Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence - PubMed (original) (raw)

. 2012 Nov 15;18(22):6260-70.

doi: 10.1158/1078-0432.CCR-12-1708. Epub 2012 Oct 3.

Emanuela Leone, Nicola Amodio, Umberto Foresta, Marta Lionetti, Maria R Pitari, Maria E Gallo Cantafio, Annamaria Gullà, Francesco Conforti, Eugenio Morelli, Vera Tomaino, Marco Rossi, Massimo Negrini, Manlio Ferrarini, Michele Caraglia, Masood A Shammas, Nikhil C Munshi, Kenneth C Anderson, Antonino Neri, Pierosandro Tagliaferri, Pierfrancesco Tassone

Affiliations

Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence

Maria T Di Martino et al. Clin Cancer Res. 2012.

Abstract

Purpose: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a.

Experimental design: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo.

Results: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity.

Conclusions: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a-based treatment strategies in MM patients.

©2012 AACR.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1. miR-34a expression and nutlin-3 response in MM cell lines

A) q-RT-PCR analysis of miR-34a using total RNA from 2 MM cell lines bearing wild type TP53 and 9 MM cell lines with mutated TP53. B) q-RT-PCR of miR-34a in nutlin-3-treated (10 μM) RPMI-8226, SKMM1 (TP53 mutated) and NCI-H929 (TP53 wild-type) cells. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values calculated respect to miR-34a levels in RPMI-8226 cells, using the comparative cross threshold method. Values represent mean observed in four different experiment ±SD. miR-34a expression was significantly higher in TP53-wt versus TP53-mutated MM cells (P=0.045). C) Western blotting of BCL2, CDK6 and TP53 protein in TP53 mutated RPMI-8226 and TP53 wild-type NCI-H929 cells 24 hours after nutlin-3-treatment. The protein loading control was γ-tubulin. A representative of three experiments is shown.

Figure 2

Figure 2. miR-34a has anti-proliferative activity and induces apoptosis in MM cell lines

Cell growth analysis of SKMM1 (A), RPMI-8226 (B) or OPM1 (C) cells transfected with miR-34a or miR-NC oligonucleotide control. Average ±SD values of three independent experiments are plotted. _P_-values calculated by Student’s t test, two-tailed, at 48 and 72 hours, respectively, after transfection, are: 0.01 and 0.005 for SKMM1; 0.001 and 0.05 for RPMI-8226; 0.006 and 0.008 for OPM1. Cell growth analysis of MM1S (D) and NCI-H929 (E) cells transfected with miR-34a inhibitor (anti-miR-34a, Life Technologies AM11030) and anti-miR-scrambled inhibitor (NC, Life Technologies AM17010) oligonucleotide control. Average ±SD values of three independent experiments are plotted. _P_-values calculated by Student’s t test, two-tailed, were 0.04 and 0.002 for MM1S cells at 48 and 72 hours after transfection; and 0.04, 0.01 and 0.0008 for NCI-H929 cells at 24, 48 and 72 hours after transfection. Annexin V/7-AAD analysis of SKMM1 (F) and RPMI-8226 (G) cells after transfection with synthetic anti-miR-34a or control. Results are shown as percentage of apoptotic cells. Data are the average ±SD of 3 independent experiments. H) Colony formation assay using RPMI-8226 and SKMM1 cells. Cells were transfected with synthetic miR-34a or miR-NC by electroporation in triplicate, and then seeded at 2000 cells per 18 well plate in methyl cellulose-based medium. After two weeks, colony formation capacity was evaluated by counting colonies including > 100 cells. Means ± SE for three independent experiments are indicated. In all the experiments, _P-_value was ≤ 0.03 comparing miR-34a versus NC. A representative image of miR-34a and NC SKMM1 colonies shows homogeneous features of colonies formed by transfected cells with miR-NC, whereas cells transfected with miR-34a form irregular heterogeneous colonies.

Figure 3

Figure 3. Molecular effects induced by transient expression of miR-34a in MM cells

q-RT-PCR of BCL2, CDK6 and NOTCH1 after transfection with synthetic miR-34a or miR-NC in SKMM1 (A) and RPMI-8226 (B) cells. The results are shown as average mRNA expression after normalization with GAPDH and ΔΔCt calculations. Data represent the average ±SD of 3 independent experiments. Western blotting of BCL2 and CDK6 protein in SKMM1 (C) and RPMI-8226 (D) cells 24 and 48 hours after transfection with synthetic miR-34a or scrambled oligonucleotides (NC). The protein loading control was γ-tubulin. Experiments were performed in triplicate. miR-34a effects on protein levels reached statistical significance (P<0.05) at all time points.

