Targeting proteasomal deubiquitinases USP14 and UCHL5 with b-AP15 reduces 5-fluorouracil resistance in colorectal cancer cells

Abstract

5-Fluorouracil (5-FU) is the first-line treatment for colorectal cancer (CRC) patients, but the development of acquired resistance to 5-FU remains a big challenge. Deubiquitinases play a key role in the protein degradation pathway, which is involved in cancer development and chemotherapy resistance. In this study, we investigated the effects of targeted inhibition of the proteasomal deubiquitinases USP14 and UCHL5 on the development of CRC and resistance to 5-FU. By analyzing GEO datasets, we found that the mRNA expression levels of USP14 and UCHL5 in CRC tissues were significantly increased, and negatively correlated with the survival of CRC patients. Knockdown of both USP14 and UCHL5 led to increased 5-FU sensitivity in 5-FU-resistant CRC cell lines (RKO-R and HCT-15R), whereas overexpression of USP14 and UCHL5 in 5-FU-sensitive CRC cells decreased 5-FU sensitivity. B-AP15, a specific inhibitor of USP14 and UCHL5, (1-5 μM) dose-dependently inhibited the viability of RKO, RKO-R, HCT-15, and HCT-15R cells. Furthermore, treatment with b-AP15 reduced the malignant phenotype of CRC cells including cell proliferation and migration, and induced cell death in both 5-FU-sensitive and 5-FU-resistant CRC cells by impairing proteasome function and increasing reactive oxygen species (ROS) production. In addition, b-AP15 inhibited the activation of NF-κB pathway, suppressing cell proliferation. In 5-FU-sensitive and 5-FU-resistant CRC xenografts nude mice, administration of b-AP15 (8 mg·kg^-1·d^-1, intraperitoneal injection) effectively suppressed the growth of both types of tumors. These results demonstrate that USP14 and UCHL5 play an important role in the development of CRC and resistance to 5-FU. Targeting USP14 and UCHL5 with b-AP15 may represent a promising therapeutic strategy for the treatment of CRC.

Keywords: 5-FU-resistant colorectal cancer; UCHL5; USP14; b-AP15; deubiquitinase; proteasome deubiquitinase inhibitors.

Fig. 1. USP14 and UCHL5 may play important roles in the 5-FU resistance of CRC cells.

a RNA expression levels are presented based on analysis of public GEO datasets (GSE8671). b Immunohistochemical results of USP14 and UCHL5 in CRC tumor and adjacent normal tissues. c The overall survival curve is presented based on analysis of public GEO datasets (GSE12945). d Western blot analysis of the indicated protein levels of USP14 and UCHL5 in 5-FU-sensitive and 5-FU-resistant CRC cells. Relative quantification of USP14 and UCHL5 expression levels is shown. *P < 0.05. RKO-R: 5-FU-resistant RKO cells. HCT-15R: 5-FU-resistant HCT-15 cells. e Western blot analysis of the indicated protein levels in the indicated USP14 and/or UCHL5 knockdown CRC cells. shControl (a nonspecific scramble shRNA) served as a negative control. shUSP14: shRNA targeting the USP14 transcript; shUCHL5: shRNA targeting the UCHL5 transcript; shUSP14+shUCHL5: shRNAs targeting the USP14 (#2) and UCHL5 (#1) transcripts. f Cell viability of USP14 and/or UCHL5 knockdown CRC cells was measured by MTS assay. Mean ± SD (n = 3). ****, P < 0.0001 versus control. g Cell viability of USP14 and/or UCHL5 knockdown CRC cells treated with the indicated doses of 5-FU for 48 h. h Western blot analysis of the indicated protein levels in USP14- and/or UCHL5-overexpressing CRC cells. An empty PCDH vector was used as a control. USP14 OE: USP14 overexpression; UCHL5 OE: UCHL5 overexpression; USP14 + UCHL5 OE: USP14 and UCHL5 overexpression. i Viability of USP14- and/or UCHL5-overexpressing CRC cells treated with the indicated doses of 5-FU for 48 h.

Fig. 2. b-AP15 effectively inhibits cell viability and cell migration of both 5-FU-sensitive and 5-FU-resistant CRC cell lines.

a HA-Ub-VS assay for testing the effects of b-AP15 on the deubiquitinase activity of USP14 and UCHL5 in CRC cells. HA-Ub-VS is a probe that binds to deubiquitinase active sites. b Western blot analysis of the indicated protein levels in CRC cells treated with various doses of b-AP15 for 6 h. c Cell viability of CRC cells treated with various doses of b-AP15 for 48 h. d Cell proliferation (OD at 490 nm) of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 1 to 5 days. Cell viability was detected by MTS assay. Mean ± SD (n = 3). **P < 0.01, ***P < 0.001. e Images and quantification of plate colony formation of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 2 weeks. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. f Migration of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 24 h. Cell migration was detected by Transwell assay. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance.

