Molecular targeting of the deubiquitinase USP14 to circumvent cisplatin resistance in ovarian carcinoma and identification of novel inhibitors

Abstract

Aim: This study aims to investigate the biological role of the proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14) in ovarian carcinoma drug resistance and to identify novel USP14 inhibitors (USP14i) for further preclinical development. Methods: USP14 expression was evaluated in clinical samples from 134 ovarian carcinoma patients and in a broad panel of human ovarian carcinoma cell lines. Functional studies, including gain- and loss-of-function assays, migration and invasion, and apoptosis induction assays, were conducted using cisplatin-sensitive IGROV-1 cells and their cisplatin-resistant derivative IGROV-1/Pt1. A library of 1,056 small molecules was screened using an optimized hydrolysis assay. Docking and molecular dynamics simulations were employed to predict binding modes of candidate inhibitors within the USP14 domain. Results: In clinical specimens, USP14 mRNA expression was associated with tumor grade. Exogenous overexpression of USP14 enhanced the survival of cisplatin-resistant IGROV-1/Pt1 cells, but not parental IGROV-1 cells, upon cisplatin exposure. USP14 knockdown by small interfering RNAs in resistant cells reduced aggressive features and restored cisplatin sensitivity, whereas no sensitization was observed in IGROV-1 cells. Medium-throughput screening identified five candidate molecules, among which ARN12502 showed the strongest inhibitory activity against USP14. ARN12502 exhibited an IC₅₀ of 18.4 µM, and molecular dynamics simulations confirmed stable binding in two distinct modes. In proteasome sensor-expressing cells, ARN12502 displayed proteasome-inhibitory activity. Conclusion: USP14 contributes to the aggressiveness of ovarian carcinoma, particularly to the cisplatin-resistant phenotype, and represents a relevant promising druggable target. ARN12502 serves as a starting point for chemical optimization toward the development of more potent USP14i.

Keywords: Deubiquitinases; cisplatin; drug resistance; proteasome.

Figure 1

Relative expression of USP14 in ovarian carcinoma patients and USP14 protein expression and activity in different ovarian carcinoma cell lines. Box plots show USP14 expression according to (A) tumor grade, (B) diagnosis, and (C) stage. Each box represents the 25th-75th percentile range, with the horizontal line indicating the median and whiskers showing the extreme values; (D) Western blot analysis of USP14 protein expression in various ovarian carcinoma cell lines and their resistant variants. Actin or β-tubulin served as loading controls. Band intensities were quantified using ImageJ, normalized to the loading control, and expressed relative to IGROV-1. The histogram below reports mean ± standard deviation (SD) from three technical replicates; (E) USP14 activity in IGROV-1 and IGROV-1/Pt1 cells. Deubiquitinase labeling with HA-Ub-VS was followed by SDS-PAGE and immunoblotting with USP14. +, samples incubated with HA-Ub-VS; -, samples incubated without HA-Ub-VS. Vinculin served as loading control; the active band is indicated by the arrow. USP14: Ubiquitin-specific protease 14; HA-Ub-VS: hemagglutinin-ubiquitin-vinyl sulfone.

Figure 2

Colony-forming ability of IGROV-1 and IGROV-1/Pt1 cells overexpressing USP14. (A) Western blot analysis of USP14 protein expression in IGROV-1 and IGROV-1/Pt1 cells stably transfected with lentiviral particles carrying USP14 or a lentiviral control (ctr) vector; (B) IGROV-1 and (C) IGROV-1/Pt1 cells were seeded in 6-well plates for colony-forming assays on plastic. Twenty-four hours after seeding, cells were treated with cisplatin and incubated for 10 days. IC₅₀ value is the concentration inhibiting cell growth by 50% (IGROV-1: lenti ctr vector, IC₅₀ = 0.2 ± 0.01 µM; USP14 vector, IC₅₀ = 0.203 ± 0.087 µM; IGROV-1/Pt1: lenti ctr vector, IC₅₀ = 1.43 ± 0.245 µM; USP14 vector, IC₅₀ = 2.79 ± 0.29 µM; one-sided KW_E P = 0.05). USP14: Ubiquitin-specific protease 14.

