miR-122 Regulates p53/Akt Signalling and the Chemotherapy-Induced Apoptosis in Cutaneous T-Cell Lymphoma

Advanced cutaneous T-cell lymphoma (CTCL) is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs) in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs), we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132). miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL

p53 amplifies the chemotherapy-induced apoptosis.

<p>A) p53 protein level was assessed by western blot analysis in SeAx cells treated for 4 h with 6 nM bortezomib or 30 nM MG132. ß-actin was used as housekeeping gene, and relative p53 expression is reported as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#pone-0029541-g003" target="_blank">Figure 3</a>. B) miR-122 mimic counteracts the GSI-1-induced p53 activation. Cells transfected with either a control siRNA or miR-122 mimic for 24 h were exposed to GSI-1 for 4 h, at indicated concentrations. p53 expression was determined by western blot as in (A). C–D) Regulation of p53 by Akt. SeAx cells were treated for 24 h with 20 µM Ly294002 or 10 µM Akt Inhibitor X (C) or transfected with PTEN or the scrambled, control siRNA (D). p53 was measured as in (A). E–F) p53 activation by nutlin-3a increases the chemotherapy-induced apoptosis. SeAx were exposed to 5 or 10 µM nutlin-3a for 4 h followed by 20 h treatment with Ly294002 (at the concentrations 10, 20 and 40 µM) (E) or GSI-1 (0.4, 0.8, 1.2 µM) (F, left panel)), bortezomib (2, 4, 6 nM) (F, middle panel), MG132 (15, 30, 45 nM) (F, right panel). Cell viability was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#pone-0029541-g002" target="_blank">Figure 2</a>. <i>Points</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05, versus untreated cells.</p

Reciprocal regulation between miR-122 and Akt.

<p>A) miR-122 activates Akt signalling in SeAx cells. Immunoblots on whole cell-lysates from scrambled siRNA (control) or miR-122 mimic-transducted cells exposed to GSI-1 (0.8 µM; 4 h) and probed for the indicated proteins of the Akt signalling pathway. ß-actin was used as housekeeping gene, and relative protein expression levels are reported below the corresponding western blot bands. The blots shown are representative from three independent experiments. B) Inhibition of Akt by bortezomib and MG132. SeAx cells were treated with 6 nM bortezomib and 30 nM MG132 for 4 h and Akt activity was assessed in whole cell lysates by western blotting. C) Akt activation protects against GSI-1-induced apoptosis. Decrease in apoptotic, annexin V positive cells in SeAx transfected with PTEN siRNA and exposed to GSI-1 (1.2 µM, 24 h), comparing to the scrambled siRNA (control). Flow cytometry was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#pone-0029541-g002" target="_blank">Figure 2A</a>. Graphs are representative of three independent experiments. D) Akt inhibition induces miR-122 expression. qRT-PCR measurement of miR-122 level in SeAx and Hut-78 cells treated with the indicated doses of Ly294002 and Akt inhibitor X for 24 h and expressed as fold change compared to those in the untreated cells. <i>Columns</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05 versus untreated cells.</p

A model of miR-122 action.

<p>Chemotherapy, such as GSI-1 or proteasome blockade (bortezomib and MG132) leads to inactivation of Akt and further to apoptosis. Akt inactivation induces p53, which further amplifies Akt-dependent apoptosis, but is unable to induce apoptosis by itself. Another function of p53 is to activate miR-122, which constitutes a cytoprotective signalling loop <i>via</i> activation of Akt, and decrease in p53.</p

miR-122 increases resistance to GSI-1 and proteasome inhibitors.

<p>A) SeAx cells were transfected for 24 h with either scrambled siRNA (control), miR-122 mimic or antagomiR-122 and treated for 48 h with GSI-1 (0.8 µM) followed by annexin V and propidium iodide staining for flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#s2" target="_blank">Methods</a>. The dot-plot graphs (annexin V: green FL1 channel, x-axis; PI: red FL3 channel, y-axis) are representative of three independent experiments. B–C) Effect of antagomiR-122 and miR-122 mimic on the apoptosis induced by GSI-1 (at the concentrations 0.4, 0.8 and 1.2 µM; 48 h) (B), bortezomib (1, 5, 10 nM; 48 h) (C, left panel) and MG132 (5, 10, 50 nM; 48 h) (C, right panel) in SeAx cells. Cell viability is expressed as percentage of the PI-negative cells calculated by flow cytometry. <i>Points</i>, mean (n = 3); <i>bars</i>, SEM. *<i>p</i>-value<0.05 compared to cells transfected with scrambled oligonucleotide. D) MyLa2000 cells were transfected for 24 h with either scrambled siRNA (control) or antagomiR-122 and treated for additional 24 h with GSI-1 (at the concentrations 0.4, 0.8 and 1.2 µM) (D, left panel) bortezomib (2, 4, 6 nM) (D, middle panel) and MG132 (15, 30, 45 nM) (D, right panel). The percentage of the PI-negative cells was calculated as in (B). <i>Points</i>, mean (n = 3); <i>bars</i>, SEM. *<i>p</i>-value<0.05 compared to cells transfected with scrambled oligonucleotide.</p

