Loss of the proteasomal deubiquitinase USP14 induces growth defects and a senescence phenotype in colorectal cancer cells.

The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype. Transcriptomic analysis revealed altered gene expression related to cell division and cellular differentiation. USP14 knockout cells also exhibited changes in morphology, actin distribution, and expression of actin cytoskeletal components. Increased ubiquitin turnover was observed, offset by upregulation of polyubiquitin genes UBB and UBC. Pharmacological inhibition of USP14 with IU1 increased ubiquitin turnover but did not affect cellular growth or morphology. BioGRID data identified USP14 interactors linked to actin cytoskeleton remodeling, DNA damage repair, mRNA splicing, and translation. In conclusion, USP14 loss in colon cancer cells induces a transient quiescent cancer phenotype not replicated by pharmacologic inhibition of its deubiquitinating activity.

Transient interdomain interactions in free USP14 shape its conformational ensemble.

The deubiquitinase (DUB) ubiquitin-specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin-like (Ubl) domain separated by a 25-residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full-length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full-length USP14 by nuclear magnetic resonance spectroscopy and used small angle x-ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.

Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action.

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a "target" not necessarily connected to the "mechanism of action" thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.