Deubiquitinylase USP47 Promotes RelA Phosphorylation and Survival in Gastric Cancer Cells

Every year, gastric cancer causes around 819,000 deaths worldwide. The incidence of gastric cancer in the western world is slowly declining, but the prognosis is unpromising. In Germany, the 5-year-survival rate is around 32%, and the average life span after diagnosis is 6 to 9 months. Therapy of gastric cancer patients comprises a gastrectomy and perioperative or adjuvant chemotherapy. However, resistance of gastric cancer cells to these agents is widespread; thus, improved chemotherapeutic approaches are required. Nuclear factor kappa B (NF-κB) transcription factors are associated with anti-apoptosis, carcinogenesis, and chemoresistance, and thus, constitute attractive targets for therapeutic intervention. In immunoblots, we show that ubiquitin specific protease 47 (USP47) promotes β-transducin repeat-containing protein (βTrCP) stability and phosphorylation of RelA. Furthermore, after knockdown of USP47 by RNA interference, we analyzed in gastric cancer cell lines metabolic activity/viability in an MTT assay, and apoptotic cell death by Annexin V staining and poly(ADP-Ribose) polymerase (PARP)-1, caspase 3, and caspase 8 cleavage, respectively. We found that USP47 contributes to cell viability and chemoresistance in NCI-N87 gastric carcinoma cells treated with etoposide and camptothecin. Inhibition of USP47 might be a suitable strategy to downregulate NF-κB activity, and to overcome chemoresistance in gastric cancer.


Introduction
With 1.3 million incident cases and 819,000 deaths recorded globally, gastric cancer was the fifth most common malignancy, and the third leading cause of cancer deaths in 2015 [1]. Surgical gastrectomy remains the only curative therapy. However, relapse occurs in 40-60% of cases [2]. Diagnosis of gastric cancers often occurs when tumors are inoperable and patients have median survival times of three to five months. First-line or adjuvant chemotherapy (or chemoradiation) extends patient survival times by 6-7 months [3]. The high number of deaths from gastric cancer, low cure rates, and tumor relapse after gastrectomy therefore demand the development of better chemotherapeutic agents to improve patient survival. In particular, overcoming chemoresistance in tumor cells that otherwise could result in relapse or metastasis poses a major challenge [4,5].
As novel therapeutic targets, deubiquitinylases are receiving increased attention, due to their association with cancer and neurodegenerative diseases [12,13]. Deubiquitinylases (DUBs) as antagonists to E3 ubiquitin ligases are an integral part of the ubiquitin-proteasome-system. E3s conjugate ubiquitin moieties to substrate proteins, and thus, regulate their stability, protein interactions, or subcellular localization. In contrast, to the more than 600 E3 ligases [14] in humans with distinct substrate specificities, less than 100 DUBs reverse the conjugation and cleave ubiquitin moieties from substrate proteins. In particular, within the NF-κB pathway, E3s and DUBs regulate IKK complex activation, IκBα degradation, or NF-κB activity [15][16][17]. Because the NF-κB pathway constitutes an attractive target for therapeutic intervention in gastric cancer [5,18], we performed an siRNA screen aiming to identify potential DUB targets that are essential for NF-κB activity. As one such target, we identified the ubiquitin specific protease 47 (USP47), that was previously shown to be essential for the proliferation of gastric carcinomas [19]. Furthermore, depletion of USP47 sensitized osteosarcoma and breast cancer cell lines towards drug and UV radiation-induced apoptosis [20].
In this study, we addressed the molecular details of NF-κB regulation using USP47, and examined the viability of USP47 as a promising target for drug intervention to enhance the action of current drugs, and to overcome chemoresistance in gastric cancer cells. Our results show that depletion of USP47 in AGS gastric cancer cells results in decreased protein levels of phospho-RelA and β-transducin repeat-containing protein (βTrCP). Even though USP47 depletion failed to increase apoptotic cell death in AGS cells treated with camptothecin (CPT) and etoposide (Eto), it overcame chemoresistance in NCI-N87 gastric carcinoma cells. Therefore, targeting of USP47 represents a suitable strategy to overcome drug resistance in gastric carcinomas.

SDS-PAGE and Immunoblotting
Proteins were separated by SDS-PAGE in Tris-Glycine buffer gels and transferred onto PVDF membranes (Merck, Darmstadt, Germany), followed by 1 h blocking at room temperature using 5% skim milk in TBS containing 0.1% Tween (TBS-T). The PVDF membranes were incubated overnight at 4 • C, with the primary antibodies in either 5% BSA or 5% skim milk/TBS-T. The membranes were washed thrice with TBS-T, and incubated with the appropriate HRP-conjugated secondary antibody diluted 1:5000 in 5% skim milk/TBS-T for 1 h at room temperature. All antibodies used in this study are listed in the Supplementary Materials Table S1. After washing the membranes thrice with TBS-T, the blots were developed using a chemiluminescent substrate (Merck), and protein bands were visualized using the ChemoCam Imager (Intas, Göttingen, Germany).

