulation of TS by HDACi leads to the enhancement of the downstream cytotoxic mechanisms of action of the fluoropyrimidines.
In this study, we investigated the mechanisms governing the downregulation of TS by HDACi and determine the cellular and molecular effects of the HDACi on the metabolic pathways of the fluoropyrimidines to evaluate the therapeutic potential of combining HDACi and fluoropyrimidines in the clinic. Because of the broad use of fluoropyrimidine-based regimens for the treatment of colon, breast and other common malignancies, strategies to improve the efficacy of the fluoropyrimidines is of significant clinical importance.
In this study, we demonstrated that TS protein was downregulated in a dose-dependent manner in multiple colon cancer cell lines by 2 clinically relevant HDACi, vorinostat and LBH589. By the use of tumor-derived and engineered colon cancer cell lines bearing relevant genetic aberrations, it was demonstrated that downregulation of TS was independent of p53, p21 and HDAC2 status. The observation that downregulation of TS by HDACi was observed in all cell lines tested is of particular importance because of the fact that over 50% of colon cancers harbors p53 mutations among other genetic abnormalities.37 We also demonstrate for the first time that the downregulation of TS protein is not just a phenomenon related to in vitro treatment and is achievable in vivo within HCT116 mouse xenografts specifically by LBH589 following a once daily administration (Supporting Information Fig. 1). This observation is of particular importance if an HDACi is to be used as targeted approach to sensitize tumors to 5-FU-mediated cytotoxicity. Pharmacokinetic data from clinical trials following a standard daily dose of vorinostat determined that the half life was in the order of 60–120 min and the maximal serum concentration obtained was 2 μM.38 These observations raise questions as to whether the concentration required to downregulate TS in vivo would be achieved and sustained following treatment with vorinostat based on a once daily administration. However, vorinostat is currently being evaluated in multiple clinical trials using twice and thrice administration daily, which is likely to significantly reduce the troughs in serum concentrations associated with its rapid metabolism and is likely to significantly increase tumor cell exposure. However, the half life of LBH589 was determined to be in the order of 10–14 hr and serum concentrations of 400–700 nM are achievable at well-tolerated doses.39 The increased potency of LBH589 in our cell line models and the more favorable pharmacokinetic properties support our observations in vivo where LBH589 was effective at significantly downregulating TS protein using a once daily administration.
In addition to TS protein expression, both vorinostat and LBH589 induced a dose-dependent downregulation of TS mRNA in HCT116 and HT29 colon cancer cell lines. We provide evidence indicating that HDAC inhibition induces a specific and potent transcriptional repression of the TS gene.38, 40 One of the most exciting findings in this report is the observation that HDACi are able to enhance the downstream mechanistic effects of TS- inhibition. Although it has been shown previously that the HDACi PXD101 is synergistic with 5-FU in vitro and in vivo, there is currently no evidence linking the mechanism between the observed synergy and the molecular consequences of TS downregulation. Here, we demonstrate for the first time that the combination of either vorinostat or LBH589 with the fluoropyrimidine 5-FU results in the abrogation of acute TS induction accompanied with a reduction in the free unbound TS enzyme. Moreover, we observed synergistic drug interactions at various drug concentrations, and increased accumulation of the cytotoxic dUTP pool. Overall, data presented in this report demonstrate that TS downregulation by HDACi is associated with the enhancement of the downstream effects of the fluoropyrimidines that is consistent with the synergistic interactions observed between these agents in colon cancer cells.
The molecular mechanisms governing normal TS expression are complex and reported evidence indicates that TS is regulated at the levels of transcription, mRNA stability, translation and protein stability. This study provides evidence demonstrating that HDACi treatment leads to potent transcriptional repression of the TS gene. The precise mechanism by which HDACi initiate the transcriptional downregulation of TS are unknown. Possible explanations include the acetylation-dependent inactivation of a transcriptional activator or the recruitment or activation of a transcriptional repressor. Previous studies have demonstrated that HDACi can induce transcriptional repression through increased degradation of a transcription factor, such as Hif-1α.41 Alternatively, TS downregulation may be initiated by alterations in chromatin structure in proximity to the TS gene. Although increased histone acetylation is frequently associated with increased transcriptional activity, the opposite effect has also been observed in specific model systems. A recent genome wide analysis of the effects of HDACi on acetylation at transcriptional start sites revealed that HDACi treatment can in fact decrease acetylation at various loci.42 A previous study reported that treatment with the HDACi TSA did not induce alterations in histone 3 or 4 acetylation status in the region of the TS promoter.18 This would suggest that the potent transcriptional suppression of TS observed in our study may be a consequence of the altered acetylation status of components of activating/deactivating transcriptional complexes. Although we provide evidence suggesting that downregulation of TS by HDACi is occurring at the level of transcription, we cannot rule out the possibility that decreased mRNA stability is also a contributing factor. Future studies will focus on elucidating the precise molecular mechanisms responsible for HDACi-induced transcriptional repression of TS mRNA.
