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Acta Pharmacologica Sinica logoLink to Acta Pharmacologica Sinica
. 2023 Aug 1;44(12):2537–2548. doi: 10.1038/s41401-023-01136-0

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

Wa Ding 1,2,#, Jin-xiang Wang 3,#, Jun-zheng Wu 1,#, Ao-chu Liu 1,#, Li-ling Jiang 1, Hai-chuan Zhang 1, Yi Meng 1, Bing-yuan Liu 1, Guan-jie Peng 1, En-zhe Lou 1, Qiong Mao 1, Huan Zhou 1, Dao-lin Tang 4, Xin Chen 1,✉, Jin-bao Liu 1,✉, Xian-ping Shi 1,✉
PMCID: PMC10692219  PMID: 37528233

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: USP14, UCHL5, deubiquitinase, 5-FU-resistant colorectal cancer, proteasome deubiquitinase inhibitors, b-AP15

Introduction

Colorectal cancer (CRC) is a major cause of cancer-related mortality and incidence worldwide [1]. However, with the development of comprehensive treatment methods, such as surgery, chemotherapy, radiotherapy, and immunotherapy, the overall survival rate for CRC patients has significantly improved [2]. Among these treatments, 5-fluorouracil (5-FU) is considered a standard chemotherapy agent for high-risk stage II and III CRC [3]. 5-FU works as an antimetabolic chemotherapeutic agent by converting into active metabolites such as FdUMP, FdUTP, and FUTP, which can cause RNA and DNA damage [4]. The use of 5-FU has led to an increase in overall and disease-free survival rates. In advanced CRC, combining 5-FU with irinotecan and oxaliplatin has enabled a significant increase in the overall response rate, from 10%–15% for single-dose 5-FU administration to 40%–50% [5, 6]. However, poor tolerance to 5-FU and the development of acquired resistance to it are still significant challenges in cancer treatment [7]. The potential mechanisms of 5-FU resistance include overexpression of antiapoptotic proteins, epithelial-mesenchymal transition, and abnormal activation of proliferation pathways such as NF-κB signaling [8]. A better understanding of 5-FU resistance may aid in the development of new approaches to improve patient outcomes.

Deubiquitinases play a key role in the protein degradation pathway, which is involved in cancer development and chemotherapy resistance [9]. In mammalian cells, the 19 S proteasome contains three deubiquitinases: USP14, UCHL5, and RPN11. Both USP14 and UCHL5 are cysteine-protease deubiquitinases, while RPN11 is a metalloprotease. USP14 is involved in malignant progression and worse prognosis of survival in various types of cancer, including prostate cancer, non-small cell lung cancer, and hepatocellular carcinoma [10]. Inhibition of USP14 decreases cell proliferation, migration, and invasion and induces cell growth arrest, apoptosis, and autophagy in cancer cells [11–13]. While there is limited evidence pertaining to UCHL5’s involvement in tumor development, it has been found to accelerate the growth of endometrial cancer and is linked to poor clinical outcomes in lung adenocarcinoma [14, 15] but is associated with improved survival in gastric and rectal cancer [16, 17].

b-AP15 is a proteasome inhibitor that works by selectively blocking the deubiquitinating enzyme activity of USP14 and UCHL5 [18]. It has been found to be effective in treating various types of cancer, including head and neck squamous cell carcinoma, large B-cell lymphoma, and pancreatic adenocarcinoma, by inducing a proteotoxic stress response, which results in suppressed proliferation and induced apoptosis [19–22]. Moreover, it has demonstrated promising results in overcoming resistance to cisplatin in urothelial carcinoma and tyrosine kinase inhibitors in chronic myeloid leukemia [23, 24]. However, the effects of inhibiting USP14 and UCHL5 on the development of CRC and resistance to 5-FU have not yet been investigated.

In this study, we uncovered potential roles of USP14 and UCHL5 in the development of 5-FU resistance in CRC. We demonstrated that treatment with b-AP15, which targets USP14 and UCHL5, effectively induced cell death in both 5-FU-sensitive and 5-FU-resistant CRC cells, both in vitro and in vivo. Thus, b-AP15 may hold promise as a therapeutic agent for reducing 5-FU resistance in CRC.

