In vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in multiple myeloma

Abstract

A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/ NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. miR-221 and miR-222 expression in primary CD138+ normal plasma cells, primary MM and PCL cells and established MM cell lines

A) Differential expression of miR-221 and miR-222 in immunoselected CD138+ cells from 3 healthy donors, 38 MM and 2 PCL, by microarray analysis. Expression values were normalized by aroma.light-package for Bioconductor. MM were TC classified according to the presence of the recurrent IGH chromosomal translocations and cyclins D expression as previously described (30). miR-221 and miR-222 are reported as raw expression values. Statistical significance was assessed by SAM multi-class analysis, (q-value=0). N(1-3): CD138+ cells from normal healthy donors. MM and PCL were numbered referring to individual patients in the original data set. B) Differential expression of miR-221 and miR-222 in 16 MM cell lines by Affymetrix GeneChip® miRNA 1.0 Array. Histogram bars indicate miR-221 or miR-222 expression values normalized by miRNA QC Tool (Affymetrix).

Figure 2. Biological effects induced by transient expression of miR-221/222 in MM cell lines

A) Cell cycle perturbation in U266 cells induced by transient pre-miR221/222 enforced expression. At least 20,000 events for each point were analyzed in 3 independent experiments. Results are representative of one out of 3 experiments 72 hours after transfection. B) Effects induced on BrdU uptake of RPMI-8226 cells by transfection with pre-miR-221/222 mimics or scrambled sequences. Averaged values ±SD from 3 independent experiments are plotted. C) p27Kip1 protein expression 48-72 and 96 hours after transfection of U266 and RPMI-8226 cells with pre-miR-221/222 mimics or scrambled controls (NC) was assessed by Western blotting assay. D) Inverse correlation between p27Kip1 mRNA and miR-221 levels in a dataset of 40 (38 MM + 2 PCL) patients is shown. The red bars represent mature miR-221 expression (vertical axis on right side) and the spots represent target gene (p27Kip1 mRNA) expression (vertical axis on left side). Horizontal axis: patient samples ordered according to increasing p27Kip1 expression.

Figure 3. In vitro anti-proliferative activity of miR-221 and miR-222 inhibitors on MM cell lines

A-B) Cell growth analysis of OPM2 (A) and NCI-H929 (B) cells transfected with miR-221/222 inhibitors or scrambled oligonucleotides (NC). Analysis was performed by trypan blue exclusion assay. Averaged values of 3 independent experiments are plotted in each frame including +SD. Significant P values (P

Figure 4. Molecular effects induced by miR-221/222 inhibitors in MM cells

A) miR-221 and miR-222 q-RT-PCR 24 hours after transfection with miR-221/222 inhibitors and NC in OPM2 cells. The results are shown as miRNA expression level after normalization with RNU44 and ΔΔCt calculations. Data represent the average of 3 independent experiments +SD. B) q-RT-PCR of p27Kip1, PUMA, PTEN and p57Kip2 mRNA expression 24 hours after transfection with miR-221/222 inhibitors or NC in OPM2 cells. The results are shown as average mRNA expression after normalization with GAPDH and ΔΔCt calculations. Data represent the average of 3 independent experiments +SD. (*) P

Figure 5. Whole Gene Expression Profiles of MM cells after miR-221/222 knockdown

A) Hierarchical clustering of differentially expressed genes between miR-221/222 inhibitors or NC treated OPM2 cells by Gene 1.0 ST array chip (Affymetrix) and DChip software. Genes that underwent a 0.5-fold change as compared to control, were selected and clustered. Assays performed in triplicate are shown. B) Pathways modulated by miR-221/222 inhibitors in OPM2 MM cells are shown. Analysis was performed by Ingenuity Pathway Analysis on genes significantly modulated (FC>0.5). The bar graphs show pathways most modulated by miR-221/222 inhibitors as compared to control, based on statistical significance (P-value and ratio). The yellow line indicates the threshold of significance. C) Functional network scenario with principal nodes in the most perturbed pathways are showed. Up- and down-regulated genes are indicated in red and green, respectively. Continuous and discontinuous lines represent direct and indirect functional and physical interactions between genes as previously reported.

Figure 6. In vivo anti-tumor activity of miR-221/222 inhibitors in MM xenografted SCID/NOD mice

A) Effects of formulated or unformulated miR-221/222 inhibitors. Subcutaneous OPM2 xenografts were treated every 2 days with 20 μg of formulated(NLE)-miR-221/222 inhibitors or unformulated-miR221/222 inhibitors. As control 2 separate groups of tumor-bearing animals were injected with formulated(NLE)-NC or unformulated-NC. B) Effects of treatments with formulated miR-221 or miR-222 individual inhibitors. Subcutaneous OPM2 xenografts were repeatedly treated every 2 days, with 20 μg of NLE-miR-221 inhibitors, or NLE-miR-222 inhibitors, or NLE-NC. C) Effects of unformulated-miR-221 inhibitors or NC. Subcutaneous OPM2 xenografts were repeatedly treated every 2 days with 20 μg of unformulated-miR-221 inhibitors, or unformulated-NC. The tumor volumes averages of each group +SD are reported. In each experiment P values were calculated for miRNA inhibitors versus scrambled oligonucleotides (NC) and significant P values (P<0.05) are indicated by stars.

Figure 7. miR-221 activity and targets silencing in retrieved MM xenografted tumors

A) q-RT-PCR of miR-221 and miR-222 in retrieved tumors treated with unformulated-miR-221 inhibitors or unformulated-NC. The results are shown as averaged miRNA expression after normalization with RNU44 and ΔΔCt calculations as compared to control (miRNA expression in NC treated animals). B) H&E (20-fold magnification) and immunohistochemistry (40-fold magnification) staining of xenografted tumors retrieved from treated animals. C) q-RT-PCR of p27Kip1 and PTEN mRNA levels in treated tumors retrieved from mice. Raw Ct values were normalized to GAPDH housekeeping mRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method. D) Western blot analysis of p27Kip1 and PTEN protein in retrieved tumors from mice treated with miR-221 inhibitors or NC. D-E) Western blot analysis of total AKT and p-AKT (D) and total ERK and p-ERK (E) in retrieved tumors from mice treated with miR-221 inhibitors or NC. Representative experiments are shown in figure. γ-tubulin was the protein loading control.