Cell Viability Assay
Cells were seeded in 96-well plates with 50,000 cells per well, and transfected as described above. Twenty-four hours after camptothecin or etoposide, treatment viability was assessed using the MTT Cell Viability Assay Kit (Abnova, Taipei, Taiwan). Assays were performed according to instructions, and absorption was measured at 570 nm on a Spectramax M5 plate reader (Molecular Devices GmbH, Biberach an der Riss, Germany). The average absorption value for the control cells was set to 100% viability.

Statistics
All quantitative data were presented as mean ± S.D (standard deviation). Statistical analysis was performed using Student's t-test (Excel, Microsoft Office Plus 2010). p < 0.05 was regarded as significant.

Depletion of USP47 Decreased Phospho-RelA and βTrCP Protein Levels
Based on the observation that USP47 promotes the proliferation of gastric carcinomas [19], we performed a USP47 knockdown in AGS gastric carcinoma cells. Stimulation with tumor necrosis factor (TNF) for up to 20 min in USP47 knockdown cells resulted in decreased Ser536-phosphorylated RelA protein levels in the cytosol, and to a minor extent, also in the nucleus ( Figure 1A). In contrast, IκBα degradation appeared unaffected, even though a minor decrease in IκBα phosphorylation was noticed after USP47 knockdown ( Figure 1A). Analysis of regulatory NF-κB pathway components revealed that USP47 depletion constitutively decreased βTrCP levels ( Figure 1A). βTrCP is the substrate adaptor of the E3 ligase complex that facilitates IκBα ubiquitinylation after phosphorylation. Inhibition of protein translation through cycloheximide (CHX) treatment showed that USP47 depletion decreased the basal expression levels of βTrCP ( Figure 2). Similar to AGS cells, knockdown of USP47 in gastric cancer cell line (NCI-N87) constitutively decreased βTrCP levels, and reduced nuclear translocation of RelA ( Figure 1B).

Depletion of USP47 Decreased Phospho-RelA and βTrCP Protein Levels
Based on the observation that USP47 promotes the proliferation of gastric carcinomas [19], we performed a USP47 knockdown in AGS gastric carcinoma cells. Stimulation with tumor necrosis factor (TNF) for up to 20 min in USP47 knockdown cells resulted in decreased Ser536-phosphorylated RelA protein levels in the cytosol, and to a minor extent, also in the nucleus ( Figure 1A). In contrast, IκBα degradation appeared unaffected, even though a minor decrease in IκBα phosphorylation was noticed after USP47 knockdown ( Figure 1A). Analysis of regulatory NF-κB pathway components revealed that USP47 depletion constitutively decreased βTrCP levels ( Figure 1A). βTrCP is the substrate adaptor of the E3 ligase complex that facilitates IκBα ubiquitinylation after phosphorylation. Inhibition of protein translation through cycloheximide (CHX) treatment showed that USP47 depletion decreased the basal expression levels of βTrCP ( Figure 2). Similar to AGS cells, knockdown of USP47 in gastric cancer cell line (NCI-N87) constitutively decreased βTrCP levels, and reduced nuclear translocation of RelA ( Figure 1B).

USP47 Promotes Chemoresistance and Cell Viability in NCI-N87 Gastric Carcinoma Cells
TNF can induce diverse cellular responses, including cell survival and apoptosis [21]. Gastric cancer patients have low cure rates after chemotherapy; we therefore asked whether USP47 knockdown could enhance apoptosis in gastric cancer cells when treated with chemotherapeutic drugs. We selected the topoisomerase I and II inhibitors CPT and Eto to treat different gastric carcinoma cell lines, because their mode of action is similar to drugs used in current chemotherapy regimens [3], and because they are known to also promote NF-κB-dependent apoptosis resistance [22,23]. Eto or CPT treatment of AGS cells increased apoptotic cell death by ca. 18% or 38% in a concentration dependent-manner, respectively, ( Figure 3A-C), promoted caspase 8, caspase 3, and poly(ADP-Ribose) polymerase (PARP)-1 cleavage ( Figure 3D), and decreased cell viability by ca. 20% (Figure 4). Combining USP47 knockdown with Eto or CPT treatment did not further enhance apoptotic cell death, and resulted in comparable levels of apoptotic cells and cell viability ( Figures  3C,D and 4).

USP47 Promotes Chemoresistance and Cell Viability in NCI-N87 Gastric Carcinoma Cells
TNF can induce diverse cellular responses, including cell survival and apoptosis [21]. Gastric cancer patients have low cure rates after chemotherapy; we therefore asked whether USP47 knockdown could enhance apoptosis in gastric cancer cells when treated with chemotherapeutic drugs. We selected the topoisomerase I and II inhibitors CPT and Eto to treat different gastric carcinoma cell lines, because their mode of action is similar to drugs used in current chemotherapy regimens [3], and because they are known to also promote NF-κB-dependent apoptosis resistance [22,23]. Eto or CPT treatment of AGS cells increased apoptotic cell death by ca. 18% or 38% in a concentration dependent-manner, respectively, ( Figure 3A-C), promoted caspase 8, caspase 3, and poly(ADP-Ribose) polymerase (PARP)-1 cleavage ( Figure 3D), and decreased cell viability by ca. 20% (Figure 4). Combining USP47 knockdown with Eto or CPT treatment did not further enhance apoptotic cell death, and resulted in comparable levels of apoptotic cells and cell viability ( Figure 3C,D and Figure 4).

