USP14 inhibition by degrasyn induces YAP1 degradation and suppresses the progression of radioresistant esophageal cancer.

Introduction

Esophageal cancer (ESCA) is a major aggressive gastrointestinal malignancy and a significant contributor to tumor-related deaths worldwide, reducing the quality of life of patients undergoing therapy [ 1 , 2 ]. Esophageal squamous cell carcinoma (ESCC) is the most common type of ESCA, but its prognosis is still unsatisfactory. Only 20 % of ESCA patients can undergo surgical treatment. Other therapeutic options for ESCA include radiotherapy, chemotherapy, chemoradiotherapy, immunotherapy, and targeted therapy [ 3 , 4 ]. Although great advancements have been made in diagnostic techniques and comprehensive treatments, the overall 5-year survival rates of this disease remain low due to recurrence, severe invasion, and metastasis [ 5 ]. Among all these therapeutic strategies, radiation therapy plays a crucial role in the treatment of cancer patients and is frequently used as the first-line treatment for ESCA, either as the main therapeutic approach or in combination with chemotherapies. Irradiation can induce cell death through several different signaling pathways [ 6 , 7 ]. Among these cellular processes, DNA damage caused by irradiation is considered as a key mechanism in cell death. After irradiation exposure, tumor cells produce damaged DNA such as single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) [ 8 ]. However, radioresistance remains a significant challenge, thereby affecting the prognosis of patients [ 9 ]. Therefore, it is urgently necessary to investigate the underlying molecular mechanism of ESCA radioresistance and develop effective strategies to improve the irradiation tolerance of ESCA cells.

Deubiquitinating enzymes (DUBs) are ubiquitin proteases that bind to ubiquitin tagged substrates, ensuring the completion of numerous biological events, such as cell proliferation, growth, cell death and immune response [ 10 ]. Ubiquitin-specific protease 14 (USP14, also known as UBP6), a member of the DUB family, belongs to the USPs and reversibly binds to the 26S proteasome, restricting proteasomal degradation of ubiquitinated proteins [ 11 ]. An increasing amount of evidence suggests that Aberrant USP14 causes various pathological conditions, including tumor growth, neurodegenerative diseases, autophagy, and viral infections [ [12] , [13] , [14] ]. Recently, numerous studies have identified that USP14 is overexpressed in most types of tumors, and its deubiquitinating activity can be activated by the proteasome. USP14 can reduce the anchoring time of ubiquitin conjugates with the proteasome and induce the deubiquitination of its target proteins for substrate protein stabilization [ 15 ]. For example, USP14 regulates cell cycle progression in breast cancer by inducing the deubiquitination of CyclinB1, exerting an oncogenic function in breast cancer [ 16 ]. USP14 also maintains hypoxia-induced factor 1-α (HIF-1α) stabilization through its deubiquitination activity in hepatocellular carcinoma (HCC) [ 17 ]. USP14 overexpression is also closely related to chemoresistance during ovarian cancer progression by stabilizing the BCL6 oncoprotein [ 18 ]. In addition, USP14 expression is increased in oral squamous cell carcinoma (OSCC) and promotes radioresistance in OSCC treatment. Its inhibition strongly triggers apoptosis to inhibit OSCC progression [ 19 , 20 ]. USP14 positively mediates head and neck squamous cell carcinoma (HNSCC) tumorigenesis through HSF1 stabilization [ 21 ]. Furthermore, USP14 plays a crucial role in mediating the activation of NF-κB activity and the response to radiation therapy in HNSCC cells [ 22 ]. Recently, USP14 was identified as a critical DDR factor to induce DNA damage response in non-small cell lung cancer (NSCLC) and might be a target for radio-sensitization [ 23 ]. USP14 also regulates DNA damage repair by targeting RNF168-dependent ubiquitination in human prostate cancer [ 24 ]. These findings indicate that USP14 dysfunction is closely correlated with tumor progression and the outcome of irradiation therapy. USP14 is also involved in the progression and metastasis of ESCC [ [25] , [26] , [27] , [28] ]. Briefly, USP14 can mediate the autophagic degradation of GPX4 to confront ferroptosis, promoting the growth of ESCC [ 26 ]. Additionally, in vitro studies suggest that suppression USP14 exhibits inhibitive effects on the expression levels of β-catenin, Cyclin D1, and c-Myc in ESCC cells, thereby inhibiting the proliferation and EMT processes in cells [ 27 ]. USP14 can mediate the deubiquitination of K48-linked poly-ubiquitination of PIK3IP1 to weaken its stability, which contributes to the EMT and tumorigenesis of ESCC [ 28 ]. These data suggest that targeting USP14 for its suppression may be a potentially useful therapeutic strategy for ESCC treatment. However, the molecular mechanism by which USP14 inhibits ESCC has not been fully understood, especially in the study of radioresistant ESCC cells under radiation therapy.

Identifying inhibitors targeting USP14 has received increasing attention for the development of efficient therapeutic approaches in the treatment of cancer [ 29 , 30 ]. Degrasyn (DGS, also known as WP1130) is a cell-permeable DUB inhibitor that directly inhibits the DUB activities of USP14, USP5, and USP9X [ 31 ]. For example, USP14 is a direct target of DGS in murine macrophages, exerting antiviral activity [ 32 ]. Furthermore, DGS can decrease the anti-apoptotic signals Bcr-Abl and Janus-activated kinase (JAK) [ 33 ]. Previously, DGS was shown to exert anti-leukemia functions through ubiquitin-regulated c-Myc degradation [ 34 ]. DGS also inhibits metastasis in pancreatic ductal adenocarcinoma by targeting the USP5/WT1/E-Cadherin signaling pathway [ 35 ]. DGS promotes TRAIL-triggered apoptotic cell death via USP9X-dependent miR-708-regulated decrease of c-FLIP [ 36 ]. However, the effects of inhibiting USP14 by DGS on the development of ESCA and resistance to radiotherapy have not yet been investigated. Here, in our present study, we attempted to explore the potential and underlying mechanism of USP14 suppression in human ESCA with radioresistance in vitro and in vivo . We mainly found that USP14 overexpression may affect the ESCA radioresistance and progression through interacting and regulating YAP1 stability and further modulating tumor cell proliferation, migration, invasion and DNA damage signaling, which could be significantly suppressed by low dose of DGS combined with irradiation therapy, contributing to the reduction of irradiation tolerance and tumor growth inhibition in ESCA cells and xenograft mouse models without apparent side effects.

Materials and methods

Human tissue specimens

A total of 64 ESCA patients (34 males and 30 females, aged 42-75 years, a mean age of 59.62 ± 8.05 years) with squamous cell carcinoma or adenocarcinoma pathological confirmation after operation were included in this study from December 2015 to January 2019 at Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences (Jinan, China). Patients received no radiotherapy or chemotherapy ahead of surgical operation. The clinicopathological information for these ESCA patients were provided in Supplementary Table 1. Informed consents were obtained from all patients or their family members. Human studies were performed in accordance with the Declaration of Helsinki, and the use of these human samples and data for research purposes was approved by the Ethics Committee of the Hospital.

Cells culture and treatments

Human ESCA cell lines including ECA109 and TE-1, and human esophageal epithelial cells (HEEC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human ESCA cell lines TE-10 and KYSE-150 were purchased from ATCC (Manassas, Virginia, USA). Human ESCA cell lines TE-12 and OE-19 were purchased from Procell (Wuhan, China). All cell lines were cytogenetically tested by STR-Promega and authenticated. All cell lines were grown in RPMI-1640 or DMEM culture medium (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Procell, Wuhan, China) in a humidified incubator at 37°C with 5 % CO 2 . The radioresistant cell lines KYSE-150R and TE-12R were established from KYSE-150 and TE-12 cells, respectively, through fractionated irradiation as previously described [ 37 ]. Briefly, KYSE-150 and TE-12 cells were cultured in RPMI-1640 and DMEM culture medium (Gibco), respectively, containing 10 % FBS (Gibco) in a humidified incubator with 5 % CO 2 (v/v). After cells grew to approximate 60 % density, the cells were treated with 2 Gy X-ray irradiation (2.5 Gy/min) using a Varian-6/100 high linear accelerator (Varian Medical Systems, CA, USA). Then, the cells were changed to new culture medium every two days. When large number of dead cells were observed, culture medium with 15 % FBS (Gibco) was used for cell culture. When the irradiated cells reached approximate 80 % confluence, the above irradiation process was repeated until the total radiation dose reached 60 Gy. In the end, the obtained cells with radioresistance were defined as KYSE-150R and TE-12R cells, which continued to be cultured for ≥14 days before further analysis. Degrasyn (Cas.no: #856243-80-6), an inhibitor of cell-permeable deubiquitinase (DUB), and the protein synthesis inhibitor cycloheximide (CHX, Cas.no: #66-81-9) were purchased from MedChemExpress (USA). USP14 inhibitor IU1-47 (Cas.no: #ajcx38630) was purchased from Wuhan Anjiekai Biological Medicine Technology Co., Ltd (Wuhan, Hubei, China).

