Histone deacetylase (HDAC) inhibitors are used in treating certain human malignancies. Our laboratories demonstrated their capability in enhancing antitumor effect of DNA vaccine driven by an RNA polymerase II (RNA pol II) promoter. However, it is unknown whether HDAC inhibitors enhance the therapeutic short hairpin RNA (shRNA) expressed by an RNA polymerase III (RNA pol III) promoter. We investigated whether HDAC inhibitors augmented antitumor effect of indoleamine 2,3 dioxygenase (IDO) shRNA. HDAC inhibitor OSU-HDAC42 and suberoylanilide hydroxamic acid enhanced RNA pol III-driven U6 and H1 promoter activity in three different cell types in vitro: 293, NIH3T3 and dendritic cell line DC2.4. Subcutaneous injection of OSU-HDAC42 enhanced U6 and H1 promoter activity on abdominal skin of mice in vivo. Combination of IDO shRNA and OSU-HDAC42 increased antitumor effect of IDO shRNA in MBT-2 murine bladder tumor model. IDO shRNA induced tumor-infiltrating CD8+ and CD4+ T cells, whereas OSU-HDAC42 treatment induced tumor-infiltrating CD4+ T cells. Combination of OSU-HDAC42 and IDO shRNA further induced tumor-infiltrating natural killer cells and enhanced interferon-γ in lymphocytes, but suppressed interleukin (IL)-4 expression of lymphocytes. In addition, OSU-HDAC42 treatment did not alter mRNA expression of IL-12 and tumor necrosis factor-α. In conclusion, HDAC inhibitor OSU-HDAC42 may serve as adjuvant of the therapeutic shRNA expressed by an RNA pol III promoter.
Histone acetylation, one type of histone codes, affects chromatin structure and regulates gene expression. The balance between acetylation and deacetylation serves as a key regulatory mechanism for gene expression and has a role in disease progression.1, 2 Histone acetyltransferases and histone deacetylases (HDACs) are two families of enzymes that modify the acetylation status, whereas HDACs remove acetyl groups from lysine. As histone acetylation status of promoter region is affected by HDAC, inhibition of HDACs by HDAC inhibitors alters multiple gene expression through enhancing many kinds of promoters. Therefore, HDAC inhibitors have diverse effects including induction of cell death, upregulation of tumor suppressor genes and suppression of pro-inflammation cytokine expression.2, 3
DNA vaccine is a powerful approach to induce adaptive immune responses against diseases, including infectious diseases and cancer. In recent years, several strategies have been used to improve potency of DNA vaccine by increasing antigen expression and immunogenicity.4, 5 A constitutive, mammalian and high-level expression promoter, such as human cytomegalovirus (CMV) promoter, is widely used in DNA vaccine.6 However, CMV promoter might be silenced by histone acetylation in vivo.7 In recent years, HDAC inhibitors have demonstrated to influence CMV promoter-driven DNA vaccine in vitro and in vivo. For example, intramuscular injection of HDAC inhibitor enhanced immune responses of DNA vaccine through elevation of antigen expression.8 Skin administration of HDAC inhibitor and Her2/neu DNA vaccine exhibited stronger antitumor effect and cellular immune responses.9 It indicates that the local administration of HDAC inhibitor on the vaccination site of DNA vaccine is a potential strategy to enhance efficiency of DNA vaccine.
Short hairpin RNA (shRNA) technique is generally used for silencing specific genes and has been applied to immunization of DNA vaccine. Mice immunized with human papillomavirus type 16 E7-DNA vaccine and shRNA targeting Fas ligand had higher antitumor effects against E7-expressing tumors.10 Administration Her2/neu DNA vaccine and FOXO3 shRNA improved therapeutic effect in Her2/neu-overexpressing animal tumor model.11 Indoleamine 2,3 dioxygenase (IDO)-expressing dendritic cells induced proliferation of regulatory T cells in tumor-draining lymph nodes.12 Skin delivery of IDO shRNA delayed tumor growth and induced tumor-specific cytotoxic activity and Th1-bias immune responses in subcutaneous and orthotopic tumor models.13, 14 These results indicate that shRNA against specific gene is a powerful technique in the field of DNA vaccine.
