Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

Abstract Objectives Targeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated. Materials and Methods The effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca2+ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting. Results PR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca2+ and then stimulated autophagy through Ca2+‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619. Conclusions Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.


| INTRODUC TI ON
Oesophageal cancer is one of the common malignancies with high mortality rates in the world. 1,2 Histologically, oesophageal squamous cell carcinoma (ESCC) is the primary subtype in Asia, including China.
Although there are rapid developments in diagnosis and therapies, the prognosis of oesophageal cancer patients is still abysmal, and the 5-year relative survival rates are only 30% in China 1 and 19% worldwide. 2 Therefore, it is crucial to investigate the mechanism of cancer progression and find novel candidates for targeted therapeutic strategies.
Deubiquitinases (DUBs) are essential components of the ubiquitin-proteasome system (UPS). By hydrolysing the isopeptide bond that present at the C-terminus of ubiquitin molecule, DUBs remove ubiquitin chains, regulate the activation and localization of target proteins and thus participate in the regulation of various cellular processes including apoptosis, senescence and autophagy. 3,4 Recent reports showed that many DUBs overexpressed in solid tumours, leukaemias and myelomas. [5][6][7][8] USP14 overexpressed in epithelial ovarian cancer, 9 colorectal cancer, 10 lung cancer 11 and oesophageal cancer, 12,13 and it was closely related to more inferior overall survival rate and prognosis. The increased expression of USP7/HAUSP was also reported to participate in the development of multiple cancers, such as multiple myeloma, 14 oesophageal cancer, 15 gliomas 16 and ovarian cancer. 17,18 The relationship between abnormally activated or expressed of DUBs and cancers was reviewed in detail in several recent papers. [5][6][7][8] These reviews highlight that DUBs represent novel candidate targets for drug development.
Inactivation of DUBs by inhibitors showed promising antitumour activity in multiple tumours. Chauhan et al 14

discovered that P5091
was a specific inhibitor of deubiquitylating enzyme USP7. P5091 significantly inhibited cell growth of ovarian cancer, 19 oesophageal cancer 15 and colorectal cancer 20 in vitro and in vivo. Meanwhile, P5091 induced apoptosis in multiple myeloma cells resistant to conventional and Bortezomib therapies. 14 D´Arcy et al first reported b-AP15 and described it as an inhibitor of USP14 and UCH37/UCHL5. 21 And then, b-AP15 was identified as an anti-cancer deubiquitinase inhibitor in many cancers. 13,[22][23][24] Using activity-based chemical proteomics, Altun et al characterized the small molecule PR-619 as a broad-range DUB inhibitor. 25 PR-619 treatment led to the striking accumulation of poly-ubiquitinated proteins and components of the 26S proteasome complex without direct impairment of proteasomal proteolysis. 25 Subsequently, PR-619 was widely used to investigate the role of ubiquitination in various cell and physiological processes.
PR-619 participated in the trafficking of Ca 2+ -activated K + channel (KCa3.1), 26 dynein localization during mitosis, 27 oocytes mature 28 and HIV-1 replication. 29 PR-619 affected the microtubule network and caused protein aggregation in neural cells. 30 Administration of PR-619 attenuated renal fibrosis in vitro and in vivo by reducing Smad4 expression. 31 PR-619 induced autophagy in oligodendroglia cells 32 and sensitized normal human fibroblasts to TRAIL-mediated cell death. 33
Cell viability was detected using the Cell Counting Kit-8 (CCK-8) kit (Beyotime Institute of Biotechnology, China). Cell growth was also examined by colony formation assay. Five hundred cells were seeded into 6-well plates in triplicate, treated with DMSO (0.1%) or PR-619 and then incubated for 10 days. The colonies were fixed with 4% paraformaldehyde (Solarbio, China) and stained with crystal violet (Beyotime, China). Colonies comprising 50 cells or more were counted as previously described. 35

