Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Clin Cancer Res. 2015 Nov 2;22(5):1222–1233. doi: 10.1158/1078-0432.CCR-15-0489

A 13 mer LNA-i-miR-221 inhibitor restores drug-sensitivity in melphalan-refractory multiple myeloma cells

Annamaria Gullà 1, Maria Teresa Di Martino 1, Maria Eugenia Gallo Cantafio 1, Eugenio Morelli 1, Nicola Amodio 1, Cirino Botta 1, Maria Rita Pitari 1, Santo Giovanni Lio 2, Domenico Britti 1, Maria Angelica Stamato 1, Teru Hideshima 3, Nikhil C Munshi 3,4, Kenneth C Anderson 3, Pierosandro Tagliaferri 1, Pierfrancesco Tassone 1,5
PMCID: PMC4775414  NIHMSID: NIHMS733332  PMID: 26527748

Abstract

Purpose

The onset of drug-resistance is a major cause of treatment failure in multiple myeloma (MM). While increasing evidence is defining the role of microRNAs in mediating drug-resistance, their potential activity as drug-sensitizing agents has not yet been investigated in MM.

Experimental Design

Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory MM cells to melphalan.

Results

MiR-221/222 expression inversely correlated with melphalan-sensitivity of MM cells. Inhibition of miR-221/222 overcame melphalan-resistance and triggered apoptosis of MM cells in vitro, in the presence or absence of human bone marrow stromal cells. Decreased MM cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx-efflux transporters SLC7A5/LAT1 and the ATP-binding cassette (ABC) transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory MM xenografts with systemic LNA-i-miR-221 plus melphalan overcame drug-resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice.

Conclusions

Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan-resistance in preclinical models of MM, providing the framework for clinical trials to overcome drug resistance and improve patient outcome in MM.

Keywords: LNA-miR-221 inhibitors, melphalan, drug-resistance, microRNA, multiple myeloma

Introduction

Multiple Myeloma (MM) is characterized by the abnormal proliferation of malignant plasma cells in the bone marrow (BM) (1, 2). Despite recent advances in MM biology (3), pre-clinical models (4–6), and translation of novel agents which have markedly improved the outcome of MM patients, the development of drug-resistance remains an obstacle to long-term survival (7). MM commonly progresses to drug-refractory end-stage disease (8), and novel therapeutic strategies are urgently needed.

For more than 30 years, melphalan has been the mainstay of MM treatment (9). Presently its therapeutic value is in younger patients who undergo high-dose melphalan (HDM) prior to autologous stem cell transplantation (ASCT), as well as in non-transplant candidates or elderly patients as part of first-line combination regimens (10, 11). The use of melphalan combination regimens with new agents, such as bortezomib or lenalidomide has significantly prolonged progression-free (PFS) and overall survival (OS); however, development of drug-resistance leads to relapse of disease (12). Recently a renewed scientific interest on melphalan is emerging, and major efforts have been devoted to delineate the mechanisms underlying primary or acquired melphalan-resistance (13). These efforts have already led to the design of novel regimens to overcome melphalan-resistance or to improve its anti-tumor activity (14).

Currently, there is a growing interest for the therapeutic potential of strategies aimed to target microRNAs (miRNAs) network. MiRNAs are a class of short non-coding RNA that function as post-transcriptional gene regulators. MiRNAs mainly act through complete or partial binding to 3′ untranslated region (3′ UTR) of their mRNA targets, inducing either mRNA degradation or translational repression (15). By targeting driver genes involved in critical cellular pathways miRNAs can function as oncogenes or tumor suppressor genes, playing a key role in tumorigenesis, as well as in cancer progression and aggressiveness (15). A variety of studies have to date demonstrated the potential relevance of miRNA mimics/inhibitors as therapeutic tools, and the promising results from the first Phase-2 trial in patients with HCV infection treated with Locked Nucleic Acid (LNA)-miR-122 inhibitors have further stimulated studies for the treatment of human cancer (16). In MM, miRNA-based strategies are presently emerging as promising approaches (17–25). Moreover, recent findings have emphasized the role of miRNAs in the development of drug-resistance in a variety of malignancies (26). In particular, miRNAs have been shown to regulate drug efflux transporters, induction of apoptosis, cell cycle progression, DNA repair mechanisms, and other alterations of drug targets (27). Among miRNAs involved in development of drug-resistance, miR-221/222 plays a key role: inhibition of miR-221/222 has been reported to overcome resistance to cisplatin (28), tamoxifene (29), fulvestrant (30), temozolamide (31), tirosin Kinase Inhibitors (32), and TRAIL (33) in a variety of cancers. We recently provided the first evidence that silencing of miR-221/222 by specific inhibitors exerts anti-tumor activity in MM cells bearing t(4;14) translocations in vitro and in vivo (34), and that naked LNA-inhibitors of miR-221 (LNA-i-miR-221) are suitable for systemic delivery in animals (35). Here we investigated the role of miR-221/222 in melphalan-refractory MM, and demonstrate restoration of melphalan-sensitivity in refractory cells after exposure of MM cells to a novel 13 mer LNA-i-miR-221. Our findings provide therefore the rationale for clinical trials investigating LNA-i-miR-221 plus melphalan in drug-refractory MM.

Materials and Methods

Cell cultures, reagents and drugs

Multiple Myeloma cell lines NCI-H929 t(4;14), RPMI-8226 t(14;16) and U266 t(11;14) were purchased from DSMZ (Germany) which certified authentication performed by Short Tandem Repeats DNA typing. These cells were immediately frozen and used from the original stock within 6 months. Melphalan-resistant U266/LR7 t(11;14) cells were kindly provided by Dr. A. Pandiella (University of Salamanca, Spain). AMO1 t(12;14) and bortezomib-resistant AMO1 Abzb t(12;14) cells were kindly provided by Dr. C Driessen (University of Tubingen, Germany). U266/LR7, AMO1 and AMO1 Abzb were not further authenticated but confirmed for the described drug-resistant phenotype. All cells were cultured in RPMI-1640 (Gibco®, Life Technologies), as previously described (36, 37). Human stromal HS-5 cells were purchased from ATCC, which certify authentication by Short Tandem Repeats profiling. Also these cells were immediately frozen and used from the original stock within 6 months. HS-5 were cultured in Dulbecco’s modified Eagle’s medium (Gibco®, Life Technologies) supplemented with 10% heath inactivated Fetal Bovine Serum (FBS) and 1% P/S (Penicillin/Streptomycin). Following informed consent and Istitutional Ethical Comeettee approval, peripheral blood mononuclear cells (PBMCs) and primary CD138+ MM cells from BM aspirates of 3 MM patients, were isolated as previously described (38). LNA-i-miR-221 was designed and synthesized as previously described (35). Melphalan and Bortezomib were purchased from Sigma Aldrich and Selleck Chemicals, respectively.

In vitro transfection of MM cells

Synthetic mirVana® miR-221 and miR-222 inhibitors or mimics were purchased (Life Technologies); mirVana™ miRNA mimic and inhibitor Negative Control #1 (Life Technologies) were used as experimental negative controls (NC). A total of 1 x 106 MM cells were transfected at 100 nM miRNAs concentrations by the Neon® Transfection System (Life Technologies) (1050 v, 2 pulse, 30 a); transfection efficiency, evaluated by flow-cytometry analysis relative to a FAM dye–labeled anti-miR–negative control, reached 85% to 90%. Similar conditions were applied for transfection of MM cells with Silencer® Select siRNA for PUMA/BBC3 (siPUMA) or with Silencer® Select siRNA control (siCNT) (Life Technologies), which was used at final concentration of 50 nM even in co-transfection experiments with miRNAs inhibitors.