Figure 3

Molecular targeting of USP14 in IGROV-1/Pt1 cells. (A) Quantitative RT-PCR analysis of USP14 mRNA levels after siRNA transfection; untransfected cells were used as calibrator, with GAPDH as housekeeping gene; (B) Western blot analysis of USP14 protein levels at different time points after siRNA transfection, with actin as loading control; (C) Colony-forming ability on plastic (left) and in soft agar (right) 48 h after transfection. For the plastic clonogenic assay, cells were continuously exposed to cisplatin and counted after 10 days. IC₅₀ is the concentration inhibiting cell growth by 50%: untransfected, 2.9 µM; negative control, 4.1 µM; USP14 siRNAa, 1.95 µM; USP14 siRNAb, 2.9 µM. For the agar assay, cells were treated with 100 µM cisplatin for 1 h, seeded in 0.33% agarose on a 0.5% agarose bed, and incubated for 2 weeks. Cell survival rates (treated versus control): 70% (untransfected), 60% (negative control), 33% (USP14 siRNAa), and 31% (USP14 siRNAb). Histograms show mean ± SD of at least three technical replicates; (D) Migratory and invasive ability of USP14-silenced cells 48 h after transfection using transwell chambers in serum-free medium. After 24 h, cells were fixed in ethanol, stained with 0.4% SRB, and counted under an inverted microscope. Graphs report mean ± SD of at least three technical replicates; error bars are not visible when values are nearly identical; (E) Quantitative RT-PCR of Axl mRNA levels after siRNA transfection. Untransfected cells were used as calibrator, and GAPDH served as housekeeping gene; (F) Apoptosis analysis in USP14-silenced cells. IGROV-1/Pt1 cells were seeded 48 h after transfection start and treated with cisplatin at IC₅₀ and IC₈₀ doses for 1 h, and apoptosis was assessed 24 h later by Annexin-V binding assay. Columns indicate total apoptosis. USP14: Ubiquitin-specific protease 14; RT-PCR: real-time PCR; siRNA: small interfering RNA; siRNAa: Silencer Select s17358; siRNAb: Silencer Select s17360; SD: standard deviation.

Figure 4

Molecular targeting of USP14 in IGROV-1 cells. (A) Quantitative RT-PCR analysis of USP14 mRNA levels after siRNA transfection; untransfected cells were used as calibrator, with GAPDH as housekeeping gene; (B) Western blot analysis of USP14 protein levels at different time points after siRNA transfection, with actin as loading control; (C and D) Colony-forming ability on plastic; (C) and in soft agar (D) 48 h after transfection. Graphs report mean ± SD of at least three technical replicates. For the plastic assay, cells were continuously exposed to cisplatin and counted after 10 days. IC₅₀ is the concentration inhibiting cell growth by 50%: untransfected, 0.27 µM; negative control, 0.36 µM; USP14 siRNAa, 0.20 µM; USP14 siRNAb, 0.19 µM. The histogram (right) shows colony numbers of untreated cells (mean ± SD, ≥ 3 technical replicates). For the agar assay, cells were treated with 3 µM cisplatin for 1 h, then seeded in 0.33% agarose on a 0.5% agarose bed, and incubated for 2 weeks. Cell survival (treated versus control): 45% (untransfected), 46% (negative control), 51% (USP14 siRNAa), and 66% (USP14 siRNAb); (E) Migratory ability of USP14-silenced cells assessed 48 h after transfection using transwell chambers in serum-free medium. After 24 h, cells were fixed in ethanol, stained with 0.4% SRB, and counted under an inverted microscope. Columns show mean ± SD of three technical replicates. USP14: Ubiquitin-specific protease 14; siRNA: small interfering RNA; siRNAa: Silencer Select s17358; siRNAb: Silencer Select s17360; SD: standard deviation.

Figure 5

Identification of new USP14i. Medium-throughput screening for USP14i was performed using Ub-Rho110-G hydrolysis assay. (A) Primary screening results of 1,056 compounds at 20 µM; (B) Chemical structure of ARN12502; (C) Representative inhibition curve of ARN12502. The dotted red line indicates basal Ub-VS 26S activity (~20% of total measured activity). The dose-response curve is representative of three independent experiments; (D) USP14 activity in IGROV-1/Pt1 cells after treatment with USP14i. Deubiquitinases were labeled with HA-Ub-VS, followed by western blotting. Cells were treated with 100 µM IU1-47 or ARN12502 for 24 h. +, samples incubated with HA-Ub-VS; -, samples incubated without HA-Ub-VS. Vinculin served as loading control. The histogram reports percentage inhibition of USP14 activity (mean ± SD, three technical replicates); (E) Proteasome perturbation analysis in U2OS/pZS cells using flow cytometry after treatment with USP14i for 24 h. IU1-47 was used as reference. Values represent mean ± SD of three technical replicates. USP14: Ubiquitin-specific protease 14; USP14i: USP14 inhibitors; SD: standard deviation; HA-Ub-VS: hemagglutinin-ubiquitin-vinyl sulfone.

Figure 6

Contact analysis of the ARN12502-USP14 complex from molecular dynamics (MD) simulations. (A) Bar plots showing interactions of ARN12502 with USP14, averaged over three replicates; error bars represent standard error. The left panel shows the persistence of contacts (% simulation time) between the ligand and the top 20 residues. The middle and right panels report the percentage of trajectory frames in which each interaction type was present, globally and by residue (top five mostly contacted), respectively; (B) 3D illustration of the most representative structure obtained by RMSD clustering, with ARN12502 in cyan and surrounding USP14 residues in grey, showing the coordination of the ligand inside the binding pocket; hydrogen atoms are omitted for clarity; (C) Schematic 2D representation corresponding to panel B, showing specific contacts; only interacting hydrogens of ARN12502 are displayed. Interaction types and residue classes are indicated in the legend. For clarity, representation is split into two panels. USP14: Ubiquitin-specific protease 14; 3D: three-dimensional; 2D: two-dimensional.