miR-122 induction depends on p53.

<p>A) p53 immunoblots of whole-cell lysates from Hut-78 (<i>P53</i>^mut) and SeAx cells treated with nutlin-3a (10 µM; 24 h). B) Dose-dependent effect of GSI-1 (at the concentrations 0.4, 0.6, 0.8, 1 and 1.2 µM), bortezomib (2, 4, 6, 8, 10 nM) and MG132 (20, 40, 60, 80, 100 nM) on apoptosis in SeAx and Hut-78 cell lines. Cell viability was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#pone-0029541-g002" target="_blank">Figure 2B</a>. <i>Points</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05 versus untreated cells. C) Quantification of miR-122 expression by qRT-PCR in SeAx and Hut-78 cells treated with the indicated doses of nutlin-3a for 24 h. miR-122 levels after treatment are expressed as fold change compared to the mock-treated cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#pone-0029541-g001" target="_blank">Figure 1D</a>. <i>Columns</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05, versus untreated cells. D) p53 siRNA counteracts the miR-122 induction in SeAx cells. qRT-PCR measurement of miR- 122 level in SeAx cells transfected with p53 siRNA or scrambled oligonucleotide and treated with 0.8 µM GSI-1, 30 nM MG132, 6 nM bortezomib and 1 µM doxorubicin for 24 h. Data show fold-changes in miR-122 level after treatment as compared to the control, mock-treated cells. Columns, mean (n = 3); bars, SEM. * <i>p</i>-value<0.05 versus untreated cells, ⁺<i>p</i>-value<0.05 versus cells transfected with scrambled oligonucleotide. E) SeAx cells were sampled at various time points during the exposure to 5 µM nutlin-3a and the expression of the mature miR-122 or miR-122 primary transcript (pri-miR-122) was quantified by qRT-PCR. <i>Points</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05, versus untreated cells.</p

Expression of miR-122 in CTCL.

<p>A–B) Analysis of miR-122 expression using ISH and miR-122-specific LNA probe in lesional skin sections from patients with plaque/patch (n = 6) or tumour-stage (n = 5) MF. A) Representative ISH staining using miR-122 or scrambled (control) probes in MF skin sections (plaque/patch-stage MF, left panels; tumour-stage MF, right panels). The blue-purple color indicates miR-122 expression. Magnification: 20× and 40×. Abbreviation: Epi., epidermis. B) Skin biopsies were classified into three categories depending on the percentages of miR-122 positive tumour cells, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029541#s2" target="_blank">Methods</a>. Comparison of the miR-122 expression profile showed an increase of miR-122 expression in the tumour-stage MF (<i>p</i>-value = 0.046). C) qRT-PCR measurements of miR-122 expression in human liver, in a panel of CTCL cell lines (MyLa2000, SeAx and Hut-78) and <i>in vivo</i> in leukaemic cells from two patients with Sézary syndrome (Sézary cells) and in lesional skin from 10 patients with MF. miR-122 expression levels were normalized to the relative expression in quiescent T-cells. <i>Columns</i>, mean (n = 3); <i>bars</i>, SEM. D) Induction of miR-122 during chemotherapy-induced apoptosis in CTCL cell lines (MyLa2000 SeAx, Hut-78) and in malignant lymphoma cells isolated from peripheral blood of a patient with Sèzary syndrome (Sézary cells). The cells were incubated with 0.8 µM GSI-1, 30 nM MG132, 6 nM bortezomib and 1 µM doxorubicin for 4 and 24 h, as indicated, and miR-122 expression was quantified by qRT-PCR. Because the Sézary cells could not be purified in large quantities, the experiments were only performed with 0.8 µM GSI-1. Data show fold-changes in miR-122 level after treatment as compared to the control, mock-treated cells. <i>Columns</i>, mean (n = 3); <i>bars</i>, SEM. * <i>p</i>-value<0.05 versus untreated cells.</p