A growing body of evidence suggests that TS is being downregulated at multiple levels by HDACi. In addition to the transcriptional mechanism demonstrated here, a previous study reported that HDACi downregulation of TS protein may also occur in part through destabilization of the TS protein.18 It has been shown that HDACi induce the acetylation of Hsp90 that results in decreased chaperone activity and consequently destabilizes the TS protein. Our observation that TS transcription is potently suppressed by HDACi is also important, as it would effectively eliminate the production of TS mRNA and thus limit the cell's ability to synthesize new TS protein. It is unclear at present which of these mechanisms may be the dominant pathway and cellular context could influence the relative contribution of the mechanisms described. Future questions will also include understanding the individual HDAC isozymes in the regulation of each of these effects.
To establish a direct mechanistic link between TS downregulation and enhanced cytotoxicity, we measured the downstream events following TS inhibition to determine if the synergistic interactions were reflected in the mechanistic consequences of enhanced TS inhibition. We also report the enhanced accumulation of dUTP, a downstream effect of TS inhibition, following LBH589 treatment alone and with HDACi and fluoropyrimidine combination treatment in the HT29 colon cancer cell line. Accumulation of dUTP has been demonstrated to enhance uracil misincorporation into genomic DNA resulting in increased TS-inhibitor-induced DNA damage and cytotoxicity.35, 43 The increase in dUTP accumulation with LBH589 treatment alone could be attributed to the severe depletion of cellular TS enzyme and the resultant accumulation of the TS substrate dUMP, which can be further phosphorylated to dUTP by nucleoside diphosphate kinases. Furthermore, Western blot analysis revealed that combination treatment resulted in an increase in dUTPase protein expression despite the accumulation of dUTP within the cell. It would be reasonable to postulate that the dUTP pool would be reduced with combination treatment as a result of the increase in dUTPase protein. However, the fact that dUTPase catalytic activity is overwhelmed leading to a dramatic increase in the dUTP pool provides strong evidence supporting a more complete TS enzyme blockade promoting dUTP accumulation. Overall, the mechanistic evidence presented here for the first time strongly suggests a direct role for TS downregulation as a contributing factor for the observed synergy between these classes of agents. Although we demonstrate that 5-FU is synergistic with HDACi, the mechanistic basis for this could also be resulting from numerous possibilities that stem from the pleotropic effects that HDACi have on various cellular processes.
Over the past 10 years, there have been substantial advancements in colon cancer treatment including the approval of a number of efficacious agents. Despite these advancements, the response rates for chemotherapy in patients with metastatic colon cancer remains only 50%.1 TS overexpression is a well-documented mechanism of resistance to 5-FU-based therapy in colon cancer.44 A 3,000-patient meta-analysis demonstrated, with high statistical significance, that high intratumoral TS expression was associated with decreased overall survival.45 This present study adds significant mechanistic rationale to a growing body of evidence demonstrating that HDACi downregulate TS gene and protein expression in colon cancer cells. In the emerging era of personalized medicine and individualized chemotherapy, combining HDACi with fluoropyrimidines has the potential to enhance the efficacy of fluoropyrimidine-based therapy by overcoming TS-mediated drug resistance and providing additional therapeutic options for the treatment of this disease.
We would like to thank Novartis Pharmaceuticals for kindly providing LBH589 and the laboratory of Dr. Bert Vogelstein for providing the HCT116 p53+/+, HCT116 p53−/− and HCT116 p21−/− cell lines. We would also like to thank Dr Dongyun Yang for statistical analysis and Margaret Kornacki for her technical expertise with the nucleotide pool analyses.