Materials and methods

Materials

b-AP15 was purchased from Selleck Chemicals (#S4920, Houston, TX, USA). MitoSOX red mitochondrial superoxide (ROS) indicator was purchased from Yeasen (#40778ES50, Shanghai, China). The DCFDA cellular ROS detection assay kit was purchased from Abcam (ab113851, Cambridge, UK). The following antibodies were purchased from Cell Signaling Technology [CST], Boston, MA, USA): PARP (Cell Signaling Technology [CST], #9532 S), USP14 (CST, #11931 S), caspase-3 (CST, #9662 S), caspase-8 (CST, #9746 S), caspase-9 (CST, #9508 S), c-MYC (CST, #18583 S), XIAP (CST, #2042 S), survivin (CST, #2808 S), cyclin D1 (CST, #2926p), p65 (CST, #8242 S), XIAP (CST, #2042 S), IκBα (CST, #4814 S), p-IKBα (CST, #2859p), p-Ikkα/β (CST, #2697p), lamin B1 (CST, #13435 S), Ki67 (CST, #9449 S). The following antibodies were purchased from Proteintech (Wuhan, China): G6PD (Proteintech, #25413-1-AP), catalase (Proteintech, #19792-1-AP), Mcl-1 (Proteintech, #16225-1-Ap), Bcl-xl (Proteintech, #66020-I-Ig), GAPDH (Proteintech, 10494-i-ap), β-actin (Proteintech, 205365-i-ap). The following antibodies were purchased from Abcam (Cambridge, UK): cleaved caspase-3 (Abcam, #ab2302), cleaved caspase-9 (Abcam, #ab3629), UCHL5 (Abcam, #ab133508). Ubiquitin was purchased from Santa Cruz Biotechnology (#SC-8017, Helena, MT, USA) and p-p65 was purchased from Bioworld Tech (#BS4138, MN, USA).

Cell culture

The colorectal cancer (CRC) cell lines RKO and HCT-15 were purchased from ATCC (Manassas, VA, USA), while RKO-5-FU-resistant (RKO-R) and HCT-15 5-FU-resistant (HCT-15R) CRC cell lines were generated as previously described [25, 26]. Briefly, 5-FU-resistant cell lines were established by stepwise exposure of their parental cell lines to increasing doses of 5-FU, from 1 μM to 100 μM. To maintain drug resistance, 5-FU (1 μM) was routinely included in the culture medium for 5-FU-resistant cells. The 5-FU-resistant cells were replaced with 5-FU-free medium at least 2 weeks before the experiments. They were incubated in RPMI-1640 medium supplemented with 10% fetal calf serum.

Cell viability assay

Cell viability was detected by MTS reagent (Promega, #G3581, Madison, WI, USA). Briefly, 3 × 103 CRC cells were treated with DMSO as a control or b-AP15 for the indicated time periods. Four hours before cell viability determination, 20 μL of MTS mixed with 80 μL of RPMI-1640 medium was added to each well. The absorbance density was read at a wavelength of 490 nm using a microplate reader.

Cell death assay

CRC cells were treated with various concentrations of b-AP15 for the indicated time periods. Flow cytometry with Annexin V/PI double staining (Sungene Biotech, #AO2001-02P-G, Tianjin, China) was applied to measure cell apoptosis. CRC cells were sequentially collected, washed, and resuspended in a binding buffer. After staining with Annexin V and PI, samples were detected by a FACSCalibur flow cytometer and analyzed by CellQuestPro software.

Clone formation assay

In total, 500 RKO or RKO-R cells and 1000 HCT-15 and HCT-15R cells were plated into six-well plates and cultured in RPMI-1640 medium with 10% FBS for 10–14 days. Then, the cells were immobilized with 4% paraformaldehyde and stained with 2% crystal violet (Beyotime, #C0121, Beijing, China) at room temperature for 30 min. The colonies were counted under an optical microscope.

