MicroRNA-424 impairs ubiquitination to activate STAT3 and promote prostate tumor progression
Cecilia Dallavalle, … , Carlo V. Catapano, Giuseppina M. Carbone
Cecilia Dallavalle, … , Carlo V. Catapano, Giuseppina M. Carbone
Published November 7, 2016
Citation Information: J Clin Invest. 2016;126(12):4585-4602. https://doi.org/10.1172/JCI86505.
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Research Article Oncology

MicroRNA-424 impairs ubiquitination to activate STAT3 and promote prostate tumor progression

Abstract

Mutations and deletions in components of ubiquitin ligase complexes that lead to alterations in protein turnover are important mechanisms in driving tumorigenesis. Here we describe an alternative mechanism involving upregulation of the microRNA miR-424 that leads to impaired ubiquitination and degradation of oncogenic transcription factors in prostate cancers. We found that miR-424 targets the E3 ubiquitin ligase COP1 and identified STAT3 as a key substrate of COP1 in promoting tumorigenic and cancer stem-like properties in prostate epithelial cells. Altered protein turnover due to impaired COP1 function led to accumulation and enhanced basal and cytokine-induced activity of STAT3. We further determined that loss of the ETS factor ESE3/EHF is the initial event that triggers the deregulation of the miR-424/COP1/STAT3 axis. COP1 silencing and STAT3 activation were effectively reverted by blocking of miR-424, suggesting a possible strategy to attack this key node of tumorigenesis in ESE3/EHF–deficient tumors. These results establish miR-424 as an oncogenic effector linked to noncanonical activation of STAT3 and as a potential therapeutic target.

Authors

Cecilia Dallavalle, Domenico Albino, Gianluca Civenni, Jessica Merulla, Paola Ostano, Maurizia Mello-Grand, Simona Rossi, Marco Losa, Gioacchino D’Ambrosio, Fausto Sessa, George N. Thalmann, Ramon Garcia-Escudero, Andrea Zitella, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. Carbone

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Figure 7

COP1 interacts with STAT3 and mediates STAT3 ubiquitylation and degradation.

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COP1 interacts with STAT3 and mediates STAT3 ubiquitylation and degradat...
(A) Immunoprecipitation of STAT3 and IB of COP1 and STAT3 (B) Immunoprecipitation of COP1 (p-COP1) with anti-FLAG and IB with anti-STAT3 in cells treated with PS-341 (10 μM for 6 hours). Asterisk: aspecific band. TUB, tubulin. (C) IB of STAT3 and COP1 in HEK293T cells transfected with indicated constructs. IP of COP1 with anti-FLAG and IB using indicated antibodies. Cells were treated with IL-6 (10 ng/ml for 45 minutes) and with 10 μM PS-341 for 3 hours. (D) STAT3 ubiquitination in RWPE1 cells transfected with siCOP1#1 or siGL3 for 48 hours. Lysates were immunoprecipitated with anti-STAT3 or IgG followed by IB with indicated antibodies. UBI,ubiquitin. (E) STAT3 ubiquitination in DU145 cells transfected for 48 hours and treated with PS-341 (10 μM for 3 hours). Lysates were immunoprecipitated with anti-STAT3 or IgG followed by IB with indicated antibodies. (F) IB of STAT3 and p-STAT3 following sequential transfection of miR-424 or control and p-COP1, the mutant construct (pRING), or empty vector (EV). (G) SFE in cells transfected as described in F. (H and I) RLA of STAT3 reporter in RWPE1 cells following transfection of siCOP1#1 or negative control (NC) (H) and in DU145 cells transfected with p-COP1 or EV (I). (J) STAT3 reporter activity following sequential transfection of miR-424 or control and p-COP1, mutant construct (pRING), or EV. (K) STAT3 reporter activity following transfection with siCOP1#1 or NC with or without IL-6. (L) IB of indicated proteins following IL-6 with and without COP1 knockdown. (M) STAT3 reporter activity following transfection of miR-424 or control and pCOP1, pRING, or EV in presence of IL-6. *P ≤ 0.05, **P ≤ 0.01 by 2-tailed Student’s t test. The experiments were performed in triplicate, and data are shown as mean ± SD.