Virus generation and infection of Human Stromal HS-5 cells

HS-5 cells cells stably expressing green fluorescent protein transgene were obtained as previously described (39) (see Supplementary Methods for detailed information).

Reverse transcription and quantitative real-time PCR

Total RNA extraction from MM cells and quantitative real-time PCR were performed as previously described (see Supplementary Methods for detailed information). (38)

Cell proliferation and survival assay

Cell growth inhibition was evaluated by Cell Counting Kit-8 (CCK-8) colorimetric assay (Dojindo Molecular Technologies, Inc.), according to the manufacturer’s instructions. For melphalan dose-response experiments, MM cells were seeded in 24-well plates at a density of 2.5 x 105 cells per well in 1 ml of culture medium and incubated for 24 hours in the presence of different μM melphalan concentrations; after incubation, MM cells were inoculated in 96-well plates for CCK-8 assay. Final optical density (O.D.) was measured at 450 nm using GloMax (Promega). Wells without cells (culture medium alone) were used as blank. For combination experiments with miRNAs, 1 x 106 electroporated cells with NC or miR-221/222 inhibitors were incubated for 24 hours in 6-well plates; after harvesting, cells were inoculated in 24-well plates at a density of 2.5 x 105 cells/ml and incubated in the presence or absence of different μM melphalan concentrations. Twenty-four hours after beginning drug exposure, cells were seeded in 96-well plates for CCK-8 assay and O.D. measurement. For co-culture experiments, 2.5 x 105 U266/LR7 cells transfected with miR-221/222 inhibitors or NC were adhered to a monolayer of GFP+ HS-5 cells at 50% confluence in 12-well plates for 24 h. After incubation, co-cultured cells were treated with 100 μM of melphalan, and collected 24 hours later by gentle pipetting for 7-aminoactinomycin D (7-AAD) staining and flow cytometry analysis. Each experiment was repeated at least three times. Data represent the mean ± SD of at least 3 independent experiments.

Apoptosis detection by fluorescence microscopy

A total of 5 x 105 U266/LR7 cells transfected with NC or miR-221/222 inhibitors were incubated for 24 hours in 12-well plates; after harvesting, each group was incubated in the presence or absence of 100 μM melphalan. Twenty-four hours later, harvested cells were plated in 96-well plates and visualized directly in the culture dishes after staining with Hoechst 33342 and Propidium Iodide (PI). Briefly, treated cells were washed and suspended in phosphate-buffered saline (PBS) 1X at a density of 1 x 106/ml with the addition of 5 μM Hoechst 33342 (Sigma Aldrich). After incubation at 37° C for 15 minutes, cells were washed and re-suspended in Binding Buffer 1X (BD Pharmigen) additions with 0.5 μM PI (BD Pharmigen) and incubated at room temperature for 15 minutes avoiding light exposure. After incubation, cells were washed and re-suspended in PBS for fluorescence microscopy analysis using EVOS FLAuto Cell Imaging System (Life Technology). Representative fields of cells were visualized (200X magnification). For apoptosis analysis by flow cytometry see Supplementary Methods.

Luciferase reporter experiments

Validation of miR-221/222 direct targeting of PUMA/BBC3 3′ UTR was performed as previously described (see Supplementary Methods for detailed information). (38)

Protein extraction and Western blot analysis

Total proteins were extracted with NP40 Cell Lysis Buffer (10 mmol/L Tris–HCl [pH 7.5], 150 mmol/L NaCl, 1% NP-40) (Life Technologies) with the addition of Halt Protease Inhibitor Single-Use Cocktail 100 x (Thermo Scientific). For Western blot analysis, 50 μg per line of the whole lysates were separated by electrophoresis on NuPAGE® precasted Gels (4%–12%) (Invitrogen, Life Technologies) and electro-transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer packs, Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). After protein transfer, the membranes were blotted with the primary antibodies (see Supplementary Methods for detailed information).

Immune precipitation

Lysates were prepared by homogenizing retrieved tumor xenografts in NP-40 lysis buffer containing Protease Inhibitor by using gentleMACS Dissociator (Miltenyi Biotec). Immune precipitation was performed by incubating 1 mg lysates with 2 μg of antibody and 30 μl of protein A/G-conjugated agarose beads (Santa Cruz Biotechnology) overnight at 4°C. The precipitates were resuspended in loading buffer and resolved by NuPAGE electrophoresis, followed by immune blotting afte electrotransfer.

Hoechst dye exclusion assay

U266/LR7 cells were collected 48 hours after transfection with miR-221/222 inhibitors or NC and resuspended in PBS (1X) at 1 x 106/ml concentration. Cells were pre-incubated at 37°C for 10 min in the presence or absence of verapamil 100 μM (Sigma Aldrich) to inhibit ABC transporters and then were incubated for 90 min at 37°C with Hoechst 33342 5 μg/ml (Sigma Aldrich), with intermittent shaking. Subsequently, cells were washed and re-suspended in ice-cold PBS (1X). 7-AAD was added (5 μl) to each tube prior to acquisition to exclude dead cells from the analysis. The cells were filtered to obtain single cell suspension for flow cytometry analysis performed by FACSAria III (Becton Dickinson). The Hoechst 33342 dye was excited at 357 nm, and its fluorescence was dual-wavelength analyzed (402–446 nm for Hoechst 33342-Blue; 650–670 nm for Hoechst 33342-Red).

Animals and in vivo model of human MM

Six to eight-week old female SCID/NOD mice (Harlan laboratories, Inc.) were housed and monitored in Animal Research Facility at Magna Graecia University. All experimental procedures and protocols had been approved by the Institutional Ethical Committee (Magna Graecia University). In accordance with the institutional guidelines, mice were sacrificed when their tumors reached 2 cm in diameter or in the event of paralysis or major compromise in their quality of life. Specifically, we evaluated the in vivo ability of LNA-i-miR-221 (35), specifically designed for systemic delivery, to enhance the anti-MM activity of melphalan. Briefly, a cohort of 24 mice for each in vivo study were subcutaneously inoculated with 5 x 106 U266/LR7 cells in 100 μl of RPMI-1640 medium; treatments started when tumors became measurable, approximately 3 weeks after cells were injected. In a first in vivo experiment, mice were randomized to receive 4 different treatments: i) LNA-i-miR-221 intraperitoneally (i.p.) injected at a dose of 25 mg/kg at day 1–4–8–15–22 from the beginning of the experiments; ii) scrambled control with the same schedule; iii) 0.75 mg/kg of i.p. melphalan twice weekly for 3 weeks; iiii) melphalan plus LNA-i-miR-221 with the above described schedules. In a subsequent in vivo study, treatment with LNA-i-miR-221 (25 mg/kg) plus melphalan (0.75 mg/kg) was given with for 4 consecutive days every 10 days. Tumor sizes were measured as previously described (35). In vivo detection of the tumor mass in xenografted mice was performed using IVIS LUMINA II Imaging System (Caliper Life sciences, Hopkinton, MA, USA). At the end of observation, tumors were retrieved from animals and placed in 10 formalin for histology or stored at −80° C for protein analysis.