Measurement of mitochondrial membrane permeability

The mitochondrial membrane potential was detected by a commercial kit (Sigma‒Aldrich, #MAK-148, St Louis, MO, USA). CRC cells were treated with b-AP15, harvested, and resuspended in RPMI-1640 medium. Then, the cells were incubated with mitochondrial potential dye (5 μL) at 37 °C for 30 min. After centrifugation, the cells were resuspended in 500 μL assay buffer, followed by monitoring of the cells using flow cytometry.

Western blot analysis

Cells were lysed by RIPA buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× Roche Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Western blotting was performed by using proper primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies as we previously described [27].

HA-Ub-VS assay

CRC cells were harvested and lysed using DUB buffer (25 mM Tris-HCl, 20 mM NaCl, 5 mM MgCl2, 200 μM ATP). The lysate was incubated with b-AP15 for 1 h. After that, 1 μM HA-Ub-VS (R&D System, #U-212-025, Minneapolis, MN, USA) was added and incubated at 37 °C for 1 h. The lysates were mixed with SDS‒PAGE loading buffer and analyzed using Western blot analysis.

Cell migration assay

CRC cells were treated with different concentrations of b-AP15 for 24 h. Sequentially, cells were collected and resuspended in serum-free RPMI-1640 medium at a concentration of 1 × 106 cells/mL. Thereafter, the upper chambers with a pore size of 8 μm were filled with 100 μL of cell suspension (1 × 105 cells), while the lower chambers were supplemented with 600 μL of complete medium. Living cells in the lower chambers were calculated.

Plasmids and shRNA infection

USP14 and UCHL5 overexpression plasmids were purchased from IGEbio company (Guangzhou, China). Briefly, USP14 and UCHL5 cDNA were subcloned into the PCDH-CMV-MCS-EF1-puro plasmid. The lentiviral vector was digested by EcoRI and BamHI. Lentiviral vector particles were produced by transfection of plasmids harboring the packaging construct, the transfer vector, and the envelope-expressing construct into 293 T cells using the DNAfect reagent polyethylenimine (Polysciences, #239661, Niles, IL, USA). Viral supernatants were harvested and used for infection assays. Stable populations were selected with an appropriate concentration of puromycin (Sigma‒Aldrich, #P7255, St Louis, MO, USA).

A lentiviral U6-based expression vector containing PLKO.1-EF1a-copGFP-T2A-puro was used to express shRNAs. The lentiviral vector was digested by Age I and EcoR I, followed by annealed shRNA oligo insertion into cloned shRNA expression plasmids. The sequences of the shRNAs (purchased from IGEbio Corporation) are as follows:

USP14-shRNA #1,

5′-CCGGCCTTGGTAACACTTGTTACCTCGAGGTAACAAGTGTTACCAAGGTTTTTGAATT-3′;

USP14-shRNA #2,

5′-CGGCGCAGAGTTGAAATAATGGCTCGAGCCATTATTTCAACTCTGCGTTTTTGAATT-3′.

UCHL5-shRNA #1,

5′-CCGGCATTTAGGCGAGACATTATCTCGAGATAATGTCTCGCCTAAATGTTTTTGAATT-3′;

UCHL5-shRNA #2,

5′-CGGGCTCATTAAAGGATTCGGTCTCGAGACCGAATCCTTTAATGAGCTTTTTGAATT-3′.

Nude mouse xenograft model

The experimental nude mice were purchased from laboratory animal center of Guangzhou University of Chinese Medicine. All mice experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Guangzhou Medical University (GY2018‐019). Balb/c mice (5 weeks old) were bred at the animal facility of Guangzhou Medical University and maintained in barrier facilities with light/dark (12 h/12 h) cycles, with food and water available ad libitum. RKO and RKO-R cells (4 × 106) were injected into the armpits of the mice. When the average tumor volume reached 50 mm3, mice were randomly separated into the vehicle group (Cremophor EL: PEG400: saline = 2: 2: 4, n = 6) and b-AP15 group (8 mg·kg−1·d−1, n = 6) and treated for the indicated days. Tumor volumes were calculated as a2 × b × 0.4 (“a” is the smallest diameter and “b” is the diameter perpendicular to “a”). The mice were sacrificed when the maximum tumor reached about 1500 mm3, then the tumor tissues were isolated from the mice body and weighed. The tumor tissues were lysed for Western blot analysis or fixed for immunohistochemical staining.

