MicroRNA-424 impairs ubiquitination to activate STAT3 and promote prostate tumor progression
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
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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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 6

miR-424 induces STAT3 stability and activity by targeting COP1.

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miR-424 induces STAT3 stability and activity by targeting COP1. (A) Pie ...
(A) Pie chart showing the number of STAT3 targets among the genes induced by miR-424 extracted with Enrich (A, top) and functional annotation analysis by DAVID (A, bottom) of genes induced by miR-424 in RWPE1 cells. (B) COP1, STAT3, and p-STAT3 level (left) and relative luciferase activity (RLA) of STAT3 reporter (right) following transfection of miR-424 or control (Ctrl) in RWPE1 cells. (C) Immunoblot (IB) of COP1, STAT3, and p-STAT3 in indicated cell lines. (D) STAT3 level in LNCaP-424 stable cells by IB (bottom) and immunocytochemistry (ICC; top). (E) IB of COP1 and STAT3 in LNCaP cells after transient transfection of miR-424 or control (Ctrl) at the indicated time points. (F) IB of COP1, STAT3, and p-STAT3 in control (EV) and LNCaP-424 monoclonal cells with or without IL-6 (10 ng/ml) stimulation. (G) RLA of STAT3 reporter in cells transfected with negative control (Ctrl) or miR-424 with or without IL-6 stimulation. (H) IB of COP1, STAT3, and p-STAT3 (left) and RLA of STAT3 reporter (right) in DU145 cells following transfection with Anti-424 or Scr. (I and J) IB of COP1, STAT3, and p-STAT3 in RWPE1 (I) and LNCaP (J) cells after COP1 silencing. (K) IB of COP1, STAT3, and p-STAT3 in DU145 cells after transfection with full-length FLAG-COP1 (p-COP1) or empty vector (EV). (L) IB of DU145 cells transfected with p-COP1 with or without treatment with proteasome inhibitor PS-341 (10 μM) for 5 hours. c-Jun used as control. (M) IB of STAT3 and p-STAT3 in DU145 transfected with EV, p-COP1, or a deletion mutant (pRING). *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.