Trichostatin a Sensitizes Hepatocellular Carcinoma Cells To

CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017) doi:10.21873/cgp.20045

Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes SANGSU SHIN 1, MIOK KIM 2,3 , SEON-JIN LEE 2, KANG-SEO PARK 4 and CHANG HOON LEE 2,3

1Department of Animal Biotechnology, Kyungpook National University, Sangju, Republic of Korea; 2Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Republic of Korea; 3Bio & Drug Discovery Division, Center for Drug Discovery Technology, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea; 4Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea

Abstract. Background/Aim: Hepatocellular carcinoma (HCC) mediated killing while increasing the expression of NKGD2 is the second leading cause of cancer-related death worldwide. ligands, including ULBP1/2/3 and MICA/B. TSA also induced The ability of HCC to avoid immune detection is considered one direct killing of HCC cells by stimulating apoptosis. of the main factors making it difficult to cure. Abnormal histone Conclusion: TSA likely increases killing of HCC cells indirectly deacetylation is thought to be one of the mechanisms for HCC by increasing NK cell-directed killing and directly by increasing immune escape, making histone deacetylases (HDACs) apoptosis. attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, Hepatocellular carcinoma (HCC) is the fifth most common on HCC (HepG2) gene expression and function. Materials and malignancy and the second leading cause of death of cancer Methods: A genome wide-transcriptional microarray was used patients in the world (1). Although a lot of progress has been to identify genes regulated by TSA in HepG2 cells. Gene made in the clinical management of HCC, its prognosis still Ontology was used to identify pathways regulated by TSA, and remains poor owing to high chances of metastasis and these changes were confirmed by qPCR. The effect of TSA on recurrence, leading to low overall survival (2). Treatment natural killer (NK) cell-mediated killing of HCC cell lines were options for advanced HCC are extremely limited owing to analyzed by both flow cytometry and LDH cytotoxicity assay. A the shortage of effective therapeutic agents. Currently, study was also conducted in a Balb/c nude mice xenograft sorafanib is the only approved therapeutic agent for model to assess the anti-tumor activity of TSA. Results: TSA advanced HCC (3), so there is an urgent need for novel regulated the transcription of numerous innate immunity & therapeutic drugs for this disease. tumor antigen recognition-associated genes, such as ULBP1 Recently, epigenetic modulation of gene transcription and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell through acetylation and deacetylation has been reported to growth in an NK cell-dependent manner. In vitro, TSA treatment play a role in the pathogenesis of advanced HCC (4-6). of HepG2 cells rendered them more susceptible to NK cell- Specifically, histone deacetylases ( HDAC )1-3 are up- regulated in primary human HCC and the enhanced HDACs levels play a critical role in malignant growth and immune escape (7). Importantly, the deacetylation process is This article is freely accessible online. reversible and can be targeted by new drugs such as HDAC inhibitors (8, 9). Thus, the biology of HDACs in HCC makes Correspondence to: Chang Hoon Lee, Ph.D., Bio & Drug the use of HDAC inhibitors to treat HCC an attractive option Discovery Division, Center for Drug Discovery Technology, Korea (9-11). HDAC inhibitors have been shown to have efficacy Research Institute of Chemical Technology, 141 Gajeongro, as anti-cancer agents in numerous phase I/II studies Yuseong, Daejeon 305-600, Republic of Korea. Tel: +82 428607414, Fax: +82 428614246, e-mail: [email protected] (http://www.clinicaltrials.gov), conducted in many different types of cancer (12, 13). Key Words: Natural killer cells, trichostatin A, immunotherapy, Trichostatin A (TSA) was originally discovered as an anti- hepatocellular carcinoma. fungal drug, but was later found to be a potent non-selective