Ethics approval and patient consent

This present study was approved by the Ethics Committee of The Sixth Affiliated Hospital of Guangzhou Medical University. Patients were recruited with CRC signed consent forms.

Statistical analysis

All tests were repeated at least three times and expressed as the mean ± SD where applicable. Comparison of multiple groups was performed with one-way ANOVA followed by Tukey’s test or Newman‒Keuls test. P < 0.05 was considered statistically significant.

Results

USP14 and UCHL5 contribute to 5-FU resistance in CRC cells

To investigate the role of the proteasomal deubiquitinases USP14 and UCHL5 in CRC, we analyzed GEO datasets and observed higher mRNA expression levels of USP14 and UCHL5 in CRC tissues than in normal colorectal tissues (Fig. 1a). Immunohistochemistry staining of tumor tissues and adjacent normal tissues from CRC patients also confirmed upregulated USP14 and UCHL5 protein levels in CRC tissue (Fig. 1b). Additionally, the expression of USP14 and UCHL5 was negatively correlated with the survival of CRC patients (Fig. 1c). We then investigated the relationship between these two genes and 5-FU resistance. Using two pairs of 5-FU-sensitive cells (RKO and HCT-15) and their resistant counterparts (RKO-R and HCT-15R) [25, 26], we assessed the expression of USP14 and UCHL5. Consistent with our hypothesis, USP14 and UCHL5 were highly expressed in 5-FU-resistant CRC cells compared to their 5-FU-sensitive counterparts (Fig. 1d and Supplementary Fig. S1).

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

Fig. 1

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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.

To examine the role of USP14 and UCHL5 in 5-FU resistance, we conducted experiments on both 5-FU-sensitive and 5-FU-resistant cell lines by individually and simultaneously knocking down USP14 and UCHL5, given their functional cooperation [28] (Fig. 1e). The results of MTS assays revealed a significant decrease in cell proliferation in both 5-FU-sensitive and 5-FU-resistant cell lines when USP14 and/or UCHL5 were knocked down (Fig. 1f). Interestingly, the knockdown of both USP14 and UCHL5 led to an increase in 5-FU sensitivity in 5-FU-resistant CRC cell lines (Fig. 1g). Conversely, overexpression of USP14 and UCHL5 in 5-FU-sensitive CRC cells decreased 5-FU sensitivity (Figs. 1h and i). These findings indicate that the overexpression of USP14 and UCHL5 may play a role in the resistance of CRC cells to 5-FU.

b-AP15 inhibits malignant phenotypes in both 5-FU-sensitive and 5-FU-resistant CRC cells

To demonstrate that inhibiting USP14 and UCHL5 could reduce 5-FU resistance, we investigated the effects of b-AP15, a specific inhibitor of these deubiquitinases [18], on both 5-FU-sensitive and 5-FU-resistant CRC cells. Initially, we evaluated whether b-AP15 targets USP14 and UCHL5 in CRC cells and monitored their activity using an HA-Ub-VS assay. As depicted in Fig. 2a, b-AP15 inhibited the interaction of HA-Ub-VS with both USP14 and UCHL5, indicating its binding ability with these deubiquitinases. Furthermore, b-AP15 treatment induced a dose-dependent increase in ubiquitinated proteins and the proteasome substrate IκBα (Fig. 2b), suggesting its ability to suppress proteasome function in CRC cells. These results suggest that b-AP15 is a potent and effective inhibitor of USP14 and UCHL5 in CRC cells.

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

Fig. 2

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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.

