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. 2009 Nov 13;139(4):693-706.
doi: 10.1016/j.cell.2009.10.014. Epub 2009 Oct 29.

An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation

Dimitrios Iliopoulos  1 , Heather A Hirsch, Kevin Struhl
Affiliations

An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation

Dimitrios Iliopoulos et al. Cell. .

Abstract

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. We show that transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal.

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Figures

Figure 1
Figure 1
Epigenetic switch from untransformed to a transformed phenotype (A) Number of mammosphere formed/1000 seeded transformed (TAM-treated for 36h; mean ± SD) ER-Src cells during 12 serial passages. (B) Levels of Src-Y419 phosphorylation (ELISA assay; mean ± SD) in untreated, and TAM-treated (36h), and 1st, 2nd, 4th, and 8th generation mammospheres derived from TAM-treated ER-Src cells. (C) Kinetics of cellular transformation as a function of TAM exposure. ER-Src cells were treated with 1 μM TAM for the indicated times, and then analyzed for cellular transformation (defined by morphological changes) 24, 48 and 72h post TAM treatment. (D) Phase-contrast images and Src-Y419 phosphorylation levels (ELISA assay; mean ± SD) in ER-Src cells 36 and 120h post TAM treatment. Cells were treated with TAM for 36h and then TAM was removed from the medium.
Figure 2
Figure 2
Src induces an inflammatory response mediated by NF-κB (A) Heatmap representation of RNA levels for the indicted inflammatory genes at the indicated time points after TAM induction. (B) IL6 levels (ELISA assay; mean ± SD)) at the indicated times after TAM treatment. The black arrows show the biphasic induction of IL6. (C) Phosphorylation status of IκBα-serine 32 (ELISA assay) in cells treated with TAM for the indicated times. (D) NF-κB/p65 ActivELISA assay (NF-κB nuclear localization) cells treated with TAM for the indicated times. (E) Soft agar colony assay (mean ± SD) in untreated and TAM-treated cells in the presence or absence of NF-κB inhibitors (5 μM BAY-117082 and 6 μM JSH-23).
Figure 3
Figure 3
Let-7a is down-regulated during transformation by NF-κB. (A) RNA levels of individual let-7 family members in cells treated with TAM for the indicated times. (B) Levels of let-7a RNA in untreated and TAM-treated (36h) cells in the presence or absence of NF-κB inhibitor (5 μM BAY-117082 and 6 μM JSH-23). (C) Lin28B protein levels (western blot) in ER-Src cells at the indicated times after TAM treatment. (D) Lin28B mRNA levels (mean ± SD) during transformation in the presence or absence of an NF-κB inhibitor (5 μM BAY-117082). (E) Levels of precursor RNAs (pri-let-7a and pri-let-7d/f) in untreated and TAM-treated cells. (F) NF-κB occupancy (fold-enrichment) at the Lin-28B and let-7a loci as determined by chromatin immunoprecipitation of crosslinked cells that were or were not treated with TAM. (G) Luciferase activity (mean ± SD)) of Lin28B vector containing the NF-κB binding site during transformation. (H) Luciferase activity of Lin28B vector (wt, mutated or deleted in NF-κB site) in TAM-treated (36h) cells. (I) Anchorage-independent growth assays (microscopic counting of 50 μm colonies) of untreated and TAM-treated cells transfected with the indicated let-7 family members or siRNA against Lin28B.
Figure 4
Figure 4
Let-7a regulates IL6 expression during transformation (A) Let-7a binding site in 3′UTR of IL6 with sequence complementarity and phylogenic conservation (yellow) of IL6 target sequence indicated. (B) Luciferase assay using a reporter containing the 3′UTR of IL6 (wt or mutant in let-7a binding site) 24h after transfection with let-7a or a scrambled miR control. (C) Physical interaction between let-7a and IL6 mRNA in the context of the RISC complex. IL6 RNA levels in HA-Ago1 immunoprecipitates in HEK-293 cells co-transfected with a plasmid expressing HA-Ago1 and let-7a or scrambled microRNA control. (D) IL6 RNA levels after treatment for 24h with antisense-let-7a (100 nM) or Lin28B. (E) Phosphorylation of STAT3-Y705 ELISA assay; mean ± SD) in untreated and TAM-treated (36h) cells in addition to treatment with increasing concentrations (50, 80, 100 nM) of let-7a and siRNA against Lin28B (80 nM). (F) VEGF and IL6 production (ELISA assay; mean ± SD) in TAM-treated cells before and after transfection (100 nM) of the indicated let-7 microRNAs. (G) Western blot analysis of RAS protein expression in 36h TAM-treated cells after let-7a (100 nM) overexpression; GAPDH levels were used as a loading control. (H) IL6 production (ELISA assay during transformation after transfection with negative control siRNA or siRNA against Ras or let-7a (100 nM). (I) Soft agar colony assay in TAM-treated cells after treatment with 2 μg/ml of monoclonal antibody against IL6 (Ab-IL6) and IgG isotype antibody (Ab-IgG) or siRNA negative control and siRNA against Ras (100 nM).