349 CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017) inhibitor of HDACs (14). Although the anti-HCC effect of were conducted using an Affymetrix whole-genome expression TSA has been reported in previous studies (15, 16), the microarray performed according to the manufacturer’s instruction. mechanism by which TSA regulates HCC function is not Data were exported and normalized with the robust multi-average method implemented in Affymetrix Expression Console software. fully understood. In this study, we attempted to reveal how Statistical significance was determined using the local pooled error TSA induced its anti-cancer effects in HCC, focusing on test and gene expression fold change, in which the null hypothesis immune modulation, because the participation of immune was that no difference existed among groups. The false discovery cells in HCC progression is known to be important (17, 18). rate was controlled by adjusting the p-values, using the Benjamini- For this purpose, we analyzed TSA-mediated transcriptional Hochberg algorithm. For a differentially expressed gene set, a changes in HepG2 cells, a well-known HCC cell line, using hierarchical cluster analysis was performed using complete linkage a microarray. As a result of this genome-wide transcriptional and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analyses for the analysis, we found that innate immunity, MHC class I/II-like significant probe list was performed using DAVID antigen-recognition protein, and chemotaxis-associated genes (http://david.abcc.ncifcrf.gov/home). Before analyzing by DAVID, were altered by TSA treatment in HepG2 cells. Furthermore, genes that were duplicated or were not identified in the database we analyzed the immune modulatory ability of TSA, were excluded. The analyses and visualization of differentially focusing on its effect on natural killer (NK) cells, the major expressed genes using a heatmap were conducted using R software innate immunity cells responsible for tumor killing. (www.r-project.org). Compared to previous studies, which have focused on the Tumor-bearing mouse model. Athymic BALB/c nude mice were cytotoxic and inhibitory effect of TSA on HCCs, this study purchased from Dooyeol Biotech; BALB/c nude mice harbor demonstrated that TSA has immune-modulatory effects by functional NK cells, but do not develop functional T cells. One regulating the expression of a broad range of genes in HCC million viable Hepa1-6 cells were subcutaneously implanted on the cells both in vitro and in vivo . right flank of mice. Mice were examined regularly for tumor growth, using a metric caliper. Mice were randomly divided into Materials and Methods three groups: a control group, TSA with control IgG (BioLegend, San Diego, CA, USA)-treated group, and TSA with an anti-asialo- GM1 antibody (BioLegend) group (n=6 per group). TSA (30 mg/kg) Mice. Athymic BALB/c nude mice were purchased from Dooyeol was administered every day for 18 days via an intraperitoneal ( i.p. ) Biotech (Daejeon, Republic of Korea) and housed in a pathogen- route. For NK cell depletion, mice were injected intraperitoneally free animal facility. Female mice (6-8 week-old) were used in all (i.p. ) with 100 μl of an antibody to asialo-GM1, or with the control