Unlike the majority of antigen expression are driven by RNA polymerase II (RNA pol II), most of shRNA are driven by U6 small nuclear promoter (U6) or the human H1 promoter (H1), which belongs to type III RNA polymerase III (RNA pol III).15, 16 Histone acetylation is demonstrated to enhance both RNA pol II and RNA pol III transcription.17 Trichostatin-A, which is one of HDAC inhibitor, increased acetylation of histone H3 and enhanced transcription of type I, type II and type III RNA pol III, and resulted in increased level of 5S rRNA and transfer RNA.18, 19 On the basis of the observations above, we hypothesized that HDAC inhibitors may activate U6 or H1 promoter (type III RNA pol III) in vitro and improve antitumor effect of shRNA-based DNA vaccine (driven by RNA pol III) in vivo.
OSU-HDAC42 (AR-42) is a novel hydroxamate-tethered phenylbutyrate derivative HDAC inhibitor and acts on both classes I and II HDAC enzymes.20 Oral treatment of OSU-HDAC42 suppressed progression of prostate cancer and hepatocarcinoma in murine tumor models,21, 22, 23 Therefore, OSU-HDAC42 was employed to study whether combination of HDAC inhibitors enhanced the cancer therapeutic effects of RNA pol III-driven shRNA. In the present study, we first demonstrated that HDAC inhibitors enhanced U6 and H1 promoter in vitro and in vivo. In addition, combination of IDO shRNA and OSU-HDAC42 induced stronger antitumor effect than single treatment. Combination treatment further enhanced interferon-γ (IFN-γ) expression and suppressed interleukin-4 (IL-4) expression. These results indicated that OSU-HDAC42 enhanced therapeutic efficiency of RNA Pol III promoter-driven DNA immunization.
Materials and methods
MBT-2 is a mouse bladder tumor cell line.24 NIH/3T3 and 293 cells were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). Murine dendritic cell line DC2.4 was a kind gift from Dr Huan-Yao Lei.25 All cell lines were maintained in culture medium that contained low-glucose Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 100 units per ml penicillin and 100 μg ml−1 streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37 °C with 5% CO2 incubator.
HDAC inhibitors, OSU-HDAC42 and SAHA (suberoylanilide hydroxamic acid; Vorinostat), were obtained from Dr Ching-Shih Chen and were dissolved into dimethyl sulfoxide (DMSO). For in vitro experiments, inhibitors were treated at final concentration of 2.5, 1, 0.1 and 0.01 μM. For in vivo treatment, stock OSU-HDAC42 (10 mg ml−1) was dissolved in DMSO and then diluted to one part in one hundred double distilled water.
The inbred female C3H/HeN mice (6–8 weeks old) were obtained and maintained in the Laboratory Animal Center of National Cheng Kung University. All animal experiments were approved by the Animal Welfare Committee at National Cheng Kung University.
Plasmid construction and preparation
U6 promoter was cloned from pHsU6 vector, and H1 promoter was cloned from pSuper vector (Oligoengine, Seattle, WA, USA). U6 promoter-driven luciferase (U6-Luc) and H1 promoter-driven luciferase (H1-Luc) were constructed into pGL3-Basic (Promega, Madison, WI, USA) at the KpnI and XhoI restriction sites. IDO shRNA plasmid and scramble IDO shRNA plasmid (Scramble IDO sh) were described before.13 All plasmids were purified by Endofree Qiagen Plasmid Mega Kits (Qiagen, Hilden, Germany) and were dissolved in endotoxin-free water.
Promoter reporter assay
5 × 104 293, NIH/3T3 or 1 × 105 DC2.4 cells were seeded into 24-well plate for 18 h. Before transfection, culture medium was replaced with fresh culture medium containing 0.1% dimethyl sulfoxide (DMSO) or HDAC inhibitor (OSU-HDAC42 or SAHA). Two hundred and ninety-three cells were transfected with 0.5 μg of U6-Luc by Turbofect transfection reagent (Fermentas, Vilnius, Lithuania) for 24 h and then cells were collected for promoter reporter assay by luciferase detection system (Promega). Luciferase activity was measured in luminometer (Lumat LB9506; Berthold Technologies, Bad Wildbad, Germany). The luciferase activity was normalized to the protein concentration of the lysate. For in vivo promoter activity assay, C3H/HeN mice will be administrated with 10 μg of vector control, U6-Luc and H1-Luc. Forty-eight hours after gene gun bombardment, mice were intraperitonally injected with 100 μl D-luciferin in saline at a dose of 100 mg kg−1 (Synchem OHG, Altenburg, Germany). The in vivo imaging was taken and the images were quantitated by IVIS Spectrum Noninvasive Quantitative Molecular Imaging System (Xenogen, Alameda, CA, USA).