Figure 5
Figure 5
IL6 signaling pathway regulates MCF10A ER-Src transformation (A) Representative phase contrast images of ER-Src cells that were or were not treated with TAM and an antibody against IL6 (2 μg/ml). (B) Kinetics of cellular transformation (mean ± SD) in untreated and TAM-treated (5 minutes) cells together with 0.5 and 2 ug/ml of a monoclonal antibody against IL6 (Ab-IL6) or an isotype (Ab-IgG). (C) Soft agar colony assay in untreated and IL6-treated (1h) MCF10A cells. (D) Tumor growth of MCF10A and IL6-treated (1h) MCF10A cells in nude mice (5 mice/group). All the mice in the IL6-treated group developed tumors. (E) Kinetics of cellular transformation in IL6-treated (1h) MCF10A cells. (F) Soft agar colony assay in IL6-transformed MCF10A cells after treatment with 2ug/ml Ab-IL6 or Ab-IgG (control). (G) Let-7a expression in untreated and IL6-transformed MCF10A cells in the presence or absence of NF-κB inhibitors (5 μM BAY-117082 and 6 μM JSH-23). (H) RNA levels for the indicated genes in cells treated with TAM or an IL6 antibody. (I) Levels of phosphorylated STAT3 (pStat3; ELISA assay and western blot) in cells that were or were not treated with TAM and an antibody against IL6 2 (νg/ml Ab-IL6). (J) Colony formation assay of untreated and TAM-treated cells in the presence of absence of an siRNA against Stat3 (80nM). (K) Tumor incidence of subcutaneously injected MCF10A ER-Src cells treated for 24h with Stat3 inhibitor (7uM JSI-124). The number of palpable tumors at 4 weeks is shown.
Figure 6
Figure 6
The positive feedback regulatory circuit is required for maintenance of the transformed phenotype and cancer stem cell population. (A) Kinetics of cellular transformation in TAM-treated (5 minutes and 36h) cells. (B) Levels of Src phosphorylation (ELISA assay), (C) Lin28B mRNA (real-time PCR), (D) let-7a (real-time PCR), (E) IL6 protein (ELISA assay) and (F) Stat3 phosphorylation (ELISA assay) in TAM-treated (5 minutes and 36h) cells (G) Colony formation assay in ER-Src cells (15 days post TAM treatment) treated with 2 μg/ml monoclonal antibody against IL6 (Ab-IL6), 80 nM siRNA against Lin28B, STAT3 inhibitor (7 μM JSI-124) and 80 nM siRNA against NF-κB (p65). Treatments with 2 μg/ml isotype antibody (Ab-IgG) and 80 nM negative control siRNA were used as controls. (H) Expression levels of the indicated RNAs (mean ± SD) in untreated cells and sorted CD44high/CD24low and CD44low/CD24high TAM-treated cells. (I) Let-7a and Lin28B RNA levels (mean ± SD) in untreated and sorted CD44high/CD24low and CD44low/CD24high TAM-treated cells. (J) Number of mammospheres/1000 seeded cells formed by TAM-treated cells in the presence or absence of 2 μg/ml Ab-IgG or Ab-IL6; 100 nM negative control siRNA or siRNA against IL6; 4 μg/ml Ab-IL6R; 5 μM BAY-117082.
Figure 7
Figure 7
Positive inflammatory feedback loop in cancer cells, xenografts and cancer patients. (A) Colony formation assay in A549 (lung), HepG2 (hepatocellular), MDA-MB-231 (breast), PC3 (prostate) and Caco2 (colon) cancer cell lines treated with 2 μg/ml Ab-IgG (control), 2 μg/ml Ab-IL6, 80 nM siRNA control, 80 nM siRNA against Lin28B, 100 nM microRNA scrambled control and 100nM let-7 microRNA for 24h. (B) Tumor growth of ER-Src cells after i.p treatment (days 15, 20, 25) with siRNA negative control, siRNA against Lin28B, BAY-117082, Ab-IgG and Ab-IL6. (C) Expression levels of Lin28B, let-7 and IL6 from tumors derived from the experiment described above. (D) Lin28B, let-7a and IL6 expression in breast, prostate, hepatocellular and lung cancer and normal tissues. Each data point represents an individual sample, and a correlation coefficient (r) between let-7a and IL6 expression is shown. (E) Schematic overview of inflammatory positive feedback loop during cellular transformation.
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