In vitro transfection

The shRNAs targeting USP14 (shUSP14), USP5 (shUSP5), and USP9x (shUSP9x), as well as the scramble control (shNC) were purchased from GeneCopoeia (Guangzhou, China). For stable shRNAs-regulated transfection, lentiviral particles were generated by HEK-293T cells and the filtered supernatant was transferred to target ESCA cell lines. Stable clones were maintained by the use of puromycin (Sigma Aldrich, USA). The USP14 overexpression plasmid (oeUSP14), Myc-Ub, HA-YAP1, Flag-USP14 and Flag-YAP1 plasmids constructed by the use of pcDNA3.1 were purchased from GeneChem (Shanghai, China). Cells were transfected with the indicated plasmids using Lipofectamine 3000 (Invitrogen, Life Technologies, USA) according to the manufacturer's instructions. The efficiency of gene knockdown or overexpression was measured using RT-qPCR and western blot assays.

Bioinformatics analysis

The TIMER: Tumor IMmune Estimation Resource platform ( https://cistrome.shinyapps.io/timer/ ) was used to analyze the expression of USP14 in ESCA patients. The Human Protein Atlas platform was used to identify USP14 protein expression levels by immunohistochemical staining ( https://www.proteinatlas.org/ ).

Real time-quantitative PCR (RT-qPCR) analysis

Total RNA was extracted from cells and tissue using the RNA Easy Fast Tissue/Cell Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. Aliquots of 1 μg RNA were then used as a template for reverse transcription to obtain cDNA with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Shanghai, China) following the provider's protocols. RT-qPCR analysis was conducted with a ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) on a Roche light cycle 480 system (Roche Diagnostics, Switzerland). The GAPDH gene was served as the internal control. The 2 −∆∆Ct analysis methods were utilized to evaluate the gene expression levels. The primers used in the study were provided by GeneScript Biotech Corporation (Nanjing, China) and listed in Supplementary Table 2.

Cell proliferation assay

ESCA cells (8.0 × 10 3 cells/well) were planted onto 96-well plates containing 200 μl culture medium per well, and cultured overnight in the incubator. Then, the cells were treated separately as described in the figure legend section. After 24, 48, or 72 h of treatments, all cells were treated with 10 μL of CCK-8 solution (Beyotime, Shanghai, China) for another 2 h. Then, the optical density was measured using a microplate reader (Thermo Scientific, Rockford, USA) at 450 nm. The 5-Ethynyl-2′-deoxyuridine (EdU) assay was also conducted for cell proliferation evaluation with a Click-iT EdU Kit (BeyoClick™ EdU-647, Beyotime, Shanghai, China) following the supplier's protocols. Briefly, ESCA cells after each treatment were incubated with 2 × EdU solution for 2 h. Thereafter, the cells were fixed with 4 % paraformaldehyde, permeabilized by Triton X-100 (Solarbio, Beijing, China) for 15 min, and incubated with 500 μL Click reaction cocktail for 30 min in dark at room temperature. The cells were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio, Beijing, China) for nuclear staining and observed under a fluorescent microscope (Olympus, Japan).

Clonogenic assay

The ESCA cells were planted on a 6-well plate. After each treatment as described in figure legends, the cells were cultured for another 10 days. Subsequently, the cells were fixed with 4 % paraformaldehyde and stained using crystal violet. The colonies >50 cells were observed and numbered under a light microscope (Nikon, Japan).

Western blotting

Total protein was extracted from cells or tissues using RIPA buffer (Beyotime, Shanghai, China). BCA assay (Beyotime, Shanghai, China) was used to examine the protein concentration levels following the manufacturer's instructions. A total of 40 μg protein from each sample was subjected to 10 % or 12 % sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to PVDF membranes (Millipore, MA, USA). After blocking the membranes with 5 % skim milk in Tris-buffered saline containing 0.1 % Tween-20 (TBST) for 1.5 h at room temperature, the membranes were incubated with the corresponding primary antibodies overnight at 4°C. After washing five times with TBST, the membranes were incubated with corresponding secondary antibodies for 1.5 h at room temperature. Thereafter, the targeting signals were visualized using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Shanghai, China). The density of protein bands was quantified by the Image J software (NIH ImageJ, NIH, Bethesda, MD, USA).

Primary and secondary antibodies used for western blotting analysis were listed as following: anti-USP14 (Cas.no: #MA5-24727; 1:1000 dilutions) and anti-YAP1 (Cas.no: #PA1-46189; 1:1000 dilutions) were obtained from Thermo Fisher Scientific (Shanghai, China). Primary antibodies including anti-γ-H2AX (Cas.no: #ab22551; 1:1000 dilutions), anti-CHK1 (Cas.no: #ab32531; 1:1000 dilutions) and anti-p-CHK1 (Cas.no: #ab79758; 1:500 dilutions) were purchased from Abcam (Cambridge, UK), and anti-polyubiquitin (Cas.no: #abs146503; 1:1000 dilutions), anti-ATR (Cas.no: #abs145433; 1:1000 dilutions) and anti-p-ATR (Cas.no: #abs173235; 1:1000 dilutions) were purchased from Absin Biotech (Shanghai, China). Primary antibodies including anti-CTGF (Cas.no: #86641; 1:1000 dilutions), anti-Myc (Cas.no: #2276; 1:1000 dilutions), anti-HA (Cas.no: #3724; 1:1000 dilutions), anti-Flag (Cas.no: #14793; 1:1000 dilutions) and anti-GAPDH (Cas.no: #5174S; 1:1000 dilutions) were purchased from Cell Signaling Technology (MA, USA). Secondary antibodies including Goat anti-Rabbit IgG-HRP Antibody (Cas.no: #abs20040; 1:8000 dilutions) and Goat anti-Mouse IgG-HRP Antibody (Cas.no: #abs20002; 1:8000 dilutions) were obtained from Absin Biotech (Shanghai, China).

HA-Ub-VS assay

ESCA cells after treatments were collected and lysed using DUB buffer (50 mM Tris-HCl, pH-7.5, 50 mM NaCl, and 5 mM DTT). The cell lysate was then centrifuged at 20,000 × g for 15 min at 4°C. After the protein concentration calculation by the BCA method, the lysate was incubated with 2.5 μM HA-tagged ubiquitin-vinyl sulfone (HA-Ub-VS) probe (Cas.no: #U212; Boston Biochem, USA) was added and incubated at 37°C for 1 h. The lysates were then boiled in reducing sample buffer and resolved by SDS-PAGE, followed by immunoblotting analysis.

Transwell analysis

For migration evaluation, the ESCA cells (5 × 10 4 ) were inoculated onto Transwell plates (Corning, New York, USA) with 200 μL serum-free medium, and complete culture medium containing 10 % FBS (Gibco) was added to the lower chamber. Migrated cells were fixed using 4 % paraformaldehyde after 24 h, stained by 0.1 % crystal violet, and observed under a light microscope (Nikon, Japan). For invasion analysis, the upper chamber was firstly coated with Matrigel (Corning, New York). Next, the ESCA cells were seeded on the upper chamber and cultured in 200 μL serum-free medium. Simultaneously, about 700 μL complete culture medium supplemented with 10 % FBS (Gibco) was added to the lower chamber. After 24 h, migratory cells were fixed, stained, and captured with a light microscope (Nikon, Japan).

Immunofluorescence (IF)

Cells on the coverslips after treatments were fixed by 4 % paraformaldehyde for 15 min, permeabilized using 0.3 % Triton X-100 (Solarbio, Beijing, China) for 10 min, and blocked in 5 % bovine serum albumin (BSA) (Solarbio, Beijing, China) in PBS for 45 min. After careful washing with PBS, the cells were incubated with primary antibodies against γ-H2AX (Cas.no: #ab22551; 1:200 dilutions), p53-binding protein 1 (53BP1) (Cas.no: #ab175933; 1:200 dilutions; Abcam), USP14 (Cas.no: #MA5-24727; 1:200 dilutions) and YAP1 (Cas.no: #PA1-46189; 1:200 dilutions) at 4°C overnight. After rinsing with PBS, the cells were incubated with a fluorescence-conjugated secondary antibodies, including Alexa Fluor 647-Labeled Goat Anti-Mouse IgG (Cas.no: #abs20165; 1:200 dilutions; Absin, Shanghai, China) and Alexa Fluor 488-Labeled Goat Anti-Rabbit IgG (Cas.no: #abs20025; 1:200 dilutions; Absin, Shanghai, China). After nuclear staining with DAPI (Solarbio, Beijing, China), images were captured using a confocal laser-scanning microscope (Zeiss, Germany). Fluorescence density was quantified by use of the Image J 1.48 V software.

RNA-Seq analysis

RNA was extracted from KYSE-150R cells with or without DGS/X-ray treatment versus the control group using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the sequencing and preliminary data analysis were conducted with the assistance by Aksomics Inc. (Shanghai) for subsequent mRNA library construction and sequencing. An adjusted p value of a gene <0.05 and a log2(FC) > 1.5 were used as the criteria for differentially expressed genes (DEGs) for further data analysis. A gene expression heatmap was generated with an R package (v3.6.3). The GO analysis and KEGG analysis were conducted using the R software (version 4.0.1). GSEA was conducted using the Broad GSEA software (version 4.0.2) with the hallmark gene sets (h.all.v.7.4.symbols.gmt).

Animals and treatments

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences (Jinan, China). All animal experiments were conducted following the Guidelines for the Care and Use of Laboratory Animals (NIH publications revised 1996). The 6-8-week-old female BALB/c athymic nude mice, weighing 18–20 g, were purchased from Vital River Experimental Animal Center (Beijing, China) for the Xenograft study, and were maintained under special pathogen-free (SPF) conditions.

