Ibrutinib targets microRNA-21 in multiple myeloma cells by inhibiting NF-κB and STAT3

Abstract

The oncogenic microRNA-21 contributes to the pathogenesis of multiple myeloma. Ibrutinib (also referred to as PCI-32765), an inhibitor of Bruton’s tyrosine kinase, while its effects on multiple myeloma have not been well described. Here, we show that microRNA-21 is an oncogenic marker closely linked with progression of multiple myeloma. Moreover, ibrutinib attenuates microRNA-21 expression in multiple myeloma cells by inhibiting nuclear factor-κB and signal transducer and activator of transcription 3 signaling pathways. Taken together, our results suggest that ibrutinib is a promising potential treatment for multiple myeloma. Further investigation of mechanisms of ibrutinib function in multiple myeloma will be necessary to evaluate its use as a novel multiple myeloma treatment.

Multiple myeloma (MM) is a genetically complex hematologic malignancy. It is characterized by abnormal infiltration of clonal plasma cells in the bone marrow and is the second most common hematological malignancy after non-Hodgkin’s lymphoma. ¹ Despite many therapeutic options, MM remains incurable, with a 5-year survival rate of 40%. Therefore, novel treatments are urgently needed. ² The gene copy number changes, chromosomal translocations, mutations, transcriptional changes, and epigenetic changes that occur during MM development result in the profound deregulation of micro-RNA (miRNA) transcription.^3,4 This, in turn, leads to aberrant messenger RNA (mRNA) translation and cell signaling. ⁵

miRNAs are small single-stranded noncoding RNA that function in RNA silencing and post-transcriptional regulation of gene expression. An increasing number of studies have shown that miRNAs play an important role in tumorigenesis, and understanding the regulatory mechanism of miRNAs in this gene regulatory network will help elucidate the complex biological processes at play during malignancy. For example, microRNA-21 (miR-21) is over-expressed in a variety of malignancies and is implicated in cell proliferation, apoptosis, invasion, and metastasis. ⁶ Increasingly, studies in vitro and in vivo support a role for miR-21 in the growth, survival, and drug resistance of MM cells.^7–9

Bruton’s tyrosine kinase (BTK), an essential element of the B-cell receptor signaling pathway, can be targeted with remarkable efficacy and an acceptable safety profile in B-cell malignancies. Activation of BTK triggers a cascade of signaling events that transcriptional regulation involving nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3), and the BCR-dependent NF-κB signaling pathway requires functional BTK, while BTK can mediate STAT3 upregulation in a TLR9-dependent manner.^9,10 Ibrutinib is a selective, irreversible, and orally bioavailable BTK inhibitor with excellent pharmacodynamics. Many clinical trials of ibrutinib treatment for relapsed non-Hodgkin’s lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia have produced high response rates. Therefore, ibrutinib shows promise as a novel therapy for MM, since it has the potential to target both MM cells and their supportive bone marrow microenvironment.^11,12 Furthermore, ibrutinib potently enhances the activity of drugs (bortezomib and lenalidomide) currently used to treat MM by targeting the nuclear trans-activation of NF-κb. ¹³ However, the link between BTK and miR-21 in MM cells has not been investigated. In this report, we demonstrate that ibrutinib significantly blunts the growth of BTK-expressing MM cells and downregulates expression of miR-21. Furthermore, NF-κB and STAT3 signaling pathways underlie transcriptional regulation of miR-21 and the effects of ibrutinib.

Materials and methods

Patients and sample preparation

Bone marrow mononuclear cells (BMMNCs) were obtained from eight patients undergoing treatment in the Tianjin Cancer Institute and Hospital. Their clinical characteristics are presented in Table 1. This research project was approved by the Ethics Committee of Tianjin Cancer Institute and Hospital. Written consent was obtained from each patient.

Table 1.

The clinical pathologic characteristics of MM patients in our study.

