Presentation of Telomerase Reverse Transcriptase, a Self-Tumor Antigen, Is Down-Regulated by Histone Deacetylase Inhibition

Research Article

Presentation of Telomerase Reverse Transcriptase, a Self-Tumor Antigen, is Down-regulated by Histone Deacetylase Inhibition

Ilenia Pellicciotta,1 Xochitl Cortez-Gonzalez,1 Roman Sasik,2 Yoram Reiter,3 Gary Hardiman,2 Pierre Langlade-Demoyen,4 and Maurizio Zanetti1

1The Laboratory of Immunology, Department of Medicine and Moores Cancer Center and 2Department of Medicine and BIOGEM, University of California, San Diego, La Jolla, California; 3Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel; and 4Unite’ de Retrovirologie Moleculaire, Institut Pasteur, Paris, France

Abstract chromatin structure influence antigen presentation in human Histone deacetylases (HDAC) modify the architecture of tumor cells. chromatin, leading to decreased gene expression, an effect Here,westudiedtheeffectofHDACinhibitononthe that is reversed by HDAC inhibition. The balance between presentation of telomerase reverse transcriptase (TRT), a bona fide deacetylation and acetylation is central to many biological universal antigen in human cancer cells (10, 11) using trichostatin events including the regulation of cell proliferation and cancer A (TSA), a natural product hydroximate with high affinity for the but also the differentiation of immune T cells. The effects of catalytic site of class I and II HDAC (12). We monitored the surface HDAC inhibition on the interaction between antitumor density of complexes formed between the major histocompatibil- effector T cells and tumor cells are not known. Here, we ity/Human Leukocyte Antigen (HLA)-A2 molecule and two high- affinity 9mer hTRT peptides: 540ILAKFLHWL548 (p540) and studied presentation of a universal self-tumor antigen, 865 873 telomerase reverse transcriptase, in human tumor cells during RLVDDFLLV (p865; ref. 11). Because telomerase is expressed HDAC inhibition. We found that HDAC inhibition with in >85% of human cancers (13) and CD8 T cells specific for p540 trichostatin A was associated with a decreased presentation are found in the blood of cancer patients at high frequency (14), and diminished killing of tumor cells by CTLs. Using gene array the model system studied herein is relevant to better understand- analysis, we found that HDAC inhibition resulted in a decrease ing the relation between cancer cells and the antitumor immune of genes coding for proteasome catalytic proteins and for response in humans. Our findings show a reduction of HLA-A2/ tapasin, an endoplasmic reticulum resident protein involved hTRT peptide complexes at the cell surface of TSA-treated human in the MHC class I pathway of endogenous antigen presenta- tumor cells. More importantly, using a highly specific human tion. Our findings indicate that epigenetic changes in cytotoxic T-lymphocyte (CTL) clone, we found that TSA-treated tumor cells decrease self-tumor antigen presentation and cancer cell killing was reduced compared with untreated cells. contribute to reduced recognition and killing of tumor cells This finding may be relevant to understand how immunosurvel- by cytotoxic T lymphocytes. This mechanism could contribute liance of cancer cells can be affected in vivo. to tumor escape from immune surveillance. [Cancer Res 2008;68(19):8085–93] Materials and Methods Cell Lines and Inhibitors Introduction The human B-lymphoblastoid JY cell line and prostate cancer cells LnCap Epigenetic modifications influence gene activity without altering (the kind gift of Dr. A. Vitiello, The R.W. Johnson Pharmaceutical Research DNA sequences from the transcriptional activity of selected genes Institute, San Diego, CA), melanoma cells 629.38 (the kind gift of Dr. S. to the individual’s susceptibility to disease (1), and include both Rosenberg, Surgery Branch, National Cancer Institute, Bethesda, MD), and transient modifications and stable heritable changes (2). Acetyla- TAP-deficient T2 cells (the kind gift of Dr. P. Creswell, Yale University, New Haven, CT) were cultured in complete RPMI 1640 supplemented with 10% tion and deacetylation of histones impart modification on the fetal bovine serum (FBS). TSA, Lactacystin, Sodium butyrate, and BFA were architecture of chromatin (3), which reflect on increased or purchased from Sigma. decreased gene expression through the opposing activities of histone acetyltransferases and histone deacetylases (HDAC). Immunochemical Detection of hTRT, HLA-A2/hTRT Acetylation plays a critical role in the regulation of cell proliferation Complexes, and HLA-A2 (4) and transcriptional silencing associated with a generalized loss Detection of HLA-A2/hTRT complexes at the cell surface. HLA-A2/ of acetylation has been found in cancer (5). Levels of acetylation p540 and HLA-A2/p865 complexes were detected with the human Fab 4A9 A also play a role in the differentiation of T cells (6, 7) and in the and 3H2 (15), respectively. Briefly, cells were treated with TSA (1 g/mL), BFA (10 Ag/mL), or lactacystin (10 Amol/mL). After washing, cells were expression of ligands for natural killer (NK) cells (8). Although incubated with human Fab 4A9 or 3H2 (2 Ag per reaction) for 1.5 h at +4jC. mouse tumor cells treated with high dose HDAC inhibitors are After washing, cells were counterstained with FITC-conjugated goat immunogenic and confer partial protection from tumor growth antibody to human Fab (Jackson Laboratories) for 45 min. Surface staining in vivo (9), little is known on whether epigenetic changes of for HLA-A2 was performed using monoclonal antibody BB7.2 (ATTC) followed by a FITC-conjugated goat antibody to mouse IgG (eBioscience). Cells were analyzed in a FACSCalibur (Becton Dickinson) using the BD Note: I. Pellicciotta and X. Cortez-Gonzalez contributed equally to this work. CellQuest data acquisition and analysis software. Requests for reprints: Maurizio Zanetti, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0815. Phone: 858-822-5412; Fax: 858-822-5421; E-mail: [email protected]. Cytotoxicity Assays I2008 American Association for Cancer Research. Buffy coats from HLA-A2.1 healthy donors were purchased from the doi:10.1158/0008-5472.CAN-08-1014 Blood bank of Hopital Henry Mondor in accordance with an approved www.aacrjournals.org 8085 Cancer Res 2008; 68: (19). October 1, 2008

