Generated by Bortezomib Immune Mechanism of the Antitumor Effects

The Journal of Immunology

Immune Mechanism of the Antitumor Effects Generated by Bortezomib

Chih-Long Chang,*,†,‡ Yun-Ting Hsu,† Chao-Chih Wu,† Yuh-Cheng Yang,* Connie Wang,x T.-C. Wu,x,{,‖,# and Chien-Fu Hungx,{

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8+ T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-speciﬁc CD8+ T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-speciﬁc CD8+ T cells. CD11c+ DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c+ DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects. The Journal of Immunology, 2012, 189: 3209–3220.

ortezomib has emerged as a potent chemotherapeutic agent D and IL-6 transcription, alteration of adhesion molecule ex- that can be used to treat advanced-stage cancer (for review, pression, release of mitochondrial cytochrome c, induction of p53 B see Refs. 1 and 2). It is the first of a novel class of anti- and MDM2 protein expression, downregulation of gp130, and cancer drugs, known as proteasome inhibitors, to be approved for activation of caspase activity (23–25). These mechanisms exert clinical use. Since its discovery, bortezomib has been used to treat immediate antitumor effects by inhibiting tumor cell proliferation a variety of human tumors, such as multiple myeloma (3–8), or promoting tumor cell apoptosis. lymphoma (9, 10), non-small cell lung cancer (11, 12), renal The antitumor immune response that develops as a result of cancer (13, 14), prostate cancer (15–17), pancreatic cancer (18), bortezomib treatment has yet to be fully explored. The bortezomib- melanoma (19), breast cancer (20, 21), and ovarian cancer (22). induced tumor cell apoptosis provides an opportunity for gener- Bortezomib inhibits a key regulator of intracellular protein deg- ating tumor-specific immunity through cross-priming mechanisms radation, the 26S proteasome, which has the downstream effect (26). However, the precise manner by which bortezomib instigates of inducing apoptotic cancer cell death by means of multiple tumor-specific immunity remains undetermined. It is known that mechanisms. These mechanisms include the prevention of NF-kB the uptake and cross-presentation of tumor Ags by dendritic cells activation, inhibition of cell growth via downregulation of cyclin (DCs) are necessary to produce tumor-specific T cell immunity. Typically, apoptosis that occurs in the developmental stages is regarded as immunologically tolerizing and leads to T cells *Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei City, that are unresponsive to Ags from normal tissues. In contrast, Taiwan 104; †Department of Medical Research, Mackay Memorial Hospital, Tamsui bortezomib-induced apoptosis is regarded as immunostimulatory. District, New Taipei City, Taiwan 251; ‡Institute of Biomedical Science, Mackay x It has been shown in vitro that the upregulation of maturation Medical College, Sanzhi District, New Taipei City, Taiwan 252; Department of Pathology, Johns Hopkins Hospital, Baltimore, MD 21231; {Department of Oncol- markers occurs in murine DCs that were fed bortezomib-treated ‖ ogy, Johns Hopkins Hospital, Baltimore, MD 21231; Department of Obstetrics and cancer cells. However, the underlying mechanism(s) by which DC Gynecology, Johns Hopkins Hospital, Baltimore, MD 21231; and #Department of Molecular Microbiology and Immunology, The Johns Hopkins University, Baltimore, maturation occurs and whether these mature DCs can produce MD 21205 T cell immunity remains uninvestigated (27). What is known is Received for publication January 4, 2012. Accepted for publication July 12, 2012. that, for T cell immunity to be produced, there must be cell-to-cell Address correspondence and reprint requests to Dr. Chien-Fu Hung or Dr. Chih-Long contact between the DC and dying cell, and furthermore, the Chang, Department of Pathology, School of Medicine, Johns Hopkins University, surface expression of heat shock protein (HSP) 90 by the dying CRB II Room 307, 1550 Orleans Street, Baltimore, MD 21231 (C.-F.H.) or Depart- cell may represent a distinct immunogenic stimulus (28, 29). ment of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei City, Taiwan 104 (C.-L.C.). E-mail addresses: [email protected] (C.-F.H.) and clchang@mmc. These in vitro reports suggest bortezomib-mediated tumor cell edu.tw (C.-L.C.) apoptosis may be an immunogenic event. The online version of this article contains supplemental material. In this study, we established an in vivo system to characterize the Abbreviations used in this article: CRT, calreticulin; DC, dendritic cell; ETB, epo- mechanisms of immune-mediated antitumor effects generated by lactaene; GA, geldanamycin; HPV, human papillomavirus; HSP, heat shock protein; bortezomib in a murine ([C57BL/63C3/He] F ) OV-HM ovarian MS, mass spectrometry; MS/MS, tandem mass spectrometry; PI, propidium iodide. 1 cancer model (30). We observed the best antitumor effects in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 tumor-bearing mice that were treated with 0.5 mg/kg bortezomib. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103826 3210 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

Furthermore, these antitumor effects were completely abolished In vivo tumor growth following bortezomib treatment with or in nude mice, which is suggestive of immune involvement in the without depletion of lymphocyte subpopulation antitumor effects of bortezomib. To determine whether the de- (C57BL/63C3/He) F1 mice were s.c. inoculated in the left thigh with 1 velopment of a tumor cell-based immunotherapy vaccine is million HM-1 cells on day 0. On day 5, mice received a single i.p. injection possible, we characterized the irradiated OV-HM tumor cells that of bortezomib (0.5 mg/kg). One week before tumor challenge, lymphocyte were treated or untreated with bortezomib. Mice that were vac- subset depletion was initiated with the injection of 100 mg rat mAb GK1.5 cinated with bortezomib-treated tumor cells developed tumor- (anti-CD4), 2.43 (anti-CD8), or PK136 (anti-NK1.1), which occurred ev- specific CD8+ T cell immunity, thereby indicating the presence ery other day for the first week and then once per week afterward. One day after the fourth administration of Abs, depletion was confirmed by flow of immunologically significant differences between the treated cytometry analysis of spleen cells stained with 2.43, GK1.5, or PK136. and untreated tumor cells. We found bortezomib could induce Depletion was maintained by weekly injections of Abs for the duration of the upregulation of chaperone proteins, HSP60 and HSP90, on the the tumor growth follow-up. tumor cell surface. These chaperone proteins are considered to be immunologically relevant because the immunogenicity of Intracellular cytokine staining and flow cytometry analysis bortezomib-treated tumor cells could be attenuated by HSP60 and/ Splenocytes from tumor-bearing mice were harvested 2 wk (day 19) after or HSP90 chemical inhibitors. These data suggest that the up- the bortezomib injection. For tumor-infiltrating lymphocyte analysis, HM-1 regulation of HSP60 and/or HSP90 on the surface of bortezomib- tumors from mice given 0.5 mg/kg bortezomib or control PBS were dis- treated tumor cells may play an important role in the immune- sected, weighed, chopped into small pieces, and washed with HBSS. Tissues were incubated for 15 min at 37˚C with a mixture of enzymes dissolved in mediated antitumor effects generated by bortezomib. HBSS. After digestion, single cells were harvested, and pooled splenocytes (5 3 106) from each group were cultured in vitro with 2 3 105 live HM-1 Materials and Methods tumor cells for 16 h with 2 mg Golgistop (BD Pharmingen, San Diego, Mouse and cell line CA). Cells were stained with allophycocyanin-conjugated monoclonal rat anti-mouse CD8a (1:100; eBioscience, San Diego, CA) for 20 min. Fix-