experiments. All animal experiments were approved by the IgG, 2 days prior to tumor cell implantation and twice afterwards Institutional Animal Use and Care Committee of the Korea (at days 2 and 5). NK cell depletion was evaluated by flow Research Institute of Bioscience and Biotechnology and were cytometry using a PE-coupled anti-mouse CD49b antibody and an performed in accordance with the Guide for the Care and Use of APC-coupled anti-mouse NK1.1 antibody (BioLegend). Laboratory Animals published by the US National Institutes of Health. NK cell infiltration. Eighteen days after the initiation of TSA treatment in tumor-bearing mice, all mice were sacrificed and Cell culture and reagents. HepG2 cells were purchased from the tumor tissues were removed, measured, and digested for 1 h in PBS American Type Culture Collection (ATCC, Manassas, VA, USA), containing 1 mg/ml collagenase D and 0.5 mg/mL DNase I. Total and Huh7 cells and SNU449 cells were purchased from Korean Cell cells from the tumor were resuspended in FACS buffer (PBS Line Bank (Seoul, Korea). HepG2 cells were cultured in Eagle’s containing 2% bovine serum albumin) by forcing the tissue through Minimum Essential Medium (EMEM) (ATCC) containing 10% FBS a 40- μm nylon cell strainer (BD Biosciences, San Jose, CA, USA). (Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine, and Cells from the mouse tumors were stained with a brilliant violet penicillin-streptomycin (Life Technologies) at 37˚C in 5% CO 2. 421 conjugated anti-mouse CD3 (BioLegend), a PE-cy7 conjugated Huh7 and SNU449 were cultured in RPMI1640 (Life Technologies) anti-mouse Ly6G (BioLegend), a phycoerythrin-conjugated anti- containing 10% FBS (Life Technologies), 2 mM L-glutamine, and mouse NK1.1 antibody (BioLegend,) and an allophycocyanin penicillin-streptomycin (Life Technologies) at 37˚C in 5% CO 2. The (APC)-conjugated anti-mouse CD49b antibody (BioLegend) with HDAC inhibitor, TSA (chemical structure shown in Figure 1a) was an Fc-blocking anti-mouse CD16/32 antibody (BioLegend). purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved The cells were washed, resuspended in FACS buffe and The at a concentration of 10 mM in DMSO as a stock solution, stored number of CD3-NK1.1+CD49b+ NK cells was determined using at –20˚C, and diluted in medium before each experiment. The final MACSQuant ®VYB (Miltenyi Biotec, Cologne, Germany) with DMSO concentration did not exceed 0.1% throughout this study (all flow cytometry counting beads (ThermoFisher Scientific, Waltham, control groups were administered 0.1% DMSO). Antibodies against MA, USA). caspase 3, PARP, and β actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Total RNA isolation and real-time RT-PCR. Cells were prepared as described above. Total RNA was isolated using TRIzol reagent Microarray and data analysis. HepG2 cells (5×10 6) in 10 ml (Invitrogen, Carlsbad, CA, USA). Real-time RT-PCR was complete RPMI culture medium were incubated for 48 h at 37˚C performed with 20 ng of RNA as a template, using qScript™ with DMSO alone (control) or with 1 μg/ml of TSA. Total RNA was cDNA SuperMix for reverse transcription and PerfeCTa ® qPCR isolated from the HepG2 cells, using Trizol (Invitrogen). Analyses FastMix ®, UNG, and ROX™ for PCR (Quanta Biosciences,