Institutional Review Board protocol. HTERT-specific human CTL lines were DNA Microarray generated according to published procedure (11). Briefly, peripheral blood Biotinylated cRNA was prepared using the Illumina RNA Amplification mononuclear cells were stimulated with hTERT peptides in vitro in 24-well kit (Ambion, Inc.) according to the manufacturer’s directions starting with plates using autologous irradiated adherents cells as antigen-presenting cells 250 ng total RNA. The labeling approach uses a modified Eberwine and interleukin (IL)-2 and IL-7. After three to four rounds of culture, CTLs protocol (17) by which mRNA is converted to cDNA, followed by an were screened in a standard 51Cr-release assay. The cold target inhibition amplification/labeling step mediated by T7 DNA polymerase. The cDNA assay (11) was performed to ensure the specificity of the CTL clone. T2 cells and cRNA filter cartridges (Ambion) were used according to the were pulsed with either p540 or the HIV p17 gag 77-85 peptide as a control. manufacturer’s instructions for RT and IVT cleanup, respectively. For An adaptation of this assay was used to investigate killing inhibition where microarray analysis, the Illumina Human Expression BeadChip was used 51Cr-labeled targets treated with TSA were used (Fig. 6C and D). In these (Illumina). Hybridization of labeled cRNA to the BeadChip, and washing experiments, the E:T ratio was kept constant at 20:1, whereas the cold/hot and scanning were performed according to the Illumina BeadStation 500 inhibitor ratio varied as indicated in the legend to the figure. manual. Essentially, the amplified, biotin-labeled human cRNA samples Proteasome Activity Assay were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) A A JY cells were treated with TSA (1 Ag/mL), Sodium Butyrate (20 Ag/mL), formamide at a final concentration of 25 ng/ L. cRNA (1.5 g of each) Lactacystin (3.8 Ag/mL), or 5 AL of DMSO (untreated) in RPMI supplemented were hybridized. Hybridization was allowed to proceed at 55 C, for 18 h after which, the bead array matrix was washed for 10 min with with 5% FBS for 18 h. Sodium Butyrate was used as a control based on the fact that to the best of our knowledge it is the only HDAC inhibitor reported 1 High temperature buffer (Illumina), followed by a subsequent 10-min wash in Wash E1BC buffer. The arrays were then washed with 100% to reduce proteasome activity beside TSA (16). Lactacystin served as a ethanol for 10 min to strip off any remaining adhesive on the chip. A generic inhibitor of proteasome function. Cells were harvested, washed, and 2-min E1BC wash was performed to remove residual ethanol. The arrays resuspended in complete medium. Proteasome activity assay was performed were blocked for 5 min with 1% (w/v) casein-PBS (Pierce). The array using the cell-based luminescent assay Proteasome-Glo (Promega) in white signal was developed via 10-min incubation with Streptavidin-Cy3 at a flat-bottomed 96-well plates (Costar) with 20,000 cells per well. Lumines- final concentration of 1 Ag/mL solution of (GEHealthcare) in 1% casein- cence was measured in a plate reader Infinite M200 (TECAN). Assays were PBS blocking solution. The Human Expression BeadChip was washed a done in triplicate and repeated twice with similar results. final time in Wash E1BC buffer for 5 min and subsequently dried via Deconvolution and Fluorescence Microscopy centrifugation for 4 min at a setting of 275 rcf. The arrays were scanned Cells treated with TSA and immunostained as described above were also on the Illumina BeadArray Reader, a confocal-type imaging system with used for deconvolution microscopy studies. Briefly, after staining with 532 (Cye3) nm laser illumination. The bead signals were computed with antibodies, cells were incubated with 4¶,6-diamidino-2-phenylindole-HCl for weighted averages of pixel intensities, and local background is subtracted. 15 min, and then cytospun onto glass slides. Slides were air dried and Sequence-type signal was calculated by averaging corresponding bead mounted in glycerol using coverslips (Fisher Scientific). The slides were signals with outliers removed (using median absolute deviation). examined by epifluorescence microscopy (Nikon T200 microscope) Preliminary data analysis and QC was carried out using the BeadStudio mounted on Delta Vision deconvolution microscope (Applied Precision, software (Illumina). Simultaneous normalization of multiple microarrays LLC). In some cases, where indicated, deconvolution was performed on the was done using the mloess method (18). resultant image stacks using SoftWorx as supplied by the manufacturer (Applied Precision, LLC). Images shown in Figs. 2 and 3 were volume projections of several consecutive optical sections taken from the center of Results the image stacks after deconvolution. In experiments where comparative HDAC inhibition down-regulates hTRT presentation. Previ- quantitation of fluorescence was performed, samples to be compared were ously, it was shown that TSA treatment increases the transcription stained under identical conditions with the same reagents at the same time. and expression of hTRT in normal (19, 20) and cancer (21) cells. Samples were imaged under identical conditions. Camera exposure time was identical and pixel intensities were kept within the linear range of the Consistent with this report, we found that treatment with TSA camera (CoolSnap HQ; Princeton Instruments). Quantitation was per- induced an increase in the transcriptional activity of the hTRT gene + + formed on volume projections composed of several consecutive optical in the HLA-A2 /hTRT lymphoblastoid JY cell line. In repeated sections. The resultant volume projections were scaled identically. independent experiments, specific mRNA amplification was Quantitation was performed using SoftWorx at Data Inspector. consistently visible after 30 minutes, peaked at 18 hours, and Reverse Transcriptase-PCR persisted for an additional 24 hours after TSA removal (data not hTRT mRNA was detected by reverse transcriptase (RT)-PCR analysis. shown). We reasoned that the increase in hTRT transcription could Briefly, 106 cells were treated with TSA (1 Ag/mL) for the indicated time, yield an increase in the number of HLA-A2/hTRT peptide and total RNA was isolated using the RNAeasy Mini kit (Qiagen). The complexes displayed at the cell surface (antigen presentation). RNA was reverse transcribed into cDNA by RT-Omniscript (Qiagen) for 1 h Therefore, we investigated complex formation between the HLA-A2 at 37jC. cDNA amplification was induced using specific primers (forward molecule and two high affinity (f4 nmol/L) hTRT peptides, p540 5¶-ACCAAGAAGTTCATCTCCCTGG-3¶ and reverse 5¶-GCTCATCTTC- and p865. Detection of the two complexes was performed using ¶ CACGTCAGCTC-3 ). Amplification of the glyceraldehydes-3-phosphate two human recombinant Fab antibodies, 4A9, which is specific for dehydrogenase (GAPDH) mRNA served as a control for RNA extraction. the HLA-A2/p540 complex, and 3H2, which is specific for the HLA- Quantitative RT-PCR A2/p865 complex (15). Notably, each antibody only recognized the cDNA was generated from 200 ng of total RNA using Omniscript Reverse complex formed with the homologous hTRT peptide. Complexes Transcriptase (Qiagen). Quantitative RT-PCR (qRT-PCR) was carried out with hybrid peptides comprising 5 amino acids residues of the using the Taqman method on a LightCycler 480 real-time PCR system homologous peptide and 4 amino acid residues from a different (Roche) and analyzed by efficiency-corrected relative quantification method. hTRT peptide did not mediate binding (Fig. 1A). Surprisingly, in Sequences for probes and primers were as follows: tapasin (NM_003190.3): Forward, CCAGAGCCTCAGCAGGAG; Reverse, GGGTGAGGACAGTCAG- several independent flow cytometry analyses, surface staining by TACCA; Universal ProbeLibrary probe #9 (Roche Applied Science): GADPH, either 4A9 or 3H2 was substantially reduced (Fig. 1B) in TSA- Forward AGCCACATCGCTGAGACA; Reverse, GCCCAATACGACCAAATCC; treated JY cells compared with untreated cells. Diminished surface Universal ProbeLibrary probe #60 (Roche Applied Science). Results were expression of the HLA-A2/hTRT peptide complex persisted for expressed as fold change compared with untreated cells. 24 h after TSA removal (data not shown). Surface expression of

