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Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

Palagani A, Op de Beeck K, Naulaerts S, Diddens J, Sekhar Chirumamilla C, Van Camp G, Laukens K, Heyninck K, Gerlo S, Mestdagh P, Vandesompele J, Berghe WV - PLoS ONE (2014)

Bottom Line: Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells.Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir.Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerp, Belgium; Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University, Ghent, Belgium.

ABSTRACT
Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

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Dexamethasone decreases cell viability in a dose and time dependent manner in GC sensitive MM1S but not GC resistant MM1R cells.(A–B) dose response curves of MM1S and MM1R cells treated with dexamethasone at different concentrations for 24, 48 and 72 h as determined by MTT colorimetric assay. Data represent (mean ± SEM) values of three independent experiments. (C) time dependent changes in GRα mRNA levels upon 1 µM dexamethasone treatment. Data represent (mean ± SEM) values of three independent experiments normalized to 28sRNA housekeeping gene and relative to MM1S untreated condition (S UT). Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 and ns are not significantly different as determined by one-way ANOVA (Dunnett’s Post-test) (D) Time dependent changes in GRα protein levels and PARP cleavage in MM1S and MM1R cells following treatment of 1 µM Dex. (E) Densitometric analysis of western blot signals (n = 2) of protein levels of GR (92 kDa), β-Actin (40 kDa), full length PARP (116 kDa) and cleaved PARP (89 kDa) in MM1S and MM1R, following treatment of 1 µM Dex. Significant differences between western signals in MM1R and MM1S are indicated.
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pone-0113842-g001: Dexamethasone decreases cell viability in a dose and time dependent manner in GC sensitive MM1S but not GC resistant MM1R cells.(A–B) dose response curves of MM1S and MM1R cells treated with dexamethasone at different concentrations for 24, 48 and 72 h as determined by MTT colorimetric assay. Data represent (mean ± SEM) values of three independent experiments. (C) time dependent changes in GRα mRNA levels upon 1 µM dexamethasone treatment. Data represent (mean ± SEM) values of three independent experiments normalized to 28sRNA housekeeping gene and relative to MM1S untreated condition (S UT). Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 and ns are not significantly different as determined by one-way ANOVA (Dunnett’s Post-test) (D) Time dependent changes in GRα protein levels and PARP cleavage in MM1S and MM1R cells following treatment of 1 µM Dex. (E) Densitometric analysis of western blot signals (n = 2) of protein levels of GR (92 kDa), β-Actin (40 kDa), full length PARP (116 kDa) and cleaved PARP (89 kDa) in MM1S and MM1R, following treatment of 1 µM Dex. Significant differences between western signals in MM1R and MM1S are indicated.

Mentions: Before investigation of the regulatory role of GC inducible microRNA in NR3C1/GR responsive gene expression and cell death responses, we first characterized the GC therapy response in MM1S and MM1R cells. Briefly, MM1S and MM1R cells were left untreated or treated with 1 µM of the synthetic GC agonist dexamethasone (Dex) for respectively 6 h, 24 h, 48 h, 72 h. mRNA and proteins were isolated at each time point and analysed for corresponding NR3C1/GRα expression. Treating the cells with different concentrations of Dex for 24 h, 48 h and 72 h followed by measuring cell viability by a MTT colorimetric assay, revealed a dose and time dependent decrease in cell survival of MM1S cells upon GC exposure (Fig. 1A & 1B). In contrast Dex lacks the capacity to induce cytotoxicity in MM1R cells and rather triggers a weak growth stimulatory effect [28], [35], [36], [37]. In line with the different GC response in both cell lines, QPCR analysis demonstrates significant NR3C1/GRα mRNA transcription in MM1S, but near background levels of NR3C1/GRα mRNA in MM1R cells (Fig. 1C). Western analysis finally confirms significant levels of GRα protein expression in MM1S and undetectable levels of GRα protein in MM1R cells (Fig. 1D–E). As can be observed from Fig. 1D, MM1S cells express multiple translational isoforms of GRα [38], [39], of which expression decreases following GC treatment.


Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

Palagani A, Op de Beeck K, Naulaerts S, Diddens J, Sekhar Chirumamilla C, Van Camp G, Laukens K, Heyninck K, Gerlo S, Mestdagh P, Vandesompele J, Berghe WV - PLoS ONE (2014)

Dexamethasone decreases cell viability in a dose and time dependent manner in GC sensitive MM1S but not GC resistant MM1R cells.(A–B) dose response curves of MM1S and MM1R cells treated with dexamethasone at different concentrations for 24, 48 and 72 h as determined by MTT colorimetric assay. Data represent (mean ± SEM) values of three independent experiments. (C) time dependent changes in GRα mRNA levels upon 1 µM dexamethasone treatment. Data represent (mean ± SEM) values of three independent experiments normalized to 28sRNA housekeeping gene and relative to MM1S untreated condition (S UT). Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 and ns are not significantly different as determined by one-way ANOVA (Dunnett’s Post-test) (D) Time dependent changes in GRα protein levels and PARP cleavage in MM1S and MM1R cells following treatment of 1 µM Dex. (E) Densitometric analysis of western blot signals (n = 2) of protein levels of GR (92 kDa), β-Actin (40 kDa), full length PARP (116 kDa) and cleaved PARP (89 kDa) in MM1S and MM1R, following treatment of 1 µM Dex. Significant differences between western signals in MM1R and MM1S are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256227&req=5

pone-0113842-g001: Dexamethasone decreases cell viability in a dose and time dependent manner in GC sensitive MM1S but not GC resistant MM1R cells.(A–B) dose response curves of MM1S and MM1R cells treated with dexamethasone at different concentrations for 24, 48 and 72 h as determined by MTT colorimetric assay. Data represent (mean ± SEM) values of three independent experiments. (C) time dependent changes in GRα mRNA levels upon 1 µM dexamethasone treatment. Data represent (mean ± SEM) values of three independent experiments normalized to 28sRNA housekeeping gene and relative to MM1S untreated condition (S UT). Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 and ns are not significantly different as determined by one-way ANOVA (Dunnett’s Post-test) (D) Time dependent changes in GRα protein levels and PARP cleavage in MM1S and MM1R cells following treatment of 1 µM Dex. (E) Densitometric analysis of western blot signals (n = 2) of protein levels of GR (92 kDa), β-Actin (40 kDa), full length PARP (116 kDa) and cleaved PARP (89 kDa) in MM1S and MM1R, following treatment of 1 µM Dex. Significant differences between western signals in MM1R and MM1S are indicated.
Mentions: Before investigation of the regulatory role of GC inducible microRNA in NR3C1/GR responsive gene expression and cell death responses, we first characterized the GC therapy response in MM1S and MM1R cells. Briefly, MM1S and MM1R cells were left untreated or treated with 1 µM of the synthetic GC agonist dexamethasone (Dex) for respectively 6 h, 24 h, 48 h, 72 h. mRNA and proteins were isolated at each time point and analysed for corresponding NR3C1/GRα expression. Treating the cells with different concentrations of Dex for 24 h, 48 h and 72 h followed by measuring cell viability by a MTT colorimetric assay, revealed a dose and time dependent decrease in cell survival of MM1S cells upon GC exposure (Fig. 1A & 1B). In contrast Dex lacks the capacity to induce cytotoxicity in MM1R cells and rather triggers a weak growth stimulatory effect [28], [35], [36], [37]. In line with the different GC response in both cell lines, QPCR analysis demonstrates significant NR3C1/GRα mRNA transcription in MM1S, but near background levels of NR3C1/GRα mRNA in MM1R cells (Fig. 1C). Western analysis finally confirms significant levels of GRα protein expression in MM1S and undetectable levels of GRα protein in MM1R cells (Fig. 1D–E). As can be observed from Fig. 1D, MM1S cells express multiple translational isoforms of GRα [38], [39], of which expression decreases following GC treatment.

Bottom Line: Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells.Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir.Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerp, Belgium; Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University, Ghent, Belgium.

ABSTRACT
Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

Show MeSH
Related in: MedlinePlus