| Gene silencing using siRNA
Kyse30 and Kyse450 cells were transfected with siRNA oligonucleotides (final concentration: 100 nmol/L) synthesized F I G U R E 2 PR-619 induced intrinsic apoptosis. Kyse30 and Kyse450 cells were treated with PR-619 for 48 h. A, Apoptosis was determined by FACS analysis using Annexin V-FITC/PI double-staining kit (left panel), and Annexin V + cell populations were defined as apoptosis (right panel). B, CASP3 activity was also analysed with FACS (left panel). The percentage of cells with active caspase3 was shown in the right panel. C, PR-619 induced mitochondrial membrane depolarization. Cells were treated with PR-619 and analysed by FACS as described in Materials and Methods section. D, Effect of PR-619 on the expression of apoptotic proteins, pro-apoptotic and anti-apoptotic proteins. Kyse30 and Kyse450 cells were treated with PR-619 for 48 h, and cell extracts were prepared for Western blotting analysis. GAPDH was used as the loading control. All data were representative of at least three independent experiments (n = 3; error bar, SD)

| Acridine orange (AO) staining for autophagy detection
Acridine orange staining was performed to detect autophagy according to published protocol. 36,37 Briefly, cells were treated with DMSO or PR-619 (8 µmol/L) for 24 hours and stained with acridine orange (Solarbio, China) in PBS containing 5% FBS at 37°C for 30 minutes.

| Measurement of intracellular Ca 2+
The levels of intracellular Ca 2+ were detected using the Fluo-3 AM fluorescence working solution (Beyotime, China) as the instruction

| Statistical analysis
The statistical significance of differences between groups was assessed using GraphPad Prism5 software. (GraphPad Software, Inc, La Jolla, CA, USA). The t test was used for the comparison of parameters between groups. For all tests, * represented the difference between the two groups was significant.

| PR-619 suppressed the growth of ESCC cells
As suggested, PR-619 inhibited the majority of DUBs at a concentration of 5-20 μmol/L. 25

| PR-619 induced G2 phase cell cycle arrest in oesophageal cancer cells
To elucidate the growth suppression mechanism of PR-619, the cell cycle profile was examined after treatment with PR-619.
As shown in Figure 1C

| Noxa played a vital role in PR-619-induced intrinsic apoptosis
Next, we examined whether apoptosis was also responsible for the growth inhibition effect of PR-619. Results showed that PR-619 treatment triggered apoptosis and significantly increased the

ATF4-Noxa Axis to induce intrinsic apoptosis
Noxa is a crucial mediator of chemotherapy-induced apoptosis, which is transactivated by several transcription factors (TFs). 43 Firstly, we examined the expression level of Noxa regulationrelated TFs after PR-619 treatment. Results showed that PR-619 treatment significantly increased the expression of ATF4 and slightly decreased the expression of FOXO3a, while other TFs changed slightly ( Figure 3C). Furthermore, knockdown of ATF4 rescued PR-619-induced apoptosis ( Figure 3D), downregulated the expression of Noxa and led to a lower level of c-PARP ( Figure 3E).
ATF4 is an essential factor in ER stress, so the expression of ER stress-related proteins was examined. Results showed that PR-619 treatment induced the accumulation of poly-ubiquitinated proteins ( Figure 3F) and increased the expression of ER stress-related proteins, including BiP and p-eIF2α ( Figure 3F). These results indicated that PR-619 triggered ER stress and induced ATF4-Noxa-mediated apoptosis in ESCC cells.