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Figure 1. Flow cytometry analysis of decreased presentation of HLA-A2/hTRT peptide complexes by TSA treatment. A, flow cytometry analysis of the specificity of 4A9and 3H2 antibodies for HLA-A2/hTRT complexes. Left, 4A9antibody (2 Ag) in 100 AL was used to stain unpulsed TAP-deficient T2 cells or cells pulsed with 10 Ag/mL of p540 or the hybrid peptides p540/p865 (ILAKFFLLV) and p540/p49(ILAKFRRRV). Right, 3H2 antibody was similarly used to stain unpulsed TAP-deficient T2 cells or cells pulsed with p865 or the hybrid peptides p865/p540 (RLLVDDLHWL) and p865/p49(RLVDDRRRV). B, flow cytometry analysis of surface staining for the HLA-A2/p540 and HLA-A2/p865 complex in TSA-treated JY cells and stained by either antibody 4A9or 3H2 (2 Ag per reaction). JY cells were treated for 8 or 18 h as indicated. Analyses were performed as detailed in Materials and Methods. C, flow cytometry analysis of surface staining for the HLA-A2 in TSA-treated JY cells. Analyses were performed as detailed in Materials and Methods. Results are representative of multiple independent experiments.

HLA-A2 molecules was similar in treated and untreated cells (Fig. dent experiments. A higher magnification of single cells further 1C), although in 3 of 10 experiments, a decrease (10–20%) in mean shows this phenomenon (Fig. 2C). fluorescence intensity was observed after TSA treatment. Decreased antigen presentation was not unique to JY cells To better visualize the reduction in antigen presentation, we because TSA treatment (18 hours) of the HLA-A2+/hTRT+ used deconvolution microscopy focusing on the HLA-A2/p540 melanoma cell line 629.38 resulted again in transcriptional complex because this has a longer half-life (f6 hours) than the activation of hTRT (Fig. 3A) and, importantly, in diminished surface HLA-A2/p865 complex (11). In untreated JY cells, HLA-A2/p540 expression of the complex (Fig. 3B). TSA treatment increased complexes were visible with a bright surface granular staining somewhat the detection of HLA-A2 molecules at the cell surface (Fig. 2A, left). However, fewer complexes were seen in TSA-treated (Fig. 3C). Collectively, whereas a decrease in presentation is not versus untreated cells (Fig. 2A, right). A comparative, quantitative supported by a decrease in surface expression of HLA-A2 molecules, analysis of the corresponding images showed a 3-fold decrease in the data show that chromatin changes after HDAC inhibition by the intensity of complex staining in TSA-treated cells (Fig. 2B). The TSA promote hTRT transcription but decrease presentation of MHC same quantitative difference was documented in three indepen- complexes formed with high affinity hTRT peptides. www.aacrjournals.org 8087 Cancer Res 2008; 68: (19). October 1, 2008

