Background
Multiple myeloma (MM) is a deadly hematologic cancer characterized by latent accumulation of clonal secretory plasma cells in the bone marrow. Despite advances in therapeutic strategies, MM remains an incurable disease with a median survival around 4–5 years in adults [
1]. However, in the past decade, the use of autologous hematopoietic stem cell transplantation (HSCT) and the introduction of new drugs, such as bortezomib and IMiDs, have improved survival [
2-
5].
Increasing evidence in myeloma patients has shown that Natural Killer (NK) cells can elicit potent allogeneic and autologous responses to myeloma cells, strongly supporting their anti-tumor potential in response to immunomodulatory drugs or following allogeneic stem cell transplantation [
6-
8]. In this regard, several studies have shown that triggering of different activating receptors, such as DNAX accessory molecule-1 (DNAM-1), NK group 2D (NKG2D) and Natural Cytotoxicity Receptors (NCRs), is involved in the recognition and killing of MM cells by NK cells [
9-
11]; moreover, MM cells can express the DNAM1-ligands (DNAM1Ls) PVR/CD155 and Nectin-2 (Nec-2) [
12] and the NKG2D-ligands (NKG2DLs) MICA/B and ULBPs on the cell surface [
9,
12,
13].
Nitric oxide (NO) is a reactive radical, highly diffusible pleiotropic regulator of many different biological pathways, including vasodilatation, neurotransmission and macrophage-mediated responses to infections. It is generated from molecular oxygen and the amino acid
L-arginine through the action of the nitric oxide synthase (NOS) enzymes; three isoforms of NOS have been identified, a neuronal form (nNOS/NOS1) and endothelial form (eNOS/NOS3) which are both constitutively expressed enzymes producing physiological levels of NO, and an inducible form (iNOS/NOS2) which produces high levels of NO in a sustained manner [
14-
16]. In the last years, the relationship between NO and the pathology of malignant disorders has been the subject of numerous studies; although the three NOS isoforms are known to be present in most tumors and generally expressed at higher levels compared to their normal tissue counterparts, their functional role still remains incompletely elucidated [
17,
18]. In this regard, a concentration-dependent dual nature of NO has been revealed, where low concentrations of NO can promote invasion and metastases in different tumor models or, on the contrary, high NO levels (e.g. immune cell-generated NO) and the different reactive nitrogen species (RNS) produced can inhibit tumor growth and metastases (reviewed in [
17,
19,
20]). Thus, NO may play different roles in regulating cancer microenvironment and progression, which can be cell-type and context specific.
These observations suggest that tumor immune rejection through NO-dependent mechanism(s) can represent an interesting promise for future tailored immunotherapeutic anticancer strategies.
Our laboratory has recently shown that suboptimal doses of different drugs, such as genotoxic chemotherapeutics, inhibitors of the HSP-90 protein or of the GSK3 kinase, can increase the expression of several NK activating ligands on MM cells, via induction of specific regulatory transcriptional pathways [
12,
21,
22]; the up-regulation of these ligands on MM cells is associated with their ability to trigger increased NK cell degranulation. At this regard, expression of DNAM-1 ligands and in particular PVR/CD155 can be regulated by activation of a DNA damage response (DDR) pathway induced by anticancer drugs (e.g. doxorubicin or melphalan) or, in a different context, by monocyte-derived reactive oxygen species (ROS) in Ag-induced T cell proliferation [
23].
Here, we analyzed the possibility that treatment of MM cells with different NO-donors could regulate the expression of the NK cell activating ligand PVR/CD155 and, in turn, modify NK cell recognition and cytotoxicity against these cancer cells.
Our results indicate that increased levels of NO can enhance surface expression of PVR/CD155 on MM cell lines, rendering these cells more susceptible to NK cell mediated killing via DNAM-1 recognition. We found that activation a DDR by NO is critical for these mechanisms since pharmacological inhibition of ATM/ATR or Chk1/2 kinases as well as knockdown of E2F1, a transcription factor activated in response to DNA damage, significantly reduced NO-induced upregulation of PVR/CD155.