To investigate the functional role of USP14 and UCHL5 in 5-FU-sensitive and 5-FU-resistant cell lines, we employed MTS assays to measure the cell viability of b-AP15-treated cells. b-AP15 significantly reduced the viability of RKO, RKO-R, HCT-15, and HCT-15R cells, with IC50 values of 1.649, 0.987, 1.453, and 0.858 μM, respectively (Fig. 2c). In addition, we treated RKO and RKO-R cells with a low dose of b-AP15 (0.25 and 0.5 μM) for 1–5 days and observed a dose- and time-dependent decrease in cell proliferation (Fig. 2d). To further confirm the effect of b-AP15 on CRC cell proliferation, we performed colony formation assays, which revealed that b-AP15 significantly reduced the number of colonies formed by both 5-FU-sensitive and 5-FU-resistant cells (Fig. 2e). Furthermore, Transwell assays demonstrated that b-AP15 effectively inhibited the migration of both 5-FU-sensitive and 5-FU-resistant cells (Fig. 2f). Taken together, our findings indicate that b-AP15 inhibits the viability and migration of both 5-FU-sensitive and 5-FU-resistant CRC cells.

b-AP15 induces apoptosis in both 5-FU-sensitive and 5-FU-resistant CRC cells

To evaluate the anticancer effect of b-AP15 in RKO and RKO-R cells, an Annexin V/PI staining assay was performed. The results demonstrated that b-AP15 triggered cell death in a dose- and time-dependent manner, affecting both 5-FU-sensitive and 5-FU-resistant CRC cells (Fig. 3a, b, Supplementary Fig. S2). As the antitumor effects of b-AP15 are associated with the induction of apoptosis [29], it is reasonable to assume that b-AP15 triggers mitochondrion-linked apoptosis in CRC cells. Indeed, treatment with b-AP15 resulted in the suppression of the mitochondrial membrane potential in a dose-dependent manner in both types of CRC cells (Fig. 3c, d). Moreover, b-AP15 treatment led to the inhibition of ATP synthesis in mitochondria (Fig. 3e).

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

Fig. 3

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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.

To further elucidate the mechanism underlying b-AP15-induced apoptosis in CRC cells, we examined whether b-AP15 activated caspases, the key executioners of both extrinsic and intrinsic apoptotic pathways. b-AP15 treatment caused marked cleavage of caspase-3, −8, and −9 in both types of CRC cells in a dose- and time-dependent manner (Fig. 3f and Supplementary Fig. S3), indicating the activation of caspase-dependent apoptosis. This was further supported by the cleavage of PARP, a classical substrate of caspase-3 (Fig. 3f and Supplementary Fig. S3). Moreover, the expression of antiapoptotic proteins, such as XIAP and Mcl-1, was suppressed by b-AP15 treatment (Fig. 3g). These findings suggest that b-AP15 induces apoptosis in both 5-FU-sensitive and 5-FU-resistant CRC cells.

To investigate whether b-AP15 exhibits a synergistic effect with 5-FU, 5-FU-resistant CRC cells were treated with 5-FU and b-AP15, either individually or in combination. The results showed that the combination treatment resulted in higher levels of apoptosis and ROS production compared to treatment with the compounds alone (Supplementary Fig. S4a, S4b). However, in normal human cell lines (including colon epithelial NCM460, bronchial epithelial 16HBE, and liver LO2 cells), the combination treatment of 5-FU and b-AP15 resulted in a relatively smaller proportion of cell death (Supplementary Fig. S4a), suggesting that the combination treatment of 5-FU and b-AP15 has an acceptable safety profile.

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

As stated previously, b-AP15 increased the expression of IκBα (Fig. 2b), which is a protein inhibitor known to retain NF-κB in the cytoplasm [30]. Given that the NF-κB pathway plays a crucial role in chemotherapy resistance, including 5-FU resistance [31], we investigated the involvement of NF-κB inhibition in the effects of b-AP15. Compared to 5-FU-sensitive cells, 5-FU-resistant RKO-R and HCT-15R cells had higher expression of phosphorylated p65 at Ser 276, which is essential for NF-κB activation [32] (Fig. 4a). Additionally, 5-FU treatment increased the phosphorylation of p65 in 5-FU-sensitive RKO and HCT-15 cells (Fig. 4a), supporting our hypothesis that NF-κB activation promotes 5-FU resistance.