Firstly, to examine the USP14 expression suppression on radioresistant ESCA progression, KYSE-150R cells (3 × 10 6 ) with or without stable USP14 knockdown were subcutaneously injected to the right flank of nude mice. Then, the mice were randomly divided into four groups with four in each, including the shNC group served as the control, shNC plus X-ray (shNC/X-ray) group, shUSP14-#1 plus X-ray (shUSP14-#1/X-ray) group and shUSP14-#2 plus X-ray (shUSP14-#2/X-ray) group. When tumor masses became visible (approximate 100 mm 3 ), the mice received a 10 Gy dose (2 Gy/day for five consecutive days) of radiotherapy at a dose rate of 1 Gy/min and were sacrificed 30 days later. To examine the effects of DGS, radiotherapy or their combination on radiosensitive and radioresistant ESCA progression, all mice were randomly allocated to eight groups (n=4 per group) with subcutaneous injection of 3 × 10 6 KYSE-150 and KYSE-150R cells, respectively, including the control group, DGS group (10 mg/kg), X-ray group or the two in combination group. When tumor masses reached to about 100 mm 3 , the mice received intraperitoneal (i.p.) injection of DGS (10 mg/kg) every other day. As for these mice with radiotherapy, a 10 Gy dose (2 Gy/day for five consecutive days) of radiotherapy at a dose rate of 1 Gy/min. For DGS/X-ray group, mice received the administration of DGS and X-ray simultaneously as described above. The tumor tissues were measured with a caliper, and the tumor volume was evaluated with following formular: Volume = tumor length × width 2 × 0.5236. After treatments for 30 days, all mice were weighed, blood samples were collected and then killed. Tumor tissues and major organs including liver, kidney, heart, lung and spleen were dissected. Tumor specimens were weighed. Subsequently, all major organs and some parts of the tumor tissues were fixed in 4 % paraformaldehyde and embedded in paraffin. Then, these sections were subjected to hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) staining. Some parts of the tumor tissues were placed in liquid nitrogen and then stored at -80°C for further analysis. Additionally, the collected blood samples were used for renal, hepatic, and cardiac toxicity analysis in our hospital. Briefly, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CR) levels, and creatine kinase-MB (CK-MB) were recorded on a Biochemical Auto-analyzer (Roche, Switzerland).

Immunohistochemical (IHC) analysis

Tissue sections (8 μm thick) after deparaffinization and dehydration were subjected to antigen retrieval using citrate buffer for 5 min. After blocking the endogenous peroxidase and alkaline phosphatase activities, the sections were incubated with primary antibodies against USP14 (Cas.no: #MA5-32821; 1:200 dilutions; Thermo Fisher Scientific, Shanghai, China), KI-67 (Cas.no: #abs171448; 1:200 dilutions; Absin Biotech, Shanghai, China) or YAP1 (Cas.no: #PA1-46189; 1:200 dilutions; Thermo Fisher Scientific, Shanghai, China) overnight at 4°C, followed by rinsing and incubation with HRP-conjugated secondary antibody (Absin Biotech, Shanghai, China) for 1 h at room temperature. Thereafter, the tissue slides were stained with a diaminobenzidine (DAB) kit (Solarbio, Beijing, China) and haematoxylin. Images were finally observed and captured under a light microscope (Nikon, Japan).

Co-immunoprecipitation (Co-IP) assay

For Co-IP experiments, total cell lysates were extracted using NP-40 lysis buffer (Beyotime, Shanghai, China) containing a protease-inhibitor cocktail (Roche, Switzerland). The supernatants were then collected. For immunoprecipitation, the cell lysates were incubated with the shown antibodies at 4°C overnight, followed by incubation with protein A/G agarose (Santa Cruz Biotechnology, USA) for 6 h. Subsequently, the immunoprecipitates were rinsed three times and analyzed by immunoblotting assay.

Ubiquitination assay

For ubiquitination analysis, cells were transfected with the indicated plasmids for 24 h, and then cells were co-treated with DGS and irradiation. Proteasome inhibitor MG132 (20 μM; MedChemExpress, Shanghai, China) were added 6 h before lysis using ubiquitination buffer supplemented with 1 % SDS and boiled at 95°C for 15 min, followed by dilution with NTEN lysis buffer containing protease inhibitors (Roche, Switzerland). After 20 min, the cell lysates were immunoprecipitated using the shown antibodies and protein A/G beads for 4h at 4°C. Next, the beads were rinsed three times with RIPA buffer, the immunocomplexes were collected and subjected to immunoblotting analysis.

Statistical analysis

All data were shown as mean ± standard deviation (SD) of at least three independent experiments and analyzed using GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA, USA). The unpaired Student's t -tests were used for comparisons between two groups, and one-way analysis of variance (ANOVA) with Tukey's multiple comparison test was applied for comparisons among three or more groups. P-values less 0.05 ( p <0.05) were considered statistically significant.

Results

USP14 is overexpressed in ESCA tissues and cell lines

To explore the USP14 mRNA expression level in ESCA, we utilized the TCGA database and found that USP14 was highly expressed in human ESCA tissue samples, which was comparable to the normal tissues in the TCGA ( Fig. 1A and B). Moreover, higher USP14 protein expression levels were detected in ESCA specimens through IHC staining from the human protein atlas ( https://www.proteinatlas.org/ ) ( Fig. 1C ). We also observed significantly increased expression of USP14 from mRNA and protein levels compared with the paired normal samples from our hospital cohort by RT-qPCR and IHC staining assays ( Fig. 1D -F). Consistently, markedly increased USP14 mRNA and protein expression levels were detected in several ESCA cell lines such as KYSE-150, TE-12, TE-1, TE-10, OE19 and ECA109 when compared with the non-tumor cell line HEEC ( Fig. 1G and H), which were confirmed by IF staining ( Fig. 1I ), as indicated by the more intense fluorescence intensity. These data above initially revealed the potential of USP14 in ESCA progression. Fig. 1 USP14 is overexpressed in ESCA tissues and cell lines. (A) The expression levels of USP14 in different human tumor types (red columns) and normal tissue samples (blue columns) based on the TCGA database. (B) Measurements of USP14 expression levels in ESCA primary tumor samples with squamous cell carcinoma or enocarcinoma pathological types, and normal tissues from the TCGA database. (C) Representative IHC images of USP14 expression levels in ESCA and normal tissues published by the human protein atlas ( https://www.proteinatlas.org/ ). (D) USP14 mRNA expression levels were examined in 64 ESCA tissues from patients with squamous cell carcinoma or enocarcinoma pathological types, and the adjacent non-tumor tissues as analyzed by RT-qPCR (n=64 in each). (E) USP14 expression levels in ESCA tissues (patients with squamous cell carcinoma or enocarcinoma pathological types), and the matched adjacent nontumor samples were evaluated using IHC assay (n=64 per group). Scale bar, 50 µm. (F) Analysis of IHC staining results of USP14 expression levels in ESCA. (G) RT-qPCR, (H) western blotting, and (I) IF staining analysis of USP14 mRNA and protein expression levels in ESCA cell lines and human non-tumor normal esophageal epithelial cell line HEEC (n=3 or 4 in each group). Scale bar, 10 µm. Data were presented as the mean ± SD. *p <0.05, ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001 versus the Normal or HEEC group. Statistical significances in (B), (D) and (F) were assessed by unpaired Student's t test; Statistical significances in (G) and (H) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 1

USP14 expression is up-regulated in radioresistant ESCA cells

Due to the significantly higher expression of USP14 in KYSE-150 and TE-12 cells, these two cell lines were chosen for the following experiments. Subsequently, through RT-qPCR and western blotting assays, we discovered that KYSE-150 and TE-12 cells exposed to X-ray irradiation showed significantly lower USP14 expression in a dose-dependent manner ( Fig. 2A and B). When KYSE-150 and TE-12 cells were irradiated with a single dose of X-ray (4 Gy), the expression of USP14 was strongly down-regulated in a time-dependent manner ( Fig. 2C ). Notably, compared to the radiosensitive KYSE-150 and TE-12 cell lines, USP14 mRNA and protein expression levels were markedly up-regulated in our constructed irradiation-resistant cell lines KYSE-150R and TE-12R, as determined by RT-qPCR and western blotting analysis ( Fig. 2D and E). Collectively, these data implied that the increase in USP14 expression might be related to the irradiation tolerance of ESCA cells and therefore be involved in the process of X-ray radiotherapy. Fig. 2 USP14 expression is up-regulated in radioresistant ESCA cells. After treatment with varying doses of X-ray (from 0 to 6 Gy), the mRNA and protein expression levels of USP14 in KYSE-150 and TE-12 cells were examined using (A) RT-qPCR and (B) western lotting assays. (C) After exposure to 4 Gy of X-ray for the indicated time, the USP14 mRNA expression levels in KYSE-150 and TE-12 cells were assessed by RT-qPCR analysis. (D) RT-qPCR and (E) western blot analysis of USP14 mRNA and protein expression levels in radiosensitive and the constructed radioresistant KYSE-150 (KYSE-150R) and TE-12 (TE-12R) cell lines were performed. Data were presented as the mean ± SD (n=3 or 4 in each group). *p <0.05, ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001 versus the Control 0 Gy group; +++ p <0.001, ++++ p <0.0001. Statistical significances in (C) to (E) were assessed by unpaired Student's t test; Statistical significances in (A) and (B) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 2