Histology and immunohistochemistry

Retrieved tumors from animals were fixed in 4% buffered formaldehyde and 24 h later washed, dehydrated, and embedded in paraffin. For light microscopy analysis by an optical microscope Nikon i55 (Nikon Corporation, Tokyo, Japan), we performed staining with Hematoxylin-Eosin on 4 μm tumors sections mounted on poly-lysine slides. For immunohistochemistry staining, 2 μm thick tumor slices were de-paraffinized and pre-treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH 8.8) at 98°C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer. All procedures were performed using the Bond Max Automated Immunohistochemistry. Tissues were washed and incubated with the primary antibody directed against Ki-67 (Dako, clone MIB-1; 1:150) or caspase-3 (Novocastra, clone JHM62; 1:500). Subsequently, tissues were incubated with polymer for 10 min and developed with DAB-Chromogen for 10 min. Slides were counterstained with hematoxylin.

Statistical analysis

Each in vitro experiment was carried out at least 3 times, and all values are reported as means ± SD. Comparisons between groups were made with Student t test, whereas statistical significance of differences among multiple groups was determined by GraphPad software (www.graphpad.com). Differences were considered significant with p ≤ 0.05. The Synergistic Index (SI) was determined as previously described (40) with the following formula: SI = (effect induced by miR-221/222 inhibitors and melphalan in combination)/(effect of miR-221/222 inhibitors)+(effect of melphalan). Interactions were considered synergistic when SI was >1.

Results

1. Melphalan-induced modulation of miR-221/222 in MM cells

We first evaluated the melphalan-sensitivity of our MM cell line panel including U266/S, NCI-H929, AMO1, RPMI-8226, AMO1 Abzb and U266/LR7. NCI-H929, AMO1 and U266/S were sensitive at low melphalan concentrations; RPMI-8226 and the bortezomib-resistant AMO1 Abzb showed resistance at doses up to 20 μM, while the melphalan-resistant U266/LR7 cells were resistant up to 200 μM drug concentration (IC50 not found) (Fig. 1a). In parallel, we measured the miR-221/222 melphalan-induced expression in MM cells. As evaluated by q-RT-PCR, miR-221/222 expression after drug exposure inversely correlated with melphalan-sensitivity (Fig. 1b–c). In particular, melphalan-resistant U266/LR7 cells showed the highest miR-221/222 expression after drug exposure. Significant miR-221 upregulation was also observed in RPMI-8226 and AMO1 Abzb cells, which are moderately resistant to melphalan, whereas those cell lines sensitive to melphalan including NCI-H929, U266/S, and AMO1 cells did not show any change in miR-221/miR-222 expression following treatment. Moreover, q-RT-PCR analysis of miR-221 and miR-222 pre-melphalan treatment in U266/LR7 showed a 1.5-fold increase for miR-221 (Fig. S1a) and a 3.5-fold increase for miR-222 (Fig. S1b) compared to the parental sensitive counterpart U266/S (Fig. S1). Altogether, these findings demonstrate an inverse correlation of drug-induced miR-221/222 expression with melphalan sensitivity of MM cells.

Fig. 1. Correlation of melphalan sensitivity with induction of miR-221 and miR-222 expression in MM cell lines.

Fig. 1

Open in a new tab

A) U266/LR7, U266/S, RPMI-8226, NCI-H929, AMO1 and AMO1 Abzb cells were treated with increasing melphalan concentrations. Cell viability, relative to the untreated controls, was measured after 24h by CCK-8 assay. Values represent mean ± SD of 3 different experiments. B-C) qRT-PCR analysis of miR-221 (B) and miR-222 (C) expression was performed in all the six MM cell lines exposed to different melphalan concentrations (0–10–20 μM). Raw Ct data were normalized to RNU44 houskeeping gene and expressed as ΔΔCt values, calculated using the comparative cross threshold (Ct) method. Values represent average ± SD of 3 independent experiments. P values (* p<0.05) were obtained using two-tailed t test.

2. MiR-221/222 inhibition overcomes melphalan-resistance of MM cells in vitro

We previous reported a strong in vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in MM cells bearing t(4;14) translocation (34). Since modulation of miR-221/222 seems to be affected by melphalan treatment independently from chromosome translocation, we investigated whether miR-221/222 plays a mechanistic role in MM cell melphalan-sensitivity/resistance regardless translocation status. To this aim, we first transfected U266/LR7, RPMI-8226, and AMO1 Abzb melphalan-resistant cells with miR-221/222 inhibitors or scramble controls, and then exposed cells to increasing concentrations of melphalan. As shown in Fig. 2a, miR-221/222 inhibitors synergistically enhanced the anti-MM activity of melphalan in a dose-dependent manner. At a fixed concentration (100 nM) of miR-221/222 inhibitors with increasing doses of melphalan, the synergistic index was measured: the highest synergistic effect was reached with 100 μM of melphalan (SI = 5.6) in U266/LR7 cells, and with 5 μM of melphalan (SI = 4.12) in RPMI-8226 cells, and in AMO1 Abzb cells (SI = 1.36) (Table S1). Conversely, enforced expression of synthetic miR-221/222 mimics in melphalan-sensitive U266/S cells, which previously we reported to exert a growth promoting activity on MM cells (34), resulted in a reduced susceptibility to the drug (Fig. S2a). Moreover, we evaluated whether miR-221/222 sensitizing effects to melphalan could be due to induction of apoptosis. As shown in Fig. 2b using Annexin V/7-AAD assay, we found that the combined treatment triggered greater apoptosis in all MM cell lines than each single agent. By western blotting analysis, we showed activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as increased PARP cleavage, in U266/LR7 cells exposed to combination treatment (Fig. 2c, top panel). An increase of cleaved caspase-3 was also confirmed by western blotting in RPMI-8226 cells (Fig. 2c, bottom panel). Conversely, enforced expression of synthetic miR-221/222 mimics in U266/S cells exposed to melphalan resulted in a decrease of cleaved caspase-3 (Fig. S2b). Using Hoechst 33342 and PI staining, we showed induction of apoptosis in cells after combination treatment, with a significant increase in late apoptosis (Fig. 2d). Importantly, inhibition of miR-221/222 enhanced the cytotoxic effect of melphalan even on primary CD138+ cells from 3 MM patients (Fig 3a). No effects on cell viability were instead observed on PBMCs from 3 healthy donors (Fig 3b). We next studied whether BMSCs protected against the synergistic combination therapy of miR-221/222 plus melphalan. By q-RT-PCR, a slight increase of miR-221/222 expression (2 and 4-folds, respectively) was detected in U266/LR7 cells adherent to the HS-5 BMSCs, as compared to cells cultured alone (Fig. S3). Importantly, anti-MM activity of miR-221/222 inhibitors plus melphalan against U266/LR7 cells adherent to HS5 BMSCs was similar to that against non adherent U266/LR7 cells (Fig. 3c). Taken together, these findings demonstrate that miR-221/222 inhibitors enhance the therapeutic activity of melphalan in drug-resistant cells by induction of apoptosis, even in the presence of BMSCs.

Fig. 2. Anti-proliferative and apoptotic effects in MM cells of miR-221/222 inhibitors combined with melphalan.