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

Fig. 4

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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.

We then verified whether b-AP15 could reverse 5-FU resistance by inhibiting the NF-κB pathway. b-AP15 decreased TNF-α-triggered p65 phosphorylation at Ser 276 and phosphorylation of IκBα (Fig. 4b). Furthermore, b-AP15 also suppressed TNF-α-triggered phosphorylation of Ikkα/β, kinase subunits that phosphorylate IκBα [33] (Fig. 4b). Similarly, immunofluorescence staining assays revealed that b-AP15 inhibited TNF-α-induced nuclear translocation of p65 in both RKO and RKO-R cells (Fig. 4c). Additionally, b-AP15 treatment resulted in a significant decrease in TNF-α-induced luciferase transcriptional activity of NF-κB in a dose-dependent manner (Fig. 4d).

Given these results, we assessed the levels of Bcl-xl, c-Myc, survivin, and cyclin D1, which are downstream target genes of NF-κB signaling [34–36]. As shown in Fig. 4e, b-AP15 clearly suppressed the expression of NF-κB downstream targets such as Bcl-xl, c-Myc, survivin, and cyclin D1 activated by TNF-α. Moreover, overexpression of USP14 and UCHL5 induced activation of the NF-κB pathway (Fig. 4f and Supplementary Fig. S5), while overexpression of USP14 and UCHL5 reversed the NF-κB inhibition effects induced by b-AP15 (Fig. 4f and Supplementary Fig. S5).

Furthermore, the combination of b-AP15 and 5-FU significantly reduced p65 phosphorylation and luciferase activity of NF-кB compared to b-AP15 or 5-FU single treatment in 5-FU-resistant CRC cells (Supplementary Fig. S6). Overall, our findings suggest that inhibition of USP14 and UCHL5 suppresses NF-κB activation in both 5-FU-sensitive and 5-FU-resistant CRC cells.

b-AP15 induces ROS generation

To explore whether inhibiting USP14 and UCHL5 could enhance the production of ROS, we investigated the effect of proteasome inhibition on oxidative stress [37]. First, we used flow cytometry with Mito-SOX staining to measure the baseline level of mitochondrial ROS in 5-FU-sensitive and 5-FU-resistant CRC cells. Our results showed that 5-FU-resistant cells had higher levels of mitochondrial ROS than 5-FU-sensitive cells (Fig. 5a). Subsequently, we found that treatment with b-AP15 led to a dose-dependent increase in mitochondrial ROS levels in both 5-FU-sensitive and 5-FU-resistant CRC cells (Fig. 5b). Additionally, we measured total cellular ROS levels using DCFDA, a commonly used probe for detecting cellular hydrogen peroxide, and observed that b-AP15 treatment increased ROS levels in both 5-FU-sensitive and 5-FU-resistant CRC cells (Fig. 5c).

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

Fig. 5

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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.

To determine whether ROS production contributes to b-AP15-induced apoptosis, we evaluated the effect of the antioxidant N-acetylcysteine (NAC) on b-AP15-treated CRC cells. As expected, NAC restored b-AP15-induced ROS production in HCT-15 and HCT-15R cells (Fig. 5d). Meanwhile, NAC significantly attenuated b-AP15-induced apoptosis in RKO and RKO-R cells (Fig. 5e). We also investigated whether inhibition of USP14 and UCHL5 affects the antioxidant system by monitoring the expression of antioxidant proteins such as catalase and G6PD. The results showed that b-AP15 downregulated the levels of catalase and G6PD in RKO and RKO-R cells (Fig. 5f). Consistent with the above results, the knockdown of USP14 and/or UCHL5 triggered ROS generation in 5-FU-sensitive and 5-FU-resistant CRC cells (Fig. 5g) and decreased the expression levels of catalase and G6PD in CRC cells (Fig. 5h). Thus, these findings demonstrate that inhibition of USP14 and UCHL5 induces apoptosis in 5-FU-sensitive and 5-FU-resistant CRC cells through the generation of ROS.

