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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 triggers GC specific transcriptional changes in cell cycle, DNA damage/repair, cell death and immunological pathways in MM1S but not MM1R.(A) The heat map represents relative gene expression levels of MM1S and MM1R cells, left untreated (solvent) or treated with 1 µM Dex for 72 h. Gene expression changes >2-fold and with a p-value <0.05 are plotted on the heat map. (B) Left panel: Realtime quantitative PCR (QPCR) validation of Dex dependent activation of GILZ (TSC22D3) mRNA transcription Right panel: Realtime quantitative PCR (QPCR) validation of Dex dependent repression of CDK2 mRNA in MM1S but not in MM1R. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) as compared to control setups, determined by one-way (Dunnett’s Post-test) (C–D) Realtime quantitative PCR (QPCR) validation and Illumina microarray observation of the top GC regulated genes involved cell cycle, DNA damage/repair and cell death in S = MM1S but not R = MM1R. All the QPCR validation studies have been normalized to the 28S RNA housekeeping gene and are represented relative to MM1S untreated condition (S UT). Bar graphs represent relative mRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) from control setups as determined by two-way ANOVA (Bonferroni post-tests).
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pone-0113842-g002: Dexamethasone triggers GC specific transcriptional changes in cell cycle, DNA damage/repair, cell death and immunological pathways in MM1S but not MM1R.(A) The heat map represents relative gene expression levels of MM1S and MM1R cells, left untreated (solvent) or treated with 1 µM Dex for 72 h. Gene expression changes >2-fold and with a p-value <0.05 are plotted on the heat map. (B) Left panel: Realtime quantitative PCR (QPCR) validation of Dex dependent activation of GILZ (TSC22D3) mRNA transcription Right panel: Realtime quantitative PCR (QPCR) validation of Dex dependent repression of CDK2 mRNA in MM1S but not in MM1R. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) as compared to control setups, determined by one-way (Dunnett’s Post-test) (C–D) Realtime quantitative PCR (QPCR) validation and Illumina microarray observation of the top GC regulated genes involved cell cycle, DNA damage/repair and cell death in S = MM1S but not R = MM1R. All the QPCR validation studies have been normalized to the 28S RNA housekeeping gene and are represented relative to MM1S untreated condition (S UT). Bar graphs represent relative mRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) from control setups as determined by two-way ANOVA (Bonferroni post-tests).

Mentions: To evaluate specific gene expression changes linked to GC therapy response in multiple myeloma, mRNA was isolated from three independent experiments from MM1S and MM1R cells exposed for 72 h to the GC Dex (1 µM). After mRNA isolation, genome-wide transcription profiles were determined by Illumina array hybridisation. Results were analysed with the R-package “Limma” (v3.14.1) [40], [41], which resulted in the identification of 268 downregulated and 151 upregulated genes in MM1S, after filtering for a minimal two-fold transcriptional change in Dex treated samples versus control setup (absolute fold change >2) and adjusted P-value <0.05 (Gene Expression Omnibus (GEO) database GSE59805). In MM1R, no significant Dex responsive changes could be detected, in line with the GC resistant phenotype and the absence of GRα in MM1R cells (see Fig. 1). The heatmap (Fig. 2A) represents a graphical presentation of Dex specific transcriptional changes in MM1S and lack of Dex response in MM1R cells in three biological replicates, revealing highly reproducible and robust patterns of gene expression. By QPCR analysis, we confirmed GC dependent transactivation of GILZ (TSC22D3) target gene as well as GC specific (trans)repression of CDK2 in MM1S but not in MM1R, in line with the Illumina array data and previous reports [37], [42], [43], [44] (Fig. 2B). Remarkably, prolonged GC exposure (72 h) of MM1R cells reveals weakly increased levels of CDK2 according to QPCR analysis, which could not be detected by less sensitive microarray analysis. This effect may be attributed to residual activity of anti-apoptotic GR isoforms in MM1R, which may also contribute to weak growth stimulatory effects (Fig. 1A) upon prolonged exposure [38]. Integrated pathway analysis (IPA) further revealed that GCs trigger gene expression changes which promote cell death, decrease cell proliferation (cell cycle, DNA replication) and regulate immunological haematological pathways in MM1S but not MM1R (Table 1), in line with the reduced cell viability assay in MM1S (see Fig. 1). Next, we further validated microarray results of some additional target genes involved in cell cycle, DNA damage/repair and cell death. As can be observed from (Fig. 2C & 2D), the QPCR validation results correlate with the obtained microarray data. Furthermore, by analyzing patterns of coregulated genes, IPA software allows to identify activation or inhibition of possible upstream regulators (kinases, receptors, transcription factors) involved in gene expression changes [45]. The latter analysis predicts highly significant activation of Dex liganded NR3C1 and TP53 transcription factors, in regulating MM1S cell cycle arrest and/or apoptosis [46] (Table 2). Furthermore, predicted activation of IKK and cyclin dependent kinases CDKN2A, CDKN1A may be indicators of DNA damage/cell death and cell cycle events triggered by Dex [47], [48], [49], [50]. Finally, possible inhibition of anti-apoptotic prosurvival signals stimulated by TNF and AKT kinases may also contribute to Dex induced cell death in MM1S (Table 2).