Fig. 2

Open in a new tab

A) CCK-8 cell proliferation assay in U266/LR7, RPMI-8226 and AMO1 Abzb tranfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with increasing melphalan concentrations. Data shown are the average of three independent experiments, and p values were obtained using two-tailed test. (* p<0.05). B) Annexin V/7-AAD staining of U266/LR7, RPMI-8226 and AMO1 Abzb electroporated cells (miR-221/222 inhibitors or NC) 24h after treatment with different melphalan concentrations. The percentage of Annexin-V positive cells is plotted. Values represent the mean ± SD of 3 independent experiments. * p<0.05 C) Western blot analysis of total and cleaved Casp-8, Casp-9, Casp-3 and Cleaved PARP in U266/LR7 cells (top panel) and Casp-3 levels in RPMI-8226 cells (bottom panel). Cells lines were transfected with 100 nM of miR-221/222 inhibitors or NC and then exposed to melphalan (100μM for resistant U266/LR7 cells and 5μM for RPMI-8226 cells). Loading control was performed using GAPD or γ-tubulin. D) Fluorescence microscopy after Hoechst 33343 and PI staining of melphalan resistant U266/LR7 cells transfected with miR-221/222 inhibitors or NC, and then exposed to 100 μM of melphalan. DNA dye Hoechst stains the nucleus (blue color emission) of both viable and dead cells, allowing identification of nuclear morphology. Apoptotic nuclei appear fragmented and condensed, with greater signal intensity. Apoptotic and necrotic cells are identified as positive PI staining cells (red color emission) with loss of membrane integrity.

Fig. 3. Effects of miR-221/222 inhibitors plus melphalan on primary cells and on MM cell lines in the presence of BMSCs.

Fig. 3

Open in a new tab

A) CCK-8 cell proliferation assay in primary CD138+ MM patients cells tranfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with 100 uM of melphalan. Data shown are the average of two independent experiments, and p values were obtained using two-tailed test. (* p<0.05). B) CCK-8 cell proliferation assay in primary hPBMCs cells transfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with increased melphalan concentrations. Data shown are the average of three independent experiments, and p values were obtained using two-tailed test. (* p<0.05). C) 7-AAD staining of U266/LR7 cells transfected with 100 nM of miR-221/222 inhibitors or NC, and then cultured in the presence or absence of GFP+ hBMSCs. 24 hours after co-culture cells were treated with 100 μM of melphalan; 24h later, they were collected and stained for 7-AAD flow cytometry analysis. The percentage of 7-AAD positive cells is represented. Values represent the mean ± SD of 3 independent experiments. * p<0.05

3. Genome-wide expression patterns triggered by miR-221/222 inhibitors plus melphalan

To analyze the transcriptome perturbations induced in MM cells by the combination of miR-221/222 inhibitors plus melphalan treatment, we performed a whole gene expression analysis (GEP) by GeneChip® Affymetrix arrays. Data are available through GEO accession number GSE66618. Since we demonstrated that inhibition of miR-221/222 restores sensitvity to melphalan in resistant MM cells, we next examined whether this effect may be associated with changes in the expression of genes involved in the most common mechanisms of drug-resistance. We performed a GEP on both untreated or drug-exposed melphalan-resistant U266/LR7 cells and melphalan-sensitive parental U266/S MM cells. By hierarchical clustering analysis, data points were grouped based on overall similarity in gene expression patterns. Clustering analysis revealed a similar profile in melphalan-resistant U266/LR7 MM cells treated with miR-221/222 inhibitors plus melphalan as was observed in the sensitive parental cells U266/S cells treated with melphalan alone (data not shown). Accordingly, we next performed fold change (FC) analysis, which yielded a list of 1487 genes modulated after combination treatment in melphalan-resistant MM cells. Notably, differentially expressed genes involved in apoptosis (PUMA/BBC3; BCL2L1), drug transport (ABCC1/MRP1, SLC7A8, SLC7A5), and DNA repair mechanisms (XRCC4, PRKDC, CNOT6L, DDB2) were found (Fig. S4a). By Ingenuity Pathway Analysis® (IPA) software, we evaluated the perturbation of biological response pathways and regulatory networks. After IPA annotation, a list of 739 genes was used for “Core Analysis” and identification of different canonical pathways with highly significant perturbation scores, primarily in the miR-221/222 inhibitors plus melphalan treated U266/LR7 cells (p<0.05) (Fig. S4b). Specifically, IPA revealed modulation of pathways: associated with alkylating agents-induced response (“NRF2-medited Oxidative Stress Response”); induced by micro-environmental stimuli (“HGF Signaling”, “Chemokine Signaling”, “IL-3 Signaling”, “Jak/Stat Signaling”, “STAT3 Pathway”); associated with tumor proliferation (“PTEN Signaling”, “EIF2 Signaling” and “PI3K Signaling in B Lymphocytes”); related to DNA repair mechanisms (“Cell Cycle:G2/M DNA Damage Checkpoint Regulation” and “DNA Double-Strand Break Repair by Non-Homologous End Joining”); as well as “Glucocorticoid Receptor Signaling”. Based on the IPA annotation analysis, we also characterized genes involved in these signaling pathways (Fig. S4c, inserted table). Interestingly, we detected downregulation of ABCC1, PIK3CB, XRCC4, PRKDC, AKT3 and STAT6; as well as upregulation of CDKN1A and E2F7 after miR-221/222 inhibitor plus melphalan treatment compared to either untreated- or either single agent treated- U266/LR7 cells. Altogether, these findings define a transcriptome profile that underlies the response to the combination treatment in MM cells, suggesting further investigation of miR-221/222 involvement in resistance mechanisms.

4. PUMA/BBC3 is a target of miR-221/222 in MM cells

We validated GEP data on XRCC4, CDKN1A, PRKDC, SLC7A8/LAT2, SLC7A5/LAT1, ABCC1/MRP1, and PUMA/BBC3 by q-RT-PCR, (Fig. S5a–b). In silico search for target prediction using mirDIP software indicates Bcl-2-binding component 3, PUMA/BBC3, as a strongly predicted target of miR-221/222. On the basis of target prediction, we co-transfected U266/LR7 cells with synthetic miR-221 or miR-222 mimics together with firefly luciferase constructs, in which either the wild-type or mutant 3′ UTR of PUMA/BBC3 mRNA are cloned downstream. A marked decrease in luciferase activity (42% for miR-221 and 38% for miR-222) indicated direct interactions between the miRNAs and PUMA/BBC3 3′UTR; moreover, target gene repression was rescued by deletions in the mutant clone (Fig. 4a). These findings provide validation of direct miR-221/222 targeting of 3′UTR of PUMA/BBC3 mRNA in MM cells.

Fig. 4. Inhibition of miR-221/222 enhances anti-MM activity of melphalan by modulation of PUMA/BBC3 expression.