Patient	Gender	Age (years)	Curative effect	β2-microglobulin	Ratio of plasma cells (%)	MM type	Clinical stages
MM 1	Male	55	New diagnosed	2.8	3	IgG-λ	IIIA
			Complete response	1.5	1
			Relapse	5.8	7	IgG-λ	IIIA
MM 2	Male	66	New diagnosed	2.3	5	IgG-κ	IIIB
			Complete response	1.4	0
			Relapse	8.9	10	IgG-κ	IIIB
MM 3	Male	65	New diagnosed	3.9	15	κlight chain	IIA
			Complete response	0.8	2
			Relapse	12.6	17	κlight chain	IIIA
MM 4	Female	56	New diagnosed	14.6	24	IgG-κ	IIIB
			Partial response	4.3	6
			Relapse	14.1	25	IgG-κ	IIIB
MM 5	Female	67	New diagnosed	21.9	32	IgG-λ	IIIA
			Complete response	2.3	1
			Relapse	18.6	26	IgG-λ	IIIA
MM 6	Male	64	New diagnosed	6.7	6.5	IgG-κ	IIIB
			Complete response	2.3	0
			Relapse	8.9	11.5	IgG-κ	IIIB
MM 7	Male	62	New diagnosed	6.3	15	κlight chain	IIB
			Complete response	3.5	2
			Relapse	12.6	29	κlight chain	IIIB
MM 8	Male	69	New diagnosed	11.6	24	IgG-κ	IIIA
			Partial response	4.3	6
			Relapse	15.1	30	IgG-κ	IIIA

MM: multiple myeloma.

Materials

RPMI-1640 medium, fetal bovine serum (FBS), and other cell culture reagents were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). Anti-NF-κB antibodies and anti-STAT3 (Tyr705/Ser727) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); all other antibodies were purchased from Boster (Wuhan, China). Trizol, Lipofectamine^TM RNAiMAX, and Opti-MEM^® I medium were purchased from Invitrogen Inc. (Carlsbad, CA, USA). Real-time polymerase chain reaction (RT-PCR) kits were purchased from TaKaRa Bio, Inc. (Tokyo, Japan). Ibrutinib, the STAT3 inhibitor Stattic, S31-201, the STAT3 agonist IL-6, the NF-κB agonist tumor necrosis factor alpha (TNF-α), and the NF-κB inhibitor BAY11-7082 were purchased from Selleckchem (Houston, TX, USA). Immunofluorescence staining and chromatin immunoprecipitation (ChIP) assay kits were purchased from Beyotime Co. (Jiangsu, China). miR-21 mimics, inhibitors, and negative controls were purchased from gene-pharma (Shanghai, China). Stattic, S31-201, and Bay 11-7082 were dissolved in dimethyl sulfoxide (DMSO; Solarbio Science and Technology, Beijing, China).

Cell lines and cell culture

Human MM cell lines, LP-1, U266, MM.1S, and MM.1R were generously provided by Dr Changhong Li from the Institute of Dermatology, Peking Union Medical College. Cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO₂ at 37°C. Primary patient MM (pp MM) cells were isolated from bone marrow aspirates by Ficoll–Hypaque density gradient sedimentation followed by antibody-mediated positive selection using anti-CD138 magnetic-activated cell separation micro beads (Miltenyi Biotech, Klner, Germany) as previously described. ⁷ Purity of selected cells was assessed by flow cytometry using a phycoerythrin-conjugated CD38 monoclonal antibody (mAb; Imgenex, San Diego, USA).

Cell viability assay

Cell survival was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma, Santa Clara, USA) assay in 96-well plates. Briefly, cells were seeded at a density of 1 × 10⁵ cells per well in 200 µL culture medium. Ibrutinib (0–20 µM) was applied for 24 h. After 24 h, 20 µL MTT (5 mg/mL) was added to each well and cells were incubated for 4 h at 37°C. Medium was then removed and 150 µL DMSO was added to each well. Plates were shaken for 10 min to dissolve the formazan. The viability of cells was determined by a micro-plate reader with a 452-nm filter (Synergy HT; Bio-Tek Instruments, Winooski, VT, USA). Experiments were repeated at least three times. Data are displayed as mean ± standard deviation (SD) of three independent experiments. Vehicle control cells were treated with 0.1% DMSO.

Soft-agar colony formation assay

Two layers of agarose containing 3 mL RPMI-1640 medium, 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin per layer were prepared in 60-mm Petri dishes. The bottom layer contained 0.6% low-melt agarose, and the top layer contained 0.3% low-melt agarose plus 5 × 10³ cells. Plates were incubated at 37°C in 5% CO₂ in a humidified incubator for approximately 2 weeks. Colony formation was then evaluated by counting colonies containing more than 100 cells. Experiments were repeated at least three times. Data are displayed as mean ± SD of three independent experiments.

