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. 2013 Jul 1;12(13):2144-53.
doi: 10.4161/cc.25251. Epub 2013 Jun 11.

Attenuation of dexamethasone-induced cell death in multiple myeloma is mediated by miR-125b expression

Megan Y Murray  1 , Stuart A Rushworth, Lyubov Zaitseva, Kristian M Bowles, David J Macewan
Affiliations

Attenuation of dexamethasone-induced cell death in multiple myeloma is mediated by miR-125b expression

Megan Y Murray et al. Cell Cycle. .

Abstract

Dexamethasone is a key front-line chemotherapeutic for B-cell malignant multiple myeloma (MM). Dexamethasone modulates MM cell survival signaling but fails to induce marked cytotoxicity when used as a monotherapy. We demonstrate here the mechanism behind this insufficient responsiveness of MM cells toward dexamethasone, revealing in MM a dramatic anti-apoptotic role for microRNA (miRNA)-125b in the insensitivity toward dexamethasone-induced apoptosis. MM cells responding to dexamethasone exhibited enhanced expression of oncogenic miR-125b. Dexamethasone also induced expression of miR-34a, which acts to suppress SIRT1 deacetylase, and thus allows maintained acetylation and inactivation of p53. p53 mRNA is also suppressed by miR-125b _targeting. Reporter assays showed that both these dexamethasone-induced miRNAs act downstream of their _target genes to prevent p53 tumor suppressor actions and, ultimately, resist cytotoxic responses in MM. Use of antisense miR-125b transcripts enhanced expression of pro-apoptotic p53, repressed expression of anti-apoptotic SIRT1 and, importantly, significantly enhanced dexamethasone-induced cell death responses in MM. Pharmacological manipulations showed that the key regulation enabling complete dexamethasone sensitivity in MM cells lies with miR-125b. In summary, dexamethasone-induced miR-125b induces cell death resistance mechanisms in MM cells via the p53/miR-34a/SIRT1 signaling network and provides these cells with an enhanced level of resistance to cytotoxic chemotherapeutics. Clearly, such anti-apoptotic mechanisms will need to be overcome to more effectively treat nascent, refractory and relapsed MM patients. These mechanisms provide insight into the role of miRNA regulation of apoptosis and their promotion of MM cell proliferative mechanisms.

Keywords: NFκB; SIRT1; mir-125b; mir-34a; p53.