Overall, our data demonstrate that NO can regulate DNAM-1 ligand expression on MM cells, suggesting novel roles of NO in immune response(s) to multiple myeloma.
Methods
Cell lines
The human MM cell lines SKO-007(J3), U266, OPM-2, ARK, RPMI-8226 and LP1 were kindly provided by Prof. P. Trivedi (Sapienza University of Rome, Italy). SKO-007(J3) cells transduced with a lentiviral vector expressing shRNAs targeting E2F1 have been already described [
24]. The erythroleukemia cell line K562 and MM cell lines were maintained at 37°C and 5% CO2 in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 2 mM glutamine and 100 units/ml penicillin-streptomycin (complete medium). All cell lines were mycoplasma-free (EZ-PCR Mycoplasma Test Kit, Biological Industries).
Reagents and antibodies
The nitric oxide donors DETA-NO [2,2′-(hydroxynitrosohydrazono) bis-ethanimine], NCX4040 (NO-aspirin), JS-K [O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate], caffeine, LY294002 and the inhibitor of nitric oxide-sensitive guanylyl cyclase ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) and Bafilomycin A1, were purchased from Sigma-Aldrich (St. Louis, MO). The Chk1/2 pharmacologic inhibitors SB218078 and UCN-01 were purchased from Calbiochem, EMD Chemicals (Darmstadt, Germany). C12FDG was from Invitrogen (Frederick, MD). The nitric oxide donor DETA-NO (2 moles of NO• per mole of compound and a half-life of 20 h at 37°C), is ideal for the treatment of cells over long periods of time (e.g. 24–48 h). JS-K (an anti-cancer agent belonging to the diazeniumdiolate family of compounds), is designed to release nitric oxide (NO) in a sustained and controlled manner within a cell, when metabolized by glutathione S-transferases (GSTs).
The following monoclonal antibodies (mAbs) were used for immunostaining or as blocking Abs: anti-PVR/CD155 (SKII.4) kindly provided by Prof. M. Colonna (Washington University, St Louis, MO), anti-CD56 (C218) mAb was provided by Dr. A. Moretta (University of Genoa, Genoa, Italy), anti-DNAM-1 (DX11) from Serotec (Oxford, UK), anti-Nec-2 (R2.525) from BD Biosciences (San Jose, CA), anti-TIGIT (MBSA43) from eBioscience Inc. (San Diego, CA). APC Goat anti-mouse IgG (Poly4053), anti-CD3/APC (HIT3a), anti-CD56/PE (HCD56), mouse IgG1/FITC, /PE or /APC isotype control (MOPC-21) were purchased from BioLegend (San Diego, CA). Anti-CD107a/FITC (H4A3) was purchased from BD Biosciences (San Jose, CA).
Immunofluorescence and flow cytometry
MM cell lines were cultured in 6-well tissue culture plates for 48 h at a concentration of 2 × 105 cells/ml in the presence of different concentrations of drugs. The expression of PVR/CD155 on MM cells was analyzed by immunofluorescence staining using an anti-PVR/CD155 unconjugated mAb, followed by secondary GAM-APC. In all experiments, cells were stained with Propidium Iodide (PI) (1 μg/ml) in order to assess cell viability (always higher than 90% in the different treatments). Nonspecific fluorescence was assessed by using an isotype-matched irrelevant mAb (R&D System) followed by the same secondary antibody. Fluorescence was analyzed using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA) and FlowJo Flow Cytometric Data Analysis Software (Tree Star, Inc. Ashland, OR).
Intracellular NO• levels were measured by flow cytometry in cells loaded with the NO-sensitive dye DAF2-DA [4,5-Diaminofluorescein-diacetate (Molecular Probes, Invitrogen, San Diego, CA)]. Cells were gated by forward/side scatter and fluorescence was recorded on the FL-1 channel according to the manufacturer’s protocol.