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351 CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017)

Table I. List of genes associated with innate immune response, MHC classes I/II-like antigen recognition protein, and chemotaxis and differentially regulated by TSA treatment in HepG2 cells.

Down-regulated genes Up-regulated genes

Innate immune response APOBEC3B, AXL, C1QBP, C4BPA, C4BPB, CASP4, CFH, ATG12, C1S, CARD9, IGHV1OR21-1, KLRG1, DDX60, eif2ak2, ELF4, GPER1, GSDMD, HMGB2, IFI16, MSRB1, SARM1, SERINC3, SLPI, TRIM15, VIP IFIH1, IFIT2, IFIT3, ifitm1, IFITM2, IFITM3, IRAK1, KLRC2, MATK, MB21D1, MIF, MST1R, MX1, NFKB1, NLRX1, OAS1, OAS3, PCBP2, POLR3C, POLR3E, PPP1R14B, PTK6, RNF135, TMEM173, TRAF3, TRIM56, TRIM8, ZC3HAV1 MHC classes I/II-like - HLA-DMA, HLA-DMB, hla-dra, RAET1G, ULBP1 Chemotaxis CMTM4, HMGB2, CXCL16, PARVA CCL20, SLIT2, ROBO3, CXCL8, antigen recognition protein CYR61, CXCL2, CXCL10

Gaithersburg, MD, USA). Primer and probe sets (FAM/VIC- from the NK cells and tumor cells co-culture. Spontaneous LDH labeled) were purchased from Applied Biosystems (Waltham, MA, release activities from tumor and NK cells were obtained from USA). Results were normalized based on the values obtained for individual cultures respectively. The maximal release of LDH GAPDH . Real-time qPCR was performed on duplicate samples, activity was obtained following lysis of the tumor cells, using using the ABI 7700 Sequence Detection System (Applied ultrasound (Soniprep 1500, Behring, Germany). Biosystems). For cells from each donor, the relative levels of expression based on 2 −ΔCT values are shown as percentages of the DAPI staining assay. The effect of TSA on nuclear morphology was subset with the highest value. evaluated by DAPI staining. HepG2 cells were incubated with TSA (0, 1, 2 and 5 μM) for 48 h. The cells were then exposed to fixative Peripheral blood and NK cell sorting. Peripheral whole blood from for 20 min and stained with DAPI (Life Technologies) for 10 min healthy donors was obtained from the Red Cross Blood Center in the dark. The labeled cells were visualized under a fluorescence (Daejeon, Republic of Korea) and the study protocol was approved microscope and then photographed. by the Institutional Review Board of the Red Cross. NK cells were isolated from whole blood to >95% purity by negative selection Cell viability and proliferation assay. Cell proliferation was using RosetteSep Human Monocyte Cell Enrichment Cocktail & assessed using the EZ-Cytox Enhanced cell viability assay kit Human NK Cell Enrichment Cocktail (StemCell Technologies) from ITSBIO (Seoul, Republic of Korea). Briefly, the appropriate according to the manufacturer’s protocol. The isolated NK cells number of cells were plated in each well of a 96-well plate and were washed in HBSS plus 4% FBS (Life Technologies), and re- exposed to different concentrations of TSA for 24 and 48 h. suspended in MEM- α ( Life Technologies ) containing 20% FBS Following this, the tetrazolium salt reagent was added and the (Life Technologies), and penicillin-streptomycin (Life Technologies) cells were incubated for 2 h. At the end of this time, the at 37˚C in 5% CO 2. absorbance in each well was measured at 450 nm using a microplate reader. The relative cell viability (%) was calculated NK cell cytotoxic assay. Evaluation of NK-cell activity was using the equation OD T/OD C ×100% (where OD T represents the performed using an LHD Cytotoxicity Assay Kit (ThermoFisher). absorbance of the treatment group, and OD c represents the Cell suspensions (100 μl) from the TSA-treated or untreated absorbance of the control group). The median inhibitory 6 tumor cell lines HepG2, SNU449, or Huh7 (1.0×10 /ml) were concentration (IC 50 ) was defined as the drug concentration 6 6 added to 100 μl of NK cells, at either 8.0×10 /ml, 4.0×10 /ml, required to inhibit 50% of the cells relative to the controls. IC 50 and 2.0×10 6/ml, in RPMI 1640 medium (Life Technologies) to values were estimated from the dose-response curve. provide effector to target cell ratios (E:T) of 0.5:1 and 1:1, and the cells were incubated in 96-microwell plates (Life Apoptosis assay . Apoptotic cells were detected by annexin V-FITC Technologies) for 4 h at 37˚C in a humid atmosphere containing and propidium iodide (PI) staining using the FITC-Annexin V 5% CO 2. Following this, the plates were centrifuged for 5 min at apoptosis detection kit with PI (BioLegend) according to the 200 × g and 100 μl of supernatant from each well were manufacturer’s instructions. The stained cells were analyzed using transferred to a 96-well flat-bottomed plates and 100 μl of lactic MACSQuant ®VYB (Miltenyi Biotec). acid dehydrogenase substrate mixture was added. Color development (5 min in the dark), was measured at 492-630 nm Statistical analysis. Analysis of variance was performed for using a microtiter plate reader (TECAN, Melbourne, Australia). multiple comparisons. Unpaired t- tests were performed to analyze The mean cytotoxicity percentage was calculated as follows: the differences between two groups. Statistical analyses were (LDH experimental - LDH spontaneous - LDH from NK performed using the Prism software package (GraphPad Software, cells)/(LDH maximal - LDH spontaneous) ×100 (%). LDH San Diego, CA, USA). Differences were considered significant at experimental release represents the LDH release activity arising p≤0.05.