Fig. 4

Open in a new tab

A) Dual-luciferase assay of U266/LR7 cells co-transfected either with NC or synthetic miR-221 or miR-222 mimics, together with firefly luciferase constructs containing the wild-type or mutant 3′ UTR of PUMA/BBC3 mRNA. The firefly luciferase activity was normalized to renilla luciferase activity. Data are represented as relative luciferase activity of either miR-221 or miR-222 mimics electroporated cells as compared to control. Values represent the mean ± SD of 3 independent experiments. * p<0.05 B) q-RT-PCR (bottom panel) and immunoblot (top panel) of PUMA/BBC3 in resistant U266/LR7 cells after transfection with NC or miR-221/222 inhibitors and treatment with 100 μM of melphalan. q-RT-PCR results are shown after normalization with GAPDH and ΔΔCt calculation and represent an average ± SD of 3 independent experiments. Protein loading control for immunoblot was performed using GAPDH. C) q-RT-PCR (bottom panel) and immunoblot (top panel) of PUMA/BBC3 in sensitive U266/S cells after transfection with NC or miR-221/222 mimics and treatment with 60 μM of melphalan. q-RT-PCR results are shown after normalization with GAPDH and ΔΔCt calculation and represent an average ± SD of 3 independent experiments. Protein loading control for immunoblot was performed using γ-tubulin. D) CCK-8 cell proliferation assay on resistant U266/LR7 cells (left panel) treated with either NC or miR-221/222 inhibitors co-transfected with siCNT or siBBC3/PUMA and subsequently exposed to 100 μM of melphalan. Effective knockdown was confirmed by immunoblot of PUMA/BBC3 protein and γ-tubulin normalization (right panel). Percentages of growth inhibition are plotted compared to control. Values represent the mean ± SD of 3 independent experiments. * p<0.05 E) CCK-8 cell proliferation assay on sensitive U266/S (left panel) transfected with siCNT or siBBC3/PUMA and treated with 60 μM of melphalan. Effective knockdown was confirmed by immunoblot of PUMA/BBC3 protein and GAPDH normalization (right panel). Cell viability is shown as percentage of control. Values represent the mean ± SD of 3 independent experiments. * p<0.05

5. MiR-221/222 inhibitors upregulate PUMA/BBC3 protein levels triggering melphalan-sensitivity in drug-resistant MM cells

We next analyzed PUMA/BBC3 expression both at the mRNA and protein levels in U266/LR7 cells transfected with miR-221/222 inhibitors and then exposed to melphalan. By western blotting, a strong upregulation of PUMA/BBC3 protein was found in cells treated with miR-221/222 inhibitors plus melphalan; conversely, exposure to melphalan alone led to upregulation of PUMA/BBC3 at mRNA, but not at protein, levels (Fig. 4b). Moreover, enforced expression of synthetic miR-221/222 mimics in melphalan-treated U266/S cells abrogates PUMA/BBC3 protein translation induced by melphalan alone (Fig. 4c). To evaluate the role of PUMA overexpression in enhancing melphalan activity, we exposed the U266/LR7 cells transfected with miR-221/222 inhibitors together with siRNA for PUMA/BBC3 (siPUMA) or siRNA control (siCNT) to melphalan. Importantly, in resistant cell lines silenced for PUMA/BBC3 (Fig. 4d, right panel), we found a significant antagonism (30%) of the sensitizing activity of miR-221/222 inhibitors compared to co-transfection with siCNT (Fig. 4d, left panel). As a further confirmation, PUMA silencing led to a 20% reduction of melphalan activity of in drug-sensitive U266/S cells (Fig. 4e, right panel). Based on these findings, we conclude that the sensitizing activity of miR-221/222 inhibitors to melphalan in MM cells is, at least in part, dependent on PUMA/BBC3 protein upregulation.

6. MiR-221/222 inhibition modulates expression of drug influx-efflux transporters in MM cells

Since GEP analysis showed modulation of drug transporter mRNA transcripts, we next studied the effects of miR-221/222 inhibitors plus melphalan on their expression. L-Type Amino Acid Transporter SLC7A5/LAT1 and ATP-binding cassette (ABC) transporter ABCC1/MRP1 were evaluated at the protein level in miR221/222 inhibitor or scramble transfected U266/LR7 cells. As shown in Fig 5a, miR-221/222 inhibition plus melphalan resulted in a significant upregulation of SLC7A5/LAT1 protein, as well as a marked downregulation of ABCC1/MRP1 protein (Fig. 5a). Moreover, based on the known ability of ABCC1/MRP1 pump to exclude drugs as well as fluorescent dyes such as Hoechst 33342 (41), we next evaluated by Hoechst dye exclusion assay whether miR-221/222 inhibitor treatment may affect dye cellular uptake. As expected, the cell fraction in U266/LR7 cells that exhibits low level of Hoechst fluorescent intensity, identified as Side Population (SP), decreased by 65% after treatment with miR-221/222 inhibitors, and by 74% after treatment with ABC transporters inhibitor Verapamil (Fig. 5b,c). Taken together, these findings identified a role of miR-221/222 in regulation of drug transporter expression.

Fig. 5. Effects of miR-221/222 inhibitors on the expression of drug influx-efflux transporters levels in resistant U266/LR7 cells.

Fig. 5

Open in a new tab

A) Western blot analysis of SLC7A5/LAT1 and ABCC1/MRP1 in U266/LR7 cells transfected with 100 nM of miR-221/222 inhibitors or NC and then exposed to 100μM of melphalan. ABBC1/MRP1 was immunoprecipitated from cell lysates (1mg) following incubation with 30 μl of protein A/G-conjugated agarose beads at 4°C overnight, and then detected by Western blot. Loading control was performed using γ-tubulin. B) Hoechst dye exclusion assay on U266/LR7 cells after treatment with either NC or miR-221/222 inhibitors or NC with Verapamil. The blue circles show the Side population (SP) with low levels of Hoechst fluorescence intensity. Dot plots represent one of three independent experiments. C) Bar column representation of percentage of SP affected after miR-221/222 inhibitors and Verapamil treatment as compared to NC. Values represent the mean ± SD of 3 independent experiments. * p<0.05

7. LNA-i-miR-221 inhibitors overcome melphalan-induced drug resistance in MM cells in vivo

Finally, we evaluated the anti-tumor activity of the combination in SCID/NOD mice bearing human melphalan-resistant MM xenografts in 2 independent in vivo studies. For each study, animals were randomized to receive 4 different treatments. As shown in Fig. 6, i.p. treatment with LNA-i-miR-221 (25 mg/kg) on days 1–4–8–15–22 plus melphalan (0.75 mg/kg) twice weekly for 3 weeks overcame drug-resistance, as evidenced by a significant tumor growth inhibition compared to control groups (p < 0.05) (Fig. 6a). In a subsequent in vivo study, we optimized the treatment schedule to improve the sensitizing activity of LNA-i-miR-221 and to adapt the LNA-i-miR-221 administration to a common melphalan schedule used in MM patients. Specifically, the treatment with LNA-i-miR-221 plus melphalan was given for 4 consecutive days every 10 days. This approach strengthened the anti-tumor activity of the combination (p < 0.001) (Fig. 6b). Moreover, no changes in body weight or mice behaviors were observed; H&E staining on retrieved organs from treated animals did not show any organ toxicity (data not shown). Additionally and consistent with our in vitro findings, analysis of retrieved tumors confirmed upregulation of PUMA/BBC3 protein (Fig. 6c bottom panel) and inhibition of ABCC1/MRP1 protein (Fig. 6c top panel) in mice treated with LNA-i-miR-221 plus mephalan. IHC analysis on excised tumors demonstrated extended necrosis following combination treatment, with reduction of Ki-67 proliferation index (Fig. 6d). Altogether, these in vivo findings further indicate the translational relevance of LNA-i-miR-221 as a sensitizing agents in melphalan-resistant MM.