Degranulation assay
NK cell-mediated cytotoxicity was evaluated using the lysosomal marker CD107a as previously described [
21]. As source of effector cells, we used primary NK cells obtained from PBMCs isolated from healthy donors by Lymphoprep (Nycomed, Oslo, Norway) gradient centrifugation and then co-cultured for 10 days with irradiated (30 Gy) Epstein-Barr virus (EBV)-transformed B-cell line RPMI 8866, without the addition of recombinant IL-2, at 37°C in a humidified 5% CO2 atmosphere as previously described [
25]. Informed consent in accordance with the Declaration of Helsinki was obtained from all donors, and approval was obtained from the Ethics Committee of the Sapienza University of Rome, Italy. On day 10, the cell population was routinely more than 90% CD56
+CD16
+CD3
−, as assessed by immunofluorescence and flow cytometry analysis. Drug-treated MM cells were washed twice in complete medium and then incubated with NK cells at the effector:target (E:T) ratio of 2.5:1, in a U-bottom 96-well tissue culture plate in complete medium at 37°C and 5% CO2 for 2 h. Thereafter, cells were washed with PBS and incubated with anti-CD107a/FITC (or cIgG/FITC) for 45 min at 4°C. Cells were then stained with anti-CD3/APC, anti-CD56/PE to gate the CD3
−CD56
+ NK cell population. In some experiments, cells were pre-treated for 20 min at room temperature with anti-DNAM-1 or anti-TIGIT blocking mAb. Fluorescence was analyzed using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA) and FlowJo Flow Cytometric Data Analysis Software (Tree Star, Inc. Ashland, OR).
Cytotoxicity assay
A standard 4-hour chromium-release assay was used as previously described [
26]. SKO-007(J3) cells stimulated as indicated above, were used as target cells and were labeled (100–200 μCi
51Cr/10
6 cells; Amersham BioSciences, Piscataway, NJ) for 90 minutes at 37°C, washed, and 5 × 10
3 cells/well were plated. As source of effector cells, we used primary NK cells as described above. The percentage of specific lysis was calculated by counting an aliquot of supernatant and using the formula: 100 × [(sample release - spontaneous release)/total release - spontaneous release)]. All determinations were made in triplicate, and E:T ratios ranged from 10:1 to 1:1, as indicated.
Cell cycle analysis
SKO-007(J3) cell cycle distribution was analyzed by PI staining after 48 h drug treatment. Cells were washed in PBS with 0.1% sodium azide and fixed for 2 h at 4°C in cold 70% ethanol. Thereafter, cells were incubated for 30 min at room temperature with 50 μg/mL of PI in PBS containing 100 μg/mL of RNAse and immediately analyzed using a FACSCalibur flow cytometer. Flow cytometric analysis was performed using FlowJo software.
Analysis of senescent cells
Senescence Associated β-galactosidase assay was performed using the fluorogenic substrate C12FDG to measure β-galactosidase activity by flow cytometry. Cells were incubated 1 h with 100 nM bafilomycin A1 to induce lysosomal alkalinization, followed by 1 h incubation with C12FDG (33 μM) and the C12-fluorescein signal of senescent cells was measured on the FL-1 detector using a FACSCalibur flow cytometer. Flow cytometric analysis was performed using FlowJo software.
RNA isolation, RT-PCR and real-time PCR
Total RNA was extracted using TRIZOL™ (Life Technologies Inc., Grand Island, NY), according to manufacturer’s instructions. The concentration and quality of the extracted total RNA was determined by measuring light absorbance at 260 nm (A260) and the ratio of A260/A280. Reverse transcription was carried out in a 25 μl reaction volume with 2 μg of total RNA according to the manufacturer’s protocol for M-MLV reverse transcriptase (Promega, Madison, WI). Real-Time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate with primers for CD155/PVR (Hs00197846_m1) conjugated with fluorochrome FAM, and β-actin (4326315E) conjugated with fluorochrome VIC (Applied Biosystems). The level of ligand expression was measured using Ct (threshold cycle). The Ct was obtained by subtracting the Ct value of the gene of interest (PVR/CD155) from the housekeeping gene (β-actin) Ct value. In the present study we used Ct of the untreated sample as the calibrator. The fold change was calculated according to the formula 2-ΔΔCt, where ΔΔCt was the difference between Ct of the sample and the Ct of the calibrator (according to the formula, the value of the calibrator in each run is 1. The analysis was performed using the SDS version 2.2 software (Applied Biosystems, Foster City, CA).
