Histone deacetylase inhibitors inhibit metastasis by restoring a tumor suppressive microRNA-150 in advanced cutaneous T-cell lymphoma

Abstract

Tumor suppressive microRNA (miR)-150 inhibits metastasis by combining with the C-C chemokine receptor 6 (CCR6) "seed sequence" mRNA of the 3'-untranslated region (3'-UTR) in advanced cutaneous T-cell lymphoma (CTCL). Because the histone deacetylase inhibitor (HDACI) vorinostat showed excellent outcomes for treating advanced CTCL, HDACIs may reduce the metastasis of CTCL by targeting miR-150 and/ or CCR6. To examine whether these candidate molecules are essential HDACI targets in advanced CTCL, we used the My-La, HH, and HUT78 CTCL cell lines for functional analysis because we previously demonstrated that their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) induced multiple metastases. We found that pan- HDACIs (vorinostat and panobinostat) inhibited the migration of CTCL cells and downregulated CCR6. The miRNA microarray analysis against CTCL cell lines demonstrated that these pan-HDACIs commonly upregulated 161 miRNAs, including 34 known tumor suppressive miRNAs such as miR-150. Although 35 miRNAs possessing the CCR6 "seed sequence" were included in these 161 miRNAs, miR-150 and miR-185-5p were downregulated in CTCL cells compared to in normal CD4+ T-cells. The transduction of 12 candidate miRNAs against CTCL cells revealed that miR-150 most efficiently inhibited their migration capabilities and downregulated CCR6. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that miR-150 was downregulated in advanced but not early CTCL primary cases. Finally, we injected miR-150 or siCCR6 into CTCL mice and found that mouse survival was significantly prolonged. These results indicate that miR-150 and its target, CCR6, are essential therapeutic targets of pan-HDACIs in advanced CTCL with metastatic potential.

Keywords: CCR6; CTCL; HDACI; metastasis; miR-150.

Figure 1. Vorinostat and panobinostat restore expression of CCR6, which inhibits migration of CTCL cells

(A) Western blot analysis of histone deacetylase (HDAC) 1, 2, 3, 4, and 6 and CCR6 in CTCL cell lines and three cases of primary and advanced CTCL, which were obtained from lymph nodes of patients with MF tumors (namely MF1, MF6, and MF22). Tubulin, control. (B) qRT-PCR analysis of CCR6 in HDACIs [vorinostat, panobinostat, Romidepsin-treated CTCL cells (My-La, MJ, HH, and HUT78 cell lines). Student's t-test was used to examine significance. Bars are means ± standard error of the mean (SEM) of three independent experiments. **0.001 ≤ P < 0.01, ***P < 0.001. NS: not significant. (C) Western blot analysis of CCR6 and acetyl-histone H3 (Acl-H3) in My-La, MJ, HH, and HUT78 cells treated with vorinostat (V), panobinostat (P), and romidepsin (R). DMSO is shown as “D”. Tubulin, control. (D) Migration of My-La, MJ, HH, and HUT78 cells treated with vorinostat (5 μM) or panobinostat (80 nM) and romidepsin (10 nM). RFU was measured 16 hr after drug treatment. Student's t-test was used to examine significance. Bars are means ± SEM of three independent experiments. Illustration of migration assay is also under bar graph. **0.001 ≤ P < 0.01, ***P < 0.001. NS: not significant.qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction; RFU, relative fluorescence units.

Figure 2. miRNA expression analysis of HDACIs-treated CTCL cell lines

(A) miRNA expression analysis of CTCL cells and normal CD4+T-cells. X- and Y-axes, intensity of miRNAs treated with dimethyl sulfoxide (DMSO) and pan-HDACIs (vorinostat and panobinostat), respectively. Normal CD4+T-cells, My-La, MJ, HH, and HUT78 cells treated with vorinostat (5 μM) or panobinostat (80 nM) for 24 hr. (B) Commonly upregulated miRNAs in My-La, HH, and HUT78 cells treated with vorinostat (left) or panobinostat (right). (C) Commonly upregulated miRNAs in My-La, HH, and HUT78 treated with vorinostat and panobinostat. (D) Heat map of My-La and HH cell lines treated with and without pan-HDACIs [vorinostat (V), panobinostat (P)]. (E) Northern blot analysis of miR-150 in the My-La and HH cell lines treated with vorinostat (V) or panobinostat (P). DMSO (D), control. 5S tRNA (5S), control.

