IL-2Rα up-regulation is mediated by latent membrane protein 1 and promotes lymphomagenesis and chemotherapy resistance in natural killer/T-cell lymphoma

Background Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive non-Hodgkin lymphoma often resistant to chemotherapy. Serum level of soluble IL-2 receptor α (IL-2Rα) is elevated in NKTCL patients and correlates significantly with treatment response and survival. In the current study we examined the potential role of IL-2Rα by over-expressing IL-2Rα in representative cell lines. Methods Levels of IL-2Rα were evaluated in the human natural killer cell line NK-92 and the NKTCL cell line SNK-6. Lentiviral vectors were used to express latent membrane protein 1 (LMP1) in NK-92 cells, and IL-2Rα in both NK-92 and SNK-6 cells. The biological effects of these genes on proliferation, apoptosis, cell cycle distribution, and chemosensitivity were analyzed. Results Expression of IL-2Rα was significantly higher in SNK-6 cells than in NK-92 cells. Expressing LMP1 in NK-92 cells remarkably up-regulated IL-2Rα levels, whereas selective inhibitorss of the proteins in the MAPK/NF-κB pathway significantly down-regulated IL-2Rα. IL-2Rα overexpression in SNK-6 cells promoted cell proliferation by altering cell cycle distribution, and induced resistance to gemcitabine, doxorubicin, and asparaginase. These effects were reversed by an anti-IL-2Rα antibody. Conclusions Our results suggest that LMP1 activates the MAPK/NF-κB pathway in NKTCL cells, up-regulating IL-2Rα expression. IL-2Rα overexpression promotes growth and chemoresistance in NKTCL, making this interleukin receptor a potential therapeutic target. Electronic supplementary material The online version of this article (10.1186/s40880-018-0334-8) contains supplementary material, which is available to authorized users.

Interleukin-2 (IL-2) is a pro-inflammatory cytokine that activates and maintains immune responses [12]. IL-2 exerts its biological effects by binding to its receptor IL-2R on the cell surface. IL-2R consists of three subunits. The α subunit, also known as CD25 or Tac antigen, does not participate on its own in IL-2-triggered signal transduction due to its very short intracellular domain. When the α subunit combines with IL-2Rβ and IL-2Rγ, the affinity of IL-2R to IL-2 increases. Proteolytic cleavage of membrane-bound IL-2Rα generates a soluble form of IL-2Rα (sIL-2Rα) [13]. In a variety of solid malignant tumors as well as hematologic malignancies, serum sIL-2Rα is elevated and correlates with poor patient prognosis [14][15][16][17][18]. In a previous retrospective analysis from this research group, we reported elevated serum sIL-2Rα in NKTCL patients than in healthy individuals [19]. We also found significant correlation between higher sIL-2Rα with poor chemotherapy response and patient prognosis [19].
In the present study, we examined the potential effects of IL-2Rα over-expression on cell proliferation, cell cycle distribution and sensitivity to chemotherapeutic drugs in representative NKTCL cell lines. Mechanistic investigation identified latent membrane protein 1 as a downstream effector.

Cell lines and culture
The human NKTCL cell line SNK-6 and human natural killer cell line NK-92 (both were generously donated by professor Zhang Ming-zhi from the First Affiliated Hospital of Zhengzhou University) were incubated at 37 °C in an atmosphere of 5% CO 2 . SNK-6 cells were cultured in RPMI 1640 (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and recombinant human IL-2 (1000 U/ml; Sigma, USA). NK-92 cells were maintained in RPMI 1640 containing 10% FBS.

Measurement of sIL-2Rα in culture supernatant
NK-92, SNK-6 and NK-92 transduced with lentivirus encoding LMP1, and NK-92 and SNK-6 transduced with lentivirus encoding IL-2Rα or negative control lentivirus were centrifuged at 382×g for 5 min. sIL-2Rα concentration in the supernatant was measured using a sandwich enzyme-linked immunosorbent assay (Fine Biological Technology, Wuhan, China).

Cell proliferation and cytotoxicity assay
Cell proliferation was assessed using the Cell Counting kit-8 (CCK-8; Dojin, Tokyo, Japan). For cytotoxicity assay, SNK-6 cells were exposed to doxorubicin, gemcitabine, or asparaginase of varying concentrations for 24 or 48 h prior to CCK-8 assay. Optical density was measured at a wavelength of 450 nm using a Multiskan microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Relative fold drug resistance was calculated using IC 50 values.