USP14 expression promotes radiation-resistance in ESCA cells

To investigate the effect of USP14 on the irradiation tolerance of KYSE-150R and TE-12R cell lines, USP14 expression was depleted in these cells by infecting them with target shRNAs. Compared to the shNC group, the expression levels of USP14 in KYSE-150R and TE-12R cells were significantly decreased in the shUSP14 groups, as shown by RT-qPCR and western blotting assays ( Fig. 3A and B). The CCK-8 results consistently demonstrated that KYSE-150R and TE-12R cells exhibited a much stronger proliferative capacity than their corresponding sensitive groups, but USP14 knockdown significantly inhibited the proliferation of radioresistant ESCA cells ( Fig. 3C ). Colony formation and EdU staining assays revealed that the proliferation of KYSE-150R and TE-12R cells was higher than that of the KYSE-150 and TE-12 cell lines, respectively. However, reducing USP14 expression by shUSP14 significantly inhibited the proliferation of radioresistant ESCA cell lines ( Fig. 3D and E). Subsequently, after exposing radioresistant ESCA cells to different doses of X-ray, the results of CCK-8 analysis indicated that USP14 knockdown significantly enhanced the sensitivity of KYSE-150R and TE-12R cells to irradiation therapy, and especially shUSP14 cells exposed to higher dose irradiation showed a faster reduction in cell proliferation ( Fig. 3F ). Xenograft mouse models bearing radioresistant ESCA were subsequently established to further explore whether USP14 knockdown had anti-cancer effects in vivo . As depicted in Fig. 4A -C, we observed that irradiation treatment had no significant impact on tumor growth in KYSE-150R transplanted mice. Nevertheless, USP14 Knockdown significantly decreased the tumor growth rates and weights in the shUSP14 groups of mice undergoing radiotherapy, along with a decreased cancer cell density and reduced KI-67 positive expression levels in tumor sections ( Fig. 4 D and E). These in vitro and in vivo findings demonstrated that suppressing USP14 could diminish the irradiation tolerance of ESCA cells. Fig. 3 USP14 expression promotes radiation-resistance in ESCA cells. The mRNA and protein expression levels of USP14 examined by (A) RT-qPCR and (B) western blot analysis in KYSE-150R and TE-12R cells to validate USP14 knockdown efficiency. (C) Survival rates of KYSE-150R and TE-12R cells after knocking down USP14 examined by CCK-8 assay compared with each radiosensitive cell lines. The proliferation ability of KYSE-150R and TE-12R cells upon USP14 knockdown calculated by (D) clonogenic assay and (E) EdU staining analysis. (F) CCK-8 analysis was adopted to evaluate the proliferation ability of KYSE-150R and TE-12R cells with or without USP14 deletion. Data were presented as the mean ± SD (n=3 or 4 in each group). ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001. Statistical significances in (A) to (F) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 3 Fig. 4 Suppressing USP14 expression exerts anti-cancer effects in radioresistant xenograft mouse model. (A) Images for tumor tissues dissected form all groups of mice were shown. (B) Tumor volume was measured. (C) Tumor weights were measured. (D) H&E staining (up panel) and IHC staining for KI-67 (bottom panel) in tumor sections were performed. Scale bar, 50 μm. (E) KI-67 positive expression levels following IHC staining analysis were quantified. Data shown were mean ± SD (n=4 per group). ⁎⁎⁎⁎ p <0.0001 versus the Control group. Statistical significances in (B) to (D) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 4

Degrasyn improves the sensitivity of radioresistant ESCA cells to irradiation treatments

DGS ( Fig. 5A ) has been reported as an inhibitor of USP14 and was thus used to further investigate whether it could be regarded as a potential therapeutic strategy for ESCA treatment through improving irradiation tolerance. The CCK-8 results in Fig. 5B showed that DGS treatments ranging from 2.5 to 10 μM significantly reduced the proliferation of radiosensitive KYSE-150 and TE-12 cells in a dose- and time-dependent manner. Furthermore, a higher concentration of DGS at 10 μM exerted significant inhibitory effects on the proliferation of KYSE-150R and TE-12R ( Fig. 5C ). RT-qPCR and western blotting assays further showed that varying doses of DGS incubation for 24 h strongly reduced the expression of USP14 from mRNA and protein levels. Similarly, higher concentration of DGS treatment resulted in a significant decrease in USP14 mRNA and protein expression levels ( Fig. 5D and E). To further identify the inhibitory effect of DGS on USP14 signaling, we introduced another USP14 specific inhibitor IU1-47. As shown in Supplementary Fig. 1A, DGS treatments resulted in a dose-dependent increase in ubiquitinated proteins, which was similar to those induced by IU1-47. Additionally, DGS obviously inhibited the activity of USP14 in a concentration-dependent manner, as proved by decreased UB-VS labeling when compared with mock vehicle-treated radioresistant ESCA cells (Supplementary Fig. 1B), which exerted similar effects to IU1-47 in the blockage of USP14. Consistently, a decreased expression of USP14 induced by higher concentrations of DGS was detected in radioresistant ESCA cell lines. Together, these results suggested that although DGS was not a specific inhibitor of USP14, it was indeed a potent and effective inhibitor of USP14 in radioresistant ESCA cells. Herein, DGS was applied in subsequent studies to uncover the role of USP14 inhibition in the treatment of ESCA with radioresistance. Fig. 5 Degrasyn improves the sensitivity of radioresistant ESCA cells to irradiation treatments. (A) Chemical structure of Degrasyn (DGS). (B) KYSE-150 and TE-12 cells, and (C) KYSE-150R and TE-12R cell lines were subjected to different doses of DGS treatments (2.5, 5 and 10 μM) for 0 to 72 h. Thereafter, all radiosensitive and radioresistant cells were collected for cell viability calculation using CCK-8 analysis. Radiosensitive and radioresistant ESCA cells were incubated with DGS (2.5, 5 and 10 μM) for 24 h, and were then collected for (D) RT-qPCR and (E) western blot analysis to examine USP14 mRNA and protein expression levels. (F) KYSE-150 and TE-12 cells, and (G) KYSE-150R and TE-12R cell lines were exposed to DGS (2.5, 5 and 10 μM) incubation for 24 h, followed by cell proliferation calculation using EdU staining assay. Scale bar, 20 µm. (H) KYSE-150R and TE-12R cells were incubated with DGS (2.5 μM) for 24 h alone, 4 Gy of irradiation or the two in combination. Thereafter, all cells were harvested for EdU staining analysis. Scale bar, 20 µm. Data were presented as the mean ± SD (n=3 or 4 in each group). *p <0.05, ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001 versus the Control group; ++++ p <0.0001. Statistical significances in (B) to (H) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 5

As expected, DGS treatments significantly reduced the proliferative capacity of KYSE-150 and TE-12 cells in a concentration-dependent manner by EdU staining ( Fig. 5F ). However, KYSE-150R and TE-12R cells were only sensitive to higher dosages of DGS, with significantly reduced cell proliferation ( Fig. 5G ). Interestingly, we found that single treatments with DGS (2.5 μM) or X-ray (4 Gy) had no significant effect on the proliferation of KYSE-150R and TE-12R cells, but their combined treatments greatly reduced the proliferation of radioresistant ESCA cells ( Fig. 5H ). These results suggest that DGS at a low concentration could enhance the sensitivity of radioresistant ESCA cells to irradiation treatment, which may be associated with the suppression of USP14 expression.

Degrasyn improves the sensitivity of radioresistant ESCA cells to irradiation by up-regulating DNA damage signaling

The efficacy of radiation therapy is closely linked to radiation-induced DSBs in tumor cells, which subsequently causes fatal damage to the tumor cells [ 8 , 9 , 38 ]. As reported, the induction of γ-H2AX and 53BP1 is a key marker for DSBs and DNA damage [ 39 , 40 ]. The IF results in Fig. 6A -C indicated that DGS and X-ray individual treatments did not affect the positive foci formation of γ-H2AX and 53BP1 in KYSE-150R and TE-12R cells; however, their combined exposure significantly enhanced the positive expression of γ-H2AX and 53BP1, suggesting that DGS/X-ray therapy could trigger DNA damage in radioresistant ESCA cells. To further verify this, western blot analysis was employed to examine the ATR/CHK1 signaling pathway, which plays a crucial role in DNA damage [ 39 , 40 ]. As expected, the protein expression levels of γ-H2AX, p-CHK1, and p-ATR were significantly up-regulated in KYSE-150R and TE-12R cells compared to the control group, DGS, and X-ray individual therapy ( Fig. 6D and E). These data clarified that DGS at a low dose in combination with irradiation could induce DNA damage to exert its anti-cancer effect in ESCA with radioresistance. Fig. 6 Degrasyn improves the sensitivity of radioresistant ESCA cells to irradiation by up-regulating DNA damage signaling. KYSE-150R and TE-12R cells were exposed to 24 h of DGS (2.5 μM), X-ray irradiation (4 Gy) or the two in both. Then, all cells were collected for subsequent assays. (A) Double IF staining for γ-H2AX and 53BP1 in ESCA cell lines. Scale bar, 10 µm. Quantification for (B) γ-H2AX and (C) 53BP1 by IF staining was exhibited. (D,E) The protein expression levels of γ-H2AX, p-CHK1 and p-ATR in radioresistant ESCA cell lines were examined using western blotting analysis. Data were presented as the mean ± SD (n=3 or 4 in each group). ++++ p <0.0001. Statistical significances in (B), (C) and (E) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 6