C57BL/63C3/He F1 origin mice and athymic nude mice were purchased ation with Cytofix/Cytoperm (BD Pharmingen) was followed by staining from BioLASCO (Taipei, Taiwan). All animals were maintained under with FITC-conjugated rat anti-mouse IFN-g (1:50; eBioscience) for 20 specific pathogen-free conditions. All procedures were performed in ac- min. Flow analysis was performed on a BD Biosciences FACScan (BD cordance to protocols and recommendations for the proper use and care of FACSCalibur). Each group was analyzed in triplicates, and representative laboratory animals. The mouse ovarian cancer cell line HM-1 (C57BL/ data were shown with mean 6 SD in a numerical bar. 63C3/He F1 origin) was cultured in MEM (Life Technologies, Gaithers- burg, MD) with 10% FBS (HyClone, Logan, UT), 100 U/ml penicillin Immunohistochemistry (Life Technologies), and 100 pg/ml streptomycin (Sigma-Aldrich, St. Louis, MO) in a humidified atmosphere of 5% CO2/95% air at 37˚C. On day 19, tumor tissues were extracted from tumor-bearing mice. Dis- sected mouse tumor tissues were briefly paraffinized. Consecutive 5-mm Cell culture and cell viability assay paraffin-embedded tissue sections were deparaffinized and rehydrated. After Ag retrieval, blocking was performed by protein blocker (DakoCy- Cell viability following bortezomib treatment in vitro (Millennium Phar- tomation, Carpinteria, CA), followed by overnight incubation with rat anti- maceuticals, Cambridge, MA) was determined by MTT assay. HM-1 cells mouse CD8a mAb (1:100 dilution; clone 53-6.7; BioLegend, San Diego, 4 seeded in 96-well plates (5 3 10 HM-1 cells/well) were incubated for 24 h CA) at 4˚C then washed. Subsequently, secondary Ab amplification and with different concentrations of bortezomib ranging from 0.1 to 1000 nM. visualization were achieved with a Rat-on-Mouse AP-Polymer kit (Biocare HM-1 cells were washed to remove the bortezomib and cultured for an- Medical, Concord, CA). The tissue sections were dehydrated and coun- other 24 h. MTT cell assay was performed in accordance to manufacturer terstained with Mayer’s hematoxylin. The primary Ab was replaced by (Sigma-Aldrich) protocol. The absorbance value of 570/630 nm for each appropriately diluted mouse IgG for negative control. well was determined by a microplate reader. Apoptotic and dead cells were detected by a Annexin V:FITC Apoptosis Detection kit (BD Biosciences Two-dimensional gel electrophoresis and image analysis Pharmingen, Oxford, U.K.). HM-1 cells were treated with either 1028 or 1027 M bortezomib and then In vivo tumor growth following bortezomib treatment harvested by trypsinization after 24 h of incubation. Cell surface proteins on these cells were separated using a Cell Surface Protein Isolation kit 3C3 (C57BL/6 /He) F1 mice were s.c. inoculated in the left thigh with (Pierce, Rockford, IL) in accordance with the manufacturer’s instructions. 1 million HM-1 cells on day 0. On day 5, mice received a single dose of The eluted proteins were further purified and precipitated by a two- bortezomib at the concentrations of 0.5, 1, and 1.5 mg/kg. Control mice received PBS. Tumor growth was monitored by calipers. Mice whose tu- dimensional clean-up kit (Amersham Biosciences, Piscataway, NJ) and mor had exceeded 2 cm in diameter prior to the end of the experiment were then resolubilized in lysis buffer containing 8 M urea and 4% CHAPS. sacrificed. After sonication, 80 mg protein was applied onto immobilized immobilized pH gradient strips (pH 3–10, linear, 13 cm; Amersham Biosciences, In vivo tumor growth following bortezomib treatment in Arlington Heights, IL) for first-dimensional isoelectric focusing (IEF) athymic nude mice using an Ettan IPGphor System (GE Healthcare, Piscataway, NJ). The strips were rehydrated overnight with reduction and alkylation steps (7 M On day 0, athymic nude mice received s.c. transplantation of 1 million HM- urea, 2 M thiourea, 4% CHAPS, 45 mM DTT, and 0.5% immobilized pH 1 cells in the left thigh. On day 5, athymic nude mice began receiving 0.5 gradient buffer). Subsequently, they were overlayered onto 10% SDS mg/kg bortezomib or PBS (control) in vivo for the specified duration. polyacrylamide gels (18 3 13 cm) for two-dimensional protein separation using a Hoefer SE 600 gel electrophoresis unit (Hoefer, San Francisco, Immunization of mice with HM-1 cell-based vaccine CA). Proteins were visualized by a Pierce Silver Stain kit (24612; Pierce). The stained two-dimensional gels were scanned with an ImageScanner I HM-1 cells were irradiated with UV light (104 mJ/cm2; Stratalinker UV 2 (GE Healthcare). The image analysis and two-dimensional gel proteome Crosslinker, Stratagene, La Jolla, CA) or treated with bortezomib (10 8, 1027, and 1026 M for 24 h) and then washed and resuspended in HBSS database management were performed with ImageMaster Two-Dimensional (treatment-induced cell death and apoptotic ratio are shown in Fig. 3A). platinum software 5.0 (GE Healthcare). For identification of proteins by mass spectrometry (MS), matching was done (by some landmark spots) Female C57BL/63C3/He F1 mice were i.p. vaccinated on days 0 and 7 with 1 3 106 of treated HM-1 cells. Ten days later (day 17), splenocytes between consecutively repeated analytical silver-stained gels, including from vaccinated and naive mice were processed for immunoassay. For preparative gel for each sample, to correlate the precise positions. Protein tumor treatment in vivo, mice were s.c. inoculated with 1 3 106 HM-1 spots with differential intensity over area (ratio . 2or,22; p , 0.01) cells on day 0. Mice received 1 3 106 of treated HM-1 cells by i.p. route were identified, upon which, 10 spots with the greatest statistical sig- on days 5 and 12. Mouse survival and tumor growth were measured over nificance were selected and excised for further analysis to identify the time. immunologically relevant proteins. The Journal of Immunology 3211

In-gel digestion and liquid chromatography/tandem MS CO2 incubator at 37˚C for 6 h. Cells were stained with allophycocyanin- analysis conjugated rat anti-mouse CD11c (MACs; Miltenyi Biotec) and subjected to flow cytometry analysis (BD FACSCalibur). In-gel digestion of protein spots was performed on silver-stained gel. Se- lected spots were excised, cut, and washed twice with 25 mM ammonium In vitro effect of bortezomib on DC phagocytosis bicarbonate buffer. The gel pieces were incubated in 100 ml 1% (v/v) 2-ME, Isolated CD11c+ DCs (2 3 105 cells/well) and HM-1 cells were treated with followed by the addition of 5% (v/v) 4-vinylpyridine. The gel pieces were indicated doses of bortezomib in vitro for 24 h. After washing the CD11c+ washed, soaked, dehydrated, and rehydrated in modified trypsin solution. DCs, either CFSE-labeled HM-1 cells or dextran–FITC (50 mg/ml) were Digested peptides were extracted and eluted with 0.1% formic acid and incubated with the CD11c+ DCs (incubation times are 5 h for bortezomib- analyzed in a linear trap quadropole-Orbitrap hybrid tandem mass spec- treated CD11c+ DCs and 1 h for dextran–FITC). DCs were stained with trometer (ThermoFisher, Houston, TX) in-lined with an Agilent 1200 allophycocyanin-conjugated rat anti-mouse CD11c (MACs; Miltenyi Bio- nanoflow HPLC system. The HPLC system was equipped with LC packing tec) and subjected to flow cytometry analysis (BD FACSCalibur). C18 PepMap100. Ten tandem MS (MS/MS) scans with linear trap quadropole were collected following a full MS scan with Orbitrap. With In vivo effect of bortezomib on DC maturation TurboSequest, all MS/MS data were searched against a mouse protein database downloaded from National Center for Biotechnology Informa- Maturation assay of tumor-infiltrating DCs obtained from tumor-bearing tion. The criterion for positive protein identification is a minimum of three mice was conducted in the following manner. HM-1 tumor-bearing mice digested peptides whose Xcorr is over 2.5 and from the same protein. were treated with 0.5 mg/kg bortezomib. A week later, tumors were dissected out and processed into single-cell preparations. Tumor-infiltrating + + Biotinylation of cell surface protein CD11c cells were isolated from the tumor tissues using CD11c microbeads (MACs; Miltenyi Biotec) and measured for their expression of Cell surface proteins of HM-1 cells were isolated using the Pierce Cell maturation markers. CD11c+ DCs were stained with FITC-conjugated rat Surface Protein Isolation kit (Thermo Scientific), according to the manu- anti-mouse CD40, CD80, CD83, or CD86 (eBioscience) and subjected to facturer’s protocol. Protein concentration was determined by bicinchoninic flow cytometry analysis. acid assay. All cell lysates were applied to a NeutrAvidin Agarose column, and biotinyl proteins were then eluted by 100 ml SDS-PAGE sample buffer Statistical analysis m with 50 mM DTT. The biotinyl proteins isolated from each 40 g total All data expressed as means 6 SE are representative of at least two proteins were analyzed by Western blots to determine the amount of cell different experiments. Comparisons between individual data points were surface Hsp90 and Hsp60 protein as described below. For total protein made using a Student t test or ANOVA. extraction, cells were lysed with protein extraction reagent (Pierce).