Figure 3. Upregulated miRNAs by HDACIs that potentially inhibit CCR6 in CTCL cells

(A) Heat map and intensity of miRNAs with seed sequence of CCR6 [FC, fold change: > 1.5]. V/D = vorinostat/DMSO, P/D = panobinostat/DMSO. (B) Northern blot analysis of candidate miRNAs with CCR6 seed sequence (miR-96-5p, miR-150, miR-183-5p, miR-194-5p, miR-301b, miR-320c, and miR-371b-5p) showing high fold change (FC: > 1.5) for normal CD4+ cells (CD4-1, and CD4-2), CTCL cell lines, and primary MF samples (n = 2, tumor phase). 5S tRNA (5S), control. (C) Western blot analysis of CCR6 expression in My-La, HH, and HUT78 cell lines transiently transduced with respective candidate miRNAs (Scr: scrambled; 96: miR-96-5p; 150: miR-150; 183: miR-183-5p; 185: miR-185-5p; 194: miR-194-5p; 301: miR-320a; 371: miR-371a-5p; 3135: miR-3135b; 3652: miR-3652; 4534: miR-4534; 4698: miR-4698; 6088: miR-6088). Tubulin, control. (D) Migration assay using miR-96-5p, miR-150, miR-183-5p, miR-185-5p, miR-194-5p, miR-320a, miR-371a-5p, miR-3135b, miR-3652, miR-4534, miR-4698, miR-6088 and scrambled control (Scr) against My-La, HH, and HUT78 cells. RFUs (relative fluorescence units) were measured 16 hr after drug treatment. Student's t test was used for examining significance. Bars are means ± standard error of the mean (SEM) of three independent experiments. *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***P < 0.001. Student's t-test was used to examine significance.

Figure 4. Examination of combined effects of vorinostat, RG108, and nutlin-3a against CTCL cells

(A) Expression of miR-150 in My-La, HH, and HUT78 cells treated with histone deacetylase inhibitor (HDACI, vorinostat, 5.0 μM), MDM2 inhibitor [nutlin-3a, 2.5 μM (My-La), 20 μM (HH and HUT78)], and/or methyltransferase inhibitor (RG108, 200 μM). ” + “: treatment of agent [vorinostat (red), nutlin3a (black), RG108 (black)] , “ – “: no treatment of agent. (B) Western blot analysis of C-C chemokine receptor 6 (CCR6) and acetyl-histone H3 (Acl-HisH3) in My-La and HH cells with treated with HDACI (vorinostat, 5.0 μM), MDM2 inhibitor [nutlin-3a, 2.5 μM (My-La), 20 μM (HH)] and/or methyltransferase inhibitor (RG108, 200 μM). Tubulin, control. (C) Migration assay of My-La and HH cells with treated with HDACI (vorinostat, 5.0 μM), MDM2 inhibitor [nutlin-3a, 2.5 μM (My-La), 20 μM (HH)] and/or methyltransferase inhibitor (RG108, 200 μM). Student's t-test was used to examine significance. Bars are means ± standard error of the mean (SEM) of three independent experiments. ***P < 0.001. NS: not significant.

Figure 5. miR-150 is downregulated during progression of primary CTCL cases

(A) Photomicrographs of early and tumor phase of mycosis fungoides (MF) samples collected from same patients (MF1 and MF22). Hematoxylin and eosin (H&E) and CD4+ staining are shown. (B) qRT-PCR analysis of miR-150 in tissue samples of normal atopic dermatitis (AD, n = 18), early MF (n = 26), and tumor phase MF (n =14). Y-axis: 2-ΔCt values for miRNA expression. P-values were calculated using Mann-Whitney U test. ***P < 0.001. NS: not significant. (C) Comparison of miR-150 expression between early and tumor MF in same patients (eight cases). #1: miR-150 expression of different clinical courses (MF1, MF6, MF7, and MF22), #2: miR-150 expression of the same patient from different biopsy regions patch/plaque and tumor (MF23, MF24, MF25, and MF28). Student's t-test was used to examine significance. **0.001 ≤ P < 0.01, ***P < 0.001. Bars are means ± standard error of the mean (SEM) of three independent experiments.

Figure 6. In vivo administration of miR-150 to CTCL xenograft mouse model

Schematic diagram of injection protocol. (A) ELISA (enzyme-linked immunosorbent assay) of IL-22 in serum of NOG mice transplanted with My-La. Serum IL-22 levels in NOG mice transplanted with My-La cells. Blood samples were collected from tail vein every 5 days after the transplantation. X-axis: day after My-La cells’ transplantation for NOG mice. Y-axis: concentration of IL-22 (pg/mL). (B) Kaplan-Meier survival curves for My-La mice administered scrambled-control (30 μM, n = 10), miR-150 (30 μM, n = 10), or si (small interfering) CCR6 (30 μM, n = 10)]. s.c.: subcutaneous injection (2 × 105 cells). i.v.: intravenous injection. Log-lank test was conducted for statistical analysis. *0.01 ≤ P < 0.05, ***P < 0.001.