Degrasyn promotes the therapeutic effects of radiotherapy in KYSE-150 and KYSE-150R xenograft mouse models without adverse effects

We then investigated the therapeutic effects of combining DGS with X-ray in the constructed xenograft mouse models with or without radioresistance. Firstly, in the mice bearing tumors driven by radiosensitive ESCA cells, we found that DGS at 10 mg/kg and 10 Gy irradiation therapy significantly reduced the tumor growth rates and tumor weights compared to the control group. Notably, their combined treatment exhibited a much stronger anti-cancer ability to suppress tumor growth, as indicated by the lowest tumor growth rates and weights. However, in mice bearing tumors induced by KYSE-150R cell transplantation, DGS and X-ray single treatment did not affect the tumor growth status, but their co-treatments significantly inhibited the tumor growth with a marked decrease in tumor weights ( Fig. 7A -C). As expected, DGS, irradiation and their combination greatly reduced KI-67 positive expression levels in tumor sections with down-regulated cancer cell density compared with the Control group of mice bearing tumors drove by KYSE-150 cells. Of note, DGS and X-ray single therapy had no significant influence on KI-67 expression changes in tumor sections from KYSE-150R/Con group, while their co-treatment obviously restrained KI-67 expression levels in tumor specimens ( Fig. 7D and E). These data identified that DGS and irradiation in combinational treatments could inhibit the irradiation tolerance of ESCA in vivo . Fig. 7 Degrasyn promotes the therapeutic effects of radiotherapy in KYSE-150 and KYSE-150R xenograft mouse models without adverse effects. (A) Images of dissected tumor samples from all groups of mice. (B) Tumor growth rates were measured. (C) Tumor weights were recorded after dissection. (D) H&E staining and IHC staining for KI-67 positive expression levels in tumor sections were conducted. Scale bar, 50 µm. (E) Quantification for KI-67 expression levels in tumor samples following IHC assay was conducted. (F) Body weights of each group of mice were recorded. Assessments of serum (G) ALT, (H) AST, (I) BUN, (J) CR and (K) CK-MB from each group of mice were performed. (L) H&E staining for major organs (heart, liver, spleen, kidney and lung) collected from all groups of mice. Scale bar, 100 µm. Data were presented as the mean ± SD (n=4 in each group). ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001 versus the Control group; ### p <0.001, #### p <0.0001 versus the DGS/X-ray group. Statistical significances in (B) to (K) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 7

Subsequently, the safety of DGS and its combination with X-ray was evaluated. First, no significant difference was observed in the changes of body weights among all groups of mice ( Fig. 7F ). Biochemistry analysis indicated that DGS, X-ray, and their combination treatments did not have significant hepatic, renal, or cardiac toxicity in all groups of mice, as evidenced by the similar serum levels of ALT, AST, BUN, CR, and CK-MB ( Fig. 7G -K). H&E staining also revealed that no significant morphological alterations were detected in major organ tissues, including the heart, liver, spleen, kidney, and lung, from all groups of mice ( Fig. 7L ). These results demonstrated that DGS or its co-treatment with irradiation could be safely administered in vivo .

USP14 overexpression abolishes the anti-tumor effects of combinational treatments of Degrasyn and radiotherapy in ESCA cells

Since DGS is a potential inhibitor of USP14, and considering the importance of USP14 in mediating irradiation tolerance in ESCA, we then overexpressed USP14 in KYSE-150R and TE-12R cells to further investigate the inhibitory effect of DGS/X-ray on cell proliferation and DNA damage processes. RT-qPCR and western blotting results confirmed the overexpression of USP14 in both KYSE-150R and TE-12R cells (Supplementary Fig. 2A and B). Results by CCK8 analysis in Fig. 8A and B indicated that compared with the Con group, USP14 overexpression markedly enhanced the cell viability of radioresistant KYSE-150R and TE-12R cell lines. Notably, the cell survival restrained by DGS/X-ray was significantly abolished upon USP14 overexpression in KYSE-150R and TE-12R cells. As expected, the proliferative capacities of KYSE-150R and TE-12R cells were strongly enhanced when USP14 was overexpressed. Promoting USP14 expression markedly abrogated the function of DGS/X-ray therapy to reduce the proliferation of KYSE-150R and TE-12R cells compared with the DGS/X-ray group by colony formation and EdU staining assays ( Fig. 8C and D). IF staining further indicated that radioresistant ESCA cells overexpressing USP14 showed much lower number of γ-H2AX and 53BP1 positive foci than that of the Con group. In expectation, USP14 overexpression strongly diminished the effects of DGS/X-ray therapy to upregulate γ-H2AX and 53BP1 in KYSE-150R and TE-12R cells ( Fig. 8E and F). Western blotting results consistently showed that facilitating USP14 could weaken γ-H2AX, p-CHK1 and p-ATR protein expression levels in radioresistant ESCA cell lines, and markedly abolish the role of DGS/X-ray in improving the expression levels of these molecules ( Fig. 8G ). These results further demonstrated that USP14 could promote the proliferative capacity of radioresistant ESCA cells, and importantly DGS/X-ray induced DNA damage might be USP14-dependent to ameliorate irradiation tolerance and subsequently inhibit the proliferation of ESCA cells with radioresistance. Fig. 8 USP14 overexpression abolishes the anti-tumor effects of combinational treatments of Degrasyn and radiotherapy in ESCA cells. (A) KYSE-150R and TE-12R cells with or without USP14 overexpression were subjected to DGS (2.5 μM) and X-ray irradiation (4 Gy) co-treatments for the shown different time points. Then, all cells were collected for CCK-8 analysis. (B-G) KYSE-150R and TE-12R cells with or without USP14 overexpression were subjected to 24 h of DGS (2.5 μM) and X-ray irradiation (4 Gy) co-treatments. Subsequently, all ESCA cells were harvested for assays as following. (B) Cell viability was measured using CCK-8 analysis. (C) Colony formation assay was conducted to examine the cell proliferation. (D) EdU staining images of ESCA cells. Scale bar, 20 µm. (E) Assessments of γ-H2Ax and 53BP1 foci formation in ESCA cells by double IF staining. Scale bar, 10 µm. (F) The number of γ-H2AX and 53BP1 positive expression foci was quantified. (G) The protein expression levels of γ-H2AX, p-CHK1 and p-ATR were detected using western blotting analysis. Data were presented as the mean ± SD (n=3 or 4 in each group). *p <0.05, ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001. Statistical significances in (A) to (G) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 8

Degrasyn combined with radiotherapy reduces EMT process in radioresistant ESCA cells through USP14 signal

Next, the malignant biological behavior in ESCA cells was examined after single DGS incubation, irradiation therapy, or their combination. The Transwell assay in Fig. 9A -C showed that KYSE-150R and TE-12R cells had stronger migratory and invasive abilities than KYSE-150 and TE-12 cell lines, respectively. In our constructed radioresistant ESCA cell lines, DGS and X-ray alone had no significant effect on the changes in cell migration and invasion compared with the KYSE-150R and TE-12R control groups. However, DGS/X-ray co-exposure significantly reduced the number of radioresistant ESCA cells in migration and invasion. E-Cadherin expression levels were highly induced in KYSE-150R and TE-12R cells after co-treatment with DGS and X-ray, but their single treatment had no such regulatory effects. In contrast, N-Cadherin expression levels were strongly suppressed in KYSE-150R and TE-12R cells co-treated with DGS/X-ray ( Fig. 9D ), which was consistent with the changes in malignant biological behavior. Importantly, all our results above further showed that USP14 overexpression markedly facilitated the migration and invasion of radioresistant ESCA cell lines, accompanied by obvious decrease of E-Cadherin and increase of N-Cadherin. More importantly, we found that promoting USP14 expression significantly abrogated the regulatory effects of DGS/X-ray on the migration, invasion, E-Cadherin and N-Cadherin expression in KYSE-150R and TE-12R cells ( Fig. 9A -D). These results above revealed that USP14 had promotive effects on the migration and invasion of radioresistant ESCA cells, and that DGS/X-ray therapy-retrained malignant biological behavior of ESCA cells with radioresistance was also dependent on USP14 suppression. Fig. 9 Degrasyn combined with radiotherapy reduces EMT process in radioresistant ESCA cells through USP14 signal. KYSE-150R and TE-12R cells with or without USP14 overexpression were subjected to 24 h of DGS (2.5 μM) and X-ray irradiation (4 Gy) co-treatments, or the two alone. Then, all cells were collected for studies as following. KYSE-150 and TE-12 cell lines were served as control. (A) Cell migration and invasion were assessed by transwell analysis. Scale bar, 50 µm. The number of migratory and invaded (B) KYSE-150R cells and (C) TE-12R were was quantified. (D) The mRNA expression levels of EMT markers including E-Cadherin and N-Cadherin in ESCA cell lines were examined using RT-qPCR analysis. Data were presented as the mean ± SD (n=4 in each group). ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001 versus the KYSE-150 group; ### p <0.001, #### p <0.0001 versus the KYSE-150R group; + p <0.05, ++ p <0.01, +++ p <0.001. Statistical significances in (B) to (D) were assessed by unpaired Student's t test and one-way ANOVA with Tukey's multiple comparisons test. Fig 9