Western blotting Results In vivo treatment with bortezomib led to inhibited tumor growth Equal amounts of proteins (25 mg surface or total) were loaded and sep- + arated by SDS-PAGE using a 10% polyacrylamide gel. The gels were kinetics through CD8 T cell-mediated immune effects electroblotted to a polyvinylidene difluoride membrane (Bio-Rad Labo- To characterize the therapeutic effect of bortezomib, we examined ratories). After blocking, membranes were probed with mouse antitumor growth in HM-1 tumor-bearing mice after bortezomib (0.5, Hsp90 Abs (1:1,000; Abcam, Cambridge, U.K.) or mouse anti-Hsp60 Abs (1:1,000; Abcam) or mouse anti–b-actin Abs (1:10,000; Sigma-Aldrich) 1, and 1.5 mg/kg) administration by i.p. injections. The greatest for 1 h and then washed and incubated with rabbit anti-mouse IgG con- antitumoral effect was observed after 0.5 mg/kg bortezomib jugated to HRP (1:3,000; PerkinElmer Life Sciences, Santa Clara, CA). (p = 0.0038; 0.5 mg/kg bortezomib versus control). These results After incubation with the secondary Abs, the membranes were rinsed. imply the therapeutic effect is not dose dependent (Fig. 1A). To Ab binding was detected with ECL kit (Amersham Biosciences). determine whether the observed antitumoral effect generated by Chemical inhibition of Hsp90 and Hsp60 expression by HM-1 bortezomib is immune mediated, we investigated bortezomib in tumor cells treated with bortezomib in vitro tumor-bearing athymic mice. Bortezomib did not impede tumor HM-1 cells were treated with bortezomib and the indicated inhibitors growth in the immunodeficient mice, thereby suggesting host in vitro. Hsp90 inhibitor was geldanamycin (GA; Sigma-Aldrich), whereas immunity is implicated in bortezomib-mediated tumor rejec- the Hsp60 inhibitors were epolactaene (ETB; Wako Chemicals, Richmond, tion (Fig. 1B). To identify the effector cell subset(s) involved in VA) and nonactin (Sigma-Aldrich). HM-1 cells were incubated with the bortezomib-mediated antitumoral effect, HM-1 tumor-bearing bortezomib for 24 h. Six hours after the initiation of bortezomib treatment, mice (C57BL/63C3/He F1) given 0.5 mg/kg bortezomib by i.p. GA (2 mM) was added (18 h incubation). Twenty-two hours after the + + + addition of bortezomib, ETB (11 mM) was added (2 h incubation). Non- injection were depleted of CD8 , CD4 , or NK1.1 lymphocyte actin (10 mg/ml) was added 24 h before bortezomib (48 h incubation). subpopulations using monoclonal neutralizing Abs. The control Treated HM-1 cells were used to immunize mice or assess phagocytosis bortezomib-treated mice were given rat IgG. The depletion of by DCs. CD8+ T lymphocytes abolished the bortezomib-mediated ther- T cell proliferation assay apeutic effect (p = 0.0038, anti-CD8 versus control; Fig. 1C), which suggests bortezomib exerts therapeutic effect through the T lymphocytes isolated from the splenocytes of C57BL/63C3/He F1 mice + + by pan T cell isolation Kit II (MACs; Miltenyi Biotec, Auburn, CA) were CD8 effectors cells. The depletion of CD4 T cells had no im- stained with 5 3 1025 M CFSE (Sigma-Aldrich) for 30 min at 37˚C. These mediate impact on the antitumoral effect elicited by bortezomib. cells were washed, resuspended, and then seeded in 96-well plates (2 3 However, long-term data indicate reduced production of memory 5 10 cells/ well) in the presence of different doses of bortezomib for 24 h. cells when CD8+ T cells were primed in the absence of CD4+ After drug treatment, T cells were washed three times to remove the drug T cells (Supplemental Fig. 1). Taken together, these data indi- and cultured for another 72 h before stimulation with 2 mg/ml PHA (Sigma-Aldrich). T cells were then collected and subjected to flow cate systemic chemotherapy with 0.5 mg/kg bortezomib can elicit + cytometry analysis (BD FACSCalibur). CD8 T cell-dependent antitumoral response in tumor-bearing mice. CD11c+ phagocytosis of bortezomib-treated HM-1 cells with or without cotreatment with HSP60 and/or HSP90 inhibitors Treatment of tumor-bearing mice with bortezomib led to activated tumor-specific CD8+ T lymphocytes and tumor- CD11c+ DCs from tumor-bearing mice were isolated using CD11c microbeads (MACs; Miltenyi Biotec) and resuspended in culture medium infiltrating lymphocytes 5 (5 3 10 cells/ml). HM-1 cells treated with bortezomib, as previously Our initial observation of the mouse tumor model encouraged us described, with or without HSP inhibitors were labeled with 5 3 1025 M CFSE (Sigma-Aldrich) for 30 min at 37˚C and then washed and resus- to investigate whether the therapeutic effect of bortezomib is pended in culture medium (1 3 105 cells/ml). Both CD11c+ DCs and HM- associated with immune responses. HM-1 tumor-bearing mice 1 cells were seeded in 24-well plate (1 ml from each/well) and incubated in were given bortezomib (0.5, 1, or 1.5 mg/kg). Two weeks later, 3212 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

splenocytes from treated mice were isolated, cultured, and stimulated with irradiated HM-1 cells. Splenocytes were then analyzed by flow cytometry to characterize activated T lymphocytes, as indicated by IFN-g secretion. Mice given 0.5 mg/kg bortezomib were found to have the highest numbers of CD8+IFN-g+ cells (Fig. 2A). The immunologic activities somewhat correlated with the observed antitumor effect in the tumor-bearing mice. The tumors were dissected and examined for tumor-infiltrating CD8+ T lymphocytes. Immunohistochemical staining revealed treatment with bortezomib resulted in more recruitment of CD8+ T lymphocytes into the tumor (Fig. 2B). Single-cell suspensions prepared from the tumors were analyzed by flow cytometry to characterize the intratumoral immune cells. Tumor-bearing mice given 0.5 mg/kg bortezomib had significantly higher amounts of tumor-infiltrating CD8+IFN-g+ and CD4+IFN-g+ Tlymphocytes (p = 0.001 and 0.05, respectively; Fig. 2C). Mice immunized with HM-1 cells treated with bortezomib in vitro developed tumor-specific CD8+ T lymphocytes and protective antitumor effects To investigate the underlying mechanisms driving the antitumor immunity, HM-1 cells were treated with 1028,1027,or1026 M bortezomib in vitro and examined for the development of immunogenicity. First, it was noted that bortezomib triggered HM-1 cell death at concentrations of 1028,1027, and 1026 Mat48h(∼10, 50, and 75%, respectively; Fig. 3A, left). However the highest numbers of apoptotic (Annexin V positive) and dead (propidium iodide [PI] positive) tumor cells were observed 24 h after 1026 M bortezomib (p , 0.0001, ANOVA; Fig. 3A, right). Mice were then immunized twice (days 0 and 7) with cell-based vaccines produced from HM-1 tumor cells that were either irradiated or treated with bortezomib in vitro. On day 17, splenocytes were analyzed for tumor-specific CD8+ T cells. Although the highest amount of apoptosis occurred at 1026 M bortezomib, cell-based vaccine produced from HM-1 cells treated with 1027 M bortezomib elicited the greatest percentage of tumor-specific CD8+IFN-g+ T lymphocytes in comparison with the control, irradiated HM-1, or HM-1 cells treated with 1028 and 1026 M bortezomib (p = 0.0002, 1027 versus 1028 M; p , 0.0001 1027 versus 1026 M; Fig. 3B). As we expected, the tumor growth curve shows that cell-based vaccine derived from HM-1 cells treated with 1027 M bortezomib in vitro induced the strongest antitumor effect (p = 0.02; Fig. 3C). The survival curve also demonstrated a similar trend in that mice immunized with HM-1 cells treated with 1027 M bortezomib had the longest survival and reduced tumor size (Fig. 3D). Two-dimensional gel analysis revealed HM-1 cells treated with 27 FIGURE 1. In vivo bortezomib treatment of tumor in immunocompetent 10 M bortezomib in vitro had increased expression of cell and immunodeficient mice. (A) Growth curves for HM-1 tumor in mice surface chaperone proteins, Hsp90 and Hsp60 given bortezomib. A total of 1 3 106 murine ovarian cancer cells, HM-1, To identify the molecules that may contribute to the immunoge- were s.c. transplanted into (C57BL/63C3/He) F1 mice on day 0. On day 5, tumor-bearing mice were i.p. given different doses (0.5, 1, and 1.5 mg/kg) nicity of bortezomib-treated HM-1 cells, cell surface proteins of of bortezomib. Maximum therapeutic effect of bortezomib was observed at treated HM-1 cells were isolated through biotinylation, separated 0.5 mg/kg (p = 0.038; 0.5 mg/kg versus control) instead of 1 and 1.5 mg/kg by two-dimensional gel electrophoresis, and analyzed by MS. The (p = NS), suggesting no dose dependence for therapeutic effect. Doses protein spots indicating differential expression between HM-1 cells 2 2 below 0.5 mg/kg yielded no appreciable therapeutic effect (data not shown). treated with 10 7 and 10 8 M bortezomib were excised for further (B) Growth curves for HM-1 tumor in nude mice given bortezomib. analysis. In total, 10 statistically significant protein spots were One million ovarian cancer cells, HM-1, were s.c. transplanted into selected, of which, only 2 of the identified proteins were found to athymic nude mice on day 0. Tumor-bearing athymic nude mice were be immunologically significant. The immunologically significant given i.p. injections of bortezomib (0.5 mg/kg) or PBS (control). The antitumor effect of bortezomib was absent in nude mice (p = 0.79; 0.5 mg/ kg bortezomib versus control), indicating the effect is dependent on host immunity. (C) Growth curve of HM-1 tumor in tumor-bearing mice de- and neutralizing Abs against CD8, CD4, NK1.1, or control rat IgG. pleted of lymphocyte subsets and given bortezomib. A total of 1 3 106 Therapeutic effect of bortezomib was abolished by CD8+ T cells depletion ovarian cancer cells, HM-1, were s.c. transplanted into (C57BL/63C3/He) (p = 0.0038; rat anti-mouse CD8 IgG versus control rat IgG), indicating + F1 mice on day 0. On day 5, mice were given 0.5 mg/kg bortezomib i.p. therapeutic effect is CD8 T cell dependent. The Journal of Immunology 3213

