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. 2016 Mar 1;8(3):268-87.
doi: 10.15252/emmm.201505495.

Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic

Nadiya M Teplyuk  1 , Erik J Uhlmann  1 , Galina Gabriely  1 , Natalia Volfovsky  2 , Yang Wang  1 , Jian Teng  3 , Priya Karmali  4 , Eric Marcusson  4 , Merlene Peter  1 , Athul Mohan  1 , Yevgenya Kraytsberg  1 , Ron Cialic  1 , E Antonio Chiocca  5 , Jakub Godlewski  5 , Bakhos Tannous  3 , Anna M Krichevsky  6
Affiliations

Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic

Nadiya M Teplyuk et al. EMBO Mol Med. .

Abstract

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR-10b regulates cell cycle and alternative splicing, often through the non-canonical targeting via 5'UTRs of its target genes, including MBNL1-3, SART3, and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR-10b ASO led to targets' derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes.

Keywords: alternative splicing; brain tumor; microRNA; oligonucleotide therapeutics; stem cells.

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Figures

Figure EV1
Figure EV1. miR‐10b expression in GBM stem‐like cells (GSC), relative to human GBM cell lines, normal human brain tissues, and brain cells
The relative expression levels of miR‐10b have been assessed by qRT–PCR analysis and normalized to levels of snRNA U6. NSCs—primary normal embryonic neural stem cells of the indicated passages.Source data are available online for this figure.
Figure 1
Figure 1. MiR‐10b inhibition reduces viability and enhances apoptosis of GSC
GSC neurospheres were dissociated to single cell suspension and transfected with either miR‐10b inhibitor (labeled “miR‐10b‐i”) or non‐targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock).

  1. Cell viability was monitored at day 5 after transfection as described in Materials and Methods.

  2. The number and size of GSC colonies were monitored at day 5 after transfection.

  3. miR‐10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta‐actin.

  4. Flow cytometry analysis of Annexin V and 7‐AAD staining of GSC GBM8 at day 5 after miR‐10b inhibition.

Data information: (A, B) Statistical significance of the difference was determined by Student's t‐test, with P‐values < 0.001 indicated by three asterisks. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure EV2
Figure EV2. Annexin V and propidium iodide staining of GSC at day 5 after miR‐10b inhibition
GSC neurospheres were dissociated to single cell suspension and transfected with either miR‐10b inhibitor (labeled “miR‐10b‐i”) or non‐targeting control oligonucleotide. Cells were stained with Annexin V and propidium iodide at day 5 after transfections. Cells were collected on microscopic slides by Cytospin, and fluorescence images were taken immediately after staining.Source data are available online for this figure.
Figure 2
Figure 2. miR‐10b regulates cell cycle‐ and splicing‐related genes in GSC
Three types of GSC (GBM4, GBM6, and GBM8) were transfected with miR‐10b ASO, and gene expression was analyzed 24 h later by the Affymetrix microarrays. The heatmaps’ colors intensity demonstrates altered expression of the genes (up‐ or down‐regulated relative to the mock‐treated samples) with the fold change > 1.2 and P < 0.05 in at least two out of the three GSC cultures.

  1. The genes associated with “cell cycle” bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR‐10b inhibitor is indicated as “miR‐10b‐i”.

  2. The genes associated with “RNA splicing” bioterm have been selected using Gene Ontology (GO). The treatment with the miR‐10b inhibitor is indicated as “miR‐10b‐i”. Arrows depict the genes selected as candidate direct targets for further study.

  3. miR‐10b‐binding motifs are enriched in 5′UTRs of the genes up‐regulated by miR‐10b ASO. The graph shows the probability that enrichment of the miR‐10b motifs in mRNAs up‐regulated vs. unchanged (P < 0.05) by anti‐miR‐10b does not occur by chance.

  4. The miR‐10b octamer motifs’ composition of the 5′UTRs was compared between transcripts up‐ and down‐regulated on the microarrays. The relative frequencies of various miR‐10b‐binding motifs are shown, indicating that mostly miR‐10b 3′‐end‐binding motifs are enriched in the up‐regulated mRNAs.

Source data are available online for this figure.
Figure EV3
Figure EV3. Regulation of cell cycle‐related genes by miR‐10b in GSC
Change in the expression of genes related to “cell cycle” bioterm upon miR‐10b inhibition in GSC, as determined by the whole‐genome expression profiling. The cell cycle‐associated genes were selected using the Ingenuity Pathway Analysis. “miR‐10b‐i” indicates the treatment with miR‐10b inhibitor.
Figure EV4
Figure EV4. Regulation of splice isoforms by miR‐10b inhibition in GSC, as determined by the whole‐genome expression profiling
Each row of the heatmap demonstrates a pair of probe sets corresponding to different splice isoforms of the same gene, and regulated inversely (with fold change > 1.2 on a left side, and < 0.8 on a right side, and P < 0.05 in at least two out of three GSC cultures). Affymetrix prob set IDs are indicated in parenthesis.
Figure 3
Figure 3. Expression analysis of splicing factor mRNAs in various GBM datasets