Fig. 6. LNA-i-miR-221 enhances anti-MM activity of melphalan overcoming drug-resistance.

Fig. 6

Open in a new tab

For the in vivo study, mice xenografted with U266/LR7 cells were randomized in 4 groups of treatment. A) Treatments were: i) i.p. LNA-i-miR-221 (25 mg/kg) at day 1–4–8–15–22; ii) scrambled control with the same schedule; iii) i.p. melphalan (0.75 mg/kg) on days 2–5–9–12–16–19; iiii) melphalan plus LNA-i-miR-221 with the above described schedules. B) mice were treated with: i) ip LNA-i-miR-221 (25 mg/kg) or ii) scramble control or iii) i.p melphalan (0.75 mg/kg) or iiii) melphalan plus LNA-i-miR-221 for 4 consecutive days every 10 days (1–4, 15–18). Arrows indicate the days of treatment. Tumors were measured with an electronic caliper every two days, and data are represented as averaged tumor volume ± SD of each group. P values were obtained using two-tailed t test and calculated by comparing LNA-i-miR-221 plus melphalan group versus each of the others. The pictures inserted show the in vivo detection of the tumor volume in a representative mouse of each group using IVIS LUMINA II Imaging System. * p < 0.05 ** p < 0.001 B) Western blot analysis of PUMA/BBC3 and ABBC1 levels in lysates from a representative retrieved xenograft from each treatment group. ABBC1/MRP1 was immunoprecipitated from cell lysates (1mg) following incubation with 30 μl of protein A/G-conjugated agarose beads at 4°C overnight. Immunoprecipitated ABCC1/MRP1 was detected by Western blot. Y-Tubulin was used as protein loading control. C) H&E (400x) and Ki-67 (400x) immunohistochemical analysis of retrieved xenografted tumors after different treatments. Representative images from each group are shown.

Discussion

Targeting the miRNA network is arising as a new promising strategy to overcome drug-resistance of human cancer (42). We previously showed that inhibition of oncogenic miR-221/222 in MM cell lines induces significant anti-MM activity via targeting key molecules involved in cell proliferation and survival (34) and that LNA-inhibitors of miR-221 are suitable for safe and effective systemic delivery in mice (35). We here report that in vitro inhibition of miR-221/222 restores melphalan-sensitivity, inducing anti-proliferative and apoptotic cell death in drug-refractory MM cells. More importantly, this anti-tumor activity has been also shown in vivo following systemic treatment with LNA-i-miR-221 plus melphalan in SCID/NOD mice bearing melphalan-resistant MM xenografts without any evidence of toxicity or side effects in treated mice. Our results further support the role of miR-221/222 as crucial mediator of tumor cell resistance to alkylating agents such as cisplatin and temozolamide (28, 31), and we here provide proof-of principle that LNA-i-miR-221 is a potent sensitizing-agent in melphalan-refractory MM.

To investigate the mechanistic role of miR-221/222 in melphalan-resistant MM, we first assessed the correlation between miR-221/222 expression and the anti-proliferative activity of melphalan. Decreased miR-221/222 expression after melphalan treatment in sensitive-cells along with increased expression in resistant-cells suggested a correlation of miR-221/222 with melphalan-resistance. This observation was also confirmed by the basal higher expression of both miRNAs pre-melphalan treatment in U266/LR7 cells as compared to the parental sensitive counterpart U266/S. Given that miR-221/222 expression has been shown to be activated by transcription factor c-jun likely as DNA-damage response following radiation therapy (43), it is tempting to hypothesize that a similar mechanism may occur in MM cells after melphalan exposure. Follow-up studies will be carried out to clarify this point.

Indeed, inhibition of miR-221/222 markedly overcame melphalan-resistance in U266/LR7, RPMI-8226 and AMO1 Abzb, evidenced by a strong synergism of these two agents; conversely, overexpression of these miRNAs antagonizes drug-activity in melphalan-sensitive U266/S cells. Moreover, the synergistic effect of the combination affected the survival of primary cells of MM patients without any cytotoxicity in PBMCs from healthy donors, suggesting a low toxicity profile of our approach. In our in vitro experimental model, miR-221/222 inhibitors enhance melphalan-induced apoptosis, as confirmed by activation of Caspase cascade. Moreover, we found that upregulation of miR-221/222 induced by adhesion of MM cells to BMSCs does not impair the effectiveness of this combination, indicating that this treatment can overcome the role of the MM microenvironment in promoting resistance to DNA-damaging agents (44).

Interestingly, functional analysis of genes and pathways significantly modulated in resistant U266/LR7 cells demonstrated a remarkable similarity in gene expression signatures of melphalan-resistant U266/LR7 MM cells treated with miR-221/222 inhibitors plus melphalan compared with the sensitive parental U266/S cells treated with melphalan. Notably, IPA analysis demonstrated significant perturbation of pathways of central relevance in cellular response to alkylating agents, frequently correlated with drug-resistance such as “NRF2-mediated Oxidative Stress Response” (45), “Cell Cycle:G2/M DNA Damage Checkpoint Regulation”, “DNA Double-Strand Break Repair by Non-Homologous End Joining” (46), as well as those pathways associated with tumor progression. Based on GEP data, we focused on pro-apoptotic PUMA/BBC3, since its downregulation has been associated with drug-resistance (47, 48) and miR-221/222-sensitizing activity to temozolamide (31). After luciferase reporter assay validation of PUMA mRNA as a specific miR-221/222 target, we demonstrated that miR-221/222 inhibitors induce mRNA and protein PUMA expression. Conversely, PUMA silencing by siRNA transfection in melphalan-resistant U266/LR7 cells antagonized the melphalan-sensitizing activity of miR-221/222 inhibitors. Of note, PUMA silencing also led to a significant reduction of melphalan-activity in drug-sensitive U266/S cells. Our results therefore highlight the role of PUMA as key player in miR-221/222-mediated induction of melphalan-resistance.

Several reports have emphasized the role of ATP-dependent ABC transporters which rapidly efflux several drugs as crucial determinants of multidrug-resistance in cancer cells (49). Of note, aberrant expression of influx and efflux transporters has been correlated with altered sensitivity to melphalan (50). Interestingly, our GEP data suggested the activity of miR-221/222 targeting ATP-dependent efflux MRP1/ABCC1 as well as influx SLC7A5/LAT1 transporters and we here confirmed that miR-221/222 inhibitors upregulate LAT1 and downregulate ABCC1 at the protein level. Furthermore, a subset of cancer stem-like cells, identified as “side population”, expresses these transporters which correlates with chemoresistance and a high tumorigenic potential (41). In this light, we performed a functional analysis to characterize cells that contribute to the SP phenotype, and we show a marked decrease of SP in cells treated with miR-221/222 inhibitors. In silico search for target prediction using mirDIP software did not indicate direct targeting of mRNAs and further studies are necessary to solve the mechanism of miR-221/222 activity on LAT1 and ABCC1 transporters. Nonetheless our results are of translational relevance since they provide proof-of-concept that miR-221/222 inhibition can antagonize resistance to melphalan through modulation of influx-efflux transporters. Finally, we validated our in vitro findings in murine xenograft models with human melphalan-resistant MM cells in 2 independent experiments: a stronger anti-tumor activity has been achieved with 4 consecutive days of treatment with LNA-i-miR-221 plus melphalan. This effect was associated with upregulation of PUMA/BBC3 and downregulation of ABCC1 proteins in tumors harvested from treated animals.