FIGURE 2. Characterization of tumor-specific CD8+ T lymphocytes in tumor-bearing mice treated with bortezomib. (A) Activation of HM-1 tumor- + 6 specific CD8 T cells. A total of 1 3 10 HM-1 were s.c. transplanted into (C57BL/63C3/He) F1 mice on day 0. Tumor-bearing mice received different doses of bortezomib (0.5, 1, and 1.5 mg/kg, i.p. single dose) on day 5. On day 19, splenocytes from treated tumor-bearing mice were analyzed for tumor- specific CD8+ T cells using intracellular cytokine staining for IFN-g with irradiated HM-1 cells as the stimulator, as described in Materials and Methods. Left, Representative of flow cytometry data. Right, The bar graph depiction of the mean and SE derived from flow cytometric data. Significantly more tumor-specific CD8+IFN-g+ T lymphocytes were produced in mice treated with 0.5 mg/kg bortezomib than mice treated with 1 or 1.5 mg/kg bortezomib (0.67 versus 0.38 and 0.36%; p , 0.0001 and 0.0003). Control mice were given PBS only. (B) Representative photographs of immunohistochemical staining for tumor-infiltrating CD8+ T lymphocytes. Tumors obtained from mice treated with bortezomib (0.5 mg/kg) or PBS (control) were stained for CD8+ T cells (identified by brown staining). Control: Top panels at magnifications 3100 (left panels) and 3400 (right). Bortezomib: Bottom panels at magnifications 3100 (left panels)and3400 (right panels). More tumor-infiltrating CD8+ T cells were found in mice given bortezomib. (C) Representative flow cytometry identification of intratumoral tumor-specific CD8+ and CD4+ T cells obtained from tumor-bearing mice given 0.5 mg/kg bortezomib. Bortezomib significantly increased the percentage of tumor-infiltrating CD8+IFN-g+ T cells (1.6 versus 5.9%, control versus bortezomib treated; p = 0.001). proteins were the chaperone proteins, Hsp90 and Hsp60 (Fig. 4A). ETB) and Hsp90 inhibitor (GA). Bortezomib-treated HM-1 cells The results were further validated by Western blot analysis, which that were cotreated with Hsp60 and/or Hsp90 inhibitors eli- showed that the HM-1 cells treated with 1027 M bortezomib had cited a smaller number of tumor-specific CD8+IFN-g+ (acti- greater amounts of cell surface Hsp90 and Hsp60. However, the vated) T lymphocytes in tumor-bearing mice (p = 0.0007 by total amounts of Hsp90 and Hsp60 in HM-1 cells treated with 1027 adding GA, p = 0.0001 by adding ETB, and p , 0.0001 by adding or 1028 M bortezomib were found to be similar (Fig. 4B). Flow nonactin; Fig. 5A). A noteworthy observation was that the inhi- cytometry analysis also confirmed HM-1 cells treated with 1027 M bition of tumor cell surface HSP60 profoundly reduced tumor bortezomib had higher levels of surface (membranous) Hsp90 immunogenicity in comparison with the inhibition of tumor cell and Hsp60 proteins in comparison with HM-1 cells treated with surface HSP90. We further investigated whether the suppression 1028 M bortezomib (Fig. 4C). Thus, treating HM-1 cells with of Hsp90 and Hsp60 expression by HM-1 cells could alter the bortezomib can increase the amount of externalized Hsp60 and susceptibility of HM-1 cells to engulfment by DCs. HM-1 cells Hsp90. treated with bortezomib in vitro were CFSE labeled and irradiated. Treated HM-1 cells were then cocultured with CD11c+ DCs that Reduced expression of cell surface HSP60 and/or HSP90 were isolated with microbeads. Subsequently, CD11c+ DCs were greatly attenuates bortezomib-induced tumor cell analyzed for CFSE staining, which indicates phagocytosis of immunogenicity and phagocytosis of tumor cells by DCs treated CFSE-labeled HM-1 cells. CD11c+ DCs showed the most HM-1 cells were treated with bortezomib in vitro in conjunction phagocytic activity when cocultured with HM-1 cells treated with with the indicated inhibitors: Hsp60 inhibitors (nonactin and 1027 M bortezomib (p = 0.002; 1027 M bortezomib versus PBS 3214 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