  1. The genes encoding splicing factors down‐regulated by miR‐10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues.

  2. In contrast, many splicing factors up‐regulated by miR‐10b are overexpressed in the GBM datasets.

Data information: (A, B) Six high‐content GBM microarray datasets from the Oncomine resource (https://www.oncomine.org/resource/login.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34), and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues.Source data are available online for this figure.
Figure 4
Figure 4. miR‐10b regulates splicing factors through the non‐canonical binding within 5′UTRs

  1. Putative miR‐10b binding sites within 5′UTRs of candidate splicing factors mRNAs.

  2. qRT–PCR analysis validates that mRNA of MBNL1‐3, SART3, RSRC1, and other splicing factors are derepressed by miR‐10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.

  3. Regulation of representative splicing‐related proteins by miR‐10b mimic in GSC, as demonstrated by Western blot analysis. The signals were quantified using ImageJ and normalized to beta‐actin. The ratios between miR‐10b mimic expressing and control samples are indicated.

  4. miR‐10b mimic regulates 5′UTR luciferase reporter containing a single miR‐10b complementary site.

  5. miR‐10b mimic regulates 5′UTR luciferase reporters of some splicing factors genes bearing wild‐type (WT) but not mutated (Mut) miR‐10b binding sites.

Data information: (B, D, and E) Statistical significance of the differences was determined by Student's t‐test, **P < 0.01 and ***P < 0.001. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure 5
Figure 5. Intratumoral injections of miR‐10b inhibitor reduce the growth of established intracranial GBM8 xenografts

  1. A schematic overview of in vivo experiments on orthotopic GBM8. The tumor growth was monitored by luciferase imaging (WBI) and expressed in photon flux per second. Mice assigned to the treatment and control groups were treated with miR‐10b inhibitors or corresponding control oligonucleotides in different formulations.

  2. 2′‐O‐MOE‐PO miR‐10b inhibitor (miR‐10b‐i) or non‐targeting control (1 μg of each) formulated with in vivo jetPEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR‐10b inhibition was assessed by qRT–PCR analysis of the resected tumors, with miR‐10b expression levels normalized to miR‐125b.

  3. qRT–PCR analysis demonstrates that miR‐10b inhibition in orthotopic GBM8 leads do derepression of its mRNA targets. mRNA expression levels were normalized to GAPDH.

  4. Inverse correlation between miR‐10b levels and expression of its mRNA targets in resected GBM8 tumors.

  5. Inhibition of miR‐10b markedly reduces tumor burden. The left panels illustrate tumor imaging in representative animals at day 29. The bars represent average signal ratios for each group at day 29 (after treatment) to day 20 (at the beginning of treatment). N = 7 animals per group at treatment initiation.

  6. Growth curves of individual tumors, based on the ratios of bioluminescence signals to the baseline signals at day 5.

  7. Each mouse was sacrificed when the tumor‐generated signal reached 1.5 × 107 photons/s, and Kaplan–Meier survival plots were built retrospectively.

Data information: (B, C, E, and F) Statistical significance of the differences was determined by Student's t‐test, with *P < 0.05, **P < 0.01, and ***P < 0.001. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure 6
Figure 6. Systemic treatment with miR‐10b inhibitor reduces the growth of established intracranial GBM8 tumors
  1. A

    Intravenously injected Cy5‐labeled 2′‐O‐MOE‐PS oligonucleotide is distributed to intracranial GBM8 tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. “T”—tumor, “B”—brain tissue. Each image is representative of three mice analyzed.

  2. B–D

    Systemic inhibition of miR‐10b markedly reduces tumor burden. Uncomplexed 2′‐O‐MOE‐PS miR‐10b inhibitor (miR‐10b‐i) or non‐targeting control of the same chemistry was injected at 80 mg/kg through the tail vein at the days indicated by arrows. (B) The left panels illustrate tumor images of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6. (C) Each mouse was sacrificed when the tumor‐generated signal reached 5 × 107 photons/sec, and Kaplan–Meier survival plots were built retrospectively. (D) Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6.

  3. E

    The efficacy of miR‐10b inhibition in intracranial tumors was assessed by qRT–PCR analysis of the resected tumor tissues, with miR‐10b expression levels normalized to miR‐125b.

  4. F

    qRT–PCR analysis demonstrates that miR‐10b targets were derepressed in orthotopic GBM8 upon systemic administration of the miR‐10b inhibitor. Seven tumors per condition and two specimens per tumor have been analyzed. mRNA expression levels were normalized to GAPDH.

  5. G

    Inverse correlation between miR‐10b levels and expression of its mRNA targets in resected GBM8 tumors.

Data information: (B, E, and F) Statistical significance of the differences was determined by Student's t‐test, with *P < 0.05, **P < 0.01, and ***P < 0.001. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure 7
Figure 7. Toxicity assessment of the systemic treatment with miR‐10b inhibitor
  1. A

    Uptake of the uncomplexed Cy5‐labeled 2′‐O‐MOE‐PS oligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy 24 h after injections.

