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MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM

Stojcheva, Nina, Schechtmann, Gennadi, Sass, Steffen, Roth, Patrick, Florea, Ana-Maria, Stefanski, Anja, Stühler, Kai, Wolter, Marietta, Müller, Nikola S., Theis, Fabian J., Weller, Michael, Reifenberger, Guido, Happold, Caroline·2016·Oncotarget
DOI 10.18632/oncotarget.7346PMC PMC4914333
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Abstract

// Nina Stojcheva 1, 2 , Gennadi Schechtmann 3 , Steffen Sass 4 , Patrick Roth 1, 2 , Ana-Maria Florea 3 , Anja Stefanski 5 , Kai Stühler 5 , Marietta Wolter 3 , Nikola S. Müller 4 , Fabian J. Theis 4, 6 , Michael Weller 1, 2 , Guido Reifenberger 3, 7 , Caroline Happold 1, 2 1 Laboratory of Molecular Neuro-Oncology, Department of Neurology, University Hospital and University of Zurich, Zurich, Switzerland 2 Neuroscience Center Zurich, University of Zurich, Zurich, Switzerland 3 Department of Neuropathology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 4 Institute of Computational Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany 5 Molecular Proteomics Laboratory, Biological and Medical Research Center (BMFZ), Heinrich Heine University Düsseldorf, Düsseldorf, Germany 6 Department of Mathematics, Technische Universität München, Garching, Germany 7 German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Heidelberg, partner site Essen/Düsseldorf, Germany Correspondence to: Caroline Happold, e-mail: caroline.happold@usz.ch Keywords: chemoresistance, temozolomide, glioblastoma, miR-138, BIM Received: December 11, 2015 Accepted: January 26, 2016 Published: February 12, 2016 ABSTRACT Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo .

