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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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Related in: MedlinePlus

Mir-150-5p transfection is associated with complex regulation of chemokine, cell cycle, UPR/mTOR signaling.Illumina BeadChip Gene Expression results of chemokine receptors or ligands (A), or regulators of cell cycle, unfolded protein stress (UPR/mTOR signaling) (B), are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S, treated for 72 h with 1 µM Dex versus control setup (UT), or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection.
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pone-0113842-g009: Mir-150-5p transfection is associated with complex regulation of chemokine, cell cycle, UPR/mTOR signaling.Illumina BeadChip Gene Expression results of chemokine receptors or ligands (A), or regulators of cell cycle, unfolded protein stress (UPR/mTOR signaling) (B), are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S, treated for 72 h with 1 µM Dex versus control setup (UT), or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection.

Mentions: Finally, we also explored possible upstream effects of mir-150-5p on receptor kinase signalling pathways. Ligand independent hormone receptor (in)activation via kinases, chemokines and cytokines is well documented nowadays [62], [63], [64], [65], [66]. With respect to chemokine signalling, mir-150-5p transfection and GC treatment were found to increase mRNA levels of CXCR4 and CX3CR1 and decrease levels of CXCR3, CCL3, CCL5 whereas other members remain unaffected (Fig. 9A). Interestingly, various reports demonstrate a positive role for CXCR4/CXCL12 in hormone independent receptor activation [65], [66], [67], [68], [69]. Surprisingly, we observed a significant increase in CXCR4 levels, upon mir-150-5p transfection, in contrast to earlier reports demonstrating CXCR4 mRNA silencing of its 3′ UTR via mir-150-5p [68], [70]. Although various studies illustrate tumor promoting and prometastatic effects of CXCR4 [71], [72], its expression also plays a pivotal role in haematopoiesis [73] and has recently been reported as a good prognostic indicator in multiple myeloma [74]. Finally, we also identified remarkable effects on effectors in cell cycle (CDKN1A) and UPR/mTOR signalling pathways (DDIT3, DDIT4, TXNIP) and the Tribble family Ser/Thr pseudokinase (TRIB3) [75] which altogether may crosstalk with glucocorticoid receptor signalling and GC therapy response in MM cells [76], [77], [78] (Fig. 9B). Of special note, involvement of C/EBP and ATF4 (Fig. 8A) has been demonstrated in transcriptional regulation of UPR stress proteins DDIT3, DDIT4, TXNIP [79], [80], further corroborating our results.


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)

Mir-150-5p transfection is associated with complex regulation of chemokine, cell cycle, UPR/mTOR signaling.Illumina BeadChip Gene Expression results of chemokine receptors or ligands (A), or regulators of cell cycle, unfolded protein stress (UPR/mTOR signaling) (B), are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S, treated for 72 h with 1 µM Dex versus control setup (UT), or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113842-g009: Mir-150-5p transfection is associated with complex regulation of chemokine, cell cycle, UPR/mTOR signaling.Illumina BeadChip Gene Expression results of chemokine receptors or ligands (A), or regulators of cell cycle, unfolded protein stress (UPR/mTOR signaling) (B), are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S, treated for 72 h with 1 µM Dex versus control setup (UT), or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection.
Mentions: Finally, we also explored possible upstream effects of mir-150-5p on receptor kinase signalling pathways. Ligand independent hormone receptor (in)activation via kinases, chemokines and cytokines is well documented nowadays [62], [63], [64], [65], [66]. With respect to chemokine signalling, mir-150-5p transfection and GC treatment were found to increase mRNA levels of CXCR4 and CX3CR1 and decrease levels of CXCR3, CCL3, CCL5 whereas other members remain unaffected (Fig. 9A). Interestingly, various reports demonstrate a positive role for CXCR4/CXCL12 in hormone independent receptor activation [65], [66], [67], [68], [69]. Surprisingly, we observed a significant increase in CXCR4 levels, upon mir-150-5p transfection, in contrast to earlier reports demonstrating CXCR4 mRNA silencing of its 3′ UTR via mir-150-5p [68], [70]. Although various studies illustrate tumor promoting and prometastatic effects of CXCR4 [71], [72], its expression also plays a pivotal role in haematopoiesis [73] and has recently been reported as a good prognostic indicator in multiple myeloma [74]. Finally, we also identified remarkable effects on effectors in cell cycle (CDKN1A) and UPR/mTOR signalling pathways (DDIT3, DDIT4, TXNIP) and the Tribble family Ser/Thr pseudokinase (TRIB3) [75] which altogether may crosstalk with glucocorticoid receptor signalling and GC therapy response in MM cells [76], [77], [78] (Fig. 9B). Of special note, involvement of C/EBP and ATF4 (Fig. 8A) has been demonstrated in transcriptional regulation of UPR stress proteins DDIT3, DDIT4, TXNIP [79], [80], further corroborating our results.

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