  2. B–D

    Systemic treatment of intracranial GBM8 tumors with uncomplexed 2′‐O‐MOE‐PS miR‐10b inhibitor (miR‐10b–i) or non‐targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects. (B) No significant difference in average mice weight was observed between the anti‐miR‐10b and control treatment groups. (C) No significant difference in average organs’ weight was observed between the anti‐miR‐10b and control treatment groups. (D) No significant difference in tissue histology using hematoxylin and eosin staining was observed between the anti‐miR‐10b and control treatment groups. The error bars (in B and C) represent Standard Deviation within each group of mice, N = 7 mice per group.

Source data are available online for this figure.
Figure 8
Figure 8. Continuous osmotic delivery of miR‐10b inhibitor reduces the growth of established orthotopic GBM8 tumor xenografts

  1. Continuous osmotic delivery of miR‐10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2′‐O‐MOE‐PO miR‐10b inhibitor or non‐targeting control, infused 2 μg of the oligonucleotides per day intratumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).

  2. Representative immunostaining of tumors for PCNA proliferation marker.

  3. Representative immunostaining of the tumors for KI67 proliferation marker.

  4. Representative tumor immunostaining for cleaved caspase 3 as a marker of apoptosis.

  5. Tumor cell invasion was examined by fluorescence microscopy for mCherry‐positive cells migrating through the tumor border.

  6. Quantitative immunostaining analysis indicates that proliferation and apoptosis markers are affected by anti‐miR‐10b treatment. No significant effect on invasion of intracranial GBM8 was observed.

Data information: (B‐E) For each staining, the immunopositive area was quantified and normalized to DAPI‐stained area using ImageJ software. The 40–50 microscopic fields were quantified within four sections per each tumor, and average values of four tumors per group are presented. (F) Statistical significance of the differences was determined by Student's t‐test, with **P < 0.01 and ***P < 0.001. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure 9
Figure 9. miR‐10b inhibition reduces the growth of mouse GL261 glioma cells in vitro and GL261‐derived intracranial tumors in immunocompetent mouse model
  1. A, B

    miR‐10b inhibition decreases GL261 cell viability. Cell viability was measured at days 3–7 after transfection with miR‐10b inhibitor, non‐targeting control, or Lipofectamine 2000 alone (mock). (A) Phase‐contrast photographs of GL261 cultures at day 6 post‐transfection. (B) Growth curves of cultured GL261 cells, based on the viability assay.

  2. C

    In vivo jetPEI‐formulated 2′‐O‐MOE‐PS/PO miR‐10b inhibitor or non‐targeting control was infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS‐containing oligonucleotides (green). GL261 tumor cells expressing M‐Cherry are red.

  3. D

    Osmotic delivery of miR‐10b inhibitor markedly reduces GL261 tumor growth in immunocompetent Black 6 Albino mice. Mice photographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumor growth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment.

  4. E

    The efficacy of miR‐10b inhibition in intracranial tumors was assessed by qRT–PCR analysis of the resected tumor tissues, with miR‐10b expression levels normalized to miR‐125b.

  5. F

    qRT–PCR analysis demonstrates that miR‐10b target p21 was derepressed in GL261 tumors upon miR‐10b inhibition. mRNA expression levels were normalized to GAPDH.

Data information: (B, D‐F) Statistical significance of the differences was determined by Student's t‐test, with *P < 0.05 and ***P < 0.001. Numbers of replicates and exact P‐values are included in Appendix Table S4.Source data are available online for this figure.
Figure EV5
Figure EV5. Systemic treatment of orthotopic GL261 tumors with miR‐10b inhibitor

  1. Systemic delivery of miR‐10b ASO to GL261 allograft tumors. About 100 mg/kg of uncomplexed 2′‐O‐MOE‐PS miR‐10b inhibitor was injected subcutaneously daily to BLACK6 mice bearing orthotopic GL261 tumors. Tumors and normal brain sections are shown. The staining for ASO is in green, DAPI nuclear staining in blue, and MCherry fluorescence in red.

  2. Treatment with miR‐10b inhibitor does not affect body and organ weight of the mice. Mice were treated daily with miR‐10b ASO (miR‐10b‐i) or non‐specific control oligonucleotide at 100 mg/kg, or saline, for 14 days, by subcutaneous injections. No difference in animal weight or weight of the organs was observed between the treatment and control groups. The error bars represent Standard Deviation within each group of mice, N = 7 mice per group.

  3. The effect of systemic treatment on viability of GL261‐bearing mice. miR‐10b ASO or non‐targeting control of the same chemistry was injected subcutaneously to the mice bearing orthotopic GL261 tumors at 100 mg/kg daily for 30 days. The mice were sacrificed upon developing lethargy or losing more than 30% of body weight, and Kaplan–Meier survival plots were built accordingly.

Source data are available online for this figure.
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