Melanoma is the deadliest skin cancer due to its heterogeneity, high metastatic potential and propensity to form resistance. Despite the current therapies, including targeted therapies against BRAF/MEK oncogenic pathway and immunotherapies, most of patients are still prone to therapeutic failure. Melanoma cell high plasticity is to blame for the rapid onset of therapeutic resistance through adaptation programs. One such program is the ability of melanoma cells to induce extracellular matrix (ECM) stiffening. This ECM remodeling will then be converted into a transcriptional program by the YAP mechanosensor that will promote a feed-forward mechanical loop leading to increase stiffess and therapy resistance. Identification and targeting of new actors of this loop of mechanoresistance can represent new opportunities to fight treatment resistance. Abnormal activation of such oncogenic pathway implicated in resistance often result in protein overexpression leading to proteostasis imbalance. To prevent this, cancer cells rely on the proteolytic activity of the ubiquitin-proteasome system on one hand. On the other hand, deubiquitinases (DUBs) contribute to proteostasis by removing ubiquitin from proteasome’s substrates thus stabilizing them. In cancers, DUBs can be hijacked to modulate oncogenic pathways, enhancing cell proliferation and/or invasion. In this context DUBs can be ideal candidate to find new actors of melanoma mechanoresistance.
To identify DUBs involved in melanoma mechanotransduction, we used cell lines cultivated on collagen matrices with various stiffnesses combined with an activity-based ubiquitin probe for profiling DUB activity. Using this approach and quantitative proteomic, we identified USP9X as a DUB whose activity is increased by collagen stiffness. In silico analysis further revealed that YAP belongs to the USP9X interactome and showed a correlation between USP9X expression and YAP transcriptional signature in melanoma. We thus hypothesized that USP9X regulates YAP levels in melanoma cells through its DUB activity. Consistently, siRNA-mediated depletion and pharmacological inhibition of USP9X decreased YAP expression at protein but not mRNA levels. Conversely knockdown of the YAP E3 ligase βTRCP increased YAP protein expression. A GST-TUBE pulldown approach revealed that combined USP9X and proteasome inhibition increased YAP poly-ubiquitination, indicating that USP9X deubiquitinates YAP to prevent its proteasomal degradation. In line with a role of USP9X in mechanotranduction upstream of YAP, USP9X targeting was found to impair stiffness-induced responses including YAP nuclear translocation and transcriptional activity, cell migration and invasion. Interestingly, we also show that the relationship between YAP and USP9X can be found in other tumor cell types, as well as in cancer-associated fibroblasts (CAFs), suggesting a broad action of USP9X in regulating tumorigenic mechanical pathways through YAP stabilization. Finally, in a melanoma syngeneic mouse model, targeting USP9X enhances BRAF/MEK inhibitor efficacy, counteracts drug-induced collagen remodeling and delays tumor relapse.
Overall, our results reveal an original role of USP9X in melanoma invasiveness and mechanoresistance through stiffness-dependent stabilization of YAP. We therefore propose USP9X as a “mechano-DUB” as well as a promising therapeutic target in refractory metastatic melanoma.
Cutaneous melanoma, Mechanotransduction, USP9X, YAP, migration
President of the jury:
Prof. Marie-Dominique GALIBERT, DR CNRS, University of Rennes
Dr Franck VERRECCHIA, DR INSERM, University of Nantes
Dr Corinne BOUSQUET, DR INSERM, University Paul Sabatier
Dr Marcel DECKERT, DR INSERM, University Côte d’Azur