Reverse transcription and quantitative RT-PCR

RNA isolation was performed according to the manufacturer’s instructions. Total cellular RNA was prepared using Trizol (Invitrogen). For mRNAs, equal amounts of total RNA were reverse-transcribed using a Prime Script RT reagent Kit (TaKaRa) per the manufacturer’s recommendations. For miR-21 analysis, bulge-loop miRNA quantitative RT-PCR (qRT-PCR) was used. Primers specific for miR-21 and control U6 were designed by RiboBio (Guangzhou, China). PCR primers for PTEN, PDCD4, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China), and the primer sequences are listed (Supplementary Table 1). qRT-PCR was performed on a CFX Connect Real-Time System (Bio-Rad, California, USA) with Fast SYBR Green Master Mix according to the manufacturer’s protocol. Samples were analyzed in triplicate. Data were analyzed using the −ΔΔCt method. PCR product specificity was confirmed by melting curve analysis.

Western blot analysis

Cells were washed twice with phosphate-buffered saline (PBS) and lysed at 10⁴ cell/µL in lysis buffer (M-PER Mammalian Protein Extraction Reagent; Thermo Inc., Saddle Brook, Germany) on ice for 10 min. After removing cell debris by centrifugation (14,000 r/min), protein concentration was determined by Bradford assay. Samples containing equal amounts of protein were mixed with loading buffer containing 5% 2-mercaptoethanol, heated (95°C, 10 min), loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel, and transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). Membranes were blocked with 5% BSA and 0.1% Tween-20 in Tris-buffered saline (TBS) and then incubated overnight at 4°C with primary antibodies. Membranes were then incubated with horseradish peroxide–conjugated secondary antibodies for 1 h at room temperature. Target proteins were detected with an enhanced chemiluminescent detection system according to the manufacturer’s protocol.

Immunofluorescence assay

MM cells were examined for NF-κB and STAT3 localization by immunofluorescence according to the manufacturer’s instructions. Briefly, cells were treated with ibrutinib (10 µM) or vehicle for 4 h, fixed with 3% paraformaldehyde, and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature. Cells were then incubated with 2% goat serum at room temperature for 1 h followed by primary antibody (specific for NF-κB or STAT3; 1:200) overnight at 4°C. Subsequently, cells were incubated with Alexa Fluor 647–Labeled Anti-Rabbit IgG (1:1000; Beyotime) for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min following secondary antibody incubation. Images were captured with the EVOS fluorescent microscope (DAPI filter: 357/44 nm excitation, 447/60 nm emission; green fluorescent protein (GFP) filter: 470/22 nm excitation, 510/42 nm emission; red fluorescent protein (RFP) filter: 531/40 nm excitation, 593/40 nm emission).

ChIP

ChIP assays were performed according to the manufacturer’s protocol (Beyotime) with slight modifications. Single-cell suspensions of adult murine testes were prepared as a positive control. Briefly, samples were prepared in RPMI-1640 medium. Chromatin in single cells was cross-linked using 1% formaldehyde for 10 min at 37°C followed by neutralization with 125 mM glycine. Cells were then lysed on ice in SDS lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% SDS) containing 1× proteinase inhibitor mixture (Roche Applied Science, Basel, Switzerland). The lysate was then sonicated on ice six times (10-s pulse, 50-s break, 20% amplitude; Sonics, Newtown, USA) to produce 100–1000 bp DNA fragments. About 2 µg primary antibody or control IgG was added to 1 mL Protein G–precleared lysate at 4°C overnight. Lysate was then washed once each with low salt, high salt, and LiCl immune complex wash buffers, and twice with Tris–EDTA buffer. DNA in the precipitate was extracted using a standard phenol–chloroform method. After amplification, PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining.

Over-expression and inhibition of miR-21 in MM cells

For transient transfection, cells were transfected at approximately 60%–70% confluence using Lipofectamine RNAiMAX Reagent (Invitrogen) with miR-21 mimics and inhibitors or negative controls in Opti-MEM medium (Invitrogen). Cells were incubated for 24 h before harvesting.

Statistical analysis

Each experiment was carried out at least three times. All values are reported as mean ± SD. Comparisons between groups were made with one-way analysis of variance (ANOVA; Graph Pad software 5.0 (www.graphpad.com)). Values of p < 0.05 were considered statistically significant.

Results

miR-21 is elevated in MM patients

We tested the hypothesis that miR-21 plays a role in MM cell growth first. Consistent with a previous report, ⁵ the expression of miR-21 in BMMNCs from MM patients was highly correlated with the progression of disease (Figure 1(a) and (b)). miR-21 expression was also positively correlated with the MM indicators β2-microglobulin and the ratio of plasma cells (Table 1, R = 0.833 and 0.758, respectively).