Degrasyn and irradiation in combination inhibits the radioresistance of ESCC cells through reducing YAP1 stabilization mediated by USP14

To further elucidate the mechanisms underlying the inhibition of radioresistant ESCA progression by DGS/X-ray, total RNA was extracted from the DGS/X-ray-exposed KYSE-150R cell line and control cells for RNA sequencing analysis to screen for differentially expressed genes (DEGs). We identified 62 upregulated and 79 downregulated genes in KYSE-150R cells upon DGS/X-ray exposure ( Fig. 10A ). Notably, the Hippo/YAP1 signaling pathway was enriched by KEGG and GSEA analyses based on these detected DEGs ( Fig. 10B and C, Supplementary Table 3). By analyzing TCGA-ESCA transcriptome data, we found a positive correlation between USP14 and YAP1 ( Fig. 10D ). Subsequently, we also observed significantly higher YAP1 protein expression levels and its downstream signal CTGF in KYSE-150R and TE-12R cells compared to KYSE-150 and TE-12 cell lines, respectively, through western blotting assays ( Fig. 10E ). Furthermore, USP14 knockdown significantly down-regulated YAP1 and CTGF expression levels in KYSE-150R cells compared to the shNC group ( Fig. 10F ). As expected, DGS and irradiation single treatments had no significant impact on the expression changes of YAP1 and CTGF, but their co-treatments notably reduced the expression levels of the two proteins in TE-12R cells ( Fig. 10G ). Notably, different with the results detected in radioresistant ESCA cells, YAP1 expression levels were significantly down-regulated by DGS from 2.5 to 10 µM in KYSE-150 and TE-12 cells with radiosensitivity via a dose-dependent manner (Supplementary Fig. 3), which were consistent with the variation tendency of USP14 in these radiosensitive ESCA cells. Furthermore, IHC and western blotting assays confirmed that DGS combined with X-ray radiotherapy significantly reduced YAP1 protein expression levels in tumor tissues collected from tumor-bearing mice drove by KYSE-150R cells (Supplementary Fig. 4A and B). YAP1 can be deubiquitinated to maintain its protein stability to mediate varying cellular events, and USP14 is a type of deubiquitin enzyme [ 15 ]. Given that USP14 suppression led to the reduction of YAP1 in ESCA cells, we then examined whether there might be a correlation between USP14 and YAP1. Co-IP analysis in Fig. 10 , Fig. 10 confirmed that YAP1 and USP14 could interact with each other in KYSE-150R and TE-12R cells. Double fluorescent images confirmed the co-localization of YAP1 with USP14 in KYSE-150R and TE-12R cells ( Fig. 10J ). Half-life analysis indicated that USP14 deletion significantly decreased the protein stability of YAP1 in KYSE-150R cells ( Fig. 10K ). Similarly, DGS/X-ray co-treatments markedly reduced the stability of YAP1 in TE-12R cells ( Fig. 10L ). However, when compared with the Con group, DGS single treatment at 2.5 μM had no significant effect on the changes of YAP1 protein degradation in TE-12R cells (Supplementary Fig. 5). These data further demonstrated that USP14 could interact with YAP1 and mediate its stabilization in ESCA cells, and the USP14/YAP1 axis might be a potential underlying mechanism for the anti-cancer role of DGS/X-ray co-therapy in ESCA. Fig. 10 Degrasyn and irradiation in combination inhibits the radioresistance of ESCC cells through reducing YAP1 stabilization mediated by USP14. (A) The differentially expressed genes (DEGs) mediated by DGS (2.5 µM)/X-ray (4 Gy) double treatments in KYSE-150R cells were depicted in the volcanic maps, with 141 genes altered versus the control group in the absence of any treatments. (|log2(FC)|>1.5 and p<0.05). (B) The Hippo signaling pathway was regulated in KYSE-150R cells after DGS/X-ray combinational treatments according to KEGG analysis based on DEGs. (C) GSEA enrichment plot for the Hippo/YAP signaling pathway enriched in DEGs. (D) Correlation between USP14 and YAP1 expression was assessed in ESCA specimens using Spearman's correlation coefficient analysis based on TCGA database. (E) YAP1 and its downstreaming signal CTGF protein expression levels in KYSE-150, TE-12, KYSE-150R and TE-12R cells were assessed by western blot analysis. (F) YAP1 and CTGF protein expression levels in KYSE-150R cells were examined after USP14 knockdown. (G) TE-12R cells were treated with 24 h of DGS (2.5 µM), X-ray (4 Gy) or the two in double. Then, the cells were collected for YAP1 and CTGF protein expression calculation using western blotting analysis. (H,I) Co‐IP assays revealing that USP14 interacted with YAP1 in both KYSE-150R and TE-12R cells. A control IgG was used as the negative control. Primary antibodies as shown were used. (J) IF staining indicating the localization of USP14 (red) and YAP1 (green) in KYSE-150R and TE-12R cells. The nuclei were stained using DAPI (blue). Scale bar, 10 µm. (K) The effect of USP14 knockdown on YAP1 stability was detected after CHX treatment (10 µM) for the shown time points in KYSE-150R cells. (L) The effect of DGS (2.5 µM)/X-ray (4 Gy) co-treatments on YAP1 protein stability was examined after CHX treatment (10 µM) for the indicated time points in TE-12R cells. Data were presented as the mean ± SD (n=3 or 4 in each group). ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001. Statistical significances in (E) was assessed by unpaired Student's t test; Statistical significances in (F) and (G) were assessed by one-way ANOVA with Tukey's multiple comparisons test. Fig 10

Degrasyn and irradiation reduces the proliferative and malignant biological behavior in radioresistant ESCA cells via the USP14/YAP1 axis

In the section, ubiquitination assay results indicated that Flag-USP49 clearly removed the ubiquitination of YAP1 in KYSE-150R and TE-12R cells, whereas USP14 knockdown facilitated YAP1 ubiquitination. Importantly, DGS/X-ray exposure also led to stronger ubiquitination of YAP1 in ESCA cell lines, but DGS single treatment at 2.5 μM showed no obvious influence on the changes of YAP1 ubiquitination ( Fig. 11A ). It has been suggested that in addition to USP14 inhibition, DGS can also restrain the activity of USP5 and USP9x [ 31 , 35 , 36 ]. We subsequently investigated that whether DGS could modulate YAP1 protein stability through suppressing USP5 or USP9x signal. The protein expression levels of YAP1 were examined in radiosensitive and radioresistant ESCA cell lines with specific knockdown of USP5 or USP9x, respectively. Western blotting results showed the efficient depletion of USP5 and USP9x, and knockdown of USP5 and USP9x did not affect the expression changes of YAP1 in radio-sensitive or -resistant KYSE-150 and TE-12 cells (Supplementary Fig. 6A and B). These results by immunoblotting assay revealed that DGS/X-ray therapy functioned as an inhibitor of USP14, which played a crucial role in retarding the deubiquitination of YAP1. Fig. 11 Degrasyn and irradiation reduces the proliferative and malignant biological behavior in radioresistant ESCA cells via the USP14/YAP1 axis. (A) Analysis of the effect of shUSP14 and DGS (2.5 µM)/X-ray (4 Gy) co-incubation on YAP1 ubiquitination with a ubiquitination experiment performed in KYSE-150R and TE-12R cells. The polyubiquitylated YAP1 protein was evaluated using anti-Myc antibody. (B-H) KYSE-150R and TE-12R cells with stable USP14 knockdown by treatment with shUSP49-#1 plasmid or with YAP1 overexpression by transfection with Flag-YAP1 plasmids alone or in combination were then subjected to DGS (2.5 µM)/X-ray (4 Gy) co-incubation for 24 h. Thereafter, all cells were collected for studies as followings. (B) CCK-8 analysis was used to examine the cell viability. (C) Colony formation of radioresistant ESCA cells was conducted. (D,E) EdU staining was performed to detect the cell proliferation status. Scale bar, 20 µm. (F-H) Transwell analysis was used to determine the migration and invasion of KYSE-150R and TE-12R cells. Scale bar, 50 µm. (I,J) Images of double IF staining for γ-H2AX and 53BP1 positive foci in cells were shown, and the number of γ-H2AX and 53BP1 positive foci was quantified. Scale bar, 10 µm. (K) Western blot analysis was used to determine YAP1, CTGF and γ-H2AX protein expression levels in cells. Data were presented as the mean ± SD (n=3 or 4 in each group). *p <0.05, ⁎⁎ p <0.01, ⁎⁎⁎ p <0.001, ⁎⁎⁎⁎ p <0.0001. Statistical significances in (B), (C), (E), (G), (H), (J) and (K) were assessed by unpaired Student's t test and one-way ANOVA with Tukey's multiple comparisons test. Fig 11