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Figures

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Figure 1. Dexamethasone fails to induce net cell death in multiple myeloma cells, despite repression of key anti-apoptotic genes. (A) Luminescent cell proliferation assays of dexamethasone-sensitive MM.1S and dexamethasone-resistant MM.1R cells with increased concentration and time of dexamethasone exposure. (B) qRT-PCR analysis of pro-survival Bcl2 and c-FLIP mRNA in response to dexamethasone (10 µM) over 24 h in MM.1S and MM.1R cells. Values indicate the mean ± SEM from three independent experiments (statistical significance calculated by Student’s t-test, with p ≤ 0.05 indicated,*). (C) Western blot analysis of total Bcl2 and c-FLIP protein and nuclear phosphorylated p65 (p-p65) in response to dexamethasone (10 μM) over 48 h in MM.1S and MM.1R cells. Membranes were re-probed for β-actin to confirm equal loading. Values indicate optical density of each band expressed as a fold change of untreated cells.
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Figure 2. miRNA profiling of multiple myeloma cells in response to dexamethasone reveals regulation of miR-125b and miR-34a. (A) qRT-PCR analysis of 92 cancer-associated miRNAs in MM.1S cells in response to 8 h dexamethasone (10 µM). Values represent change in qRT-PCR cycle threshold normalized to an endogenous control (ΔCT). Dashed line indicates no change in expression. Open circles indicate miR-125b and miR-34a (labeled). (B) qRT-PCR analysis of miR-125b and miR-34a in response to dexamethasone (10 μM) over 24 h in MM.1S and MM.1R cells. Values indicate the mean ± SEM from three independent experiments (statistical significance calculated by Student’s t-test, * indicates p ≤ 0.05).
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Figure 3. Dexamethasone-induced miR-125b and miR-34a expression are responsible for repression of SIRT1 and p53 _target mRNA. (A) qRT-PCR shows time-dependant repression of SIRT1 and (C) p53 mRNA levels in response to dexamethasone. Dexamethasone-resistant MM.1R cells were used as a control. Insets show western blotting for SIRT1 and p53 protein at 24 h. (B) Chemiluminescent plasmid reporter gene assays confirm _targeting of a fragment of the SIRT1 3′ UTR following transfection with synthetic miR-34a-mimics and (D) _targeting of the p53 3′ UTR following transfection with synthetic miR-125b-mimics, compared with a non-_target control 3′ UTR or scrambled-miR in MM.1S cells. (E) Schematic representation of the reporter constructs created and used. Values indicate the mean ± SEM from three independent experiments (statistical significance calculated by Student’s t-test; *, indicates p ≤ 0.05).
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Figure 4. miR-125b and miR-34a have divergent effects on the p53/miR-34a/SIRT1 signaling network in multiple myeloma cells. (A) qRT-PCR analysis reveals enhanced p53 and repressed SIRT1 mRNA expression in MM.1S and MM.1R cells following transfection with anti-miR-125b-transcripts or synthetic miR-34a-mimics. (B) Transfection with anti-miR-34a-transcripts or synthetic miR-125b-mimics reveals an antagonistic effect. (C) Western blot analysis of total p53 and SIRT1 protein expression correlate with mRNA in response to anti-miR-125b or miR-34a-mimics in MM.1S and MM.1R cells. Values indicate the mean ± SEM from three independent experiments (statistical significance calculated by Student’s t-test; *, indicates p ≤ 0.05).
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Figure 5. Repression of miR-125b allows dexamethasone-induced cell death by enhancing tumor suppressor p53 expression in multiple myeloma cells. (A) Luminescent cell death analysis shows significantly enhanced levels of dexamethasone-induced cell death in MM.1S cells transfected with anti-miR-125b-transcripts or synthetic miR-34a-mimics after 24 h against a scrambled-miR control and compared with dexamethasone-resistant MM.1R cells. (B) Dexamethasone-induced cell death is further enhanced with anti-miR-125b-transcript up to 72 h after dexamethasone exposure in MM.1S cells. (C) qRT-PCR reveals regulation of the p53/miR-34a/SIRT1 signaling network in MM.1S cells transfected with anti-miR-125b and exposed to dexamethasone: p53 mRNA and miR-34a levels are enhanced, while SIRT1 mRNA is repressed. (D) Western blot analysis of total p53 and SIRT1 protein confirms divergent regulation, in response to anti-miR-125b and dexamethasone. Dexamethasone-resistant MM.1R cells were used as a control. Values indicate the mean ± SEM from 3 independent experiments (statistical significance calculated by Student’s t-test; *, indicates p ≤ 0.05).
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Figure 6. miR-125b modulates expression of the p53/miR-34a/SIRT1 signaling network and dexamethasone-induced cell death in primary MM samples and is dependent on p53WT expression. (A) Luminescent cell death analysis reveals enhanced dexamethasone-induced cell death with anti-miR-125b-transcript in six primary bone marrow MM patient samples. (B) qRT-PCR reveals regulation of the p53/miR-34a/SIRT1 signaling network in the same primary MM patient samples: miR-34a levels are enhanced, while SIRT1 mRNA is repressed. (C) Luminescent cell death analysis reveals enhanced dexamethasone-induced cell death with anti-miR-125b-transcript in p53WT MM cell lines while p53MUT MM cells are unable to respond. (D) A similar lack of response to anti-miR-125b-transcripts and dexamethasone exposure is seen in the regulation of miR-34a expression and SIRT1 mRNA levels in p53MUT compared with p53WT cell lines. Values indicate the mean ± SEM from three independent experiments (statistical significance calculated by Student’s t-test; *, indicates p ≤ 0.05).
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Figure 7. A potential role for miR-125b in the p53/miR-34a/SIRT1 signaling network in MM. (A) qRT-PCR reveals regulation of the p53/miR-34a/SIRT1 signaling network following direct p53 stabilization and activation by 16 h nutlin3 pre-treatment before a further 24 h exposure to dexamethasone. (B) Luminescent cell death analysis reveals nutlin3 pre-treatment to have no effect of on dexamethasone-induced cytotoxic responses. (C) qRT-PCR reveals the effect of bortezomib treatment on miR-125b levels in MM.1S cells is dose-dependent, and can be significantly enhanced by addition of dexamethasone, compared with MM.1R cells. (D) Proposed scheme of how the p53/miR-34a/SIRT1 signaling network is regulated by dexamethasone in MM and intervention of miR-125b. Several solid tumors and hematological malignancies, display a balance between net cell survival and net cell death., Tumor suppressor p53 transcription factor transactivates pro-apoptotic miR-34a, that in turn _targets numerous 3′ UTR sites of pro-survival histone deacetylase SIRT1. Active SIRT1 deacetylates p53’s C terminus to prevent its transcriptional activity. Dexamethasone represses numerous pro-survival proteins; however, MM cells exposed to dexamethasone demonstrate only low levels of cell death. Dexamethasone induced expression of oncogenic miR-125b triggers net cell survival in MM cells by _targeting p53. We show here that repression of miR-125b activity with synthetic anti-miRs enhances p53 expression, shifting the balance back toward net cell death.
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