Figure 1.

miR-21 promoted MM cell proliferation.

miR-21 enhances MM cell growth

To investigate the role of miR-21 in MM cells in depth, we quantified miR-21 expression in pp MM cells and several MM cell lines (LP-1, U266, MM.1S, and MM.1R); using qRT-PCR, we can see that all MM patients showed a higher expression of miR-21, and U266 cell lines had the highest miR-21 expression while LP-1 cell lines showed the lowest miR-21 expression (Figure 1(c) and (d)). The 50% inhibitory concentration (IC50) values of ibrutinib in LP-1 and U266 were 9.5 and 7.5 µM (Supplementary Material Figure 1), respectively, so we choose 10 µM in our study. Inhibiting miR-21 in U266 and LP-1 cells both slowed proliferation, whereas expression of a miR-21 mimic in MM cells enhanced proliferation, and the effect of miR-21 mimics or miR-21 inhibitors was obvious on U266 (Figure 1(e) and (f)).

Ibrutinib preferentially inhibits BTK-expressing MM cell lines

A previous report ¹³ suggested that the toxicity of ibrutinib in MM cells depends on the expression of BTK. U266 cells showed the highest and LP-1 showed the lowest expression of BTK of the four cell lines tested (Supplementary Material Figure 2). Consistent with previous findings, ibrutinib significantly inhibited the pp MM cell proliferation (Figure 2(a)) and more profoundly inhibited soft-agar clonogenicity of U266 than that of LP-1 cells (Figure 2(b)). Interestingly, miR-21 expression correlated with BTK expression across cell lines. Since miR-21 is widely considered to be oncogenic in MM, ¹⁴ we asked whether ibrutinib affects miR-21 expression. Ibrutinib downregulated miR-21 in both pp MM cells and MM cell lines (Figure 2(c) and (d)), and its canonical targets PTEN and PDCD4 ¹⁵ were also regulated (Supplementary Figure 3). Moreover, the effects of ibrutinib on miR-21 were enhanced by treating cells with a miR-21 inhibitor. Conversely, addition of miR-21 mimics mitigated the inhibition of miR-21 by ibrutinib (Figure 2(e) and (f)).

Figure 2.

Ibrutinib attenuates MM cell proliferation.

Ibrutinib inhibits p65, Iκβα, and STAT3 phosphorylation in MM cells

We then investigated the mechanisms underlying the ibrutinib-mediated inhibition of MM cell growth and miR-21 expression. miR-21 transcription is regulated by the NF-κB and STAT3 signaling pathways, and we therefore tested the effects of manipulating the NF-κB and STAT3 pathways on miR-21 expression in ibrutinib-treated MM cells.^13,14 First, we found that the NF-κB inhibitor BAY-11 and ibrutinib inhibited miR-21 synergistically (Figure 3(a) and (b)). Conversely, the NF-κB agonist TNF-α attenuated the effects of ibrutinib on miR-21 (Figure 3(c) and (d)). We then treated MM cells with multiple ibrutinib concentrations and for varying exposure times. After ibrutinib treatment, we examined p65 and IκBα phosphorylation, as well as p100 and p52 expression, in U266 and LP-1 cells. Ibrutinib markedly inhibited basal p65 and IκBα phosphorylation in U266 cells compared to LP-1 cells (Figure 4(a) and (b)). These observations suggest that ibrutinib downregulates miR-21 by inhibiting the NF-κB pathway.

Figure 3.

The role of NF-κB and STAT3 in ibrutinib-mediated downregulation of miR-21 expression.

Figure 4.

The role of NF-κB and STAT3 in ibrutinib-mediated downregulation of miR-21 expression.

Next, we applied the STAT3 inhibitors S31-201 (nonspecific phosphorylation site inhibitor) or Stattic (phospho-Tyr705-specific inhibitor). Conversely, the STAT3 agonist IL-6 reversed the effects of ibrutinib on miR-21 (Figure 3(c) and (d)). Similar to the NF-κB pathway, ibrutinib inhibited STAT3 (Tyr705/Ser727) phosphorylation in U266 cells significantly more than in LP-1 cells (Figure 4(c) and (d)). These results suggest that the STAT3 pathway maybe involved in the ibrutinib-mediated downregulation of miR-21 expression.