Analysis of cell cycle distribution and apoptosis
Flow cytometry was used to determine cell cycle distribution and detect apoptosis. Upon 85% confluence, culture medium was removed and cells were suspended, centrifuged and fixed in precooled 70% ethanol for 1 h. The suspension was centrifuged again, the supernatant was removed, and the cells were washed with ice-cold PBS and stained with propidium iodide (PI; 50 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) in the presence of RNase A (100 μg/ml; Fermentas ® , Shanghai, China). The suspension was passed through a 300-mesh filter, and DNA content of stained nuclei was analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA). Each experiment was performed in triplicate.
Apoptosis was analyzed using the Annexin V-APC/7-AAD Apoptosis Detection Kit (Lianke Bio, Hangzhou, China). The percentage of apoptotic cells was determined by flow cytometry on a BD FACS Calibur flow cytometer. All experiments were performed in triplicate.

Statistical analysis
Results are expressed as mean ± SD. Statistical analysis was performed using SPSS 17.0 (IBM, Chicago, IL, USA). Inter-group differences were assessed for significance using Student's t-test. Differences were defined as statistically significant at P < 0.05 (2-sided).

Expression of IL-2Rα is higher in NKTCL cells than in natural killer cells
IL-2Rα expression was significantly higher in SNK-6 cells than in NK-92 cells at both the mRNA (Fig. 1a) and protein levels (Fig. 1b). Similarly, the level of sIL-2Rα in culture supernatant was significantly higher in SNK-6 cells (Fig. 1c).

LMP1 acts via the MAPK/NF-κB pathway to up-regulate IL-2Rα in NK-92 cells
Lentivirus encoding LMP1 and negative control lentivirus infected cells efficiently at a multiplicity of infection of 300 (Fig. 1d, e). Western blot analysis revealed significantly higher LMP1 expression in NK-92 cells expressing lentivirus-encoded LMP1 at the mRNA (Fig. 1f ) and protein levels (Fig. 1h) than in negative control lentivirus vector control. Similar results were observed with IL-2Rα (Fig. 1g, h) as well as sIL-2Rα in culture supernatant (Fig. 1i).

IL-2Rα overexpression promotes proliferation of NK-92 and SNK-6 cells
The IL-2Rα expression was significantly higher in cells expressing lentivirus-encoded IL-2Rα at the mRNA (Fig. 4a) and protein levels (Fig. 4b) in comparison to the negative lentivirus control. Cells expressing lentivirus-encoded IL-2Rα also secreted higher amount of sIL-2Rα into the supernatant (Fig. 4c), and had higher rate of proliferation (Fig. 5a, b). IL-2Rα overexpression did not significantly alter the percentage of apoptotic cells (Fig. 6).
IL-2Rα overexpression decreased the percentage of cells in the G0/1 phase and increased the percentage in the S phase (Fig. 7). These changes were caused by increases in protein levels of cyclin A1, A2, B1, D, CDK1, and CDK4 (Additional file 1: Figure S1).

Targeting of IL-2Rα to treat relapsed/refractory NKTCL
A 42-year-old man with stage IV NKTCL who relapsed after six cycles of GELOX (gemcitabine, oxaliplatin, and asparaginase) and who did not respond to two cycles of EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and adriamycin) and two cycles of SMILE (dexamethasone, methotrexate, ifosphamide, asparaginase, and etoposide) was then given two cycles of pegaspargase and anti-IL-2Rα antibody basiliximab (2500 IU/m 2 pegaspargase given on day 1 + 20 mg basiliximab given on day 1 and 8, repeated every 3 weeks). This last treatment achieved partial remission and its toxicity was tolerable (Additional file 2: Figure S2). Levels of Epstein-Barr virus DNA in plasma decreased from 4.2 × 10 3 copies/mL to 0.

