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. 2010 Aug 27;39(4):493-506.
doi: 10.1016/j.molcel.2010.07.023.

STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer

Dimitrios Iliopoulos  1 , Savina A Jaeger, Heather A Hirsch, Martha L Bulyk, Kevin Struhl
Affiliations

STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer

Dimitrios Iliopoulos et al. Mol Cell. .

Abstract

A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-kappaB, Lin28, let-7, and IL-6. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Remarkably, transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD tumor suppressors, leading to increased NF-kappaB activity required to maintain the transformed state. These STAT3-mediated regulatory circuits are required for the transformed state in diverse cell lines and tumor growth in xenografts, and their transcriptional signatures are observed in colon adenocarcinomas. Thus, STAT3 is not only a downstream target of IL-6 but, with miR-21, miR-181b-1, PTEN, and CYLD, is part of the positive feedback loop that underlies the epigenetic switch that links inflammation to cancer.

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Figures

Figure 1
Figure 1. MicroRNA Dynamics during ER-Src cellular transformation
(A) Differentially expressed microRNAs between transformed (TAM 36h) and untransformed (0h) MCF10A ER-Src cells. (B) Heatmap representation of differentially expressed microRNAs in different time points (1, 2, 4, 8, 12, 16, 24, 36h) during ER-Src transformation. Up-regulated microRNAs are shown in red, while down-regulated microRNAs in green.
Figure 2
Figure 2. MicroRNAs Important for ER-Src transformation
(A) Strategy for identifying microRNAs regulating ER-Src transformation. (B) Heatmap representation of transformation efficiency (% transformed cells as assayed by cell morphology) after transfection of microRNAs or antisense microRNAs in ER-Src cells. (C) Soft agar colony assay and (D) invasion assay in ER-Src cells transfected with sense or antisense microRNAs. Experiments performed in triplicate and data are shown as mean ± SD.
Figure 3
Figure 3. STAT3 and Myc are important regulators of transformation
(A) Lever algorithm analysis to identify transcription factor binding site motifs over-represented in promoter areas of differentially expressed microRNAs. Motifs with AUC score higher than 0.7 and z-score higher than 2 are considered statistically significant (red color) with STAT3 an Myc indicated. (B) Pairs of motifs (connected by lines) that are differentially regulated (up-regulated in red and down-regulated in blue) during transformation and over-represented in microRNA promoters. (C) STAT3 and Myc mRNA expression levels in ER-Src cells during transformation. (D) Soft agar colony assay (mean ± SD) in TAM-treated (36h) cells transfected with siRNAs against STAT3, Myc, or control. (E) Soft agar colony assay (mean ± SD) in transformed ER-Src cells (for 10 days) transfected with siRNAs against STAT3, Myc, or control.
Figure 4
Figure 4. STAT3 and MYC-regulated microRNAs during ER-Src transformation
(A) STAT3 occupancy (fold-enrichment) at the indicated miR loci in cells that were or were not treated with TAM as determined by chromatin immunoprecipitation. (B) MicroRNA expression levels in TAM-treated cells ER-Src cells transfected in the presence of STAT3 inhibitors (siSTAT3#2 or 8 μM JSI-124). (C) MicroRNA expression levels in MCF-10A cells treated with 50 ng/ml IL6. (D) Soft agar colony assay in MCF10A-IL6 transformed cells transfected with the indicated antisense-microRNAs. (E) Myc occupancy (fold enrichment) at the indicated miR loci that were or were not treated with TAM. (F) MicroRNA expression levels in TAM-treated ER-Src cells transfected with siRNA negative control or siMyc. In all experiments, data are presented as mean ± SD of 3 independent experiments.
Figure 5
Figure 5. MiR-21 and miR-181b-1 induce transformation of MCF-10A cells
(A) Colony formation assay of MCF-10A cells treated with miR-21, miR-181b-1, Lin28b, BAY-117082 (5μM) or siRNA against Lin28B. (B) Cells treated with miR-21 or miR-181b-1 were propagated for 10 or 30 days and tested for cell morphology and colony formation. (C) NF-κB activity in MCF-10A cells treated with as-miR-NC, as-miR-21, as-miR-181b-1, or BAY-117082. The data are presented as mean ± SD of three independent experiments.
Figure 6
Figure 6. MiR-181b-1 regulates CYLD expression during ER-Src transformation
(A) Sequence complementarity between miR-181b-1 and the 3’UTR of CYLD gene. (B) MiR-181b-1 and CYLD mRNA expression levels at the indicated times during ER-Src transformation. (C) Luciferase activity of a reporter containing the 3’UTR of CYLD 24h after transfection with miR-181b-1 or miR negative control. (D) CYLD mRNA levels after treatment with miR-181b-1 or miR negative control. (E) CYLD protein levels after treatment with miR-181b-1 or miR negative control. (F) CYLD mRNA levels after treatment with as-miR-181b-1 or as-miR-NC. (G) NF-κB activity (ELISA assay) in ER-Src cells untreated (NT) or treated with as-miR-181b-1, si-CYLD, or as-miR-NC. (H) IL6 production (ELISA assay) in ER-Src cells treated with as-miR-181b-1, si-CYLD or as-miR-NC. (I) Number of colonies, (J) NF-κB activity assessed by ELISA assay and (K) IL6 luciferase activity in ER-Src cells treated with two different siRNAs against CYLD. The data are presented as mean ± SD of three independent experiments.
Figure 7
Figure 7. STAT3-regulated microRNAs in cancer cells, xenografts and cancer patients
(A) Colony formation assay in colon (HCT-116, HT-29), prostate (PC3), lung (A549), cervical (HeLa), hepatocellular (Hep3B) and pancreatic (Panc1) cancer cell lines treated with antisense microRNA negative control, antisense-miR-21, antisense-miR-181b-1 or their combination. The data are presented as mean ± SD of three independent experiments. (B) Tumor growth (mean ± SD) of ER-Src cells after i.p treatment (days 10, 15, 20, 25) with Ab-IgG, Ab-IL6, siRNA negative control, siRNA against STAT3, antisense miR-NC, antisense-miR-21 and/or antisense-miR-181b-1. (C) MiR-21 and miR-181b-1 expression levels from tumors derived from the experiment described above. (D) Tumor growth (mean ± SD) of HCT-116 and HT-29 colon cancer cells after intraperitoneal treatment (days 10, 15, 20, 25) with microRNA negative control or antisense-miR-21 or antisense-miR-181b-1 or siRNA against STAT3. (E) MiR-21, miR-181b-1 and STAT3 mRNA expression levels in colon adenocarcinomas, with each data point represents an individual sample and a correlation coefficients (r) indicated. (F) MiR-21, miR-181b-1, PTEN and CYLD mRNA expression levels in colon adenocarcinomas. (G) Model of the inflammatory positive feedback loop that mediates the epigenetic switch between non-transformed and transformed cells.
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