Figure 4

Figure 4. In vivo activity of miR-34a stably expressed in MM cells

A) In vivo tumor formation of miR-34a stably expressed in MM xenografts. Tumor volumes were measured starting day 14 after cell injection (5 × 106 pMIF-34a-SKMM1 in the right flank or pMIF-SKMM1 in the left flank) in a cohort of 10 CB-17 SCID mice. Following the detection of tumors, measurements were assessed by an electronic caliper in two dimensions every 2–3 days until the date of sacrifice or death of the first animal. The tumor volume was calculated as detailed in methods. A representative mouse image is inserted in the graph. Tumor weight averages between pMIF-34a-SKMM1 or pMIF-SKMM1 xenografts retrieved from animals at the end of experiments show a significant difference between miR-34a transduced tumors versus controls (P=0.008; right panel). Means ± SD weight in grams are shown. _P-_value was calculated by Student’s t test, two tailed, of pMIF-34a versus pMIF tumor volumes. A representative image (insert) of a miR-34a retrieved tumor (left) versus control (right) is shown. B) Histologies and immunohistochemistry staining directed against Ki-67 and caspase-3 in pMIF-34a-SKMM1 or pMIF-SKMM1 tumors. Histologic and immunohistochemistry micrographs are at 20-fold magnification (H&E) and 40-fold magnification (Casp-3 and Ki-67), respectively. C) Quantitative analysis of BCL2, CDK6 and NOTCH1 mRNA (left panel) and protein (right panel) levels in retrieved pMIF-34a-SKMM1 or pMIF-SKMM1 tumors. The mRNA expression levels are shown as average after normalization with GAPDH and ΔΔCt calculations. Western blot analysis in retrieved tumors was performed as described in supplemental methods. The protein loading control was GAPDH. Experiments were performed in triplicate. miR-34a effects on protein levels reached statistical significance (P<0.05).

Figure 5

Figure 5. Intratumoral injection and systemic delivery of NLE-formulated miR-34a inhibits tumor growth in MM xenografts in SCID mice

A) Effects of formulated miR-34a in SKMM1 xenografts by intratumoral injections. Palpable subcutaneous tumor xenografts were repeatedly treated every 3 days, as indicated by arrows, with 20 μg of formulated miR-34a or miR-NC (NC). As control 2 separate groups of tumor-bearing animals were injected with vehicle alone (_MaxSuppressor_™ In Vivo RNA-LANCEr II) or phosphate buffer saline (PBS). Tumors were measured with an electronic caliper every day, averaged tumor volume ±SD of each group are shown. P values were calculated of miR-34a versus miR-NC (Student’s t test, two-tailed). (*) indicate significant _P_-values (P<0.05). B) Survival curves (Kaplan-Meier) of intratumorally treated mice show prolongation of survival in miR-34a-treated SKMM1 xenografts compared to controls (log-rank test, _P_=0.0009). Survival was evaluated from the first day of treatment until death or sacrifice. Percent of mice alive is shown. C) Mice with palpable subcutaneous SKMM1 tumor xenografts were treated with 20 μg of formulated miR-34a or scrambled oligonucleotides (NC) by intravenous tail vein injections. Caliper measurement of tumors were taken every 2 days from the day of first treatment. Averaged tumor volumes ± SD are reported. (*) indicate significant _P_-values (P<0.05). D) Survival curves (Kaplan-Meier) of systemically miR-34a treated mice show prolongation of survival compared to controls (log-rank test, P=0.041). Survival was evaluated from the first day of treatment until death or sacrifice. Percent of mice alive is shown.

Figure 6

Figure 6. miR-34a exerts anti-MM activity in co-cultures of primary MM-BMSCs and in the SCID-synth-hu model

A) MTS assay performed in CD138+ cells from a MM patient co-cultured with BMSCs, 24 hours after electroporation with synthetic miR-34a or miR-NC. Absorbance measurements ±SD of 3 independent experiments is shown; significant reduction in cell survival was observed (P<0.002, by Student’s t test, two-tailed) in co-cultures treated with miR-34a. B) In vivo analysis of miR-34a in the SCID-synth-hu model. TP53-mutated primary CD138+ MM cells were injected into 3D biopolymeric scaffolds with BMSCs. Engrafted synthetic scaffolds were directly injected in vivo by NLE formulated miR-34a or miR-NC in three mice for each group. A representative H&E and immunohistochemistry staining of Ki-67 and caspase-3 on retrieved scaffolds from treated animals is shown (magnification x40).

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