Discussion
NKTCL is a rare, highly aggressive hematologic malignancy. The frequency with which the disease is resistant to conventional chemotherapy (e.g., CHOP and EPOCH) has made radiotherapy the primary therapy [5,8,21,22]. Although novel regimens incorporating l-Asp or pegaspargase have significantly improved outcomes, 20%-40% of patients still experience treatment failure [6,7,9,10]. Therefore, there is an urgent need to develop new In previous clinical work, our group reported that sIL-2Rα levels in serum were significantly elevated in NKTCL, and that these levels correlated with chemotherapy response and prognosis [19]. The present study extends and deepens that work by describing a pathway by which IL-2Rα overexpression promotes NKTCL tumor growth, and by showing the potential of anti-IL-2Rα antibody therapy. Here we describe several major findings: (1) LMP1 acts via the MAPK/NF-κB pathway to up-regulate IL-2Rα in NKTCL, (2) IL-2Rα overexpression promotes NKTCL cell proliferation and cell cycle progression without affecting apoptosis, and (3) IL-2Rα overexpression correlates positively with chemoresistance in NKTCL cells, and anti-IL-2Rα antibody can restore chemosensitivity.
Elevation of various cytokines has been observed in both non-Hodgkin and Hodgkin lymphoma [23][24][25]. IL-2 acts as a potent immunomodulator and activates many immune cells, including antigen-specific T cells, B cells, and natural killer cells [12]. Elevated expression of the IL-2Rα subunit of the IL-2 receptor has been reported in several types of malignancy, including NKTCL [26,27]. The high affinity of IL-2Rα for IL-2 led us to hypothesize that IL-2Rα up-regulation may promote tumor cell proliferation and progression. Consistent with this idea, a preliminary study of NKTCL patients showed that in 53.8% of cases, IL-2Rα was expressed on the tumor surface (data not shown). In the present study, IL-2Rα expression was significantly stronger in NKTCL cells than in natural killer cells, and more sIL-2Rα was present in supernatants of NKTCL cultures.
We provide here evidence that IL-2Rα up-regulation in NKTCL may be mediated by the Epstein-Barr viral protein LMP1. The Epstein-Barr virus plays a pivotal role in NKTCL pathogenesis, and LMP1 regulates several cytokines and cytokine receptors in malignancies associated with the Epstein-Barr virus [28][29][30]. We found that LMP1 expression in NK-92 cells up-regulated IL-2Rα and sIL-2Rα levels, which was reversed by selective inhibitors of the MAPK/NF-κB pathway. This is consistent with previous work showing that LMP-1 acts via the NF-κB pathway to up-regulate IL-2Rα in two other lymphomas associated with Epstein-Barr virus, Hodgkin's lymphoma and Burkitt's lymphoma [31]. Elevated levels of sIL-2R predict inferior outcomes in several types of lymphoma, including B-cell and T-cell non-Hodgkin lymphomas [18,32,33]. In some hematological neoplasms, such as hairy-cell leukemia and adult T-cell leukemia, sIL-2Rα is released by tumor cells constitutively expressing IL-2Rα [34,35]. Therefore, the serum level of sIL-2Rα may reflect tumor burden and disease activity. The intracellular domain of IL-2Rα is too short to elicit signal transduction on its own, but IL-2Rα promotes IL-2 recycling back to the cell surface, creating an IL-2 reservoir at the surface, thereby potentiating its activity [36]. This activates oncogenic pathways such as JAK-STAT, MAPK, and PI3 K, with mitogenic and antiapoptotic effects [37,38]. At the same time, sIL-2Rα binds soluble sIL-2, and the complex activates tumorfriendly Treg cells rather than antitumor T cells [32]. In the present study, we report for the first time that IL-2Rα overexpression promotes NKTCL cell proliferation and cell cycle progression without affecting apoptosis. This is consistent with previous reports that IL-2 activates and promotes natural killer cell proliferation [39], and further work should clarify the mechanism(s) involved.
Resistance to chemotherapy is the major cause of treatment failure in NKTCL, especially in advanced disease [40]. While overexpression of multi-drug resistance genes may contribute to this chemoresistance [4], our previous work showed that elevated serum levels of sIL-2Rα correlated with significantly inferior response rate to chemotherapy (mainly CHOP or EPOCH regimens) [19]. Consistent with this previous work, we report here that IL-2Rα overexpression in NKTCL cells led to chemoresistance in vitro to gemcitabine, doxorubicin, and l-Asp. As further evidence for the involvement of IL-2Rα, this chemoresistance was reversed by anti-IL-2Rα antibody.   sensitivity to a, b gemcitabine, c, d doxorubicin, and e, f l-asparaginase (l-Asp) in SNK-6 cells. Cells overexpressed with IL-2Rα were treated with anti-IL-2Rα antibody (0.2 μg/ml) and different concentrations of one of the three drugs. Cell viability was determined by CCK-8 assay. IC 50 values are presented as mean (95% confidence interval)