YAP1 has been recognized to function as an oncogene in tumor growth, and its overexpression is involved in radioresistance [ 41 , 42 ]. To determine whether the oncogenic actions of USP14 mediated by DGS/X-ray were dependent on YAP1, function-reversal and blockade experiments were then conducted. Firstly, CCK-8 and colony formation analysis indicated that YAP1 upregulation markedly enhanced the proliferative capacity of radioresistant ESCA cell lines. Intriguingly, shUSP14- and DGS/X-ray-reduced cell viability and proliferation were significantly reversed by YAP1 ( Fig. 11B and C). EdU staining confirmed that YAP1 facilitation could markedly promote the proliferation of ESCA cell lines with radioresistance, and more importantly, YAP1 overexpression significantly reverse the proliferation suppression of KYSE-150R and TE-12R cells with USP14 knockdown or DGS/X-ray co-exposure ( Fig. 11D and E). In addition, the migration and invasion ability of radioresistant ESCA cells was strongly facilitated by YAP1. Furthermore, the number of cells in migration and invasion reduced by shUSP14 and DGS/X-ray was almost restored upon YAP1 overexpression ( Fig. 11F -H). IF staining then showed that the YAP1 overexpression could further reduce γ-H2AX and 53BP1 foci formation and considerably reverse the up-regulation of γ-H2AX and 53BP1 foci formation induced by USP14 knockdown and DGS/X-ray ( Fig. 11I and J). Western blotting results confirmed that γ-H2AX and CTGF protein expression levels mediated by shUSP14 and DSG/X-ray were remarkably abolished in KYSE-150R and TE-12R cells with YAP1 overexpression by western blotting assay ( Fig. 11K ). These results above demonstrated that YAP1 overexpression was indeed associated with the progression of radioresistance during ESCA growth. Notably, DSG/X-ray therapy-restrained the proliferative and malignant biological behaviors of ESCA cells mediated by USP14 was mainly dependent on the suppression of YAP1 signaling.

Discussion

Radiotherapy is an effective treatment method for ESCA; however, radiation tolerance often occurs, limiting its clinical application to a large extent [ 6 , 8 , 9 ]. Increasing evidence reports that USP14 is overexpressed in many different types of tumors, such as oral cancer, lung cancer, and ESCA, and is related to the radiosensitivity and resistance of OSCC and nasopharyngeal carcinoma [ 11 , 16 , [18] , [19] , [20] , 23 , 25 , 43 ]. However, few studies have focused on the effects of USP14 on ESCA radioresistance, and the underlying molecular mechanisms are still unclear.

In the present study, we similarly showed that USP14 was overexpressed in ESCA tissues and cell lines, especially in ESCA cells with radioresistance. Irradiation therapy dose-dependently down-regulated USP14 gene and protein expression levels in radiosensitive ESCA cells. Importantly, suppressing USP14 by shRNAs not only inhibited the proliferation of radioresistant ESCA cells but also enhanced the sensitivity of these cells to X-ray therapy, which was validated in xenograft murine models, as indicated by the decreased tumor growth rates and weights. In contrast, overexpressing USP14 significantly promoted the proliferative and migratory capacities of radioresistant ESCA cell lines. DGS also known as WP1130 is a cell-permeable DUB inhibitor. We here found that DGS functioned as a specific inhibitor of USP14 known as IU1-47. DGS dose-dependently induced a marked increase in ubiquitylated proteins and reduced the labeling of USP14 with HA-Ub-VS, confirming that DGS was an efficient DUB inhibitor for USP14. Intriguingly, we discovered for the first time that DGS exerted antitumor effects in both radio-sensitive and -resistant ESCA cells through a time- and dose-dependent manner, and importantly, sensitized the cytotoxic effects of irradiation on ESCA cell lines, which was mainly achieved by inhibiting USP14 expression levels and DUB activities. Furthermore, DNA damage signaling γ-H2AX, CHK1, and ATR was enhanced in ESCA cells with radioresistance after combinational treatments with low concentration DGS and X-ray. Additionally, malignant biological behaviors such as cell migration and invasion were significantly retarded by DGS/X-ray co-exposure in radioresistant ESCA cells. More importantly, the anti-tumor effects of DGS/X-ray cotreatments were almost abrogated in ESCA cells upon USP14 overexpression, suggesting that USP14 inhibition was necessary by DGS, which could enhance the antitumor effects of irradiation to overcome radioresistance during ESCA treatment. Animal studies confirmed that DGS combined with X-ray radiotherapy effectively restrained the tumor growth both in nude mice induced by KYSE-150 and KYSE-150R cell transplantation with undetectable adverse effects. Moreover, RNA-Seq analysis indicated that Hippo/YAP1 signaling was enriched by DGS/X-ray. YAP1 has been recognized to function as an oncogene in tumor growth, and its overexpression is involved in radioresistance [ 41 , 42 ]. Firstly, similar with the expression alterations of USP14, our data indicated that in the absence of radiation treatment, DGS treatments from 2.5 to 10 μM markedly downregulated YAP1 expression levels in KYSE-150 and TE-12 cells with radiosensitivity via a concentration-dependent manner, which revealed the potential of USP14/YAP1 signaling as a therapeutic target for DGS. It should be noticed that low dose DGS at 2.5 μM showed no significant effects on YAP1 expression changes in ESCA cells with radiation-resistance. These results suggested that radiation-resistant ESCA cells were not as sensitive to low dose DGS intervention as we observed in radiation-sensitive ESCA cells, which might be associated with the stronger proliferative and migrant capacities of radioresistant ESCA cells. Herein, an important aspect to consider was the dose range of DGS in our study. We further found that YAP1 overexpression markedly accelerated the proliferation and migration of ESCA cells with radioresistance, whereas further restrained the DNA damage process. Co-IP results showed that USP14 could interact with YAP1 in radioresistant ESCA cells, and USP14 suppression obviously reduced the stabilization of YAP1 protein. Surprisingly, YAP1 stability could also be strongly weakened upon DGS/X-ray combinational exposure. USP14 knockdown and DGS/X-ray therapy led to YAP1 ubiquitination in ESCA cells with radioresistance, thereby reducing YAP1 stability and inducing its expression suppression. Finally, we found that USP14 depletion- and DGS/X-ray therapy-mediated proliferation, EMT process and DNA damage biological processes were almost abolished upon YAP1 overexpression, uncovering that YAP1 blockage was required for DGS/X-ray therapy to depress the malignant biological behaviors of ESCA cells through USP14 signal. Increasing evidence suggests that through combination therapy, the lethality of radiation resistant ESCA cells can be improved, thus improving the therapeutic effect [ 3 , 4 , 6 ]. Notably, the small dose of DGS combined with low-intensity radiotherapy could enhance the therapeutic sensitivity of radioresistant ESCA, thereby retraining the proliferation, migration and invasion of ESCA cells with radioresistance, which was mainly through the suppression and disruption of USP14/YAP1 axis. Taken together, all these new findings disclosed that DGS combined with X-ray exerted anti-ESCA effects by targeting USP14 and its related YAP1 signaling pathway ( Fig. 12 ) and could be applied as a potential therapeutic strategy for ESCA treatment, particularly in those patients with radioresistance. Fig. 12 Overall schematic diagram. The schematic diagram illustrates that USP14 induces YAP1 deubiquitylation to improve its stability in ESCA cells, resulting in the proliferation, EMT process and radioresistance, which contribute to the progression of ESCA. Notably, degrasyn exerts its anti-cancer functions in ESCA and reduces radioresistance mainly through suppressing USP14/YAP1 axis. Fig 12

Ubiquitination, a ubiquitous posttranslational modification, participates in various biological processes, including protein sorting, degradation, DNA repair, and transcriptional activation. Numerous studies have shown that the balance between ubiquitination and deubiquitylation determines the fate of cancer cells [ 10 , 44 ]. USP14 is a member of the USP family, the largest DUBs family. The critical roles of USP14 in the development of various tumors have been identified. USP14 promotes tumorigenesis and chemoresistance in multiple myeloma by suppressing apoptosis [ 45 ]. USP14 also contributes to the initiation and progression of breast cancer. Its inhibition or silencing strongly enhances the ubiquitination of cyclin B1 and induces cells at the G2/M phase, thereby restricting the proliferative and migratory abilities of breast cancer cells [ 16 ]. Genetic and pharmacological inhibition of USP14 promote the ubiquitination and degradation of the androgen receptor (AR) and inhibit the growth of prostate cancer cells [ 46 ]. USP14 suppression increase radiosensitization in NSCLC cell lines, as evidenced by reduced survival rates [ 23 ]. Previously, USP14 was reported to be significantly upregulated in human ESCC tissues and cell lines, and its downregulation suppressed the migration and invasion of ESCC cells [ 25 , 27 ]. Consistent with previous studies, we also identified USP14 overexpression in human ESCA tissues cell lines. Although it is well-established that USP14 is overexpressed in tumor cells and tissues, few studies discuss the correlation between radioresistance and USP14 in ESCA. It's worth noting that ESCA cells with radioresistance exerted much higher USP14 expression levels than that of the radiosensitive parental cells. Overexpressing USP14 enhanced the proliferative and migratory capacities of radioresistant ESCA cell lines; however, its knockdown exhibited anti-cancer effects on ESCA, and it's worth noting that USP14 suppression markedly promoted the sensitivity of radioresistant ESCA cells in response to X-ray exposure. Therefore, collectively, these findings confirmed that USP14 was indeed a promising indicator and regulator of ESCA and functioned as an oncogene in ESCA progression.