| PR-619 induced pro-survival autophagy
Previous reports showed that DUBs were involved in the regulation of autophagy, 44 so we investigated whether broad-range DUB inhib- F I G U R E 4 PR-619 treatment activated autophagy. A, Immunofluorescence of LC3. Kyse30 and Kyse450 cells were treated with PR-619 (8 μΜ) for 24 h. Cells were then incubated with LC3 primary antibody (1:200, overnight at 4℃) and Alexa Fluor ® 488 Goat Anti-Rabbit IgG (H + L) secondary antibody (green) (1:500, 2 h at room temperature), respectively. The nuclei were stained by DAPI (blue) (5 μg/mL, 20 min at room temperature). Images were captured using fluorescence microscopy (magnification: 200×). Representative images were shown. B, AO staining after PR-619 treatment. Kyse30 and Kyse450 cells were treated with PR-619 for 24 h and then stained with AO. The formation of acidic vesicular organelles (AVOs) was examined under fluorescence microscopy. AVOs, such as autolysosomes, were orange/ red. Non-AVO areas (cytoplasm, nucleus, and nucleolus) were green. C, Detection of the expression of LC3. Kyse30 and Kyse450 cells were treated with PR-619, and cell extracts were prepared for Western blotting analysis. GAPDH was used as the loading control. D, Treatment with CQ or BafA1 suppress LC3-II degradation. Kyse30 and Kyse450 cells were treated with 8 μmol/L PR-619 alone or in combination with CQ (12 μmol/L) or BafA1 (8 nmol/L). Cell lysates were analysed by immunoblotting with antibody against LC3. Tubulin was used as the loading control. E, Blockage of autophagy enhances PR-619-induced cell growth suppression of oesophageal cancer cell. Kyse30 and Kyse450 cells were treated with 8 μmol/L PR-619 alone or in combination with CQ (8 μmol/L) or BafA1 (3 nmol/L). Cell viability was measured using the CCK-8 assay. F and G, Blockage of the autophagic response increased PR-619-induced apoptosis of oesophageal cancer cells. Kyse30 and Kyse450 cells were treated with 8 μmol/L PR-619 alone or in combination with CQ (12 μmol/L) or BafA1 (8 nmol/L). Apoptosis was determined by the Annexin V-FITC/PI double-staining analysis (F). Total PARP and caspase 3 and cleaved PARP and caspase 3 were detected by immunoblotting (G). β-actin was used as the loading control. All data were representative of at least three independent experiments (n = 3; error bar, SD) Furthermore, we found that the inhibition of autophagy with either CQ or BafA1 significantly enhanced PR-619-induced inhibition of cell viability ( Figure 4E) and PR-619-induced apoptosis ( Figure 4F).

| PR-619 induced autophagy through activation of Ca 2+ -CaMKKβ-AMPK signalling cascade
It has been well documented that ER stress can disrupt Ca 2+ homeostasis inside the ER and lead to Ca 2+ releasing into other cellular compartments. 45 Accumulating evidence also shows that cytosolic calcium is a potent inducer of autophagy by CaMKKβ-AMPK signal-