Gene profiling analysis. MHC I/peptide complexes originate in peptide generation, MHC I/peptide complex maturation, and/or the endoplasmic reticulum (ER) where nascent MHC I molecules egress from the ER. To this end, the effect of TSA on surface are loaded with peptides that are generated in the proteasome and expression of the HLA-A2/p540 complex was compared with that are translocated into the ER by the transporter associated with of lactacystin, a selective inhibitor of the proteasome, and BFA, an antigen processing (TAP). Because the MHC I/peptide cargo in the ER ! Golgi transport inhibitor that also inhibits MHC I/peptide ER is a function of peptide input from the cytosol and the local complex presentation. Lactacystin, markedly reduced presentation synthesis of MHC I molecules, we reasoned that the 3-fold lower compared with untreated as well as TSA-treated JY cells (Fig. 4A). antigen presentation concomitant with an increase in antigen BFA (18-hour treatment) blocked completely surface expression of expression in the cytosol could have several explanations. For the complex (Fig. 4A). Interestingly, BFA treatment for 4 hours did instance, TSA could inhibit the proteasome and/or TAP, hence not affect the cell surface expression of the MHC/peptide complex diminishing assembly of MHC I/peptide complexes in the ER. To (data not shown), in agreement with the half-life (f6 hours) of the this end, we tested TAP expression levels by flow cytometry in complex. This suggests that the diminished presentation associated TSA-treated cells and found no change. This lead to the further with HDAC inhibition by TSA could be due to either a partial defect possibility that proteasome, or events in the ER-Golgi, could impair of the proteasome, a defect in ER ! Golgi transport, or both.

Figure 2. Deconvolution microscopy analysis of decreased presentation of HLA-A2/hTRT peptide complexes by TSA treatment. A, deconvolution microscopy analysis of surface (Sfc) staining for the HLA-A2/p540 complex in untreated and TSA-treated (18 h) JY cells. Green, HLA-A2/p540 complexes; blue, nuclei (magnification, 60). Analyses were performed as detailed in Materials and Methods. Data are representative of four independent experiments. B, quantitation of fluorescence intensity of the optical images shown in A. The scale represents a gradient of pixel intensities. Analyses were performed as detailed in Materials and Methods. C, deconvolution microscopy visualization of surface HLA-A2/p540 complexes at higher magnification (100) comparing surface contour of untreated and TSA-treated (18 h) JY cells. Green, complexes; blue, nuclei.

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genes in malignant human cells (22), and supports the notion that TSA-mediated apoptosis of tumor cells may be mediated by ER stress. Consistent with the induction of ER stress is the transcriptional activation of proinflammatory genes such as tumor necrosis factor-a (7) and IL-23 p19 (4; data not shown), which are up-regulated during ER stress (23). ER and Golgi resident and trafficking related genes. Analysis of these genes showed an overall minor increase in ER!Golgi trafficking genes indicating that the diminished presentation of HLA-A2/p540 complexes by TSA treatment is unlikely to be due to a decrease in ER!Golgi trafficking. MHC I antigen presentation–related genes. Transcrip- tional analysis of the MHC I pathway was performed to identify changes that could explain the diminished surface expression of the HLA-A2/p540 complex. Overall, MHC I genes were unchanged, although a slight negative trend was observed (Fig. 4B). The increase in Bap31 (an ER resident type II transmembrane protein exerting quality control on MHC I/peptide complexes) and the decrease in Der1 (an ER retrotranslocation transmembrane protein) suggest a program to conserve MHC I molecules. In contrast, tapasin, a key regulator of TAP and MHC I peptide binding, and a gatekeeper of MHC I/peptide complex stability (24), was markedly diminished. Consistently, transcription of the tapasin-like gene, which shares similarity at the tridimensional level with tapasin, was also markedly down-regulated. Finally, additional members of the peptide loading complex and MHC I maturation (TAP1, TAP2, calnexin, and ERp57) showed little change, if any, with the exception of calreticulin (2 increase). HDAC inhibition affects the proteasome and tapasin. Gene profiling analysis proved of key value in assessing the possible downstream targets of TSA with respect self-tumor antigen Figure 3. TSA treatment down-regulates presentation in 629.38 melanoma presentation. First, we noted that wide spectrum HDAC inhibition cells. Melanoma cells (629.38) were treated with TSA (1 Ag/mL) for 18 h. A, RNA was extracted and tested by RT-PCR for hTRT transcription as indicated in down-regulated transcription of most genes of the proteasome Materials and Methods. B, cells were cell surface stained with antibody 4A9to complex corresponding to a global decrease in proteasome activity visualize the HLA-A2/p540 complexes as indicated in Materials and Methods. of f30% (Fig. 5A). Because hTRT proteolysis requires ubiquination Green, HLA-A2/p540 complexes; blue, nuclei (magnification, 60). C, HLA-A2 expression was assessed by flow cytometry. and is mediated by the proteasome (25), the decrease in proteasome activity by HDAC inhibition is consistent with the decrease in presentation of hTRT. Because peptide generation in To distinguish between these possibilities, a genome-wide the proteasome is the first step in antigen processing, the observed expression profiling of TSA-treated (18 hours) JY cells was diminished presentation of hTRT may reflect in part an effect on performed to see which gene pathway(s) might be involved in the proteasome. Second, HDAC inhibition promoted ER stress, reducing antigen presentation by HDAC inhibition. Proteasome something not observed previously after TSA treatment. Several genes. Peptides for the MHC I are generated mainly by the considerations suggest that ER stress may not play a crucial and proteasome and other cytoplasmic proteases. The 26S proteasome direct role in diminishing presentation of hTRT because (a) hTRT that is responsible for the degradation of poly-ubiquinated proteins transcription increases after TSA treatment, whereas the unfolded is composed of outer structural a-subunits and inner proteolytic protein response (ER stress) in general diminish transcription (26), h-subunits that are encoded by different genes. The microarray and (b) treatment with thapsigargin (an inhibitor of the Ca2+ pump analysis of the proteasome in TSA-treated JY cells showed an and inducer of ER stress) did not diminish the surface expression of overall down-regulation of h-subunit genes (Fig. 4B), with the MHC I/peptide complex (data not shown). Third, a defect in h-subunits 8 (PSMB8) and 10 (PSMB10) being down-regulated ER!Golgi transport of MHC I/peptide complexes is also unlike the most. Of note, gene expression of the cytoplasmic peptidase because most genes involved in transport and trafficking TPPII was unchanged (data not shown), suggesting that the down- underwent discrete up-regulation upon TSA treatment, ruling out modulation of the h-subunits of the proteasome could be defects in ER!Golgi transport. Finally, selected changes in the responsible, in part, for a downstream diminished loading of MHC I–related genes were observed with tapasin being markedly peptides (i.e., p540 and p865) onto the MHC I molecules in the ER. down-regulated. Tapasin down-regulation observed in the array ER stress genes. ER stress responsive genes, such as DNA damage– analysis (Fig. 4B) was confirmed by qRT-PCR (Fig. 5B). Tapasin is inducible transcript 3 (Ddit3), PP1RS15A (Gadd34), Heat shock involved in optimal loading of MHC I molecules with peptide and protein 5 (Grp78, Bip), Wolfram syndrome 1 (Wfs), activating in retaining unstable MHC I/peptide complexes in the ER, whereas transcription factor 4 (ATF4), and calreticulin, were all up- favoring egress of stable, high-affinity complexes (27), and in the regulated (Fig. 4B). This indicates that a downstream effect of retrieval of COPI vesicles containing MHC I molecules loaded with HDAC inhibition is the induction of ER stress. This is not surprising unstable peptides (28). Thus, its down-regulation can explain the because TSA is known to transcriptionally modulate many (2%) diminished antigen presentation. Because other genes related to www.aacrjournals.org 8089 Cancer Res 2008; 68: (19). October 1, 2008