Ibrutinib relieves miR-21 transcriptional repression by inhibiting NF-κB and STAT3

To determine whether the ibrutinib-mediated inhibition of STAT3 and NF-κB requires BTK, cell lines with the highest (U266) and lowest (LP-1) BTK expression were exposed to ibrutinib (10 µM) for 4 h. Nuclear translocation of NF-κB and STAT3 was detected using immunofluorescent staining. As shown in Figure 5(a) and (b), p-p65 and p-STAT3 located in both cytoplasm and nucleus in control; when exposed to ibrutinib, p-p65 and p-STAT3 located in both cytoplasm and nucleus in LP-1 (Figure 5(a)). However, compared with LP-1, either p-p65 or p-STAT3 in U266 cells was located mainly in cytoplasm in U266 when the cells were treated by ibrutinib (Figure 5(b)). These results support the idea that ibrutinib preferentially inhibits BTK-expressing MM cells.

Figure 5.

Ibrutinib relieves miR-21 transcriptional repression by inhibiting NF-κB and STAT3.

As previously suggested,^16–18 ibrutinib may inhibit the direct interaction between STAT3/NF-κB with the upstream/enhancer region of the miR-21 promoter. To test this, we performed ChIP assays in ibrutinib-treated U266 and LP-1 cells. As shown in Figure 5(c) and Supplementary Material Figure 4, immunoprecipitation using anti-p-STAT3 (Ser727; data not shown) or anti-p-P65 antibodies pulled down less miR-21 after ibrutinib treatment in U266 cells. miR-21 was detected using a primer pair-specific PCR assay for the miR-21 promoter/enhancer region containing the STAT3 and NF-κB binding sites. These findings suggest that ibrutinib can inhibit miR-21 transcription by disrupting NF-κB and STAT3 binding to the miR-21 promoter.

Discussion

MM is a progressive hematologic disease originating in bone marrow plasma cells. Despite recent treatment advances, including the novel pharmacological agents, bortezomib and lenalidomide, MM remains incurable. Development of therapy resistance and the complexity of the bone marrow microenvironment pose a problem for developing effective treatments. ¹⁹ To improve the outcome of MM therapy and overcome drug resistance, novel therapeutic approaches need to be explored.

Previous studies have established a link between miR-21 and the pathogenesis of MM.^5,20 Here, we also found that upregulation of miR-21 expression is positively correlated with MM progression and poor prognosis, lowering the expression of miR-21 suppressed MM cell proliferation, survival, and clonogenicity in vitro and in vivo, and had the largest effects on cells expressing higher amounts of miR-21. In addition, proliferation of cells with low expression of miR-21 was increased after transfection by a miR-21 mimic. In accordance with these observations, miR-21 expression correlated with tumor load–related indicators and response to treatment in patients with MM.

In recent years, increasing evidence suggests that targeting BTK may be effective for treating relapsed and refractory MM.^20,21 In this study, ibrutinib inhibited the proliferation of MM cells from patients. Ibrutinib more profoundly reduced proliferation and clonogenicity in cells with high BTK (U266 cell line) than in cells with low BTK (LP-1 cell line). In addition to modulating MM cell growth, miR-21 correlated with BTK expression in MM cell lines, and ibrutinib decreased miR-21 expression. Ibrutinib also decreased phosphorylation of NF-κB and STAT3, two transcription factors that regulate miR-21 expression.^22–24 NF-κB and STAT3 are constitutively active in MM cells and play the central role in chemotherapy resistance, cell survival, and proliferation. ²⁵ Development of drugs that suppress NF-κB and STAT3 activity in MM cells, such as bortezomib and thalidomide, has been an effective therapeutic strategy for patients with MM. ²⁶ Similar effects of ibrutinib suggest that it may also prove to be a valuable treatment for MM.

The results of this study suggest that ibrutinib prevents NF-κB and STAT3 phosphorylation by BTK, thereby downregulating miR-21 transcription and inhibiting cell growth (Figure 6). In spite of effective progress of therapies for multiple myeloma, patients suffering from this disease will eventually experience relapse and refractory. In this case, it is necessary for development of novelty therapies based on elucidating the complicated molecular pathogenesis of MM and mechanisms of resistance. Similar to previous reports, we identified that ibrutinib is effective on the BTK-expressed multiple myeloma cells. However, previous studies^9,10 showed that ibrutinib can inhibit either NF-κB or STAT3 separately, while our results indicated that ibrutinib blunted both the NF-κB and STAT3 signal pathway in BTK-expressed U266 cells simultaneously. Excessive activation of NF-κB and STAT3 signal pathway plays a key role in the progressive pathogenesis of MM;^14,25 simultaneous targeting of the two synergistic pathways may represent novel strategies on the success of potential agents. Because ibrutinib was approved by Food and Drug Administration (FDA), it is easy for the physician to explore a potential candidate for relapse and refractory MM.

Figure 6.

Schematic diagram of how ibrutinib may inhibit miR-21-induced cell death in MM.