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Figure 1 mir-138 is significantly upregulated in TMZ-resistant glioma cell lines and in recurrent glioblastoma (A) Heat-map of differentially expressed miRNA obtained by microarray-based expression profiling of parental (P) and TMZ-resistant (R) LN-18, LN-229 and LN-308 cells using GeneChip® miRNA 2.0 arrays. Results were obtained from hybridization of three independent batches of each P and R cell line. Rows and columns were clustered hierarchically using the hclust method of R. The expression profile distances were calculated based on the Pearson correlation coefficients between miRNAs and samples. (B) Box plots showing upregulation of miR-138 expression in resistant versus parental cell lines of LN-18 and LN-308 but not LN-229 based on microarray data. (C) Real-time qPCR analysis for miR-138 expression in the paired P and R cells (n = 3, unpaired Student's t-test). (D) Relative baseline expression levels of miR-138 in LTC (gray columns) and GIC (striped columns) assessed by real-time qPCR (n = 3). (E) Real-time qPCR analysis of miR-138 expression in paired primary and recurrent glioblastoma tissues of 10 patients using U6 snRNA as reference (*p < 0.05; **p < 0.01; ***p < 0.001; paired Student's t-test).
Figure 1 mir-138 is significantly upregulated in TMZ-resistant glioma cell lines and in recurrent glioblastoma (A) Heat-map of differentially expressed miRNA obtained by microarray-based expression profiling of parental (P) and TMZ-resistant (R) LN-18, LN-229 and LN-308 cells using GeneChip® miRNA 2.0 arrays. Results were obtained from hybridization of three independent batches of each P and R cell line. Rows and columns were clustered hierarchically using the hclust method of R. The expression profile distances were calculated based on the Pearson correlation coefficients between miRNAs and samples. (B) Box plots showing upregulation of miR-138 expression in resistant versus parental cell lines of LN-18 and LN-308 but not LN-229 based on microarray data. (C) Real-time qPCR analysis for miR-138 expression in the paired P and R cells (n = 3, unpaired Student's t-test). (D) Relative baseline expression levels of miR-138 in LTC (gray columns) and GIC (striped columns) assessed by real-time qPCR (n = 3). (E) Real-time qPCR analysis of miR-138 expression in paired primary and recurrent glioblastoma tissues of 10 patients using U6 snRNA as reference (*p < 0.05; **p < 0.01; ***p < 0.001; paired Student's t-test).CC BY · PubMed Central
Figure 2 miR-138 induces proliferation of glioma cells in vitro (A) Transfection efficiency of mimic-138 in LN-18, LN-308, LN-319, LN-428 and ZH-305 cells was assessed by real-time qPCR of mimic and control transfected cells (n = 3). The final concentration of mimic/control was 50 nM in LTC and 100 nM in GIC. (B) BrdU incorporation assay was performed in LN-308 cells transfected with mimic-138 or control (50 nM) (***p < 0.001, unpaired Student's t-test). (C) LN-308 (left panel) or LN-319 (right panel) cells were transfected with mimic-138/control (50 nM) and proliferation and cell death were measured by counting trypan blue stained cells at days 1, 3, 5 and 7 after reseeding the transfected cells. Assay was analyzed by unpaired Student's t-test. (D) Flow cytometric cell cycle analysis of mimic-138/control (50 nM) transfected LN-308 cells was performed by PI staining at day 3 and 6 post-transfection.
Figure 2 miR-138 induces proliferation of glioma cells in vitro (A) Transfection efficiency of mimic-138 in LN-18, LN-308, LN-319, LN-428 and ZH-305 cells was assessed by real-time qPCR of mimic and control transfected cells (n = 3). The final concentration of mimic/control was 50 nM in LTC and 100 nM in GIC. (B) BrdU incorporation assay was performed in LN-308 cells transfected with mimic-138 or control (50 nM) (***p < 0.001, unpaired Student's t-test). (C) LN-308 (left panel) or LN-319 (right panel) cells were transfected with mimic-138/control (50 nM) and proliferation and cell death were measured by counting trypan blue stained cells at days 1, 3, 5 and 7 after reseeding the transfected cells. Assay was analyzed by unpaired Student's t-test. (D) Flow cytometric cell cycle analysis of mimic-138/control (50 nM) transfected LN-308 cells was performed by PI staining at day 3 and 6 post-transfection.CC BY · PubMed Central
Figure 3 Overexpression of miR-138 confers TMZ and CCNU resistance in glioma cells (A) LN-308 and ZH-305 cells were transfected with miR-138 mimics or control and TMZ or (B) CCNU sensitivity was assessed by clonogenic survival assays in LTC and spherogenicity assays in GIC (concentration of mimic/control: 50 nM for LTC, 100 nM for GIC) (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test).
Figure 3 Overexpression of miR-138 confers TMZ and CCNU resistance in glioma cells (A) LN-308 and ZH-305 cells were transfected with miR-138 mimics or control and TMZ or (B) CCNU sensitivity was assessed by clonogenic survival assays in LTC and spherogenicity assays in GIC (concentration of mimic/control: 50 nM for LTC, 100 nM for GIC) (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test).CC BY · PubMed Central
Figure 4 BIM downregulation via miR-138 confers TMZ resistance to glioma cells (A) Immunoblot for BIM detection in LN-18, LN-229 and LN-308 parental versus resistant (R) cells (B) and in LN-308 or LN-229 cells transfected with miR-138 mimic, inhibitor or the respective control (100 nM); lysates were collected 72 h post-transfection. Quantification of protein bands was performed by densitometry, and the values stated under each band represent the normalized value to the corresponding loading control signal. (C) Luciferase reporter assay in LN-308 cells co-transfected with psiCheck2 empty vector, psiCheck2_Bim vector or one of the three mutant control vectors (psiCheck2_mut1, psiCheck2_mut2 and psiCheck2_mut3) and miR-138 mimic or control (transfection performed with 100 ng vector and 100 nM mimic/control concentration). Left panel: schematic of the two miR-138 binding sites in the 3′UTR region of the BIM mRNA, and the content of each vector construct (red cross represents mutation in the recognition site). Right panel: representative luciferase reporter assay. (D) siRNA-mediated silencing of BIM in LN-308 cells confirmed by immunoblot analysis (siRNA concentration: 100 nM). (E) Clonogenic survival assay after TMZ exposure of LN-308 cells transfected with two different siBim constructs (siBim1 and siBim2) or with siControl (siCtrl) (100 nM). (F) Clonogenic cell survival assay after TMZ treatment (37.5 μM) in combination with ABT-737 (3.3 μM) or DMSO of siBim1 or siControl transfected LN-308 cells. All values were normalized to siCtrl cells treated with DMSO (solvent control) and TMZ-untreated cells.
