STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

Abstract

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC. In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Keywords: BIC; CTCL; JAK; MF; STAT5; miR-155; miRNA.

Figures

Figure 1. BIC/miR-155 expression in CTCL. (A) miR-155 expression in non-malignant (MySi) and malignant (SeAx, SeZ4.1, MyLa2059, PB2B, MAC2A) CTCL T cell lines as measured by qPCR. (B) Expression of the miR-155 host gene BIC in the same cell lines obtained by RT-PCR. Reference GAPDH. miR-155 expression levels are significantly higher in the malignant cell lines and highest in the MF cell line MyLa2059 and the ALCL cell line MAC2A.

Figure 2. miR-155 precursor BIC is a transcriptional target of STAT5. (A) Analysis of the BIC promoter region yielded one STAT binding site (highlighted in blue). Primers flanking the binding site that were used for PCR analysis of ChIP samples are indicated in red. The beginning of the BIC transcript is highlighted in bold script. (B) ChIP-seq reads from the BIC (MIR155HG) gene promoter region in malignant MyLa2059 cells. Reads (76 bases) obtained from immunoprecipitation of STAT5, STAT3, RelA and a negative control (rabbit IgG, bottom). The chromosomal positions of MIR155HG refer to hg19. Forward reads are indicated in green and reverse reads in red. (C) PCR analysis of ChIP samples using the primer set indicated in (B). The 190 bp amplicon was only detected in the STAT5-precipitated samples and the positive control (histone H3). (D) Similar results were obtained by qPCR: STAT5-precipitated samples displayed a 10-fold enrichment of the amplified sequence in relation to a negative control, whereas detection in STAT3 precipitated samples ranged at background level. U6 rRNA was used for normalization. (E) Luciferase assay for BIC promoter activity. CHO-K1 cells were transiently transfected with pCMV-LacZ, empty pGL3 or pGL3-BIC-WT, together with STAT5A, STAT5A-CA, STAT5B or STAT5B-CA (CA, constitutively active). Transfection with the constitutively active STAT5A-CA and STAT5B-CA resulted in a 28.6- (p < 0.001) and 29-fold (p = 0.002) induction of BIC promoter activity, respectively. Data were obtained from three independent experiments.

Figure 3. miR-155 expression is regulated by the JAK/STAT pathway. (A) Representative western blot showing siRNA-mediated knockdown of STAT3, STAT5A, STAT5B and STAT5A+B in malignant MyLa2059/MAC2A cells. (B) Relative quantification of miR-155 (top) and BIC (bottom) in malignant MyLa2059 (left) and MAC2A (right) cells as measured by qPCR and determined by the ddCt method. miR-155 expression was decreased by 40% after knockdown of STAT5A and STAT5B, but not STAT3 in both cell lines; nt, non-targeting control. p values were obtained from Student’s t-test. *Indicates a significance of p < 0.05. (C) Incubation with JAK3 inhibitor (JAK3i) CP-690550 (50 µM) decreased pY-STAT5 (top) and resulted in decreased miR-155 expression in malignant MyLa2059 cells. miR-155 expression was decreased by 60–80% when cells were treated with JAK3i for 24, 48 or 72 h. Control DMSO. (D) JAK3 inhibition with CP-690550 (0.1 and 50 µM, 24 h) in PBMCs from a patient with SS resulted in a decreased STAT5 phosphorylation (top) and a 50% inhibition of miR-155 expression.

Figure 4. miR-155 expression in response to STAT5-activating cytokines. (A) Incubation with IL-2 (103U/ml) for 24 h increased expression of BIC (left) and miR-155 (right) in non-malignant MyLa1850 (top, 1.7-fold increase, p < 0.05) as well as malignant SeAx T cells (bottom, 1.5-fold increase, p < 0.02) as shown by qPCR, and was accompanied by induction of STAT5 phosphorylation. (B) In primary T cells obtained from two SS patients (P3 and P4), incubation with IL-2 (2 × 103 U/ml) for 24 h resulted in an increase of miR-155 in both malignant (CD26−) and non-malignant (CD26+) populations. The increase was stronger in T cells isolated from P4; however, both samples showed elevated levels of miR-155 in response to IL-2. (C) In PBMCs from a healthy donor, a 24 h incubation with IL-2 (2 × 103U/ml) led to a 2.3-fold increase of miR-155. (D) Incubation of non-malignant MySi T cells with IL-15 (25 ng/ml) for 24h resulted in increased levels of BIC (2-fold) and miR-155 (1.5-fold). p values were obtained from Student’s t-test. *Indicates a significance of p < 0.05.

Figure 5. miR-155 is involved in malignant proliferation. (A) siRNA-mediated knockdown of BIC in malignant MyLa2059 cells resulted in a 40% decrease of mature miR-155 levels after 48 h (p = 0.02). (B) Proliferation of malignant cells in the same samples was reduced by 20% (p = 0.01) determined by methyl-3H-thymidine incorporation. (C) Transfection of MyLa2059 with small RNA inhibitors targeting the mature miR-155 (antagomiR-155) yielded similar results, entailing a 20% reduction in cell proliferation. p < 0.001. (D) Likewise, knockdown of STAT5A (p = 0.004) or STAT5B (ns) entailed an approximately 25% decrease of proliferation as determined by 3H-thymidine uptake. nt = non-targeting control. p values were obtained by Student’s t-test. *Indicates a significance of p < 0.05.