MHC I/peptide complex biogenesis, such as calreticulin, Grp78/ p540-specific CTL as the effectors. The percent lysis of TSA-treated Bip, and COPI, were all moderately up-regulated, it seems as if the T2 cells pulsed with p540 was similar to that of untreated targets down-regulation of tapasin affected the quality control and sorting (Fig. 6A), suggesting that TSA does not interfere with cell surface of MHC I/peptide complexes in and from the ER, hence playing a MHC loading with peptide, nor does it change the susceptibility to central role in the diminished presentation of high affinity HLA-A2/ lysis of these tumor cells. Interestingly, after TSA treatment the hTRT complexes. lysis of all three tumor cells was markedly diminished compared Reduced killing of TSA-treated tumor cells by CD8 T cells. with untreated cells (Fig. 6A). The specificity of the CTL clone for Recognition of the MHC I/peptide complex is the structural basis the HLA-A2/p540 complex was shown by a canonical cold target for specific killing of tumor cells by cytotoxic T lymphocytes. To inhibition assay (11). As shown, the lysis of JY cells was specifically assess whether the decreased presentation by TSA treatment had and completely competed for by T2 cells pulsed with p540 but an effect on tumor target recognition and lysis, we performed not T2 cells pulsed with the control HIV gag peptide (Fig. 6B). killing assays using three different HLA-A2+/hTRT+ cancer cells To further prove the functional relevance of HDAC inhibition– (JY, Mel629.38, and LnCap) as targets and in vitro generated human induced reduced presentation of p540, new cold target inhibition

Figure 4. HDAC inhibition by TSA modulates the processing and presentation pathway in JY cells. A, deconvolution microscopy visualization of surface expression of the HLA-A2/p540 complex by antibody 4A9(2 Ag per reaction) on JY cells treated (18 h) with either lactacystin (LC;3.8Ag/mL), TSA (1 Ag/mL), or BFA (10 Ag/mL). B, gene array profiling of JY cells treated with TSA (18 h). Total RNA was extracted and analyzed using Illumina bead arrays. Levels of gene expression were compared with untreated JY cells. In the clustergrams, genes are grouped into proteasome genes; ER stress responsive genes; ER!Golgi resident and trafficking genes; and MHC I–related genes. The fold changes are noted on the respective color scales. Two independent experiments were performed and shown.

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Figure 5. Analysis of proteasome function and the expression of tapasin after TSA treatment. A, reduction of proteasome activity by TSA. JY cells were treated (18 h) with TSA (1 Ag/mL), sodium butyrate (20 Ag/mL), or lactacystin (10 Amol/L), and the activity of the proteasome was quantified using Proteasome-Glo assay. Cells in 5 AL of DMSO (untreated) served as a control. Tests were done in triplicate as indicated in Materials and Methods. B, levels of tapasin gene expression as determined by qPCR. Tapasin levels in JY cells were determined at 8 and 18 h after TSA treatment and compared with untreated cells. GAPDH is shown as a control.