352 Shin et al : Trichostatin A Sensitizes Hepatocellular Carcinoma Cells

Results HCC. In summary, we found a major alternation in immune- associated genes in TSA-treated HepG2 cells, particularly TSA triggers selective expression of genes associated with with respect to innate immunity-related genes and antigen- immune modulation in HCC cells. Although the anti-HCC recognition-related genes. These data also strongly suggested effect of TSA (chemical structure is shown in Figure 1a) has that TSA induced NK cell-mediated anti-tumor effects in been previously reported (15, 16), the mechanism by which HCC. We then attempted to confirm the effect of TSA on the TSA affects HCC has not been clearly defined. In this study, expression of ligands associated with the recognition of HCC we performed a microarray analysis to investigate the cells by NK cells. transcriptional changes in HepG2 cells, which are HCC cells, TSA enhanced NK cell cytotoxicity in HCC cells in the following treatment with 0.5 μM TSA compared to those in Hepa1-6 carcinoma-bearing mouse model. As described cells treated with vehicle (DMSO). The analysis identified above, TSA induced differential expression of immune- 1,216 genes that were up-regulated by ≥2-fold and 1,591 modulatory genes, in particular those related to innate genes that were down-regulated by ≥2-fold in TSA-treated immunity and MHC-classes I/II like antigen recognition versus vehicle-treated HepG2 cells. To understand the global protein and chemotaxis, which might suggest an NK cell- biological significance of these data, we further analyzed mediated anti-HCC effect of TSA. As previously reported these differentially expressed genes by functional enrichment (24), NK cells are the most potent anti-tumor innate immune analysis using the DAVID database (19). Among the 2,807 cells, and are one of the major target cells for used by differentially-expressed genes, 2,143 genes were selected for various cancers for immune escape. We hypothesized that further analysis, after removing duplicated gene names and TSA might regulate the susceptibility of HCC cells to NK genes that could not be identified in the database. Since gene cell-mediated killing and designed an in vivo study to ontology (GO) annotations are important for the address this (see the scheme in Figure 2a). To investigate NK investigation of functional significance from large datasets, cell-mediated changes in HCCs triggered by TSA, we we used the GO annotations based on the Gene ontology wished to eliminate the potential effect of other potent anti- consortium (20) and found that ‘innate immune response’ tumor immune cells, specifically T cells, which form part of was one of the major GO annotations associated with TSA the adaptive immune system. For this purpose, we selected treatment. TSA differentially affected the expression of 44 to use BALB/c nude mice that lack T cells, and as a result genes associated with innate immunity as shown in Figure do not develop an adaptive immune system. We established 1b. Among the 44 genes associated with innate immunity an HCC tumor xenograft model in BALB/c nude mice, using that were significantly differentially regulated in HepG2 cells Hepa1-6 carcinoma cells. As shown in Figure 2b, peripheral by TSA, 11 were up-regulated and 33 were down-regulated blood from BALB/c nude mice lacked CD3 + T cells, (Figure 1c). Because of the significant alternation of genes regardless of anti-asialo-GM1 antibody treatment. However, associated with innate immunity, we then explored which these mice were equipped with functional Ly6G + myeloid HCC immune escape mechanisms were regulated by TSA. cells (representing granuolocytes, macrophages, neutrophils, To escape the immune response, many types of tumor and monocytes) and F4/80 + and MHC-II + macrophages cells down-regulate antigen-recognition associated proteins (Figure 2b and c). Importantly, BALB/c nude mice have (21). Interestingly, five important genes associated with functional NK cells (CD49b + and NK1.1 +), as shown in MHC class I/II-like antigen recognition proteins were Figure 2d. Administration of an anti-asialo-GM1 antibody to significantly up-regulated by ≥2-fold in our microarray data BALB/c nude mice eliminated almost all NK cells, as shown (Figure 1d). Among these five genes, UL16-binding protein in Figure 2d. Importantly, we found that TSA significantly (ULBP)1 and RAE1G are known to be ligands for NK cells reduced the Hepa1-6 tumor size in BALB/c nude mice, as that allow for recognition and killing of target cells (22, 23). shown in Figure 2e. We investigated whether the effects of Both ULPB1 and RAE1G were significantly up-regulated by TSA on tumor suppression in the Hepa1-6 carcinoma mouse TSA (Figure 2d). These data suggested that recognition of model was mediated by NK cells by comparing the growth HCC cells by NK cells might be regulated by TSA treatment of the Hepa1-6 carcinoma in mice, with and without deletion of HCC cells. Specifically, ULBP1, one of the ligands for of NK cells, (achieved by i.v. injection of an anti-asialo-GM1 natural killer group-2 member-D (NKG2D) activator antibody). The anti-asialo-GM1 antibody injection receptors on NK cells, might be one of the critical specifically eliminated NK cells in mice (Figure 2d) and did components in the mechanism triggered by TSA that not change the percentage of myeloid cells and macrophages enhances susceptibility to NK cell killing. (Figure 2b and c). Importantly, NK cell-deletion did reduce In addition, 11 genes associated with chemotaxis were the magnitude of the TSA-induced reduction in Hepa1-6 significantly up-regulated or down-regulated by ≥2-fold in carcinoma size in these tumor-bearing mice (Figure 2e). our microarray data (Figure 1e). These data suggest that TSA However, there was not a complete reversal of the TSA also regulates immune cell migration into tumor tissue in effect. These results strongly suggest that NK cells play a