Evaluation of therapeutic effect
MBT-2 cells (1 × 106) resuspended in 200 μl of serum free Dulbecco’s modified Eagle’s medium were subcutaneously injected into C3H/HeN mice. Five micrograms of IDO shRNA or scramble IDO shRNA were bombarded to shaved, abdominal skin with low-pressure gene gun (Bioware, Taipei, Taiwan) at day 7 after tumor implantation. Meanwhile, 10 μg of HDAC-42 or vehicle (1% DMSO in double distilled water) was subcutaneously injected to the shaved skin of abdomen. Tumor volume of mice was measured by caliper twice a week and calculated by following formula: width × width × length × 0.5236 from day 7. An analysis of tumor growth was using Student’s t-test between the treated group and control group.
Analysis of tumor-infiltrating immune cells
Tumor samples were collected 3 days after the third vaccination. Immunohistochemistry was performed on 5-μm cryostat sections. Infiltrating T cells were detected by anti-CD4, anti-CD8 and anti-CD49b/Pan-natural killer (NK) antibodies (BD Pharmingen, San Diego, CA, USA). The number of T cells was randomly counted in five fields of each sample from at least three independent mice.
Detection of antigen-specific CD4+/Foxp3+ T lymphocytes in inguinal lymph nodes
Three days after the third vaccination, inguinal lymph nodes were collected. Lymphcytes (1 × 106) were stained with phycoerythrin-conjugated anti-CD4 antibody (eBioscience, San Diego, CA, USA) for 30 min. Subsequently, stained cells were fixed and permeabilized by Transcription Factor Buffer Set (BD Pharmingen) and then stained with FITC-conjugated anti-FoxP3 antibody (eBioscience) for 30 min. The CD4+FoxP3+ T lymphocytes were measured using a flow cytometry (Accuri C6, BD Biosciences, Ann Arbor, MI, USA), and data were further analyzed with Flow Jo software (Tree Star, Ashland, OR, USA).
Reverse transcription PCR and real-time quantitative PCR
Total RNA was extracted from inguinal lymphocytes of mice by using TRIZOL (Invitrogen). For complementary DNA synthesis MMLV-Reverse Transcriptase (Promega) was used according to the manufacturer’s directions. Primer sequences are in Table 1. Real-time PCR was performed on a StepOne Plus real-time PCR instrument (Applied Biosystems, Foster City, CA, USA) using Maxima SYBR Green qPCR Master Mix (Fermentas). The cycling conditions were 10 min of 95 °C, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The calculation was based on the delta-delta Ct procedure.
Graph and statistical analysis
All of the numerical data and graphs were analyzed with GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA, USA).
HDAC inhibitor enhanced RNA pol III promoter activity in vitro and in vivo
To investigate whether HDAC inhibitor enhanced RNA pol III U6 and H1 promoters activity, the U6 or H1 luciferase reporter plasmid was transiently transfected into cells and assayed for the promoter activity. HDAC inhibitor OSU-HDAC42 significantly enhanced U6 and H1 promoters activity in the 293 cell line, and enhanced U6 promoter activity in mouse fibroblast NIH/3T3 and dendritic cell line DC2.4 (Figures 1a–d). Another HDAC inhibitor SAHA, which is an Food and Drug Administration-approved drug for treatment of cutaneous T-cell lymphoma,26 also enhanced U6 and H1 promoters activity in several types of cells (Figures 1e–f).
To further determine whether HDAC inhibitors enhanced promoter activity in vivo, the reporter plasmid (U6-Luc) was delivered to abdominal skin of mice with gene gun. Ten micrograms of OSU-HDAC42 was subcutaneously injected at the bombarded area. U6 promoter-driven luciferase activity was enhanced by OSU-HDAC42 in C3H/NeN mice. H1 promoter-driven luciferase activity was also upregulated after OSU-HDAC42 injection (Figure 2). Altogether, HDAC inhibitors enhanced U6 and H1 promoter activity in vitro and in vivo.