experiments were performed using TSA-treated JY, 629, and LnCap recognition of epitopes associated with impaired peptide process- cells as hot targets. We reasoned that if TSA treatment diminishes ing (31). MHC I/peptide presentation in tapasin (/) human presentation of the HLA-A2/p540 complex rendering cells less cells is subverted qualitatively and quantitatively (32). Thus, by susceptible to lysis by CTL, inhibition by cold targets pulsed with altering the composition of the MHC I/peptide complexes in favor p540 would be more effective diminishing killing of hot targets. As of low affinity peptide complexes, and by decreasing antigen shown, killing of TSA-treated targets was markedly reduced in the presentation, HDAC inhibition reduces presentation of high- presence of p540-pulsed cold inhibitors compared with control affinity self-antigen peptides in tumor cells. This scenario assumes cold inhibitors (Fig. 6C and D). Of note, this effect was dose that the abundance of hTRT relative to other ubiquinated dependent and almost complete at the highest cold/hot ratio. proteins remains the same before and after HDAC inhibition. Collectively, these results show that HDAC inhibition decreases Furthermore, although the main effect of TSA at the transcrip- presentation, and this negatively affects killing by specific CTL. tional level is through histone acetylation, a direct effect on tapasin by acetylation of the double lysine motif at the COOH terminus (33) cannot be ruled out. Discussion That HDAC inhibition decreases the presentation of a self-tumor We show that HDAC inhibition in human cancer cells diminishes antigen is new and at variance with recent findings that mouse the presentation of high affinity peptides of telomerase reverse tumor cell lines treated with TSA undergo the up-regulation of transcriptase, a self-tumor antigen. HDAC inhibition also down- genes of the MHC Class I pathway, including TAP and tapasin regulates proteasome genes and tapasin, a key ER-associated gene (34). A discrepancy between the present report and the studies of the class I pathway. Importantly, we show that subsequent to in mouse cells is unclear and may reflect species differences. Of these changes, tumor cells are killed less efficiently by cytotoxic T note, however, antigen presentation in mouse tumor cells was lymphocytes. Collectively, our data suggest that epigenetic not studied with the aid of an anticomplex antibody as it was modifications of tumor cells can affect self-tumor antigens done here. Finally, the down-regulation of tapasin documented presentation. here is surprising in light of the mouse data. Because HDAC Although HDAC inhibitors alter 2% to 5% of the genome inhibition opens the chromatin and transcriptionally activates depending on the cell type and the inhibitor (22, 29), we could many genes, down-regulation of tapasin may be the indirect identify transcriptional changes that directly relate to the MHC I effect of genes not yet identified. Future studies will need to pathway of antigen presentation provisionally excluding defects in address this issue. transport along the secretory pathway. In light of our findings, we hTRT is a self-antigen in human cancer cells (35) and it may be propose that modifications associated with HDAC inhibition by involved in immune surveillance because cancer patients, but not TSA modulate hTRT presentation involving (a) down-regulation normal individuals, carry hTRT-specific CD8 T cells in their blood of proteasome’s genes with diminished proteasome function (14). Here, chromatin acetylation by HDAC inhibition leads (f30%) and (b) down-regulation (f80%) of tapasin. Because unexpectedly to decreased antigen presentation of hTRT and tapasin impairs the quality control and sorting mechanisms for diminished killing by human CTL (Fig. 6A), suggesting that this high-affinity peptide complexes, a decrease in its expression and may be yet another mechanism of immune evasion. Because function would effect on the MHC I peptide repertoire, favoring the previous studies in normal human cells showed that hTRT plays an egress of MHC I molecules bound to unstable (low affinity) important role in resetting chromatin architecture during DNA peptides. Several facts favor this hypothesis. Tapasin-deficient mice replication (36), the finding that HDAC inhibition diminishes hTRT have defective immune responses with an altered MHC I/peptide presentation suggests that changes in the equilibrium between repertoire (30). Deficiency of tapasin in mice results in the hTRT and chromatin that is normally in place to preserve the DNA www.aacrjournals.org 8091 Cancer Res 2008; 68: (19). October 1, 2008

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Cancer Research damage response (37), is a fail-safe mechanism to oppose epigenetic modifications to chromatin, it seems as if epigenetic recognition of tumor cells by adaptive cellular T-cell responses, changes are at the origin of this type of immune evasion by cancer which are the main effector immune defense against cancer. cells. The above considerations on potential evasion of immune Taken together, the above considerations suggest that HDAC surveillance need, however, to be seen in the broader context of the inhibition in tumor cells could lead to evasion of immune reported increase in expression of ligands for the NKG2D receptor surveillance (38). Several reports indicate that tapasin is down- on human NK cells by HDAC inhibitors (45, 46). Higher expression regulated in tumors (39–44), implying that its down-regulation may of these ligands would increase susceptibility to killing by NK cells, somehow be linked with poor presentation of tumor antigens as hence counterbalancing the diminished killing by cytotoxic T cells. well as poor tumor protection by T cells in vivo. Interestingly, Whether or not the epigenetic regulation of hTRT presentation impaired tapasin expression in tumor cells can be reversed by IFNg reported herein applies to other self-tumor antigens remains (42), excluding genetic alterations as the underlying molecular untested. Of interest, however, TSA activates the transcription of mechanism of such deficiency. Because IFNg may impart the melanoma-associated MAGE A1, A2, A3, and A12 genes in a

Figure 6. Reduced killing of human tumor cells treated by TSA by CTL specific for the HLA A2/p540 complex. CTL from an individual donor immunized in vitro against p540 (11) were used as effectors (E) against 51Cr-labeled tumor targets (T) treated (18 h) with TSA (1 Ag/mL). A, direct lysis of TSA-treated or untreated human tumor cells (JY, LnCap, and Mel 629). T2 cells pulsed with p540 served as a control for the ability of the CTL clone to lyse p540 pulsed targets. Results refer to percent (%) lysis at the E:T ratios indicated. B, specificity of CTL by cold target inhibition assay. 51Cr-labeled, TSA-treated JY cells (5 104 cells/mL) were mixed with T2 cells pulsed with p540 (10 Ag/mL; E)orT2 cells pulsed with control HIV-1 gag peptide at the indicated cold/hot target cell ratios. The E:T ratio was kept constant at 20:1. Results refer to percent lysis according to ref. 11. Tests were run in triplicate. C to D, TSA treatment of tumor cells decreases presentation of p540 and, as a result, enhances cold target inhibition. The canonical CTL reaction was set at a fixed E:T ratio of 20:1. Hot target cells were mixed at cold/hot ratio of 50:1 (C) or 250:1 (D); black columns, cold T2 cells pulsed with p540; open columns, cold T2 cells pulsed with control HIV-1 gag peptide. Results are expressed as killing of tumor cells, with or without TSA treatment, in the presence of T2 cells pulsed with p540 as a proportion of killing in presence of T2 cells pulsed with control gag peptide that was used as 100% killing.