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355 CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017) critical role in the anti-tumor effect of TSA in HCC cells in cells significantly increased the extent of HCC cell killing vivo , and that another non-NK cell mechanism also (Figure 3b) compared to the NK cell-mediated killing of contributes to the anti-tumor effect. We also analyzed the untreated HepG2, SNU449, and Huh7 cells respectively. weight of tumors from mice when they were sacrificed at the These data strongly suggest that a common mechanism exists end of experiment. Tumor weights of each treated group of to enhance the susceptibility of HCC cells to NK cell killing mice showed similar pattern from the sizes measured during that can be triggered by TSA. Thus, we attempted to the experiment (Figure 2f). We also analyzed the number of understand the TSA-mediated mechanism in HCC cells, by infiltrated NK cells in the tumor, because we found that, analyzing the genome-wide transcriptional changes in based on our microarray data, various chemokines, including TSA-treated HepG2 cells. CXCL10, a ligand for the chemokine receptor CXCR3, were up-regulated by TSA in HCC cells. Importantly, we found TSA regulated expression of NKG2D ligands on HCC cells that number of NK cells was significantly increased in toward NK cell cytotoxicity. To extend our findings, we tumors from TSA-treated mice compared to that in control measured the mRNA levels of ULPB1, ULPB2, ULPB3, and IgG-treated mice. We also found that injection of the anti- major histocompatibility complex class I-related chain asialo-GM1 antibody almost completely eliminated the (MIC)-A and B, which are known to be NKG2D ligands. For infiltrated NK cells in the tumor (Figure 2g). this purpose, HepG2 cells were cultured in the absence or presence of 0.5 and 1.0 μM TSA. Total RNA was extracted TSA enhanced NK cell cytotoxicity in HCC cells in vitro . On from the cells and the mRNA expression of ULPB1, ULBP2, the basis of the above findings, we attempted to understand, and ULBP3 were analyzed by qPCR (Figure 3c); the mRNA using an in vitro experiment, how TSA enhanced the NK levels of MICA/B were also analyzed using this method cell killing capacity toward HCC cells. First, we (Figure 3d). In addition, the protein expression levels of investigated the direct effect of TSA on NK cells. For this MICA/B were assessed by flow cytometry using an antibody purpose, we treated NK cells isolated from human against MICA/B (Figure 3e). We found that TSA treatment peripheral blood, with and without TSA, overnight and then of HepG2 cells significantly increased the expression of co-cultured the treated NK cells with HepG2, SNU449, or ULPB1, ULPB2, ULPB3, and MICA/B (Figure 3c, d and e). Huh7 HCC cells for 4 h to assess the killing capacity of the To functionally assess the role of an NKG2D ligand in NK- NK cells. Based on a previous study (25), histone mediated killing of HCC cells, we measured NK-induced deacetylase inhibitors including TSA did not enhance NK cell cytotoxicity in co-cultures with HepG2 cells that had had cell activation and viability. Moreover, some histone been treated or not with TSA, along with blockade of deacetylase inhibitors could impair NK cell viability and NKG2D with an anti-NKG2D antibody after TSA treatment. activation in the dose-dependent manner. In our study, we Cell cytoxicity was assessed using a lactate dehydrogenase found that NK cell activity might not be impaired in the activity assay (LDH) cytotoxicity assay kit. Notably, doses of our in vivo and in vitro experimental designs. blockade of NKG2D significantly reduced TSA mediated- However, we found that TSA did not enhance the killing enhanced NK cytotoxicity (Figure 3f). capacity of NK cells (data not shown). Thus, we hypothesized that the NK cell-mediated enhanced TSA is also a potent cytotoxic agent in HCC cells. The data anti-tumor effect of TSA on HCC cells might instead be obtained so far showed that TSA enhanced NK cytotoxicity caused by an increase in susceptibility of HCC cells to NK against HCC cells both in vivo and in vitro . However, we cell killing, caused by TSA-mediated changes in the were also interested in assessing the direct cytotoxic effect expression of various HCC genes. To assess this, we pre- of TSA against HCC cells. To assess this, we measured the treated HepG2 cells with TSA for 2 days, and then analyzed direct effect of TSA on cell viability and proliferation of the cytotoxicity of human primary NK cells isolated from HepG2, Huh7, and SNU449 cells, using the EZ-Cytox peripheral blood on the HepG2 cells after removal of the TSA. Enhanced cell viability assay (Figure 4a). The IC 50 values Interestingly, TSA-treated HepG2 cells showed a potent for TSA-mediated cell cytotoxicity were calculated from the increase in susceptibility to NK cell killing compared to respective dose-response curves. Importantly, the HepG2, untreated HepG2 cells when cultured at a 1:1 ratio of NK Huh7, and SNU449 were all sensitive to TSA (IC50s for cells to HepG2 cells (Figure 3a). While NK cells converted HepG2, Huh7 and SNU449 cells were 1.085 μM, 0.954 μM, only 8.38% of HepG2 cells into the apoptotic state without and 0.859 μM respectively). To confirm this cytotoxic effect TSA treatment, NK cells converted 42.3% of the HepG2 of TSA, we observed cell morphology after two days of cells that had been treated with 0.5 μM TSA. Furthermore, culture of HepG2, Huh7, and SNU449 cells in the presence the TSA-mediated increase in susceptibility to NK cells was of 2 μM TSA. We observed a TSA-mediated reduction in also seen in the other HCC cells, SNU449 and Huh7 (Figure HepG2, Huh7, and SNU449 cell densities under the light 3b). Increasing the ratio of NK cells to TSA-treated HCC microscope (Figure 4b). In addition, there was a significant