FIGURE 3. In vitro bortezomib-treated HM-1 cells induced tumor-specific CD8+ T lymphocyte production and antitumor protective immunity in HM-1 tumor-bearing mice. (A) MTT assay assessment of cell vitality and tumor cell apoptosis following bortezomib treatment for 48 and 96 h. Left, The bar graph indicating the proapoptotic effects of increasing concentrations of bortezomib on HM-1 in vitro. Right, Representative flow cytometric data indicating the percentage of tumor cell apoptosis (Annexin v positive) and cell death (PI positive) following bortezomib treatment for 24 h. Bortezomib induced HM-1 cell death at 1028 M, 1027, and 1026 M after 48 and 96 h (∼10, 50, and 75% at 48 h; 0, 15, and 35% at 96 h). Representative flow cytometric data indicate the highest number of apoptotic (Annexin V positive) and dead cells (PI positive) following 24 h of 1026 M bortezomib treatment (3.12 versus 7.94 versus 21.75% of apoptotic cells; 1.84 versus 12.19 versus 18.92% of dead cells at 1028,1027, and 1027 M bortezomib; p , 0.0001; ANOVA). (B) Representative flow cytometric data indicating CD8+IFN-g+ T cells from mice immunized with cell-based vaccine. Cell-based vaccines were produced from irradiated HM-1 cells or HM-1 cells treated with different concentrations of bortezomib in vitro. Mice were immunized with cell-based vaccine twice (days 0 and 7). Cell-based vaccine produced from HM-1 cells treated with 1027 M bortezomib resulted in the most CD8+IFN-g+ T cell activation (0.19, 0.25, 0.30, and 0.76% for unvaccinated control mice, vaccination with irradiated HM-1, and vaccination with HM-1 treated with 1028 or 1027 M bortezomib, respectively). However, the highest concentration of bortezomib (1026 M) significantly reduced the number of antitumor CD8+IFN-g+ T lymphocytes (0.24%; p , 0.0001 1027 versus 1026 M). (C) Tumor growth curve evaluation of cell-based vaccines derived from irradiated or HM-1 cells treated with bortezomib in vitro. Tumor growth curve indicates cell-based vaccine produced from HM-1 treated with 1027 M bortezomib in vitro (Figure legend continues) The Journal of Immunology 3215 and 1028 M bortezomib). However, the phagocytic activity of vulnerability of tumor cells to the actions of immune cells. The CD11c+ cells was diminished when bortezomib-treated HM-1 systemic treatment of B16 melanoma with bortezomib sensitized cells were treated with Hsp60 inhibitors (p =0.0014byadding B16 melanoma to DC-based immunotherapy, which generated ETB and p , 0.0001 by adding nonactin) or Hsp90 inhibitor activated CD8+IFN-g+ T lymphocytes (31). Previous research (p = 0.036 by adding GA). The inhibition of tumor cell surface has also indicated that bortezomib may additionally enhance the Hsp60 had a greater impact on the immunogenicity of bortezomib- susceptibility of a variety of formerly apoptosis-resistant tumor treated HM-1 cells (p = 0.0002) (Fig. 5B). Our data indicate that cells with dysfunctional mitochondrial apoptotic pathways to the both Hsp60 and Hsp90 are important mediators of bortezomib- cytolytic action of the treatment-generated CD8+ T lymphocytes induced tumor immunogenicity and the resultant increase in the (31–34). A subtoxic concentration of bortezomib was able to uptake of bortezomib-treated tumor cells by CD11c+ DCs. How- drastically favor the mitochondrial apoptotic pathway of mel- ever, it seems that Hsp60, more so than Hsp90, is responsible for anoma cells by enhancing the release of SMAC and cytochrome the ability of bortezomib-treated tumor cells to be preferentially c in response to the CTL effector molecules of caspase-8 and phagocytized by CD11c+ DCs and promote the generation of tumor- cytosolic granzyme B (32). Furthermore, some cells respond to specific CD8+ T lymphocytes. For flow histograms depicting bortezomib with increased sensitivity to the cytotoxic downstream HM-1 cell surface Hsp90 and Hsp60 expression levels following effects of TRAIL binding to its respective receptor on the tumor bortezomib treatment with or without HSP inhibitors, refer to cell surface (34). Supplemental Fig. 2. In an attempt to delineate the mechanism by which bortezomib was rendering HM-1 cells immunogenic, we discovered that the Bortezomib treatment of DCs led to enhanced phagocytic amplification of the cell surface expression of HSP60 and HSP90 activity and DC maturation proteins made bortezomib-treated HM-1 cells unique from the Following bortezomib treatment in vitro, CD11c+ DCs exhibited non–bortezomib-treated HM-1 cells. We reasoned that proteasome increased phagocytosis of CFSE-labeled HM-1 cells (39.39, 45.0, inhibition by bortezomib led to the augmented cell surface ex- 2 2 and 51.51%, control versus 10 8 versus 10 7 M bortezomib, p , pression of HSP60 and HSP90 by HM-1 cells, because the evidence 0.0001, ANOVA; Fig. 6A). Bortezomib influenced both CD11c+ that suggests proteasome inhibition can bias gene expression in DCs and HM-1 cells to promote phagocytosis. The phagocytic favor of various HSPs are plentiful (23, 35). activities of bortezomib-treated DCs increased in a dose-dependent We attempted to identify the possible role(s) that HSP60 and manner. In addition, the phagocytosis of bortezomib-treated HSP90 may have in immune signaling because the immunogenicity CFSE-labeled HM-1 cells also increased in a dose-dependent of HM-1 cells and their preferential uptake by CD11c+ DCs, in 2 manner. When both DCs and HM-1 cells were treated with 10 7 M conjunction with the subsequent potency of these tumor-loaded bortezomib, treated HM-1 cells were phagocytized by the greatest CD11c+ DCs in the priming of HM-1–specific CD8+IFN-g+ ef- percentage of treated DCs (Supplemental Fig. 3). fector cells, are directly correlated with the externalization of To examine the effect of bortezomib on DC maturation in vitro, HSPs following bortezomib treatment. The present study dem- DCs identified by a CD11c+ marker were isolated from mouse onstrates that bortezomib-treated HM-1 cells have greater sus- 2 2 splenocytes. After bortezomib treatment (10 8 or 10 7 M), ceptibility to phagocytosis and that the surface expression of CD11c+ DCs increased their expression of maturation markers Hsp60, rather than Hsp90, has the greater role in promoting CD8+ 2 (CD40, CD80, and CD86) with 10 7 M bortezomib, leading to T cell activation. Another report that highlights the importance of greater expression of maturation markers (p = 0.002, 0.0014, Hsp60 as an immune mediator demonstrates the ability of Hsp60 and0.003,ANOVA;Fig.6B).Tounderstandtherealinvivo to enhance phagocytosis by monocytic U937 cells (36), consistent effect of bortezomib on DC maturation, we studied the intra- with the observation that DC phagocytosis of bortezomib-treated tumoral CD11c+ DCs obtained from tumor-bearing mice given cancer cells, including human myeloma cells, results in mature bortezomib. In response to systemic drug treatment, tumor- DCs that are especially effective at eliciting tumor-specific CD8+ infiltrating DCs upregulated their expression of CD40, CD80, IFN-g+ T lymphocytes (26). CD83, and CD86 maturation markers (p = 0.02, 0.013, 0.006, and Although the results of the current study emphasize the rela- 0.019; Fig. 6C). tionship between the externalization of Hsp60 by tumor cells and bortezomib-mediated generation of antitumor immunity, in truth, Discussion a variety of HSPs have been determined to modulate immune The use of bortezomib in the treatment of cancer has benefits responses. APCs of several lineages have been found to express beyond the simple proapoptotic action on tumor cells, because in HSP receptors; one such is the CD91 receptor whose ligands are mice with intact host immunity, it also results in the promotion of HSPs, such as calreticulin (CRT), gp96, hsp70, and hsp90 (37, 38). antitumor immunity through several mechanisms. Tumor-specific Moreover, the translocation of endogenous HSP90 to the cell CD8+IFN-g+ T lymphocytes were observed to be the effector cells surface has already been proven to be a part of the bortezomib- by which bortezomib exerts its antitumoral action. In response to induced immunogenic cell death that induces antitumoral immu- treatment with optimal bortezomib dose, the tumor-bearing mice nity (26). were detected to have markedly higher numbers of CD8+ tumor- Even though the nature of HSP60 and HSP90 as immune stimuli infiltrating lymphocytes. with respect to their role(s) in generating protective immunity Other than the generation of tumor-specific activated CD8+ against cancer is as yet undefined, the ability of HSP60 and HSP90 T cells, bortezomib has the added capability of enhancing the inhibitors to abrogate the bortezomib-induced HM-1 cell immu-

exhibited greater therapeutic effects (p = 0.02; 1027 M bortezomib versus control) than irradiated HM-1 cells or HM-1 treated with 1028M bortezomib. Control mice were unvaccinated. (D) Survival curve indicating enhanced survival of mice following immunization with cell-based vaccine derived from bortezomib-treated HM-1 cells. The tumor size of mice with prolonged survival was smaller than that of other groups. The duration of survival experiment was 65 d, and mice whose tumor had exceeded 2 cm in diameter prior to that period were sacriﬁced. Cell-based vaccine derived from HM-1 treated with 1027 M bortezomib in vitro signiﬁcantly prolonged the survival of mice in comparison with other groups. 3216 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

FIGURE 4. Two-dimensional gel analysis identification of upregulated cell surface chaperone proteins. (A) Two-dimensional gel electrophoresis comparison of differential expression levels of HM-1 cell surface proteins following treatment with either 1027 or 1028 M bortezomib. Left, The image of the two-dimensional gel containing separated tumor cell surface HSPs. Right, The synchronized three-dimensional view comparison of protein spots with differential intensity over area. Cell surface proteins of bortezomib-treated cells were isolated using biotinylation method as described in Materials and Methods. Surface proteins were run through two-dimensional gel electrophoresis, and selected spots were analyzed by MS. The differentially expressed protein spots (encircled by red line) between HM-1 cells treated with 1027 or 1028 M bortezomib were identified to be Hsp90 and Hsp60 proteins. (B) Western blot validation of two-dimensional gel results. Surface expressions of Hsp90 and Hsp60 proteins from HM-1 cells treated with 1027 M bortezomib were higher, whereas the total Hsp90 and Hsp60 protein expressions between the two treatment groups (1027 or 1028 M bortezomib) were similar. (C) Representative flow histogram depicting surface Hsp90 and Hsp60 expression levels by HM-1 cells following bortezomib treatment. HM-1 cells were treated with either 1027 or 1028 M bortezomib and then analyzed by flow cytometry to identify externalization of Hsp90 and Hsp60. Greater amounts of surface Hsp90 and Hsp60 were seen in HM-1 cells treated with 1027 M bortezomib (black solid line) and not in the cells treated with 1028 M bortezomib (gray solid line) or control cells (black dot line). The secondary Ab is represented by gray dot line. The Journal of Immunology 3217