Fig. 3. b-AP15-induced apoptosis is related to mitochondrial dysfunction and caspase activation.

a Cell death of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 24 h. Cell death was detected by Annexin V/PI staining. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. b Cell death of 5-FU-sensitive and 5-FU-resistant CRC cells treated with 1 μM b-AP15 for the indicated time periods. Cell death was detected by Annexin V/PI staining. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. c, d Mitochondrial membrane potential of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 24 h. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MMP, mitochondrial membrane potential. e ATP content of 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 24 h. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. f, g Western blot analysis of the indicated protein levels in 5-FU-sensitive and 5-FU-resistant CRC cells treated with various doses of b-AP15 for 24 h or 1 μM b-AP15 for the indicated time periods. Cas3, caspase-3; C-C3, cleaved caspase-3; Cas8, caspase-8; Cas9, caspase-9; C-C9, cleaved caspase-9.

Fig. 4. b-AP15 suppresses NF-κB activation in both 5-FU-sensitive and 5-FU-resistant CRC cells.

a Western blot analysis of the indicated protein levels in CRC cells treated with or without 5-FU (30 μM, 12 h). b Western blot analysis of the indicated protein levels in CRC cells treated with TNF-α (25 ng/mL) for the indicated time periods. The cells were pretreated with DMSO (control) or b-AP15 (1 μM) for 12 h. c Immunofluorescence staining of p65 in RKO and RKO-R cells treated with TNF-α (25 ng/mL) for 1 h. The cells were pretreated with DMSO (control) or b-AP15 (1 μM) for 24 h. d NF-κB luciferase activity of RKO and RKO-R cells treated with TNF-α (25 ng/mL) for 1 h. The cells were pretreated with DMSO (control) or b-AP15 (1 μM) for 24 h. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Western blot analysis of the indicated protein levels in CRC cells treated with TNF-α (25 ng/mL) for 1 h. The cells were pretreated with DMSO (control) or b-AP15 (1 μM) for 24 h. f NF-κB luciferase activity of USP14- and/or UCHL5-overexpressing RKO and RKO-R cells treated with TNF-α (25 ng/mL) for 1 h. The cells were pretreated with DMSO (control) or b-AP15 (1 μM) for 24 h. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. b-AP15 reverses 5-FU resistance in CRC cells through the induction of ROS.

a The mitochondrial ROS levels of 5-FU-sensitive and 5-FU-resistant CRC cells were detected by MitoSOX staining using flow cytometry. b The mitochondrial ROS level of 5-FU-sensitive and 5-FU-resistant CRC cells treated with the indicated doses of b-AP15 for 12 h. *P < 0.05, **P < 0.01, ****P < 0.0001, versus control group. c The ROS level of 5-FU-sensitive and 5-FU-resistant CRC cells treated with the indicated doses of b-AP15 for 24 h. ROS were detected by DCFH-DA staining using flow cytometry. ***P < 0.001, ****P < 0.0001, versus the control group. d The ROS level of 5-FU-sensitive and 5-FU-resistant CRC cells treated with b-AP15 (1 µM) and/or NAC (20 mM) for 48 h. ***P < 0.001, ****P < 0.0001. e Cell death of RKO and RKO-R cells treated with b-AP15 (1 µM) and/or NAC (20 mM) for 48 h. *P < 0.05, **P < 0.01, ****P < 0.0001. f Western blot analysis of the indicated protein levels in CRC cells treated with/without the indicated doses of b-AP15 for 48 h. g The ROS level of USP14 and/or UCHL5 knockdown 5-FU-sensitive and 5-FU-resistant CRC cells. h Western blot analysis of the indicated protein levels in USP14 and/or UCHL5 knockdown 5-FU-sensitive and 5-FU-resistant CRC cells.

Fig. 6. b-AP15 restrains the growth of 5-FU-sensitive and -resistant CRC xenografts in nude mice.

Nude mice (n = 6/group) bearing RKO and RKO-R cells were treated with either vehicle or b-AP15 (8 mg·kg⁻¹·d⁻¹) for the indicated time periods. a At the end of the experiment, the mice were sacrificed, and the tumor tissues were weighed, imaged and summarized. Representative images are shown (n = 6/group). ***P < 0.001 versus control group. b The effects of b-AP15 on tumor growth. Mean ± SD (n = 6). *P < 0.05. c The effects of b-AP15 on mouse weights. Mean ± SD (n = 6). d Immunohistochemistry and H&E staining of the indicated proteins were analyzed. Representative images are shown.

Fig. 7. Scheme summarizing the mechanisms of action of b-AP15 on 5-FU-resistant CRC cells.

Proteasomal deubiquitinases USP14 and UCHL5 play a key role in 5-FU resistance in CRC cells. b-AP15 impaired proteasome function by binding with USP14 and UCHL5. Accordingly, b-AP15 or knockdown of USP14 and UCHL5 induces apoptosis and attenuates proliferation through induction of ROS and inhibition of NF-κB in 5-FU-resistant CRC cells. Images were generated using Biorender.com.