356 Shin et al : Trichostatin A Sensitizes Hepatocellular Carcinoma Cells

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357 CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017)

358 Shin et al : Trichostatin A Sensitizes Hepatocellular Carcinoma Cells reduction in the number of live HepG2, Huh7, and SNU449 Discussion cells in the presence of 2 μM of TSA, compared to that in its absence (Figure 4c). HDACs are critical enzymes for the deacetylation of histone and are thus important in the regulation of transcription of a TSA induced apoptosis in HCC cells. To understand whether broad range of genes. They are also known for their critical the inhibitory effect of TSA HCC cell proliferation was role in tumor development and malignancy (4, 26, 27). mediated by the apoptotic process, we stained untreated and Although the anti-tumor effect of TSA has been TSA-treated HepG2 cells with Annexin V and PI and demonstrated in some malignant tumors, these studies have performed flow cytometry. We found that the proportion of focused on regulation of the cell cycle and apoptosis by TSA apoptotic HepG2 cells (Annexin V-positive and PI-positive) (26-28). However, the effect of the immune system on tumor increased in a TSA-dose dependent manner (Figure 5a). development is also important (31, 32, 33, 34). There are Similar effects on apoptosis were also noted for Huh7 and several recent reports that show an unexpected effect of TSA SNU449 cells after treatment with 0, 0.5, 1, and 2 μM TSA. in regulating the immune system in various tumors (35, 36). In all cases, the percent of apoptotic cells increased in a For example, HDACs are known to play critical roles in the TSA-dose dependent manner (Figure 5b). We also analyzed immune escape mechanism of several tumor cells, such as the levels of caspase 3, a critical player in the apoptotic melanoma cells and lymphoma cells (35, 36). In melanoma process, by the western blot method. We found that TSA cells (B16F10 cells), HDACs were shown to have a role in treatment increased the levels of cleaved (active) caspase 3 the reduction of MHC class I-associated antigen processing in HepG2 and Huh7 cells (Figure 5c and d). Furthermore, and presentation. Also, HDACs inhibit the expression of PARP, a critical DNA repair protein, was also cleaved by CD1d, a non-classical MHC class I-like molecule in TSA treatment in HepG2 and Huh7 cells (Figure 5c and e). lymphoma cells (LCD1dwt cells). Inhibition of the In these experiments, β- actin expression was used as a expression of the MHC class I-associated genes, TAP1, loading control. Finally, we found that TSA treatment TAP2, LMP2, LMP7, and Tapasin, makes it difficult for increased nuclear fragmentation and apoptotic bodies in a CD8 + T cells to recognize tumor cells (36). Similarly, down- dose-dependent manner (Figure 5f). regulation of CD1d on tumor cells makes it difficult for NKT cells to respond to tumor cells (35). This immune depression in the respective tumor cells limits recognition by CD8 + T cells and NKT cells and is thought to be a critical strategy for tumor growth and survival. Thus, blocking this immune escape mechanism, by HDAC inhibition with TSA, could → block this immune escape mechanism. Compared to data from previous studies, our data Figure 5. Induction of apoptosis in HCC cells by TSA. (a) HepG2 cells suggested that TSA had a broad effect on immune response were treated with DMSO (control), or with 1 μM and 2 μM TSA for pathways. On the basis of our microarray data, we found that 48 h and apoptosis assessed. Representative dot plots showing the the largest GO annotation was immunity, with 62 percentage (%) of Annexin V and PI double-positive cells (apoptotic cells) from three different independent experiments are shown. (b) differentially-expressed genes (DEGs), and within this set, Percentage (%) of dead cells in Huh7, SNU449, and HepG2 cell 34 DEGs were involved in the innate immune response cultures treated with the indicated concentrations of TSA. Data were (Figure 1b). In fact, approximately 53% of immunity-related pooled from three independent flow cytometry experiments. **, ***, DEGs were involved in the innate immune response. In significant differences from the control (untreated) based on two-tailed addition, we found 10 more innate immune response-related unpaired Student’s t-tests at p<0.01 and p<0.005, respectively. Error bars denote SEM. (c) HepG2 and Huh7 cells were treated with DMSO, DEGs that were not captured in the immunity GO annotation or with 1 μM and 2 μM TSA for 48 h. Cell lysates were analyzed by (Figure 1b). Moreover, 10 chemotaxis and two MHC-classes immunoblot analysis with antibodies against cleaved caspase-3 and I/II-like antigen recognition protein-related DEGs, which PARP. β- actin expression was analyzed as a loading control. The data were also not captured in the immunity GO annotation, were shown are representative of three independent experiments. (d) Ratio of discovered to be induced by TSA in HCCs (Figure 1b). Thus, cleaved caspase-3 to β- actin and (e) ratio of cleaved PARP to β- actin calculated based on Western blot band intensities. Data from three we hypothesized that the innate immune response associated independent experiments were pooled. **, ***, significant differences gene alternations induced by TSA in HCCs could be a major from the control (untreated) based on two-tailed unpaired Student’s t- mechanism for TSA-mediated immune modulation. Among tests at p<0.01 and p<0.005, respectively. Error bars denote SEM. (f) the innate immune response genes found in our microarray HepG2 cells were treated with DMSO (control), or with 1 μM and 2 μM data, we noted that there was upregulation of ULBP1 and TSA for 48 h. DAPI staining was performed and photographs were taken under a fluorescence microscope. Representative images from RAET1G, which are NKG2D ligands. By qPCR, we found more than three independent experiments are shown. White arrows that the NKG2D ligands ULBP1, 2, 3, as well as MIC-A and indicate nuclei with morphology characteristic of apoptosis. B, that are important in NK cell recognition of HCCs, were