HDAC inhibitor enhanced anti-tumor effect of IDO shRNA
In our previous study, skin delivery of U6 promoter-driven IDO shRNA exhibited antitumor effect in several tumor models by gene gun bombadrment.13, 14 Gene gun-mediated vaccination caused the migration of skin-resident dendritic cells to inguinal lymph nodes.27 Furthermore, local administration of OSU-HDAC42 enhanced EGFP plasmid carried dendritic cells migration to inguinal lymph nodes.9 As HDAC inhibitor enhances the polymerase III activity in vitro, HDAC inhibitor may enhance the antitumor effects of IDO shRNA driven by RNA pol III promoter in MBT-2 bladder tumor animal model. Combination of IDO shRNA and OSU-HDAC42 treatment significantly delayed tumor growth in MBT-2 tumor model, but subcutaneous OSU-HDAC42 injection did not affect tumor growth by itself (Figure 3). The result indicated that HDAC inhibitor enhanced therapeutic effect of U6 promoter-driven IDO shRNA.
Local administration of HDAC inhibitor enhanced IDO shRNA-mediated immune responses
IDO shRNA treatment induced tumor-infiltrating immune cells in our previous study.14 Therefore, we determined whether OSU-HDAC42 influenced tumor-infiltrating immune cells. The number of tumor-infiltrating CD4+ and CD8+ T cells increased in IDO shRNA vaccination group and combination group. More infiltration of CD4+ T and NK cells, but no increase of CD8+ T-cell infiltration, was observed in OSU-HDAC42-treated group (Table 2 and Figure 4). In addition, both OSU-HDAC42 and IDO shRNA did not affect the number of CD4+FxoP3+ regulatory T cells in tumor-draining lymph nodes (Figure 5). The immune responses were in agreement with the minimal therapeutic effects of local administration of OSU-HDAC42 because CD8+ T cells have an essential role in the therapeutic effect of IDO shRNA vaccination. It was interesting to note that the combination of OSU-HDAC42 and IDO shRNA increased the number of NK cells infiltration but did not increase the number of CD4+ and CD8+ T cells.
Local HDAC inhibitor treatment enhanced IDO shRNA-induced Th1 immune responses
As oral administration of HDAC inhibitors produce anti-inflammatory effect, we evaluated the effect of subcutaneous OSU-HDAC42 injection on expression of inflammatory genes in lymph nodes by quantitative PCR. Subcutaneous OSU-HDAC42 injection did not affect tumor necrosis factor-α, IFN-γ and IL-12 but repress mRNA expression of IL-4 and IL-10 (Figure 6). IDO shRNA vaccination induced IFN-γ but suppress IL-10 expression. Combination of IDO shRNA and OSU-HDAC42 showed similar pattern with IDO shRNA vaccination. The results indicated that local administration of OSU-HDAC42 did not display anti-inflammatory effect in lymphocytes. Furthermore, OSU-HDAC42 may enhance the antitumor effect of IDO shRNA by increasing IFN-γ expression and by suppressing IL-4 expression in lymphocytes (Figure 6). Therefore, the cotreatment with OSU-HDAC42 may increase the activity of lymphocytes, but not the total number of infiltrated lymphocytes.
Enhancement of promoter activity is one of the strategies to improve therapeutic effect of DNA vaccine. Our results demonstrated that HDAC inhibitors enhanced polymerase III promoter activity in vitro and in vivo. In a mouse cancer therapeutic model, combination of HDAC inhibitor OSU-HDAC42 with IDO shRNA induced stronger antitumor effects. OSU-HDAC42 suppressed class I and class II HDAC enzymes.20 SAHA and Trichostatin-A, which are derived from hydroxamic acids, inhibit class I and class II HDAC substrates.28 We also demonstrated that SAHA treatment enhanced U6 and H1 promoter activity (Figure 1). Trichostatin-A has been reported to enhance RNA pol III transcription.18 Altogether, it suggested that certain types of HDAC inhibitors might be potential adjuvant to enhance of RNA Pol III-driven DNA vaccines. Recent evidences indicated that shRNA can be effectively expressed by both RNA pol II and RNA pol III promoter.29 As OSU-HDAC42 was shown to enhance certain RNA pol II promoter activity in our previous report and RNA pol III promoter in this report, OSU-HDAC42 may be appropriate for combination therapy with DNA vaccine driven by these two types of RNA polymerase.