Much efforts have been made to develop inhibitors of USP14. For instance, small molecule inhibitors, such as b-AP15, are identified in cell-based screening processes for compounds, which can regulate the lysosomal apoptotic signaling pathway [ 47 ]. IU1, another USP14 inhibitor, exerts anti-tumor effects on the proliferation of cervical cancer cells via modulating MDM2 degradation [ 48 ]. DGS is also a cell-permeable DUB inhibitor, and directly inhibits DUB activity of USP9x, USP5 and USP14 [ 35 , 36 , 49 ]. DGS reduces the metastasis of pancreatic ductal adenocarcinoma via targeting USP5/WT1/E‐cadherin signaling pathway [ 35 ]. DGS also exhibits suppressive effects on USP9x to inhibit oral cancer development [ 36 ]. In our present study, we further identified that DGS could significantly reduce the expression of USP14 in ESCA cell lines via a dose-dependent manner. Notably, DGS showed similar efficacy with IU1-47, a recognized more specific inhibitor of USP14 [ 50 ], in suppressing the deubiquitylating activity of USP14. Although radiotherapy is a pivotal therapeutic approach for controlling ESCA development, the therapeutic outcome is still not optimistic due to radioresistance [ 51 ]. Therefore, combining radiotherapy with chemotherapy has been widely applied and extensively studied to overcome radioresistance [ 52 , 53 ]. In our study, we newly discovered that DGS significantly reduced the survival rates of ESCA cells in a time- and dose-dependent manner with decreased expression levels of USP14. Interestingly, we unexpectedly found that a low concentration of DGS combined with 4 Gy of X-ray irradiation significantly reduced the proliferation, migration, and invasion of radioresistant ESCA cell lines with decreased expression levels of the EMT promoter N-Cadherin and increased expression of the EMT inhibitor E-Cadherin [ 54 ]. Animal studies confirmed not only the stronger inhibitory effects of the combination treatment of DGS and X-ray on the progression of radiosensitive ESCA in our established xenograft mouse models, but also improved the radiosensitivity of nude mice bearing tumor tissues driven by KYSE-150R cells in response to X-ray exposure. Hence, the co-treatment of DGS with X-ray could reduce the irradiation tolerance of ESCA both in vitro and in vivo.

The effectiveness of radiation therapy is associated with radiation-induced DSBs in tumor cells, which subsequently result in lethal damage to tumor cells. In response to DSBs caused by ionizing radiation, ataxic telangiectasia mutation/Ataxic telangiectasia and Rad3-associated protein (ATM/ATR) kinases can be recruited to the DSB lesion site and activated, thereby inducing γH2AX. Subsequently, DNA repair-related proteins accumulate at the damaged DSB sites and complete DNA repair [ 39 , 40 , 55 , 56 ]. Meanwhile, ATM/ATR can activate CHK1/2 to induce cell cycle arrest and provide sufficient time for DNA repair [ 57 ]. It is well known that the abnormal expression of these essential factors involved in DDR can lead to changes in the radiosensitivity of tumor cells. 53BP1 is also a key indicator of DSBs and is involved in DSB repair. It has been demonstrated that ATM can also phosphorylate 53BP1 at different sites to promote the recruitment of various 53BP1-interacting proteins involved in DSB repair [ 58 , 59 ]. USP14 was previously reported to mediate the DNA damage response in NSCLC. Knockdown of USP14 increased irradiation-induced foci formation by 53BP1 [ 60 ]. Similarly, in our current study, we found that USP14 suppression by DGS and X-ray co-exposure significantly induced γH2AX and 53BP1 foci formation in radioresistant ESCA cell lines, along with increased protein expression levels of p-CHK1 and p-ATR, which indicated that the DNA damage process was involved in the anti-cancer effects of DGS/X-ray on ESCA progression to improve radiosensitivity and thereby alleviate radioresistance. Notably, upon USP14 overexpression, the function of DGS/X-ray to induce DNA damage was considerably abrogated, proved by the diminished γH2AX and 53BP1 foci deterioration and inactivation of CHK1/ATR signaling. Therefore, USP14 expression suppression was indispensable for the co-exposure of DGS and irradiation against radioresistance in ESCA treatment.

The malignant growth of ESCA is a complex process involving multiple genes. To further elucidate the specific mechanism by which DGS/X-ray mediates radioresistant ESCA progression, RNA-Seq analysis was performed, and we found that the Hippo/YAP1 signaling pathway might be involved in the improvement of irradiation tolerance and tumor growth by DGS/X-ray. Accumulating evidence has reported that YAP1 functions as a proto-oncogene in various types of tumors, such as breast cancer, oral cancer, and lung cancer [ [61] , [62] , [63] ]. Importantly, YAP1 is abnormally overexpressed in ESCA and participates in the biological processes of tumors, including cell proliferation and EMT processes [ 64 , 65 ]. Suppressing YAP1 could radiosensitize breast cancer cells through targeting DNA damage response [ 66 ]. Additionally, YAP1 nuclear translocation promotes radiation resistance in nasopharyngeal carcinoma cells [ 67 ]. Recently, YAP overexpression was shown to confer radioresistance in ESCA by regulating HIF-1α [ 42 ]. Here in our study, we found significantly higher YAP1 expression levels in radioresistant ESCA cell lines, and its overexpression facilitated the proliferation and migration of ESCA cells with radioresistance. It was worth noting that USP14 knockdown- and DGS/X-ray therapy-suppressed tumor cell proliferation, EMT process and -enhanced DNA damage event was considerably eliminated upon YAP1 overexpression. These data revealed that YAP1 suppression might be involved in ESCA treatment mediated by DGS and irradiation combinational therapy. Increasing evidence has suggested that YAP1 can be degraded by ubiquitination. For instance, FBXW7, a typical tumor suppressor through targeting oncoproteins for ubiquitylation and degradation, mediates YAP1 stability by ubiquitin-proteasomal degradation in hepatocellular carcinoma [ 68 ]. In our study, we newly found that USP14 could interact with YAP1 in ESCA cell lines, and USP14 knockdown obviously reduce the stability of YAP1, revealing that USP14 could stabilize YAP1. These findings demonstrated that USP14 was a novel YAP1-interacting DUB. Furthermore, DGS combined with irradiation significantly weakened the stabilization of YAP1 in ESCA cells. Further studies revealed that USP14 might remove the ubiquitination of YAP1 to subsequently maintain YAP1 stability, while USP14 silencing and DGS/X-ray clearly induced the YAP1 polyubiquitination in ESCA cells, which might be a key for DGS/X-ray therapy to induce YAP1 degradation and thus retard its nuclear translocation and subsequent tumor growth behaviors. Importantly, we demonstrated that YAP1 overexpression could reverse the suppression of cell proliferation, malignant biological behaviors, and downregulation of CTGF expression caused by USP14 knockdown and DGS/X-ray co-exposure, accompanied by the elimination of γ-H2AX and 53BP1 foci in ESCA cells with radioresistance. Therefore, we concluded that DGS/X-ray reduced irradiation tolerance in ESCA treatments depending on the blockade of YAP1 mediated by USP14 ( Fig. 12 ). Recently, it was reported that YAP1 could induce USP14 expression in oxygen-glucose deprivation (OGD) mouse neuronal cells, indicating that YAP1 may mediate USP14 expression [ 69 ]. Based on these data, we hypothesized that USP14 might be a target gene of YAP1. Therefore, whether there is a feedback loop between USP14 and YAP1 during ESCA progression or even in other types of tumors, and whether this is involved in tumor treatment mediated by DGS and irradiation co-exposure, further studies are still needed in the future. Finally, there was still another limitation in our present study. It was noted that besides the suppressive effects of DGS on the DUB activity of USP14, DGS could also markedly reduce the mRNA and protein expression levels of USP14, which was different with other inhibitors of DUBs [ 32 ]. We supposed that the specific effects might be associated with varying concentrations, duration of action, and the types of cells. In addition, DGS might also indirectly affect the expression of deubiquitinating protein USP14. DGS might influence intracellular signaling pathways, protein stability, or transcriptional regulation, thereby modulating the expression of USP14. Therefore, to precisely understand the effects of DGS on the expression of USP14, detailed experimental studies and analysis are still warranted in our near future.

Conclusion

Our study found that USP14 was a promising DUB of YAP1 protein, which participated in ESCA radioresistance onsetting and tumor growth. Suppressing USP14 by DGS/X-ray cotreatments resulted in the inactivation of YAP1 signaling, thus depressing the proliferative and malignant processes of ESCA cells, along with promoted DNA damage behavior, contributing to the reduction of irradiation tolerance. This study uncovered that the aberrant activation of USP14/YAP1 axis played an essential role in the progression of ESCA and that DGS in combination with irradiation might provide valuable strategies for clinical management of ESCA.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences.

Consent for publication

All authors consent for publication.

Availability of data and materials

No datasets were generated or analyzed during the current study.

Funding

This work was supported by Shandong Young Scholars Science and Technology Support Program ( ZC200SDjk45 ).

CRediT authorship contribution statement

Fang Yuan: Conceptualization, Formal analysis, Writing – original draft. Juan Xu: Conceptualization, Formal analysis, Writing – original draft. Lingmei Xuan: Conceptualization, Formal analysis. Chan Deng: Formal analysis. Wei Wang: Formal analysis. Rong Yang: Software, Resources, Data curation, Formal analysis, Conceptualization, Writing – original draft.

Declaration of competing interest

The authors declare no competing interests.

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Supplementary materials

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Acknowledgments

We thank Letpub, Ltd., (London) for editing the English language of our article.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101101 .