b-AP15 restrains the growth of both 5-FU-sensitive and 5-FU-resistant CRC xenografts in vivo

To assess the efficacy of b-AP15 on 5-FU-sensitive and 5-FU-resistant CRC tumors in vivo, we established nude mouse xenograft models using RKO and RKO-R cells. Treatment with b-AP15 (8 mg·kg−1·d−1, administered via intraperitoneal injection) led to a significant decrease in tumor weight in both RKO and RKO-R xenografts (Fig. 6a), consistent with the observed tumor growth suppression (Fig. 6b). Notably, we observed no significant difference in body weight between the vehicle and b-AP15 treatment groups (Fig. 6c). Immunohistochemical analysis showed a significant increase in cleaved caspase-3 and ubiquitinated proteins in tumors treated with b-AP15 compared to vehicle-treated tumors (Fig. 6d). Conversely, b-AP15 treatment resulted in the downregulation of proliferation markers such as Ki67 and phosphorylated p65 (Fig. 6d). Taken together, these findings confirm that pharmacological inhibition of USP14 and UCHL5 by b-AP15 can attenuate the progression of both 5-FU-sensitive and 5-FU-resistant tumors in vivo.

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

Fig. 6

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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.

Discussion

The ubiquitin proteasome degradation pathway is a promising therapeutic target for various cancers. Several 20 S proteasome inhibitors, such as bortezomib, carfilzomib, and ixazomib, have been approved by the US FDA for treating multiple myeloma and mantle cell lymphoma [38]. Additionally, b-AP15 functions as a novel proteasome-targeted anticancer agent by inhibiting the activities of the 19 S proteasomal deubiquitinases USP14 and UCHL5 [18]. In this study, we demonstrate that b-AP15 can reverse 5-FU resistance and inhibit the malignant phenotype of CRC cells by inducing apoptosis and inhibiting NF-κB signaling (Fig. 7).

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

Fig. 7

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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.

To determine the roles of the proteasomal deubiquitinases USP14 and UCHL5 in CRC progression and 5-FU resistance, we analyzed their mRNA expression levels in a public GEO dataset and detected the protein levels of USP14 and UCHL5 in both 5-FU-sensitive and 5-FU-resistant cell lines and CRC patient tissues. We found that USP14 and UCHL5 were frequently overexpressed in CRC cells and exhibited significantly higher levels in 5-FU-resistant cells than in 5-FU-sensitive cells. The knockdown of USP14 and UCHL5 in CRC cells confirmed that their activation contributed to tumor survival and 5-FU resistance. While USP14 and UCHL5 have pro-tumor effects in some cancer settings, they have been reported to be potential targets for reducing proteasome inhibitor resistance in multiple myeloma and Waldenstrom macroglobulinemia tumors [29, 39]. Targeting USP14 and UCHL5 also provides a new approach for the treatment of BRAF inhibitor resistance in melanoma, cisplatin resistance in gastric cancer, and imatinib resistance in chronic myeloid leukemia [40–43]. Therefore, understanding the new roles of USP14 and UCHL5 in innate or acquired resistance to chemotherapy is critical for improving clinical outcomes in cancer patients.

b-AP15 is an inhibitor of the 19 S proteasomal deubiquitinases USP14 and UCHL5, which contain an α,β-unsaturated carbonyl group [18]. We demonstrated that b-AP15 effectively inhibited the growth and weakened the migration capacity of 5-FU-sensitive and 5-FU-resistant CRC cells by inhibiting USP14 and UCHL5. In nude mouse xenograft models, b-AP15 effectively inhibited proteasome function and tumor growth without significantly reducing mouse body weight at effective doses [20, 44]. These findings suggest that b-AP15 treatment is a potentially effective and low-toxicity approach to reduce 5-FU resistance in CRC cancer. However, a phase I/II clinical trial of VLX1570, a promising analog of b-AP15, showed severe pulmonary toxicity in patients with relapsed or refractory multiple myeloma [45]. Therefore, developing other inhibitors that target USP14 and UCHL5 with better safety profiles for human use should be a focus of future research.