Figure 4 BIM downregulation via miR-138 confers TMZ resistance to glioma cells (A) Immunoblot for BIM detection in LN-18, LN-229 and LN-308 parental versus resistant (R) cells (B) and in LN-308 or LN-229 cells transfected with miR-138 mimic, inhibitor or the respective control (100 nM); lysates were collected 72 h post-transfection. Quantification of protein bands was performed by densitometry, and the values stated under each band represent the normalized value to the corresponding loading control signal. (C) Luciferase reporter assay in LN-308 cells co-transfected with psiCheck2 empty vector, psiCheck2_Bim vector or one of the three mutant control vectors (psiCheck2_mut1, psiCheck2_mut2 and psiCheck2_mut3) and miR-138 mimic or control (transfection performed with 100 ng vector and 100 nM mimic/control concentration). Left panel: schematic of the two miR-138 binding sites in the 3′UTR region of the BIM mRNA, and the content of each vector construct (red cross represents mutation in the recognition site). Right panel: representative luciferase reporter assay. (D) siRNA-mediated silencing of BIM in LN-308 cells confirmed by immunoblot analysis (siRNA concentration: 100 nM). (E) Clonogenic survival assay after TMZ exposure of LN-308 cells transfected with two different siBim constructs (siBim1 and siBim2) or with siControl (siCtrl) (100 nM). (F) Clonogenic cell survival assay after TMZ treatment (37.5 μM) in combination with ABT-737 (3.3 μM) or DMSO of siBim1 or siControl transfected LN-308 cells. All values were normalized to siCtrl cells treated with DMSO (solvent control) and TMZ-untreated cells.CC BY · PubMed Central
Figure 5 TMZ and miR-138 induce little apoptosis but cytoprotective autophagy (A) Annexin V/PI flow cytometric analyses of LN-308 mim-138/control (left panel) or siBim/control (right panel) transfected cells treated with TMZ (100 μM) 1 and 3 days later. (B) Immunoblot analysis of cleaved caspase 3 (Casp-3) levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (left panel), and of Bcl-2, Bcl-xL, Mcl-1 and BIM levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (middle panel, right panel). (C) Immunoblot analysis of LC3 A/B I and II levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (left panel); immunoblot analysis of LC3 A/B I and II levels and BIM levels in LN-308 and LN-308_R cells; LN-308 cells were transfected with siCtrl or 4 different siBim molecules (100 nM), and mimic-ctrl (mim-ctrl) or mimic-138 (mim-138) molecules (50 nM), and cell lysates were collected 72 h after transfection (right panel). Quantification of protein bands was performed by densitometry for BIM levels in mim-ctrl and mim-138 transfected cells; values representing the normalized value to the corresponding loading control signal were 1.38 for mim-ctrl and 1.14 for mim-138, respectively (D) Clonogenic survival assay after TMZ exposure of LN-308 cells treated with the autophagy inhibitor 3-MA (2 mM) or solvent control.
Figure 5 TMZ and miR-138 induce little apoptosis but cytoprotective autophagy (A) Annexin V/PI flow cytometric analyses of LN-308 mim-138/control (left panel) or siBim/control (right panel) transfected cells treated with TMZ (100 μM) 1 and 3 days later. (B) Immunoblot analysis of cleaved caspase 3 (Casp-3) levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (left panel), and of Bcl-2, Bcl-xL, Mcl-1 and BIM levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (middle panel, right panel). (C) Immunoblot analysis of LC3 A/B I and II levels in LN-308 cells at different indicated time points after DMSO (vehicle control), TMZ (100 μM) or staurosporin (Stauro, 2 μM) stimulation (left panel); immunoblot analysis of LC3 A/B I and II levels and BIM levels in LN-308 and LN-308_R cells; LN-308 cells were transfected with siCtrl or 4 different siBim molecules (100 nM), and mimic-ctrl (mim-ctrl) or mimic-138 (mim-138) molecules (50 nM), and cell lysates were collected 72 h after transfection (right panel). Quantification of protein bands was performed by densitometry for BIM levels in mim-ctrl and mim-138 transfected cells; values representing the normalized value to the corresponding loading control signal were 1.38 for mim-ctrl and 1.14 for mim-138, respectively (D) Clonogenic survival assay after TMZ exposure of LN-308 cells treated with the autophagy inhibitor 3-MA (2 mM) or solvent control.CC BY · PubMed Central
Figure 6 Autophagy mediates TMZ resistance via the miR-138/BIM axis Long-term TMZ treatment induces miR-138 expression, which in turn suppresses BIM translation. Hence, BIM-mediated autophagy inhibition is blocked, and increased autophagy promotes cell survival of the glioma cells.
Figure 6 Autophagy mediates TMZ resistance via the miR-138/BIM axis Long-term TMZ treatment induces miR-138 expression, which in turn suppresses BIM translation. Hence, BIM-mediated autophagy inhibition is blocked, and increased autophagy promotes cell survival of the glioma cells.CC BY · PubMed Central

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Authors
Stojcheva, N., Schechtmann, G., Sass, S., Roth, P., Florea, A.-M., Stefanski, A., Stühler, K., Wolter, M., Müller, N. S., Theis, F. J., Weller, M., Reifenberger, G., & Happold, C.
Affiliations
  • Laboratory of Molecular Neuro-Oncology, Department of Neurology, University Hospital and University of Zurich, Zurich, Switzerland
  • Department of Neuropathology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
  • Institute of Computational Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany
  • Molecular Proteomics Laboratory, Biological and Medical Research Center (BMFZ), Heinrich Heine University Düsseldorf, Düsseldorf, Germany
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2016-02-12
Volume
7
Issue
11
Pages
12937-12950
ISSN
1949-2553
DOI
10.18632/oncotarget.7346
PMC
PMC4914333
Cited by
50
References
60
Language
en

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