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 triggers GC specific transcriptional changes in cell cycle, DNA damage/repair, cell death and immunological pathways in MM1S but not MM1R.(A) The heat map represents relative gene expression levels of MM1S and MM1R cells, left untreated (solvent) or treated with 1 µM Dex for 72 h. Gene expression changes >2-fold and with a p-value <0.05 are plotted on the heat map. (B) Left panel: Realtime quantitative PCR (QPCR) validation of Dex dependent activation of GILZ (TSC22D3) mRNA transcription Right panel: Realtime quantitative PCR (QPCR) validation of Dex dependent repression of CDK2 mRNA in MM1S but not in MM1R. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) as compared to control setups, determined by one-way (Dunnett’s Post-test) (C–D) Realtime quantitative PCR (QPCR) validation and Illumina microarray observation of the top GC regulated genes involved cell cycle, DNA damage/repair and cell death in S = MM1S but not R = MM1R. All the QPCR validation studies have been normalized to the 28S RNA housekeeping gene and are represented relative to MM1S untreated condition (S UT). Bar graphs represent relative mRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) from control setups as determined by two-way ANOVA (Bonferroni post-tests).
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pone-0113842-g002: Dexamethasone triggers GC specific transcriptional changes in cell cycle, DNA damage/repair, cell death and immunological pathways in MM1S but not MM1R.(A) The heat map represents relative gene expression levels of MM1S and MM1R cells, left untreated (solvent) or treated with 1 µM Dex for 72 h. Gene expression changes >2-fold and with a p-value <0.05 are plotted on the heat map. (B) Left panel: Realtime quantitative PCR (QPCR) validation of Dex dependent activation of GILZ (TSC22D3) mRNA transcription Right panel: Realtime quantitative PCR (QPCR) validation of Dex dependent repression of CDK2 mRNA in MM1S but not in MM1R. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) as compared to control setups, determined by one-way (Dunnett’s Post-test) (C–D) Realtime quantitative PCR (QPCR) validation and Illumina microarray observation of the top GC regulated genes involved cell cycle, DNA damage/repair and cell death in S = MM1S but not R = MM1R. All the QPCR validation studies have been normalized to the 28S RNA housekeeping gene and are represented relative to MM1S untreated condition (S UT). Bar graphs represent relative mRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05 respectively) from control setups as determined by two-way ANOVA (Bonferroni post-tests).
Mentions: To evaluate specific gene expression changes linked to GC therapy response in multiple myeloma, mRNA was isolated from three independent experiments from MM1S and MM1R cells exposed for 72 h to the GC Dex (1 µM). After mRNA isolation, genome-wide transcription profiles were determined by Illumina array hybridisation. Results were analysed with the R-package “Limma” (v3.14.1) [40], [41], which resulted in the identification of 268 downregulated and 151 upregulated genes in MM1S, after filtering for a minimal two-fold transcriptional change in Dex treated samples versus control setup (absolute fold change >2) and adjusted P-value <0.05 (Gene Expression Omnibus (GEO) database GSE59805). In MM1R, no significant Dex responsive changes could be detected, in line with the GC resistant phenotype and the absence of GRα in MM1R cells (see Fig. 1). The heatmap (Fig. 2A) represents a graphical presentation of Dex specific transcriptional changes in MM1S and lack of Dex response in MM1R cells in three biological replicates, revealing highly reproducible and robust patterns of gene expression. By QPCR analysis, we confirmed GC dependent transactivation of GILZ (TSC22D3) target gene as well as GC specific (trans)repression of CDK2 in MM1S but not in MM1R, in line with the Illumina array data and previous reports [37], [42], [43], [44] (Fig. 2B). Remarkably, prolonged GC exposure (72 h) of MM1R cells reveals weakly increased levels of CDK2 according to QPCR analysis, which could not be detected by less sensitive microarray analysis. This effect may be attributed to residual activity of anti-apoptotic GR isoforms in MM1R, which may also contribute to weak growth stimulatory effects (Fig. 1A) upon prolonged exposure [38]. Integrated pathway analysis (IPA) further revealed that GCs trigger gene expression changes which promote cell death, decrease cell proliferation (cell cycle, DNA replication) and regulate immunological haematological pathways in MM1S but not MM1R (Table 1), in line with the reduced cell viability assay in MM1S (see Fig. 1). Next, we further validated microarray results of some additional target genes involved in cell cycle, DNA damage/repair and cell death. As can be observed from (Fig. 2C & 2D), the QPCR validation results correlate with the obtained microarray data. Furthermore, by analyzing patterns of coregulated genes, IPA software allows to identify activation or inhibition of possible upstream regulators (kinases, receptors, transcription factors) involved in gene expression changes [45]. The latter analysis predicts highly significant activation of Dex liganded NR3C1 and TP53 transcription factors, in regulating MM1S cell cycle arrest and/or apoptosis [46] (Table 2). Furthermore, predicted activation of IKK and cyclin dependent kinases CDKN2A, CDKN1A may be indicators of DNA damage/cell death and cell cycle events triggered by Dex [47], [48], [49], [50]. Finally, possible inhibition of anti-apoptotic prosurvival signals stimulated by TNF and AKT kinases may also contribute to Dex induced cell death in MM1S (Table 2).

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