Western-blot analysis
For Western-Blot analysis, SKO-007(J3) cells were pelleted, washed once with cold phosphate-buffered saline, resuspended in lysis buffer [1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% Sodium Deoxycholate, 1 mM phenyl-methyl-sulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, COMPLETE protease1 inhibitor mixture (Roche, Indianapolis, IN) in PBS] and subsequently incubated 30 min on ice. The lysate was centrifuged at 14000 g for 15 min at 4°C and the supernatant was collected as whole cell extract. Protein concentration was determined by the BCA method (Pierce, Rockford, IL). Thirty to 50 μg of cell extract were run on 10% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NJ) and blocked in 3% milk in TBST buffer. Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-coupled goat anti-rabbit or goat anti-mouse immunoglobulins and the ECL detection system (GE Healthcare Amersham), following the manufacturer’s instructions. Antibodies against phospho-Chk1 (Ser317), phospho-Chk2 (Thr68), total Chk1 and total Chk2 were purchased from Cell Signaling (Danvers, MA). Antibody against phospho-H2A.X was purchased from Millipore (Billerica, MA). Densitometric analysis was performed using Quantity One software (Bio-Rad, Hercules, CA).
Discussion and conclusion
Anticancer immune responses may contribute to the control of tumors after conventional chemotherapy and different observations have indicated that chemotherapeutic agents (e.g. genotoxic drugs) or adjuvant radiotherapy can induce immune responses that result in immunogenic cancer cell death or immunostimulatory side effects [
47-
50]. In this regard, increasing experimental and clinical evidence highlight the importance of NK cells in immune responses toward MM and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. Recently, a novel connection between therapeutic immuno-modulation and chemotherapy has been the finding that anti-cancer drugs (e.g. genotoxic agents, inhibitors of histone deacetylases, of the proteasome or of the HSP-90 chaperone) can increase the expression of DNAM-1 and NKG2D activating ligands, thus enhancing the response of receptor-expressing lymphocytes (NK cells, NKT cells and CTLs) against tumor cells, including MM [
11,
12,
21,
24,
51-
54].
Different and contradictory results have been reported about the role of nitric oxide in cancer progression, metastases and treatment of disease (reviewed in [
19,
20]). Initial findings suggested that immune cell-generated NO can be cytostatic or cytotoxic for a number of tumors; indeed, several reports have shown that macrophages can selectively destroy different tumor types (
in vitro and
in vivo) through the production of high levels of NO [
55-
58]. Moreover, NO can also enhance the cytotoxicity of NK cells and regulate survival of dendritic cells [
59-
61] and its release in models of lung and hepatic metastases microvasculature has been associated to a natural local defense mechanism inducing tumor cell killing [
62,
63]. On the other hand, other findings highlighted opposite actions mediated by NO, leading to increased tumor growth; in this context, low concentrations of NO have been shown to promote invasion and metastases (reviewed in [
17,
20]) and production of NO within specific tumor microenvironments has been described to enhance tumor progression, mainly by stimulating angiogenesis and/or to repress T cell responses by CD11b
+/Gr-1
+ myeloid cells (reviewed in [
17]).