359 CANCER GENOMICS & PROTEOMICS 14 : 349-362 (2017) up-regulated by TSA treatment, thereby rendering the HCCs studies will be required to broaden our knowledge about how susceptible to NK cell-mediated lysis (Figure 3c, d and e). TSA induces its anti-HCC effect. In addition, the anti-tumor effect of TSA was significantly Regardless, this study revealed that TSA stimulated HCC reduced by blocking the NKG2D receptor with a blocking cells to induce changes in the transcription of many genes antibody (Figure 3f). From these data, the regulation of associated with innate immunity and tumor antigen- genes associated with innate immunity in HCC cells might recognition; i.e. , TSA changed the HCC cells to become be associated with susceptibility of the HCC cells to NK responsive to NK cell killing, and also facilitated NK cell cell-mediated killing. Even though we did not investigate the migration into tumor tissues. Because it has been an effect of TSA on other innate immune cells, such as important goal to enhance the immune function of NK cells macrophages, monocytes, and myeloid-derived suppressor in patients for successful tumor therapy, our study cells (MDSCs), we still need to study further the interaction demonstrating a broad range of transcriptional changes between macrophages, monocytes, and myeloid-derived induced by TSA, that are associated with NK cell suppressor cells (MDSCs) and TSA-stimulated HCC cells in susceptibility and migration, could be important. In addition, order to completely understand the whole innate immune TSA also has a direct effect on HCC cells by directly response. stimulating apoptosis (Figure 5c). To summarize, our study Nevertheless, NK cells are one of the main players in the suggests that the TSA-mediated anti-tumor effect against first-line defense against tumor growth among innate HCC is related to the TSA-mediated induction of anti-tumor immune cells (37). Thus, the effect of TSA on NK cell- innate immune responses (specifically, NK cell-mediated) mediated tumor killing could be critical for the anti-tumor and this contributed, at least partially, to the TSA-mediated effect of TSA. Our study investigated the effect of TSA on anti-HCC effect. These data strongly suggest that the NK cell-mediated tumor killing, using an HCC cell xenograft immuno-modulatory effect of anticancer drugs should be model in BALB/c nude mice that lack T cells (CD4 + T cells, considered for next-generation drug development. CD8 + T cells and NKT cells) and an adaptive immune system. In this experimental model, we could eliminate the Conflicts of Interest effect of the potent anti-tumor effective immune cells, namely T cells and NKT cells. Thus, this experimental The Authors declare that they have no conflict of interest. mouse model allowed us to focus on the NK-mediated effects. Importantly, using an anti-asialo-GM1 antibody to Research Involving Human Participants and/or deplete NK cells in vivo reduced the anti-tumor growth effect Animals of TSA as shown in Figure 2e. Taken together, these data All applicable international, national, and/or institutional guidelines suggest that TSA-mediated immune modulation of HCCs for the care and use of animals were followed. This article does not affects NK cell recognition, which could be one of the major contain any studies with human participants performed by any of mechanisms in TSA’s anti-HCC effect. the Authors. We also found that multiple chemotaxis-related genes were altered by TSA treatment of HepG2 cells and TSA- Funding treated mice had increased infiltration of NK cells into the tumor tissue. Taken together, these results strongly suggest This work was supported by a grant from the National Research that the TSA-mediated effect on chemotaxis molecules might Council of Science & Technology (NST) of the Korean government (MSIP; grant no. CRC-15-02-KRIBB). also be an important component for the anti-tumor effect of TSA against HCCs in vivo . Among the 11 chemotaxis related genes we found altered, the chemokines CCL20, CXCL8, References CXCL2, and CXCL10 were significantly upregulated by 1 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and TSA treatment. CXCL10 is a ligand for CXCR3, which is Jemal A: Global cancer statistics, 2012. CA Cancer J Clin 65 : abundantly expressed on NK cells (38). 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37 Martner A, Rydström A, Riise RE, Aurelius J, Brune M, Foà R, 39 Won EJ, Ju JK, Cho YN, Jin HM, Park KJ, Kim TJ, Kwon YS, Hellstrand K and Thorén FB: NK cell expression of natural Kee HJ, Kim JC, Kee SJ, and Park YW: Clinical relevance of cytotoxicity receptors may determine relapse risk in older AML circulating mucosal-associated invariant T cell levels and their patients undergoing immunotherapy for remission maintenance. anti-cancer activity in patients with mucosal-associated cancer. Oncotarget 6: 42569-42574, 2015. Oncotarget 7: 76274-76290, 2016. 38 Bernardini G, Antonangeli F, Bonanni V and Santoni A: Dysregulation of chemokine/chemokine receptor axes and NK Received June 30, 2017 cell tissue localization during diseases. Front Immunol 7: 402, Revised July 18, 2017 2016. Accepted July 20, 2017

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