FIGURE 5. Evaluation of bortezomib-induced HM-1 immunogenicity through chemical inhibition of Hsp90 and Hsp60 expressions. (A) Representative flow cytometric data of tumor-specific CD8+IFN-g+ T lymphocytes following stimulation with bortezomib-treated HM-1 cells that were or were not cotreated with HSP90 and HSP60 inhibitors. Left, Representative flow cytometric data of tumor-specific CD8+ T cell activation (IFN-g+) by HM-1 cells. Right, The bar graph of the mean and SE derived from flow cytometric data. HM-1 cells were treated with bortezomib and the indicated inhibitors (Hsp90 inhibitor, GA; Hsp60 inhibitors, ETB and nonactin) in vitro, as described in Materials and Methods. On day 0, mice were immunized once with HM-1 cells that were treated. On day 10, splenocytes from these mice were stimulated overnight by live HM-1 cells and analyzed for IFN-g+ cells. Representative flow cytometric data indicates immunization of tumor-bearing mice with HM-1 cells treated in vivo with 1027 M bortezomib leads to the highest numbers of tumor-specific CD8+IFN-g+ T lymphocytes (p , 0.0001; 1027 M versus irradiated and 1028 M), but the coadministration of Hsp60 and/or Hsp90 inhibitors could abolish the immunogenicity of bortezomib-treated HM-1 cells (p = 0.0007 by adding GA, p = 0.0001 by adding ETB, and p , 0.0001 by adding nonactin). (B) Representative flow cytometric dot plot depicting the phagocytosis of bortezomib-treated HM-1 cells by CD11c+ DCs. Left, The flow cytometric data depicting the percentage of CD11c+ DCs that have engulfed HM-1 cells that were treated with different concentrations of bortezomib, with or without coadministration with HSP90 and HSP60 inhibitors. The controls are DCs only (DCs incubated in the absence of HM-1 cells) and PBS treatment (Tx) DC (DCs coincubated with PBS-treated HM-1 cells). Right, The bar graph depiction of the mean and SE of flow cytometric data. HM-1 cells were treated with bortezomib with or without Hsp90 and Hsp60 inhibitors in vitro. Treated HM-1 cells were labeled with CFSE and irradiated. CD11c+ cells isolated with microbeads were cocultured with treated HM-1 cells. DCs were then analyzed for CFSE staining, which represents phagocytosis of HM-1 cells. The greatest percentage of CD11c+ DCs phagocytized HM-1 cells treated with 1027 M bortezomib (p = 0.002; 1027 M bortezomib versus PBS and 1028 M bortezomib). The coadministration of HSP90 inhibitors (p = 0.0014, ETB; p , 0.0001, nonactin) or Hsp90 inhibitor (p = 0.036, GA) or both inhibitors (p = 0.0002) during the in vitro treatment of HM-1 cells with bortezomib was able to diminish the phagocytic activities of CD11c+ cells. nogenicity demonstrates that bortezomib-invoked antitumor im- protective antitumor immunity that is induced by bortezomib- munity is HSP dependent. HSPs prepared from cancer cells can treated tumor cells. Inhibition of HSP60 greatly reduces the associate with endogenous tumor-specific antigenic peptide in vivo phagocytosis of bortezomib-treated HM-1 cells by CD11c+ DCs (39) and are effective immunogens capable of generating strong and, in turn, would diminish the generation of tumor-specific tumor-specific CD8+ T cell responses against syngeneic cancers CTLs. Although there is plenty of literature on the immunoge- (40, 41), which for this reason, have been used in multiple ther- nicity of Hsp90, our experimental results suggest the expression of apeutic cancer vaccine strategies (42). An important aspect of our Hsp60 on the tumor cell surface is even more essential than Hsp90 study is the identification of HSP60 as a novel mediator of the in the generation of tumor-specific CTLs. 3218 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

FIGURE 6. Flow cytometry analysis of DCs following bortezomib treatment in vivo and in vitro. (A) Representative flow cytometric histogram of the phagocytic activities of bortezomib-treated CD11c+ DCs on CFSE- labeled HM-1 cells. The greatest percentage of CD11c+ DCs phagocytized CFSE-labeled HM-1 cells after treatment with 1027 M bortezomib (39.39, 45.0, and 51.51%, control versus 1028 versus 1027 M bortezomib; p , 0.0001, ANOVA). (B) Representative flow histograms depicting CD11c+ DC maturation following bortezomib treatment in vitro. DCs were treated with either 1028 or 1027 M bortezomib. DC expression of maturation markers (CD40, CD80, and CD86) increased after treatment with 1027 M bortezomib (p = 0.002, 0.0014, and 0.003; ANOVA). (C) Representative flow histograms depicting the effect of bortezomib on DC maturation in vivo. HM-1 tumor-bearing mice were given bortezomib (0.5 mg/kg). One week later, CD11c+ cells were isolated from tumor tissues and stained for the expression of maturation markers. Bortezomib treatment in vivo promoted the expression of CD40, CD80, CD83, and CD86 in the tumor-infiltrating DCs (p = 0.02, 0.013, 0.006, and 0.019).