| D ISCUSS I ON
Low early diagnosis rate and drug resistance are the main reasons for the high death rate of ESCC. It is crucial to study the mechanistic basis for ESCC progression and develop new therapeutic strategies fighting ESCC. In previous studies, we demonstrated that inactivation of USP7 or USP14 with specific inhibitor led to the effective suppression of ESCC cell growth in vitro and in murine model. 13,15 However, it is unknown whether broad-range DUB inhibitor PR-619 could also significantly inhibit ESCC cell growth via disrupting the dynamic balance of ubiquitin conjugation. Here, our results showed that PR-619 treatment induced the accumulation of ubiquitinated proteins and led to effective suppression of ESCC cell growth by inducing G2/M cell cycle arrest and apoptosis. Meanwhile, PR-619 F I G U R E 5 Ca 2+ -CaMKKβ signalling in PR-619 treatment cells was involved in AMPKα activation. A to C, Detection of intracellular Ca 2+ . Kyse30 and Kyse450 cells were treated with PR-619 as indicated. Twenty-four h later, cells were collected and washed with PBS. Then, Fluo-3AM was added into the cells as described in the instruction and incubated for 30 min at 37℃. Cells were washed with PBS again and pictures were captured under a microscope. Represented pictures were shown in panel A. Fluo-3 + cells were detected (B) and statistically analysed (C) using FACS. D, PR-619 treatment activated AMPKα. Kyse30 and Kyse450 cells were treated with PR-619, as indicated. Cells were collected, and proteins were extracted and analysed by Western blotting with specific antibodies. GAPDH was used as the loading control. E, STO-609 inhibited the activation of AMPKα and rescued autophagy in PR-619-treated cells. Kyse30 and Kyse450 cells were treated with PR-619 single (8 µmol/L) or combined with STO-609 (10 µmol/L). Then, proteins were extracted and analysed using specific antibodies against AMPKα, p-AMPKα and LC3. GAPDH was used as the loading control. All data were representative of at least three independent experiments (n = 3; error bar, SD) Accumulating evidence shows that ER stress is a vital mechanism response to DUB inhibition. 15,52,53 Noxa is involved in ER stress-induced cell death. 43,54 Meanwhile, eIF2α-ATF4 plays an essential role in Noxa activation and Noxa-mediated apoptosis. 35,54 In the present study, we found that PR-619 treatment led to the accumulation of poly-ubiquitinated proteins and triggered ER stress in ESCC cells, as evidenced by the increased expression of Bip, p-eIF2α and ATF4. Moreover, we found that PR-619 treatment led to Noxa-mediated apoptosis, which was dependent on ATF4. PR-619 treatment increased the protein level of It is known that ER stress can disrupt Ca 2+ homeostasis, induce the influx of Ca 2+ leakage into intracellular cytosol 45,55 and thus stimulate macroautophagy by CaMKKβ-AMPK signalling pathway. 45,47,56 Multiple lines of evidence indicate that inhibition of the 26S proteasome and accumulation of ubiquitinated proteins induce protective autophagy through an AMPK-dependent pathway. 57,58 Compound C treatment could significantly block AMPK activation and downregulate the level of phosphorylated AMPK (Thr172) and autophagy, evidenced by decreased levels of LC3-II. 59  F I G U R E 6 AMPK signalling pathway was involved in the PR-619-induced autophagy and apoptosis. A, Blockage of AMPK reduced PR-619 induced autophagy. Kyse30 and Kyse450 cells were treated with PR-619 single (8 µmol/L) or combined with CC (8 µmol/L), an AMPK inhibitor. LC3 was detected using immunofluorescence assay as described in material and methods, reprehensive pictures were captured (left panel), and LC3 puncta cells were statistically analysed (right panel). B, Blockage of AMPK enhanced the inhibition of cell viability in PR-619-treated cells. Kyse30 and Kyse450 cells were treated with PR-619 single (8 µmol/L) or combined with CC (4 µmol/L) as described above. Cell proliferation was observed and captured under inverted microscope (left panel). Cell viability was detected using CCK-8 assay (right panel). C, Blockage of AMPK inhibited colony formation. Kyse30 and Kyse450 cells were treated with PR-619 single (3 µmol/L) or combined with CC (2 µmol/L) for 10 days and then fixed, stained captured (left panel) and counted (right panel). D, Blockage of AMPK accelerated PR-619 triggered apoptosis. Kyse30 and Kyse450 cells were treated with PR-619 single (8 µmol/L) or combined with CC (8 µmol/L). Apoptosis was determined by FACS analysis using Annexin V-FITC/PI double-staining kit (left panel), and Annexin V + cell populations were defined as apoptosis (right panel). E, Effect of CC and PR-619 co-treatment on protein expression. Kyse30 and Kyse450 cells were treated with PR-619 single (8 µmol/L) or combined with CC (8 µmol/L). Then, proteins were extracted and analysed by Western blotting with specific antibodies. GAPDH was used as the loading control. All data were representative of at least three independent experiments (n = 3; error bar, SD) | 15 of 17 WANG et Al.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

E TH I C A L S TATEM ENT
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ping Chen
https://orcid.org/0000-0002-8504-5552 Cell proteins were detected using specific antibodies. GAPDH was used as the loading control. All data were representative of at least three independent experiments (n = 3; error bar, SD) F I G U R E 8 Schema of the mechanism for PR-619-induced cell cycle arrest, apoptosis and autophagy in ESCC