cMyc/miR-125b-5p Signalling Determines Sensitivity to Bortezomib in Preclinical Model of Cutaneous T-Cell Lymphomas

<div><p>Successful/effective cancer therapy in low grade lymphoma is often hampered by cell resistance to anti-neoplastic agents. The crucial mechanisms responsible for this phenomenon are poorly understood. Overcoming resistance of tumor cells to anticancer agents, such as proteasome inhibitors, could improve their clinical efficacy. Using cutaneous T-cell lymphoma (CTCL) as a model of the chemotherapy-resistant peripheral lymphoid malignancy, we demonstrated that resistance to proteasome inhibition involved a signaling between the oncogene cMyc and miR-125b-5p. Bortezomib repressed cMyc and simultaneously induced miR-125b-5p that exerted a cytoprotective effect through the downmodulation of MAD4. Overexpression of cMyc repressed miR-125b-5p transcription and sensitized lymphoma cells to bortezomib. The central role of miR-125b-5p was further confirmed in a mouse model of T-cell lymphoma, where xenotransplantation of human CTCL cells overexpressing miR-125b-5p resulted in enhanced tumor growth and a shorter median survival. Our findings describe a novel mechanism through which miR-125b-5p not only regulates tumor growth <i>in vivo,</i> but also increases cellular resistance to proteasome inhibitors <i>via</i> modulation of MAD4.</p> </div

MAD4 is a direct target of miR-125b-5p.

<p>(A) The miR-125b-5p responsive element in <i>MAD4</i> gene 3′UTR and the base-paired to miR-125b-5p are showed on the top row of the schematic diagram. The psiCheck2 reporter constructs containing the <i>MAD4</i> 3′UTR (top row) or the mutated one (obtained by deleting the seed sequence of miR-125b-5p as shown in the lower row of the schematic diagram) were assayed. (B) Luciferase reporter assay (taken after 24h transfection in Hek-293 cells) showing that miR-125b-5p represses the luciferase activity of the reporter construct containing the MAD4 responsive element (psiCheck2-MAD4). Deletion of the miR-125b-5p responsive element in the MAD4 3′UTR (psiCheck2-Mut) rescued the luciferase activity. Renilla luciferase activities were normalized to the values of the internal Firefly and to the scrambled transfection control. (C) Detection of the MAD4 and cMyc protein levels in CTCL cells (SeAx and MyLa) after miR-125b-5p overexpression or downregulation. β-actin was the loading control and protein changes are showed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059390#pone-0059390-g001" target="_blank">Figure 1A</a>. (D) Changes in the relative MAD4 mRNA level detected by qRT-PCR in CTCL cells. (E) Evaluation of the efficient MAD4 silencing in SeAx cells by Western Blot. (F) Decrease of the percentage of the annexin-V positive cells after MAD4 silencing in SeAx cells treated with bortezomib (48h) comparing to scrambled siRNA. (G) A mechanistic model describing the regulative loop involving cMyc and miR-125b-5p in cell resistance to bortezomib. cMyc induces the transcriptional inhibition of miR-125b-5p that directly inhibits MAD4 and induces cMyc accumulation. All quantitative data are shown as mean ± SD (*<i>P</i>< 0.05).</p

miR-125b-5p inversely correlates with cMyc expression in mycosis fungoides.

<p>(A) Representative cases from the 17 mycosis fungoides specimens (plaque (T2), n = 11; advanced mycosis fungoides (T3), n = 6) analyzed by IHC for cMyc and by ISH for detection of miR-125b-5p. Scale bar, 10 µm. (B) The 17 mycosis fungoides specimens were classified into three categories depending on the percentages of miR-125b-5p or cMyc positive tumour cells. The Pearson coefficient (R² = 0.65; <i>P</i>< 0.05) indicates an inverse correlation between miR-125b-5p and cMyc expressions.</p

miR-125b-5p facilitates tumorigenesis <i>in vivo</i>.

<p>(A) Linear regression analysis of the <i>in vivo</i> tumor volume in 12 NSG mice (n = 6/group) after xenotransplantation of MyLa cells transfected with miR-125b-5p or scrambled oligonucleutide. The tumor growth rate represented by the slope of the linear regression was statistically different in the miR-125b-5p group compared to the scrambled one (<i>P</i> = 0.03). Each point represents a single measurement. (B) Kaplan-Meier survival curve showing a significant (Log-Rank Mantel Cox test <i>P</i> = 0.04) shorter median survival in the miR-125b-5p group compared to the scrambled one. (C) miR-125b-5p expression in miR-125b-5p and scrambled tumors was quantified by qRT-PCR (*<i>P</i>< 0.05). Each point represents the miR-125b-5p level in one tumor tissue and the line corresponds to the median value of miR-125b-5p in the miR-125b-5p and scrambled tumors. (D) A representative ISH from 6 cases of paraffin sections of miR-125b-5p or scrambled tumors using miR-125b-5p-specific LNA probe. miR-125b-5p expression was high in all tumors within the miR-125b-5p group, while it was almost undetectable in the tumors within the control group. Images were acquired using a Nikon D60 digital single-lens reflex (D-SLR) camera. Original magnification x20 for both panels. Scale bar, 10 µm.</p

miR-125b-5p induces cell resistance to bortezomib.

<p>(A) cMyc protein levels after bortezomib treatment (4 nM) at the indicated time points in CTCL cells (SeAx and MyLa). β-actin was used as a loading control and the cMyc band were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059390#pone-0059390-g001" target="_blank">Figure 1A</a>. (B) qRT-PCR showing the miR-125b-5p upregulation after 24h treatment with bortezomib (4 nM). (C) Effect of miR-125b-5p overexpression on the apoptosis induced by 48 h treatment with bortezomib. Percentages of annexin V-positive cells in the total cell population is shown. (D) Representative dot-plot graphs (annexin V: green FL1 channel, x-axis; PI: red FL3 channel, y-axis) from three independent experiments showing the reduced apoptosis after bortezomib treatment (48 h) in SeAx cells overexpressing miR-125b-5p compared to the scrambled transfected ones. All quantitative data are shown as mean ± SD (*<i>P</i>< 0.05).</p

cMyc is a transcriptional repressor of miR-125b-5p.

<p>(A, B) Schematic representation of the predicted cMyc binding motifs (black segments) in the region 10 kb upstream of the pre-miR-125b-1 (A) and pre-miR-125b-2 (B) 5′ terminus (indicated as −1). The numbers indicate the amplicon positions relative to the 5′ terminus of pre-miR-125b-1 and pre-miR-125b-2. (C) ChIP assay on the BS2 fragment. Cross-linked chromatin was precipitated from SeAx and MyLa cells using cMyc antibody. (D) Luciferase activity (measured 48h after transfection) of the pGL3-promoter reporter construct containing the BS2 fragment compared to the empty construct in Hek-293 cells. The relative Firefly luciferase activity is showed after normalization to internal control Renilla luciferase and pGL3-promoter empty vector. Data are presented as mean ± SD.</p