In conclusion, the validation of LNA-i-miR-221 as a melphalan sensitizing-agent both confirms the role of miR-221/222 in mediating drug resistance, and provides the framework for combination clinical trials to sensitize or overcome resistance to melphalan in MM.

Supplementary Material

1
2

Statement of translational relevance.

Disregulation of oncogenic microRNAs (miRNAs) is frequently involved in cancer progression up to drug-resistant stage. We investigated the role of miR-221/222 in melphalan-resistance in multiple myeloma (MM) cells. The translational relevance of our study relies in the demonstration of in vivo efficacy of a novel 13 mer LNA-i-miR-221 inhibitor in restoring melphalan-sensitivity of refractory MM cells by selective interference with relevant molecular mechanisms of resistance. Importantly, LNA-i-miR-221 exerts anti-MM activity by itself and is suitable for systemic delivery. Our findings provide therefore the rationale for clinical investigation of LNA-i-miR-221 plus melphalan in drug-refractory stage of disease.

Acknowledgments

Financial support: This work has been supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n. 9980, 2010/15. This work has also been supported by a grant from NIH PO1-155258, RO1-124929, P50-100007, PO1-78378 and VA merit grant IO1-24467. Nicola Amodio was supported by a “Fondazione Umberto Veronesi” Fellowship.

Footnotes

Conflicts- of- interest disclosure: The authors declare no competing financial interests.