The splenocyte isolated non–tumor-loaded CD11c+ DCs that reported that bortezomib adversely affects monocyte-derived DCs were treated with bortezomib in vitro responded with enhanced by inhibiting proteasomal b5 subunit-located chymotrypsin-like phagocytosis of FITC-labeled dextran, the reason for which is peptidase, which is part of the ubiquitin–proteasome pathway unknown. In an attempt to define the effect of bortezomib on the that is crucial for the preservation of normal DC processes (43). living system, mice were treated with bortezomib in vivo, and the Additional reports of unfavorable effects that bortezomib has on DC markers were characterized. The maturation level of DCs is DCs include the reduction of monocyte-derived DC phagocytic highly correlated with their ability to prime T lymphocytes, which activity (44) and the triggering of monocyte-derived DC apoptosis makes it interesting that bortezomib treatment in vivo led to the (45) with differentially greater apoptotic effect on immature than upregulated expression of costimulatory molecules by tumor- mature monocyte-derived DCs (46). infiltrating CD11c+ DCs. We expect the in vivo effect that It is also a concern that bortezomib may cause immuno- bortezomib has on tumor-infiltrating DCs is related to the matu- suppression by inhibiting the action and/or proliferation of ration signals that are provided only when tumor cells undergo T lymphocytes. Lundqvist et al. (47) demonstrated that bortezomib immunologically relevant death. It has been demonstrated that renders tumor cells resistant to Ag-specific CTLs by altering the DCs acquire maturation signals via cell-to-cell contact with proteasomal processing of the Ag for which CTLs have specificity. bortezomib-treated tumor cells expressing Hsp90 (26). When Another example of immunosuppression by bortezomib was il- immature DCs were cocultured with bortezomib-treated apoptotic lustrated in a study conducted by Sun et al. (48) in which they tumor cells, the mature DCs, identified by the upregulation of both showed bortezomib could reduce graft-versus-host-disease fol- CD40 and costimulatory molecules, were capable of generating lowing marrow transplantation via the dose-dependent anti- protective antitumor immunity in mice (27). proliferative effect that the drug has on T lymphocytes. Like many Although the current study finds bortezomib to be beneficial in chemotherapeutic drugs, bortezomib leads to immunosuppression regard to the production of CTL-mediated immunity, other studies at high concentrations. Our results show that bortezomib posi- have suggested that bortezomib can restrain the production of tively influences the host immune system through the promotion tumor-reactive CD8+ T cells by acting on the DCs. It has been of CTL-mediated antitumor immunity, with the caveat being at The Journal of Immunology 3219 a low dose that is still of sufficient amount to produce the de- 9. Strauss, S. J., L. Maharaj, S. Hoare, P. W. Johnson, J. A. Radford, sired response. Bortezomib does not compromise the proliferative S. Vinnecombe, L. Millard, A. Rohatiner, A. Boral, E. Trehu, et al. 2006. Bortezomib therapy in patients with relapsed or refractory lymphoma: potential abilities of T lymphocytes at most of the tested concentrations correlation of in vitro sensitivity and tumor necrosis factor a response with 2 2 2 (10 10 to 10 8 M) and only at higher concentrations (10 7 and clinical activity. J. Clin. Oncol. 24: 2105–2112. 26 10. Zinzani, P. L., G. Musuraca, M. Tani, V. Stefoni, E. Marchi, M. Fina, 10 M) does T cell proliferation become restricted (Supple- C. Pellegrini, L. Alinari, E. Derenzini, A. de Vivo, et al. 2007. Phase II trial of mental Fig. 4). proteasome inhibitor bortezomib in patients with relapsed or refractory cutane- Akin to bortezomib, doxorubicin stands out as a chemothera- ous T-cell lymphoma. J. Clin. Oncol. 25: 4293–4297. 11. Scagliotti, G. V., P. Germonpre´, L. Bosqueé, J. Vansteenkiste, R. Gervais, peutic drug that is capable of modifying the manner in which cancer D. Planchard, M. Reck, F. De Marinis, J. S. Lee, K. Park, et al. 2010. A ran- cells die such that their apoptosis is recognized as immunologically domized phase II study of bortezomib and pemetrexed, in combination or alone, important. Apoptotic cancer cells that were treated with doxoru- in patients with previously treated advanced non-small-cell lung cancer. Lung Cancer 68: 420–426. bicin will translocate the HSP, CRT, to the cell surface where it 12. Voortman, J., E. F. Smit, R. Honeywell, B. C. Kuenen, G. J. Peters, H. van de is seen as a prophagocytosis signal by DCs (49). Similarly, the Velde, and G. Giaccone. 2007. A parallel dose-escalation study of weekly and surface expression of Hsp60 and Hsp90 by tumor cells fol- twice-weekly bortezomib in combination with gemcitabine and cisplatin in the first-line treatment of patients with advanced solid tumors. Clin. Cancer Res. 13: lowing bortezomib treatment also stimulates DC phagocytosis. 3642–3651. Other chemotherapeutic drugs that are capable of instigating the 13. Davis, N. B., D. A. Taber, R. H. Ansari, C. W. Ryan, C. George, E. E. Vokes, apoptosis of cancer cells, such as etoposide and mitomycin C, do N. J. Vogelzang, and W. M. Stadler. 2004. Phase II trial of PS-341 in patients with renal cell cancer: a University of Chicago phase II consortium study. J. not result in the development of antitumor immunity; and perhaps Clin. Oncol. 22: 115–119. not coincidentally, the nonimmunogenic apoptosis coincides with 14. Kondagunta, G. V., B. Drucker, L. Schwartz, J. Bacik, S. Marion, P. Russo, a lack of cell surface expression of CRT (ecto-CRT) (50, 51), M. Mazumdar, and R. J. Motzer. 2004. Phase II trial of bortezomib for patients with advanced renal cell carcinoma. J. Clin. Oncol. 22: 3720–3725. which underlines the importance of identifying immunomodula- 15. Papandreou, C. N., D. D. Daliani, D. Nix, H. Yang, T. Madden, X. Wang, tory drugs that are cytotoxic to tumors and trigger the develop- C. S. Pien, R. E. Millikan, S. M. Tu, L. Pagliaro, et al. 2004. Phase I trial of the ment of tumor-specific immunity. proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J. Clin. Oncol. 22: 2108– Our findings can serve as the basis for the development of new 2121. immunotherapy regimens that can enlist the host immunity to 16. Price, N., and R. Dreicer. 2004. Phase I/II trial of bortezomib plus docetaxel in enhance the antitumor therapeutic potency of bortezomib. Aside patients with advanced androgen-independent prostate cancer. Clin. Prostate Cancer 3: 141–143. from the production of tumor cell-based vaccines, bortezomib 17. Dreicer, R., D. Petrylak, D. Agus, I. Webb, and B. Roth. 2007. Phase I/II study of has additionally been shown to augment the efficacy of antitumor bortezomib plus docetaxel in patients with advanced androgen-independent prostate cancer. Clin. Cancer Res. 13: 1208–1215. therapeutic DNA vaccines (33). The coadministration of bortezomib 18. Alberts, S. R., N. R. Foster, R. F. Morton, J. Kugler, P. Schaefer, M. Wiesenfeld, with a therapeutic human papillomavirus (HPV) DNA vaccine T. R. Fitch, P. Steen, G. P. Kim, and S. Gill. 2005. PS-341 and gemcitabine in that encoded the HSP, CRT, was able to augment the production patients with metastatic pancreatic adenocarcinoma: a North Central Cancer + Treatment Group (NCCTG) randomized phase II study. Ann. Oncol. 16: 1654– of HPV Ag-specific CD8 T lymphocytes that were capable of 1661. cytotoxically targeting HPV Ag-positive cancers (33). Further- 19. Markovic, S. N., S. M. Geyer, F. Dawkins, W. Sharfman, M. Albertini, more, bortezomib sensitized the tumor cells to the cytolytic actions W. Maples, P. M. Fracasso, T. Fitch, P. Lorusso, A. A. Adjei, and C. Erlichman. + 2005. A phase II study of bortezomib in the treatment of metastatic malignant of the tumor-reactive CD8 T lymphocytes that were generated by melanoma. Cancer 103: 2584–2589. the treatment (33). Accordingly, we believe the results of these 20. Yang, C. H., A. M. Gonzalez-Angulo, J. M. Reuben, D. J. Booser, L. Pusztai, studies suggest the antitumor effects of bortezomib in vivo is in S. Krishnamurthy, D. Esseltine, J. Stec, K. R. Broglio, R. Islam, et al. 2006. Bortezomib (VELCADE) in metastatic breast cancer: pharmacodynamics, bio- part a consequence of the augmented generation of an antitumor logical effects, and prediction of clinical benefits. Ann. Oncol. 17: 813–817. immunity and may significantly benefit the therapeutic utility of 21. Engel, R. H., J. A. Brown, J. H. Von Roenn, R. M. O’Regan, R. Bergan, this drug. S. Badve, A. Rademaker, and W. J. Gradishar. 2007. A phase II study of single agent bortezomib in patients with metastatic breast cancer: a single institution experience. Cancer Invest. 25: 733–737. Disclosures 22. Aghajanian, C., D. S. Dizon, P. Sabbatini, J. J. Raizer, J. Dupont, and D. R. Spriggs. 2005. Phase I trial of bortezomib and carboplatin in recurrent The authors have no financial conflicts of interest. ovarian or primary peritoneal cancer. J. Clin. Oncol. 23: 5943–5949. 23. Mitsiades, N., C. S. Mitsiades, V. Poulaki, D. Chauhan, G. Fanourakis, X. Gu, C. Bailey, M. Joseph, T. A. Libermann, S. P. Treon, et al. 2002. Molecular se- References quelae of proteasome inhibition in human multiple myeloma cells. Proc. Natl. 1. Adams, J. 2004. The proteasome: a suitable antineoplastic target. Nat. Rev. Acad. Sci. USA 99: 14374–14379. Cancer 4: 349–360. 24. Hideshima, T., D. Chauhan, T. Hayashi, M. Akiyama, N. Mitsiades, 2. Richardson, P. G., C. Mitsiades, T. Hideshima, and K. C. Anderson. 2006. C. Mitsiades, K. Podar, N. C. Munshi, P. G. Richardson, and K. C. Anderson. Bortezomib: proteasome inhibition as an effective anticancer therapy. Annu. Rev. 2003. Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades Med. 57: 33–47. via caspase-dependent downregulation of gp130 in multiple myeloma. Oncogene 3. Richardson, P. G., B. Barlogie, J. Berenson, S. Singhal, S. Jagannath, D. Irwin, 22: 8386–8393. S. V.Rajkumar, G. Srkalovic, M. Alsina, R. Alexanian, et al. 2003. A phase 2 study 25. Hideshima, T., C. Mitsiades, M. Akiyama, T. Hayashi, D. Chauhan, of bortezomib in relapsed, refractory myeloma. N. Engl. J. Med. 348: 2609–2617. P. Richardson, R. Schlossman, K. Podar, N. C. Munshi, N. Mitsiades, and 4. Jagannath, S., B. Barlogie, J. Berenson, D. Siegel, D. Irwin, P. G. Richardson, K. C. Anderson. 2003. Molecular mechanisms mediating antimyeloma activity R. Niesvizky, R. Alexanian, S. A. Limentani, M. Alsina, et al. 2004. A phase 2 of proteasome inhibitor PS-341. Blood 101: 1530–1534. study of two doses of bortezomib in relapsed or refractory myeloma. Br. J. 26. Spisek, R., A. Charalambous, A. Mazumder, D. H. Vesole, S. Jagannath, and Haematol. 127: 165–172. M. V. Dhodapkar. 2007. Bortezomib enhances dendritic cell (DC)-mediated 5. Richardson, P. G., P. Sonneveld, M. Schuster, D. Irwin, E. Stadtmauer, T. Facon, induction of immunity to human myeloma via exposure of cell surface heat J. L. Harousseau, D. Ben-Yehuda, S. Lonial, H. Goldschmidt, et al. 2007. Ex- shock protein 90 on dying tumor cells: therapeutic implications. Blood 109: tended follow-up of a phase 3 trial in relapsed multiple myeloma: final time-to- 4839–4845. event results of the APEX trial. Blood 110: 3557–3560. 27. Demaria, S., F. R. Santori, B. Ng, L. Liebes, S. C. Formenti, and S. Vukmanovic. 6. Uy, G. L., R. Trivedi, S. Peles, N. M. Fisher, Q. J. Zhang, M. H. Tomasson, 2005. Select forms of tumor cell apoptosis induce dendritic cell maturation. J. J. F. DiPersio, and R. Vij. 2007. Bortezomib inhibits osteoclast activity in Leukoc. Biol. 77: 361–368. patients with multiple myeloma. Clin. Lymphoma Myeloma 7: 587–589. 28. Liu, B., J. Dai, H. Zheng, D. Stoilova, S. Sun, and Z. Li. 2003. Cell surface 7. Hainsworth, J. D., D. R. Spigel, J. Barton, C. Farley, M. Schreeder, J. Hon, and expression of an endoplasmic reticulum resident heat shock protein gp96 triggers F. A. Greco. 2008. Weekly treatment with bortezomib for patients with recurrent MyD88-dependent systemic autoimmune diseases. Proc. Natl. Acad. Sci. USA or refractory multiple myeloma: a phase 2 trial of the Minnie Pearl Cancer 100: 15824–15829. Research Network. Cancer 113: 765–771. 29. Dai, J., B. Liu, M. M. Caudill, H. Zheng, Y. Qiao, E. R. Podack, and Z. Li. 2003. 8. Bringhen, S., A. Larocca, D. Rossi, M. Cavalli, M. Genuardi, R. Ria, S. Gentili, Cell surface expression of heat shock protein gp96 enhances cross-presentation F. Patriarca, C. Nozzoli, A. Levi, et al. 2010. Efficacy and safety of once-weekly of cellular antigens and the generation of tumor-specific T cell memory. Cancer bortezomib in multiple myeloma patients. Blood 116: 4745–4753. Immun. 3: 1. 3220 MECHANISM OF ANTITUMOR EFFECTS GENERATED BY BORTEZOMIB