HDAC inhibitors induced activation of multiple signaling pathways in transformed cells, including cell cycle arrest and apoptosis.30 Therefore, HDAC inhibitors serve as antitumor agents in clinical use. SAHA has been used to treat cutaneous T-cell lymphoma,26 and other kinds of inhibitors in clinical trials showed antitumor potency in several types of cancers. In this report, local administration of OSU-HDAC42 did not affect tumor growth in MBT-2 tumor model that is probably due to the lower dose used.26 OSU-HDAC42 was injected subcutaneously once a week in the present study while oral administration of HDAC inhibitor at a daily schedule is used in cancer therapy experiments. It is possible that OSU-HDAC42 by local administration was majorly restricted in subcutaneous regions of abdomen and plasma concentration of OSU-HDAC42 was too low to affect MBT-2 tumor growth. Interestingly, the number of tumor-infiltrating CD4+ T and NK cells significantly increased in the OSU-HDAC42-treated tumor (Table 2 and Figure 4), supporting the effects of OSU-HDAC42 on the skin-resident immune system. The increase of CD4+ T cells and NK cells may not be effective in delaying tumor progression because the eradicating of MBT-2 tumor cells is mainly dependent on the CD8+ T cells.23 However, an increasing number of NK cells may be in part responsible for the better therapeutic effect in combination group. On the other hand, 100 μg of OSU-HDAC42 treatment enhanced CD8+ T cells infiltration in mice vaccinated with Her2/neu DNA.9 Ten micrograms of OSU-HDAC42 treatment did not further increase the tumor-infiltrating CD4+ and CD8+ T cells in IDO shRNA and Her2/neu DNA vaccinated mice.9 Our results suggest that the dosage of OSU-HDAC42 may be a key factor to determine the T-cell infiltration. High concentration of OSU-HDAC42 may simultaneously triggers multiple pathways in epithelial cell, keratinocytes and skin-resident dendritic cells, and leads to CD4+, CD8+ T-cell and NK cell infiltration. The detail mechanism warrants further study.
HDAC inhibitors treatment also affected immune cells through multiple signaling pathways in vitro and in vivo.3, 31 Oral administration of SAHA suppressed the level of lipopolysaccaride-induced proinflammatory cytokines IL-12, tumor necrosis factor-α and IFN-γ in serum.32, 33 Moreover, IDO expression was induced by SAHA in bone-marrow-derived dendritic cells through transcription factor STAT3.34, 35 Therefore, IDO might be one of essential molecules in HDAC inhibitor-dependent immunosuppression of dendritic cells in certain situations. It implies that silencing IDO expression in dendritic cell might be beneficial for antitumor effects of HDAC inhibitors. In addition, our results showed that OSU-HDAC42 treatment did not affect the number of CD4+FoxP3+ T cells in tumor-draining lymph nodes (Figure 5). Our results indicated that low dose of OSU-HDAC42 was able to enhance antitumor effect of IDO shRNA without inducing expansion of regulatory T cells. Expression of Th1 cytokines IFN-γ and IL-12 was induced by IDO shRNA vaccination.14 Cytokine expression profiles of lymphocytes from combination group showed similar pattern with IDO shRNA group (Figure 6). It suggested that subcutaneous administration OSU-HDAC42 is sufficient to enhance U6 promoter activity locally but is not sufficient to suppress proinflammatory cytokines of lymphocytes. Thereby, OSU-HDAC42 in subcutaneous region may not contribute to induce immunosuppressive signaling. Our results further suggest that HDAC inhibitors are potential candidates in combining with other immunotherapies because HDAC inhibitors did not dampen the dendritic cell activity in our animal model.
In summary, OSU-HDAC42 treatment enhanced U6 promoter activity and improved antitumor effect of U6 promoter-driven IDO shRNA. It provided a novel strategy to enhance efficacy of shRNA-based DNA vaccine. Many types of HDAC inhibitors have been used for treatment of cancers and inflammatory diseases clinically or in clinical trial. Some of these HDAC inhibitors might be adjuvant of RNA pol III and RNA pol II promoter-driven DNA vaccine, either. It will be important to study combination of these HDAC inhibitors with DNA vaccine for clinical interests.
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This study is supported by the grant to MD Lai, NSC-100-2325-B-006-008, 101-2325-B-006-007 and 101-2320-B-006-028-MY3 from the National Science Council, Taiwan
Dr Chen conducts a clinical trial on OSU-HDAC42 currently. The remaining authors declare no conflict of interest.
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Cite this article
Yen, M., Weng, T., Chen, Y. et al. An HDAC inhibitor enhances cancer therapeutic efficiency of RNA polymerase III promoter-driven IDO shRNA. Cancer Gene Ther 20, 351–357 (2013) doi:10.1038/cgt.2013.27
- H1 promoter
- IDO shRNA
- indoleamine 2,3-dioxygenase
- RNA polymerase III
- U6 promoter
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