References

  • 1.Palumbo A, Anderson K. Multiple myeloma. The New England journal of medicine. 2011 Mar 17;364(11):1046–60. doi: 10.1056/NEJMra1011442. [DOI] [PubMed] [Google Scholar]
  • 2.Munshi NC, Anderson KC. New strategies in the treatment of multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013 Jul 1;19(13):3337–44. doi: 10.1158/1078-0432.CCR-12-1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Anderson KC, Carrasco RD. Pathogenesis of myeloma. Annual review of pathology. 2011;6:249–74. doi: 10.1146/annurev-pathol-011110-130249. [DOI] [PubMed] [Google Scholar]
  • 4.Calimeri T, Battista E, Conforti F, Neri P, Di Martino MT, Rossi M, et al. A unique three-dimensional SCID-polymeric scaffold (SCID-synth-hu) model for in vivo expansion of human primary multiple myeloma cells. Leukemia. 2011 Apr;25(4):707–11. doi: 10.1038/leu.2010.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tassone P, Neri P, Carrasco DR, Burger R, Goldmacher VS, Fram R, et al. A clinically relevant SCID-hu in vivo model of human multiple myeloma. Blood. 2005 Jul 15;106(2):713–6. doi: 10.1182/blood-2005-01-0373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Tassone P, Neri P, Burger R, Di Martino MT, Leone E, Amodio N, et al. Mouse models as a translational platform for the development of new therapeutic agents in multiple myeloma. Current cancer drug targets. 2012 Sep;12(7):814–22. doi: 10.2174/156800912802429292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rossi M, Di Martino MT, Morelli E, Leotta M, Rizzo A, Grimaldi A, et al. Molecular targets for the treatment of multiple myeloma. Current cancer drug targets. 2012 Sep;12(7):757–67. doi: 10.2174/156800912802429300. [DOI] [PubMed] [Google Scholar]
  • 8.Anderson KC. Therapeutic advances in relapsed or refractory multiple myeloma. Journal of the National Comprehensive Cancer Network : JNCCN. 2013 May;11(5 Suppl):676–9. doi: 10.6004/jnccn.2013.0199. [DOI] [PubMed] [Google Scholar]
  • 9.Bayraktar UD, Bashir Q, Qazilbash M, Champlin RE, Ciurea SO. Fifty years of melphalan use in hematopoietic stem cell transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2013 Mar;19(3):344–56. doi: 10.1016/j.bbmt.2012.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, Hawkins K, et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. The New England journal of medicine. 2003 May 8;348(19):1875–83. doi: 10.1056/NEJMoa022340. [DOI] [PubMed] [Google Scholar]
  • 11.Palumbo A, Attal M, Roussel M. Shifts in the therapeutic paradigm for patients newly diagnosed with multiple myeloma: maintenance therapy and overall survival. Clinical cancer research : an official journal of the American Association for Cancer Research. 2011 Mar 15;17(6):1253–63. doi: 10.1158/1078-0432.CCR-10-1925. [DOI] [PubMed] [Google Scholar]
  • 12.Palumbo A, Rajkumar SV, San Miguel JF, Larocca A, Niesvizky R, Morgan G, et al. International Myeloma Working Group consensus statement for the management, treatment, and supportive care of patients with myeloma not eligible for standard autologous stem-cell transplantation. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2014 Feb 20;32(6):587–600. doi: 10.1200/JCO.2013.48.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Yarde DN, Oliveira V, Mathews L, Wang X, Villagra A, Boulware D, et al. Targeting the Fanconi anemia/BRCA pathway circumvents drug resistance in multiple myeloma. Cancer research. 2009 Dec 15;69(24):9367–75. doi: 10.1158/0008-5472.CAN-09-2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chauhan D, Ray A, Viktorsson K, Spira J, Paba-Prada C, Munshi N, et al. In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013 Jun 1;19(11):3019–31. doi: 10.1158/1078-0432.CCR-12-3752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nature reviews Genetics. 2009 Oct;10(10):704–14. doi: 10.1038/nrg2634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. The New England journal of medicine. 2013 May 2;368(18):1685–94. doi: 10.1056/NEJMoa1209026. [DOI] [PubMed] [Google Scholar]
  • 17.Di Martino MT, Leone E, Amodio N, Foresta U, Lionetti M, Pitari MR, et al. Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012 Nov 15;18(22):6260–70. doi: 10.1158/1078-0432.CCR-12-1708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Amodio N, Leotta M, Bellizzi D, Di Martino MT, D’Aquila P, Lionetti M, et al. DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma. Oncotarget. 2012 Oct;3(10):1246–58. doi: 10.18632/oncotarget.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Amodio N, Di Martino MT, Foresta U, Leone E, Lionetti M, Leotta M, et al. miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1. Cell death & disease. 2012;3:e436. doi: 10.1038/cddis.2012.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Leone E, Morelli E, Di Martino MT, Amodio N, Foresta U, Gulla A, et al. Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013 Apr 15;19(8):2096–106. doi: 10.1158/1078-0432.CCR-12-3325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Amodio N, Bellizzi D, Leotta M, Raimondi L, Biamonte L, D’Aquila P, et al. miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells. Cell Cycle. 2013 Dec 1;12(23):3650–62. doi: 10.4161/cc.26585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Di Martino MT, Campani V, Misso G, Gallo Cantafio ME, Gulla A, Foresta U, et al. In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma. PloS one. 2014;9(2):e90005. doi: 10.1371/journal.pone.0090005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Leotta M, Biamonte L, Raimondi L, Ronchetti D, Martino MT, Botta C, et al. A p53-dependent tumor suppressor network is induced by selective miR-125a-5p inhibition in multiple myeloma cells in vitro. Journal of cellular physiology. 2014 May 13; doi: 10.1002/jcp.24669. [DOI] [PubMed] [Google Scholar]
  • 24.Raimondi L, Amodio N, Di Martino MT, Altomare E, Leotta M, Caracciolo D, et al. Targeting of multiple myeloma-related angiogenesis by miR-199a-5p mimics: in vitro and in vivo anti-tumor activity. Oncotarget. 2014 May 30;5(10):3039–54. doi: 10.18632/oncotarget.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Pitari MR, Rossi M, Amodio N, Botta C, Morelli E, Federico C, et al. Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts. Oncotarget. 2015 Jun 24; doi: 10.18632/oncotarget.4398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Garofalo M, Croce CM. MicroRNAs as therapeutic targets in chemoresistance. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy. 2013 Jul-Nov;16(3–5):47–59. doi: 10.1016/j.drup.2013.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zhu H, Wu H, Liu X, Evans BR, Medina DJ, Liu CG, et al. Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochemical pharmacology. 2008 Sep 1;76(5):582–8. doi: 10.1016/j.bcp.2008.06.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhao G, Cai C, Yang T, Qiu X, Liao B, Li W, et al. MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human osteosarcoma. PloS one. 2013;8(1):e53906. doi: 10.1371/journal.pone.0053906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Miller TE, Ghoshal K, Ramaswamy B, Roy S, Datta J, Shapiro CL, et al. MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. The Journal of biological chemistry. 2008 Oct 31;283(44):29897–903. doi: 10.1074/jbc.M804612200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Rao X, Di Leva G, Li M, Fang F, Devlin C, Hartman-Frey C, et al. MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways. Oncogene. 2011 Mar 3;30(9):1082–97. doi: 10.1038/onc.2010.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chen L, Zhang J, Han L, Zhang A, Zhang C, Zheng Y, et al. Downregulation of miR-221/222 sensitizes glioma cells to temozolomide by regulating apoptosis independently of p53 status. Oncology reports. 2012 Mar;27(3):854–60. doi: 10.3892/or.2011.1535. [DOI] [PubMed] [Google Scholar]
  • 32.Garofalo M, Romano G, Di Leva G, Nuovo G, Jeon YJ, Ngankeu A, et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nature medicine. 2012 Jan;18(1):74–82. doi: 10.1038/nm.2577. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 33.Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, et al. miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer cell. 2009 Dec 8;16(6):498–509. doi: 10.1016/j.ccr.2009.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 34.Di Martino MT, Gulla A, Cantafio ME, Lionetti M, Leone E, Amodio N, et al. In vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in multiple myeloma. Oncotarget. 2013 Feb;4(2):242–55. doi: 10.18632/oncotarget.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Di Martino MT, Gulla A, Gallo Cantafio ME, Altomare E, Amodio N, Leone E, et al. In vitro and in vivo activity of a novel locked nucleic acid (LNA)-inhibitor-miR-221 against multiple myeloma cells. PloS one. 2014;9(2):e89659. doi: 10.1371/journal.pone.0089659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Maiso P, Carvajal-Vergara X, Ocio EM, Lopez-Perez R, Mateo G, Gutierrez N, et al. The histone deacetylase inhibitor LBH589 is a potent antimyeloma agent that overcomes drug resistance. Cancer research. 2006 Jun 1;66(11):5781–9. doi: 10.1158/0008-5472.CAN-05-4186. [DOI] [PubMed] [Google Scholar]
  • 37.Ruckrich T, Kraus M, Gogel J, Beck A, Ovaa H, Verdoes M, et al. Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells. Leukemia. 2009 Jun;23(6):1098–105. doi: 10.1038/leu.2009.8. [DOI] [PubMed] [Google Scholar]
  • 38.Morelli E, Leone E, Cantafio ME, Di Martino MT, Amodio N, Biamonte L, et al. Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo. Leukemia. 2015 May 19; doi: 10.1038/leu.2015.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Amodio N, Scrima M, Palaia L, Salman AN, Quintiero A, Franco R, et al. Oncogenic role of the E3 ubiquitin ligase NEDD4-1, a PTEN negative regulator, in non-small-cell lung carcinomas. The American journal of pathology. 2010 Nov;177(5):2622–34. doi: 10.2353/ajpath.2010.091075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Tassone P, Neri P, Burger R, Savino R, Shammas M, Catley L, et al. Combination therapy with interleukin-6 receptor superantagonist Sant7 and dexamethasone induces antitumor effects in a novel SCID-hu In vivo model of human multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2005 Jun 1;11(11):4251–8. doi: 10.1158/1078-0432.CCR-04-2611. [DOI] [PubMed] [Google Scholar]
  • 41.Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, et al. A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proceedings of the National Academy of Sciences of the United States of America. 2004 Sep 28;101(39):14228–33. doi: 10.1073/pnas.0400067101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Mishra PJ. The miRNA-drug resistance connection: a new era of personalized medicine using noncoding RNA begins. Pharmacogenomics. 2012 Sep;13(12):1321–4. doi: 10.2217/pgs.12.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Li W, Guo F, Wang P, Hong S, Zhang C. miR-221/222 confers radioresistance in glioblastoma cells through activating Akt independent of PTEN status. Current molecular medicine. 2014 Jan;14(1):185–95. doi: 10.2174/1566524013666131203103147. [DOI] [PubMed] [Google Scholar]
  • 44.Hideshima T, Chauhan D, Podar K, Schlossman RL, Richardson P, Anderson KC. Novel therapies targeting the myeloma cell and its bone marrow microenvironment. Seminars in oncology. 2001 Dec;28(6):607–12. doi: 10.1016/s0093-7754(01)90033-8. [DOI] [PubMed] [Google Scholar]
  • 45.Cho JM, Manandhar S, Lee HR, Park HM, Kwak MK. Role of the Nrf2-antioxidant system in cytotoxicity mediated by anticancer cisplatin: implication to cancer cell resistance. Cancer letters. 2008 Feb 18;260(1–2):96–108. doi: 10.1016/j.canlet.2007.10.022. [DOI] [PubMed] [Google Scholar]
  • 46.Chen Q, Van der Sluis PC, Boulware D, Hazlehurst LA, Dalton WS. The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells. Blood. 2005 Jul 15;106(2):698–705. doi: 10.1182/blood-2004-11-4286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhang LN, Li JY, Xu W. A review of the role of Puma, Noxa and Bim in the tumorigenesis, therapy and drug resistance of chronic lymphocytic leukemia. Cancer gene therapy. 2013 Jan;20(1):1–7. doi: 10.1038/cgt.2012.84. [DOI] [PubMed] [Google Scholar]
  • 48.Yu J, Zhang L. No PUMA, no death: implications for p53-dependent apoptosis. Cancer cell. 2003 Oct;4(4):248–9. doi: 10.1016/s1535-6108(03)00249-6. [DOI] [PubMed] [Google Scholar]
  • 49.Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature reviews Cancer. 2002 Jan;2(1):48–58. doi: 10.1038/nrc706. [DOI] [PubMed] [Google Scholar]
  • 50.Kuhne A, Tzvetkov MV, Hagos Y, Lage H, Burckhardt G, Brockmoller J. Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines. Biochemical pharmacology. 2009 Jul 1;78(1):45–53. doi: 10.1016/j.bcp.2009.03.026. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS733332-supplement-1.doc (676.5KB, doc)
2
NIHMS733332-supplement-2.doc (37.5KB, doc)

RESOURCES