30. Hashimoto, M., O. Niwa, Y. Nitta, M. Takeichi, and K. Yokoro. 1989. Unstable 41. Tamura, Y., P. Peng, K. Liu, M. Daou, and P. K. Srivastava. 1997. Immuno- expression of E-cadherin adhesion molecules in metastatic ovarian tumor cells. therapy of tumors with autologous tumor-derived heat shock protein prepara- Jpn. J. Cancer Res. 80: 459–463. tions. Science 278: 117–120. 31. Schumacher, L. Y., D. D. Vo, H. J. Garban, B. Comin-Anduix, S. K. Owens, 42. Murshid, A., J. Gong, M. A. Stevenson, and S. K. Calderwood. 2011. Heat shock V. B. Dissette, J. A. Glaspy, W. H. McBride, B. Bonavida, J. S. Economou, and proteins and cancer vaccines: developments in the past decade and chaperoning A. Ribas. 2006. Immunosensitization of tumor cells to dendritic cell-activated in the decade to come. Expert Rev. Vaccines 10: 1553–1568. immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade). 43. Naujokat, C., C. Berges, A. Ho¨h, H. Wieczorek, D. Fuchs, J. Ovens, M. Miltz, J. Immunol. 176: 4757–4765. M. Sadeghi, G. Opelz, and V. Daniel. 2007. Proteasomal chymotrypsin-like 32. Seeger, J. M., P. Schmidt, K. Brinkmann, A. A. Hombach, O. Coutelle, peptidase activity is required for essential functions of human monocyte- P. Zigrino, D. Wagner-Stippich, C. Mauch, H. Abken, M. Kro¨nke, and derived dendritic cells. Immunology 120: 120–132. H. Kashkar. 2010. The proteasome inhibitor bortezomib sensitizes melanoma 44. Nencioni, A., K. Schwarzenberg, K. M. Brauer, S. M. Schmidt, A. Ballestrero, cells toward adoptive CTL attack. Cancer Res. 70: 1825–1834. F. Gru¨nebach, and P. Brossart. 2006. Proteasome inhibitor bortezomib modulates 33. Tseng, C. W., A. Monie, C. Y. Wu, B. Huang, M. C. Wang, C. F. Hung, and TLR4-induced dendritic cell activation. Blood 108: 551–558. T. C. Wu. 2008. Treatment with proteasome inhibitor bortezomib enhances 45. Nencioni, A., A. Garuti, K. Schwarzenberg, G. Cirmena, G. Dal Bello, I. Rocco, antigen-specific CD8+ T-cell‑mediated antitumor immunity induced by DNA E. Barbieri, P. Brossart, F. Patrone, and A. Ballestrero. 2006. Proteasome vaccination. J. Mol. Med. 86: 899–908. inhibitor-induced apoptosis in human monocyte-derived dendritic cells. Eur. J. 34. Shanker, A., and T. Sayers. 2007. Sensitizing tumor cells to immune-mediated Immunol. 36: 681–689. cytotoxicity. Adv. Exp. Med. Biol. 601: 163–171. 46. Subklewe, M., K. Sebelin-Wulf, C. Beier, A. Lietz, S. Mathas, B. Do¨rken, and 35. Mimnaugh, E. G., W. Xu, M. Vos, X. Yuan, J. S. Isaacs, K. S. Bisht, D. Gius, and A. Pezzutto. 2007. Dendritic cell maturation stage determines susceptibility to L. Neckers. 2004. Simultaneous inhibition of hsp 90 and the proteasome pro- the proteasome inhibitor bortezomib. Hum. Immunol. 68: 147–155. motes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic 47. Lundqvist, A., S. Su, S. Rao, and R. Childs. 2010. Cutting edge: bortezomib- vacuolization, and enhances antitumor activity. Mol. Cancer Ther. 3: 551–566. treated tumors sensitized to NK cell apoptosis paradoxically acquire resistance to 36. Goh, Y. C., C. T. Yap, B. H. Huang, A. D. Cronshaw, B. P. Leung, P. B. Lai, antigen-specific T cells. J. Immunol. 184: 1139–1142. S. P. Hart, I. Dransfield, and J. A. Ross. 2011. Heat-shock protein 60 translocates 48. Sun, K., L. A. Welniak, A. Panoskaltsis-Mortari, M. J. O’Shaughnessy, H. Liu, to the surface of apoptotic cells and differentiated megakaryocytes and stim- I. Barao, W. Riordan, R. Sitcheran, C. Wysocki, J. S. Serody, et al. 2004. In- ulates phagocytosis. Cell. Mol. Life Sci. 68: 1581–1592. hibition of acute graft-versus-host disease with retention of graft-versus-tumor 37. Binder, R. J., D. K. Han, and P. K. Srivastava. 2000. CD91: a receptor for heat effects by the proteasome inhibitor bortezomib. Proc. Natl. Acad. Sci. USA 101: shock protein gp96. Nat. Immunol. 1: 151–155. 8120–8125. 38. Basu, S., R. J. Binder, T. Ramalingam, and P. K. Srivastava. 2001. CD91 is 49. De Boo, S., J. Kopecka, D. Brusa, E. Gazzano, L. Matera, D. Ghigo, A. Bosia, a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. and C. Riganti. 2009. iNOS activity is necessary for the cytotoxic and immu- Immunity 14: 303–313. nogenic effects of doxorubicin in human colon cancer cells. Mol. Cancer 8: 108. 39. Meńoret, A., P. Peng, and P. K. Srivastava. 1999. Association of peptides with 50. Obeid, M., A. Tesniere, T. Panaretakis, R. Tufi, N. Joza, P. van Endert, heat shock protein gp96 occurs in vivo and not after cell lysis. Biochem. Biophys. F. Ghiringhelli, L. Apetoh, N. Chaput, C. Flament, et al. 2007. Ecto-calreticulin Res. Commun. 262: 813–818. in immunogenic chemotherapy. Immunol. Rev. 220: 22–34. 40. Sato, K., Y. Torimoto, Y. Tamura, M. Shindo, H. Shinzaki, K. Hirai, and 51. Apetoh, L., M. Obeid, A. Tesniere, F. Ghiringhelli, G. M. Fimia, M. Piacentini, Y. Kohgo. 2001. Immunotherapy using heat-shock protein preparations of leu- G. Kroemer, and L. Zitvogel. 2007. Immunogenic chemotherapy: discovery of kemia cells after syngeneic bone marrow transplantation in mice. Blood 98: a critical protein through proteomic analyses of tumor cells. Cancer Genomics 1852–1857. Proteomics 4: 65–70.