Activation of the NF-κB pathway is a well-known cause of drug resistance to chemotherapy [46]. To investigate whether b-AP15 could reverse 5-FU resistance through the NF-κB pathway in CRC, we assessed its effects on TNF-α-induced NF-κB activation, including the phosphorylation and nuclear translocation of p65, in both 5-FU-sensitive and 5-FU-resistant cells. We found that b-AP15 suppressed NF-κB activation and downstream targets, such as Bcl-xl, c-Myc, survivin, and cyclin D1, in both cell types. These findings suggest that b-AP15 can reduce drug resistance, at least in part, by inhibiting the NF-κB pathway.

Excessive levels of ROS can lead to cytotoxicity and apoptosis in cancer cells [47]. Previous studies suggest that treatment with b-AP15 and its analog VLX1570 results in the production of ROS, oxidative stress, and endoplasmic reticulum stress in cancer [48]. b-AP15 decreases mitochondrial oxidative phosphorylation and induces severe proteotoxic stress, which plays an important role in the induction of ROS [49]. In this study, we found that ROS levels were higher in 5-FU-resistant CRC cells than in 5-FU-sensitive cells, which is consistent with previous reports [50]. Treatment with b-AP15 dose-dependently increased ROS levels in both mitochondria and cytoplasm, which could be rescued by NAC. Furthermore, b-AP15 reduced the expression of antioxidant enzymes such as catalase and G6PD. Catalase protects cancer cells from apoptosis by decomposing hydrogen peroxide [51], while G6PD is essential for pentose phosphate pathway-mediated generation of NADPH, which plays a key role in ROS detoxification [52]. Inactivation of catalase and G6PD may contribute to b-AP15-induced cell death in both 5-FU-sensitive and -resistant CRC cells. The altered balance of antiapoptotic (e.g., XIAP and Mcl-1) and pro-apoptotic proteins leads to a decrease in mitochondrial membrane potential, triggering the release of mitochondrial pro-apoptotic proteins, activation of caspases, and ultimately apoptotic cell death.

In conclusion, we uncovered the novel functions of USP14 and UCHL5 in mediating 5-FU resistance in CRC cells. Genetic disruption and pharmacological inhibition of USP14 and UCHL5 can reduce 5-FU resistance in CRC cells by inducing apoptosis and inhibiting the NF-κB pathway, providing a theoretical basis for clinical evaluation. Through our experimental validation, we confirmed that b-AP15 exhibits therapeutic potential against 5-FU-resistant CRC by inhibiting USP14 and UCHL5. Consequently, a more effective b-AP15 analog based on the inhibition of USP14 and UCHL5 may represent a promising avenue for further research.

Supplementary information

41401_2023_1136_MOESM1_ESM.docx (1.4MB, docx)

Supplementary_figures_and_figure_legends

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82170177/H0809 and 81670154/H0812), the Foundation of Innovation Projects of General Colleges and Universities in Guangdong Province (2020KTSCX10), the Natural Science Foundation of Guangdong Province (2021A1515011334 and 2023A1515011976), Key Discipline of Guangzhou Education Bureau (Basic Medicine) (201851839), the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital (202011-203), the Innovation Team of General Universities in Guangdong Province (2022KCXTD021), and the Foundation of Guangzhou Science and Technology Innovation Committee (202201010811) to XPS.

Author contributions

XPS, JBL, and XC designed the study. WD, JXW, JZW, and ACL conducted the experiments and analyzed the data. LLJ, HCZ, YM, BYL, GJP, EZL, QM, and HZ participated in the experiments. WD and XC wrote the manuscript. DLT edited the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

These authors contributed equally: Wa Ding, Jin-xiang Wang, Jun-zheng Wu, Ao-chu Liu

Contributor Information

Xin Chen, Email: chenxin@gzhmu.edu.cn.

Jin-bao Liu, Email: jliu@gzhmu.edu.cn.

Xian-ping Shi, Email: xianping.shi@gzhmu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-023-01136-0.

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