Cancer Res 2008; 68: (19). October 1, 2008 8092 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Epigenetics and Self-Antigen Presentation variety of different cell lines (47). To conclude, the demonstration Acknowledgments that epigenetic changes by HDAC inhibition affect the presentation Received 3/17/2008; revised 6/17/2008; accepted 7/15/2008. of hTRT, a universal self-tumor antigen, suggests a potentially The costs of publication of this article were defrayed in part by the payment of page important new link between epigenetic regulation of cancer cells charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and immune surveillance (38). We thank Drs. J. Feramisco (Moores Cancer Center Imaging Facility) for help with the deconvolution microscopy experiments and invaluable advice in their interpre- tation, S. Wain-Hobson (Istitut Pasteur) for comments on the manuscript, and Disclosure of Potential Conflicts of Interest J. Lapira for technical help. X.C. Gonzalez is grateful to the UCSD Alliance for Graduate Education and the Professoriate (NSF-9978892), and the Training Program in Basic No potential conflicts of interest were disclosed. Genetics (NIH/National Institute of General Medical Sciences 5 T32 GM08666-07).

References 18. Sasik R, Woelk CH, Corbeil J. Microarray truths and MHC class I antigen presentation by melanoma cells. consequences. J Mol Endocrinol 2004;33:1–9. Cancer Immunol Immunother 2008;57:647–54. 1. Feinberg AP. Phenotypic plasticity and the epigenetics 19. Takakura M, Kyo S, Sowa Y, et al. Telomerase 35. Zanetti M, Hernandez X, Langlade-Demoyen P. of human disease. Nature 2007;447:433–40. activation by histone deacetylase inhibitor in normal Telomerase reverse transcriptase as target for anti- 2. Bird A. Perceptions of epigenetics. Nature 2007;447: cells. Nucleic Acids Res 2001;29:3006–11. tumor T cell responses in humans. Springer Semin 396–8. 20. Cong YS, Bacchetti S. Histone deacetylation is Immunopathol 2005;27:87–104. 3. Polevoda B, Sherman F. N-terminal acetyltransferases involved in the transcriptional repression of hTERT in 36. Masutomi K, Possemato R, Wong JM, et al. The and sequence requirements for N-terminal acetylation normal human cells. J Biol Chem 2000;275:35665–8. telomerase reverse transcriptase regulates chromatin of eukaryotic proteins. J Mol Biol 2003;325:595–622. 21. Hou M, Wang X, Popov N, et al. The histone state and DNA damage responses. Proc Natl Acad Sci 4. Kouzarides T. Histone acetylases and deacetylases in deacetylase inhibitor trichostatin A derepresses the U S A 2005;102:8222–7. cell proliferation. Curr Opin Genet Dev 1999;9:40–8. telomerase reverse transcriptase (hTERT) gene in 37. d’Adda di Fagagna F, Teo SH, Jackson SP. Functional 5. Fraga MF, Ballestar E, Villar-Garea A, et al. Loss of human cells. Exp Cell Res 2002;274:25–34. links between telomeres and proteins of the DNA- acetylation at Lys16 and trimethylation at Lys20 of 22. Mitsiades N, Mitsiades CS, Richardson PG, et al. damage response. Genes Dev 2004;18:1781–99. histone H4 is a common hallmark of human cancer. Nat Molecular sequelae of histone deacetylase inhibition in 38. Burnet FM. Immunological surveillance in neoplasia. Genet 2005;37:391–400. human malignant B cells. Blood 2003;101:4055–62. Transplant Rev 1971;7:3–25. 6. Zou YR, Sunshine MJ, Taniuchi I, Hatam F, Killeen N, 23. Wheeler M, Rizzi M, Sasik R, et al. KDEL-retained 39. Delp K, Momburg F, Hilmes C, Huber C, Seliger B. Littman DR. Epigenetic silencing of CD4 in T cells antigen in B lymphocytes induces a pro-inflammatory Functional deficiencies of components of the MHC committed to the cytotoxic lineage. Nat Genet 2001;29: response. A possible role for ER stress in adaptive T cell class I antigen pathway in human tumors of epithelial 332–6. immunity. J Immunol 2008;181:256–64. origin. Bone Marrow Transplant 2000;25 Suppl 7. Avni O, Lee D, Macian F, Szabo SJ, Glimcher LH, Rao A. 24. Cabrera CM. The double role of the endoplasmic 2:S88–95. T(H) cell differentiation is accompanied by dynamic reticulum chaperone tapasin in peptide optimization of 40. Li J, Schuler-Thurner B, Schuler G, Huber C, Seliger B. changes in histone acetylation of cytokine genes. Nat HLA class I molecules. Scand J Immunol 2007;65:487–93. Bipartite regulation of different components of the MHC Immunol 2002;3:643–51. 25. Kim JH, Park SM, Kang MR, et al. Ubiquitin ligase class I antigen-processing machinery during dendritic 8. Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA MKRN1 modulates telomere length homeostasis cell maturation. Int Immunol 2001;13:1515–23. damage pathway regulates innate immune system li- through a proteolysis of hTERT. Genes Dev 2005;19: 41. Seliger B, Atkins D, Bock M, et al. Characterization of gands of the NKG2D receptor. Nature 2005;436:1186–90. 