The observations described in this work can provide additional information on the role of nitric oxide in cancer and in MM. In particular, we investigated the effect of nitric oxide on the expression of the DNAM-1 ligand PVR/CD155 in MM cells. We found that treatment of MM cell lines with nitric oxide donors (DETA-NO, NitroAspirin/NCX4040 or JS-K) can increase the expression of this ligand, rendering these cells more susceptible to NK cell-mediated killing (Figure
2). Moreover, we identified one of the possible mechanism(s) involved in this up-regulation, the activation of a DNA damage response, a molecular pathway already described to regulate the expression of NK cells activating ligands in several cellular models [
12,
24,
64]. NO-generated nitrogen species [
20,
37] and the consequent production of single and/or double DNA strand breaks can activate DDR in MM cells (as shown in Figure
3B and C); in this regard, upregulation of PVR/CD155 by DETA-NO was significantly reduced by inhibitors of ATM/ATR catalytic activity (caffeine and LY294002) and by inhibitors of the Chk1/2 kinases (SB218078 and UCN-01) (Figure
4C-D). In addition, silencing of E2F1, a transcription factor activated/stabilized by ATM/ATR/Chk2 [
41-
43] and described to upregulate the expression of PVR/CD155 in MM cells exposed to genotoxic drugs [
24], resulted in a marked reduction of PVR/CD155 up-regulation (Figure
4E and F). These results indicate that NO-mediated activation of DDR is involved in the up-regulation of PVR/CD155 and that one of the mechanism(s) underlying this regulation implicates the activity of E2F1. Interestingly, and differently from our previous observation that up-regulation of PVR/CD155 is preferentially associated with a senescence-dependent G2/M cell cycle arrest [
12], NO failed to activate a senescence and G2/M cell cycle arrest in our experimental system, as indicated by the different levels of SA-
βGal activity and G2/M phase between DETA-NO and doxorubicin-treated cells (used here as positive control) (Figure
5B and C). These data suggest that specific molecular pathways activated by RNSs and/or a different strength of DDR might be induced by these drugs and that cellular senescence is not correlated or involved in up-regulation of PVR/CD155. Moreover, the three NO-donors used in this work differ in their capability to upregulate PVR/CD155 expression, at least in our experimental setting of donor concentration and duration of treatment (as shown in Figures
1 and
6); these differences might reflect the possibility that additional molecular action(s) besides NO release might contribute to donors biologic activities, in particular mediated by the aspirin-moiety (NCX4040) or by the JS-K’s arylating ability on different nucleophilic biomolecules [
65]. Further experiments will be needed to better characterize possible differences in activation of DDR by these drugs and the correlation with the expression of activating ligands.
Work by other groups has demonstrated a direct cytotoxic/anti-myeloma activity of NO as a consequence of induction of DDR, using the NO-releasing prodrug JS-K [
46], which can also affect the interaction of MM cells with bone marrow microenvironment, modulating tumor angiogenesis
in vivo and
in vitro [
66]. Moreover, NO can function as a negative feedback signal to limit pathologic osteoclastogenesis via RANKL/iNOS/NO autoregulatory pathway [
67]. In a different context, treatment with JS-K or the activation of macrophage-dependent NO expression after IL-2 + anti-CD40 immunotherapy has been shown to modulate metastatic progression in an orthotopic model of renal cell carcinoma [
68]. Similarly, local production of significant amounts of NO by iNOS
+ has been also shown to deeply affect the activity of pro-tumoral microenvironments, as demonstrated using neoadjuvant local low-doses of gamma irradiation (LDI) in a model of pancreatic carcinogenesis [
69]; in this model, LDI is able to redirect local (or pre-adoptive-transfer) macrophage differentiation from a cancer-promoting immunosuppressive state to an iNOS
+ phenotype, to normalize aberrant angiogenesis-driven vascular abnormalities and to enable infiltration of cytotoxic T cells. In this regard, local MM-associated macrophages play a crucial role in the pathophysiology of MM and can promote plasma cell growth with aberrant vasculogenesis (reviewed in [
70]); moreover, hypoxia-mediated impairment of NO signalling can also contribute to tumor escape from NK cell immunesurveillance by inducing shedding of the NKG2DL MICA, through a mechanism involving increased expression/activity of ADAM10 via HIF-1α [
71,
72].
The possibility to regulate activating ligands such as PVR/CD155 in MM cells, able to enhance the activity of cytotoxic lymphocytes (e.g. NK cells) by pharmacological delivery of NO-releasing prodrugs (also in combined immunotherapy) or local production of NO by “therapy-reprogrammed” or adoptively transferred iNOS+ macrophages, might be considered as an additional strategy to hit the tumor and to modify local microenvironment allowing and/or enhancing immuno-therapeutic applications.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CF designed research, performed experiments, and contributed to paper writing. MPA, AZ, ASo, BR, RM, RP, performed experiments. MC and ASa designed research, and contributed equally to paper writing and supervising the laboratory activities. All authors read and approved the final manuscript.