Deubiquitinating enzymes stabilizing Snail1 transcription factor : a role for USP27X in epithelial-to-mesenchymal transition

Cancer cells undergo epithelial-to-mesenchymal transition (EMT) to dissociate from the primary tumor and to survive to chemotherapy. EMT is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAFs). Snail1 is short-lived in normal epithelial cells as a consequence of the coordinated and continuous ubiquitination by several F-box specific E3 ligases. We have performed a siRNA screening to identify deubiquitinases (enzymes removing the ubiquitin chains conjugated to a substrate), increasing Snail1 stability and identified the Ubiquitin Specific Protease 27X (USP27X) as an enzyme that interacts with, deubiquitinates and stabilizes Snail1. We observed that USP27X modulate EMT features through the stabilization of Snail1, such as tumor cell invasion, cancer metastasis and chemo-resistance. In addition, USP27X is essential for the TGFβ-induced EMT and the activation of CAFs modulated by this cytokine. To summarize, we identified USP27X as a new modulator of Snail1 stabilization and its repression could prevent the metastatic and chemo-resistant proteins of the tumoral cells.Les cèl·lules canceroses inicien un procés anomenat transició epiteli-mesènquima (EMT, per l’acrònim en anglès de epithelial-to-mesenchymal transition), per escapar del tumor primari i tornar-se resistents a la quimioteràpia. Aquest procés està estrictament regulat pel factor de transcripció Snail1 que, a més a més, és requerit per l’activació dels fibroblasts que envolten el tumor. Snail1 és una proteïna amb una vida mitja molt curta en les cèl.lules epitelials; això es deu a que és contínuament ubiqüitinat per l’activitat coordinada que exerceixen diferents enzims anomenats ligases d’ubiqüitina. Fent un screening de siRNAs, vam identificar deubiqüitinases (enzims que remouen la ubiqüitina) que podrien estar involucrades a l’estabilització de Snail1 i vam caracteritzar la USP27X com un enzim que interacciona, deubiqüitina i estabilitza aquest factor. Vam observar que USP27X modula a través de l’estabilització d’Snail1 trets relacionats amb la EMT com la invasió, la metàstasi i la quimioresistència de les cèl·lules tumorals. A més a més, USP27X és crucial per la inducció de la EMT i l’activació de fibroblasts depenents de TGFβ. En resum, hem caracteritzat USP27X com una proteïna clau en el control de l’estabilització d’Snail1 i el bloqueig d’aquesta deubiqüitinasa podria contrarestar les capacitats metastàtiques i quimioresistents de les cèl·lules tumorals

TGFβ-activated USP27X deubiquitinase regulates cell migration and chemoresistance via stabilization of snail1

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFβ during EMT and was required for TGFβ-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFβ-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.This study was funded by grants awarded by Ministerio de Economía y Competitividad (MINECO) and Fondo Europeo de Desarrollo Regional-FEDER to A. García de Herreros (SAF2013-48849-C2-1-R and SAF2016-76461-R) and to V.M. Díaz (SAF2013-48849-C2-2-R). Research at the A. García de Herreros lab is supported by funds from the Fundación Científica de la Asociación Española contra el Cáncer and from the Instituto de Salud Carlos III (PIE15/00008). Research at the B.S. Atanassov lab is supported by the Rosswell Park Cancer Institute and NCI grant P30CA016056. Research at the J. Arribas lab is supported by funds from the Breast Cancer Research Foundation (BCRF-17-008) and Instituto de Salud Carlos III (PI16/00253)

TGFβ-activated USP27X deubiquitinase regulates cell migration and chemoresistance via stabilization of snail1

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFβ during EMT and was required for TGFβ-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFβ-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.This study was funded by grants awarded by Ministerio de Economía y Competitividad (MINECO) and Fondo Europeo de Desarrollo Regional-FEDER to A. García de Herreros (SAF2013-48849-C2-1-R and SAF2016-76461-R) and to V.M. Díaz (SAF2013-48849-C2-2-R). Research at the A. García de Herreros lab is supported by funds from the Fundación Científica de la Asociación Española contra el Cáncer and from the Instituto de Salud Carlos III (PIE15/00008). Research at the B.S. Atanassov lab is supported by the Rosswell Park Cancer Institute and NCI grant P30CA016056. Research at the J. Arribas lab is supported by funds from the Breast Cancer Research Foundation (BCRF-17-008) and Instituto de Salud Carlos III (PI16/00253)