776–81. human lymphocyte antigen class I antigen-processing 9. Khan AN, Magner WJ, Tomasi TB. An epigenetically 26. Harding HP, Calfon M, Urano F, Novoa I, Ron D. machinery defects in renal cell carcinoma lesions with altered tumor cell vaccine. Cancer Immunol Immun- Transcriptional and translational control in the Mam- special emphasis on transporter-associated with anti- other 2004;53:748–54. malian unfolded protein response. Annu Rev Cell Dev gen-processing down-regulation. Clin Cancer Res 2003;9: 10. Vonderheide RH, Hahn WC, Schultze JL, and Nadler Biol 2002;18:575–99. 1721–7. LM. The telomerase catalytic subunit is a widely 27. Howarth M, Williams A, Tolstrup AB, Elliott T. 42. Seliger B, Schreiber K, Delp K, et al. Downregulation expressed tumor-associated antigen recognized by Tapasin enhances MHC class I peptide presentation of the constitutive tapasin expression in human tumor cytotoxic T lymphocytes. Immunity 1999;10:673–9. according to peptide half-life. Proc Natl Acad Sci U S A cells of distinct origin and its transcriptional upregula- 11. Minev B, Hipp J, Firat H, Schmidt JD, Langlade- 2004;101:11737–42. tion by cytokines. Tissue Antigens 2001;57:39–45. Demoyen P, Zanetti M. Cytotoxic T cell immunity 28. Paulsson KM, Kleijmeer MJ, Griffith J, et al. 43. Seliger B, Wollscheid U, Momburg F, Blankenstein T, against telomerase reverse transcriptase in humans. Association of tapasin and COPI provides a mechanism Huber C. Coordinate down-regul ation of multiple MHC Proc Natl Acad Sci U S A 2000;97:4796–801. for the retrograde transport of major histocompatibility class I antigen processing genes in chemical-induced 12. Finnin MS, Donigian JR, Cohen A, et al. Structures of complex (MHC) class I molecules from the Golgi murine tumor cell lines of distinct origin. Tissue a histone deacetylase homologue bound to the TSA and complex to the endoplasmic reticulum. J Biol Chem Antigens 2000;56:327–36. SAHA inhibitors. Nature 1999;401:188–93. 2002;277:18266–71. 44. Seliger B, Wollscheid U, Momburg F, Blankenstein T, 13. Shay JW, Bacchetti S. A survey of telomerase activity 29. Johnstone RW. Histone-deacetylase inhibitors: novel Huber C. Characterization of the major histocompati- in human cancer. Eur J Cancer 1997;33:787–91. drugs for the treatment of cancer. Nat Rev Drug Discov bility complex class I deficiencies in B16 melanoma 14. Filaci G, Fravega M, Setti M, et al. Frequency of 2002;1:287–99. cells. Cancer Res 2001;61:1095–9. telomerase-specific CD8+ T lymphocytes in cancer 30. Garbi N, Tan P, Diehl AD, et al. Impaired immune 45. Armeanu S, Bitzer M, Lauer UM, et al. Natural patients. Blood 2006;107:1505–12. responses and altered peptide repertoire in tapasin- killer cell-mediated lysis of hepatoma cells via specific 15. Lev A, Denkberg G, Cohen CJ, et al. Isolation and deficient mice. Nat Immunol 2000;1:234–8. induction of NKG2D ligands by the histone deacety- characterization of human recombinant antibodies 31. van Hall T, Wolpert EZ, van Veelen P, et al. Selective lase inhibitor sodium valproate. Cancer Res 2005;65: endowed with the antigen-specific, major histocompat- cytotoxic T-lymphocyte targeting of tumor immune 6321–9. ibility complex-restricted specificity of T cells directed escape variants. Nat Med 2006;12:417–24. 46. Diermayr S, Himmelreich H, Durovic B, et al. toward the widely expressed tumor T-cell epitopes of 32. Purcell AW, Gorman JJ, Garcia-Peydro M, et al. NKG2D ligand expression in AML increases in the telomerase catalytic subunit. Cancer Res 2002;62: Quantitative and qualitative influences of tapasin on the response to HDAC inhibitor valproic acid and 3184–94. class I peptide repertoire. J Immunol 2001;166:1016–27. contributes to allorecognition by NK-cell lines with 16. Yin L, Laevsky G, Giardina C. Butyrate suppression of 33. Paulsson KM, Jevon M, Wang JW, Li S, Wang P. The single KIR-HLA class I specificities. Blood 2008;111: colonocyte NF-n B activation and cellular proteasome double lysine motif of tapasin is a retrieval signal for 1428–36. activity. J Biol Chem 2001;276:44641–6. retention of unstable MHC class I molecules in the 47. Wischnewski F, Pantel K, Schwarzenbach H. Pro- 17. Eberwine J, Yeh H, Miyashiro K, et al. Analysis of endoplasmic reticulum. J Immunol 2006;176:7482–8. moter demethylation and histone acetylation mediate gene expression in single live neurons. Proc Natl Acad 34. Khan AN, Gregorie CJ, Tomasi TB. Histone deacety- gene expression of MAGE-A1, -A2, -A3, and –A12 in Sci U S A 1992;89:3010–4. lase inhibitors induce TAP, LMP, Tapasin genes and human cancer cells. Mol Cancer Res 2006;4:339–49.

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Ilenia Pellicciotta, Xochitl Cortez-Gonzalez, Roman Sasik, et al.

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