USP47 Promotes Chemoresistance and Cell Viability in NCI-N87 Gastric Carcinoma Cells
TNF can induce diverse cellular responses, including cell survival and apoptosis [21]. Gastric cancer patients have low cure rates after chemotherapy; we therefore asked whether USP47 knockdown could enhance apoptosis in gastric cancer cells when treated with chemotherapeutic drugs. We selected the topoisomerase I and II inhibitors CPT and Eto to treat different gastric carcinoma cell lines, because their mode of action is similar to drugs used in current chemotherapy regimens [3], and because they are known to also promote NF-κB-dependent apoptosis resistance [22,23]. Eto or CPT treatment of AGS cells increased apoptotic cell death by ca. 18% or 38% in a concentration dependent-manner, respectively, ( Figure 3A-C), promoted caspase 8, caspase 3, and poly(ADP-Ribose) polymerase (PARP)-1 cleavage ( Figure 3D), and decreased cell viability by ca. 20% (Figure 4). Combining USP47 knockdown with Eto or CPT treatment did not further enhance apoptotic cell death, and resulted in comparable levels of apoptotic cells and cell viability ( Figures  3C,D and 4).  Analysis of additional gastric carcinoma cell lines showed that the NCI-N87 cell line was resistant to Eto and CPT treatment, independent of the concentration (Figure 5A,B). Interestingly, after USP47 knockdown, the NCI-N87 cell line displayed increased apoptotic cell death ( Figure 5C), increased cleavage of PARP, caspase 3, and caspase 8 ( Figure 5D), and reduced cell viability ( Figure 6). Analysis of additional gastric carcinoma cell lines showed that the NCI-N87 cell line was resistant to Eto and CPT treatment, independent of the concentration (Figure 5A,B). Interestingly, after USP47 knockdown, the NCI-N87 cell line displayed increased apoptotic cell death ( Figure 5C), increased cleavage of PARP, caspase 3, and caspase 8 ( Figure 5D), and reduced cell viability ( Figure 6).   Analysis of additional gastric carcinoma cell lines showed that the NCI-N87 cell line was resistant to Eto and CPT treatment, independent of the concentration (Figure 5A,B). Interestingly, after USP47 knockdown, the NCI-N87 cell line displayed increased apoptotic cell death ( Figure 5C), increased cleavage of PARP, caspase 3, and caspase 8 ( Figure 5D), and reduced cell viability ( Figure 6).

Discussion
After cardiovascular diseases, cancer is the second leading cause of death worldwide, and with an increasing and ageing population, it is expected that patient numbers will rise [1]. Chemoresistance constitutes a major problem of chemotherapy, because it promotes relapse and thus increases morbidity [4]. Therefore, it is necessary to improve current chemotherapy regimens and identify new drug targets, especially for malignancies like gastric carcinomas with low cure rates [3]. Deubiquitinylases represent promising novel targets for drug discovery in cancer therapy, due to their involvement in metastasis and their regulatory role in apoptosis pathways [24]. In this study we could identify USP47 as a regulator of the NF-κB pathway. Therapeutic intervention of NF-κB signaling is an intensively studied research field [18,25], because aberrant NF-κB activation in the context of cancer promotes carcinogenesis, tumor progression, metastasis, or chemoresistance [5].
Our results demonstrate that USP47 stabilizes the E3 ligase complex substrate adaptor protein βTrCP, and promotes phosphorylation of RelA at Ser536 (Figure 1). The residual βTrCP levels after USP47 depletion suffice to allow for IκBα degradation. Within the chosen time points, differences between USP47 and control cells did not become apparent, probably due to the rapidness of the process. Nonetheless, nuclear translocation of RelA is diminished after USP47 knockdown (Figure 1), which is consistent with a delayed release from IκBα.
Apart from its regulatory role in the NF-κB pathway, USP47 constitutes an interesting target for gastric cancer chemotherapy, because it promotes gastric carcinoma cell proliferation [19], and its depletion sensitize cancer cells to chemotherapy, probably due to the upregulation of Cdc25a [20]. We therefore addressed the question of whether USP47 inhibition could affect cell survival/proliferation of gastric cancer cells. For instance, USP47 inhibition could be beneficial in attenuating NF-κB-dependent chemoresistance in response to DNA double strand break inducing agents [22]. AGS cells displayed pronounced apoptotic cell death when treated with CPT or Eto ( Figure 3A-D), but no further enhancement was observed after USP47 depletion. It is likely that the already strong response masks additional contributions to the apoptosis caused by the USP47 knockdown. Interestingly, in the CPT-and Eto-resistant gastric carcinoma cell line NCI-N87 ( Figure  5A,B) USP47 depletion resulted in pronounced apoptosis induction after CPT and Eto treatment ( Figures 5C,D and 6). The molecular mechanisms responsible for the drug-resistance are currently unknown. However, it is likely that in addition to the attenuation of NF-κB activity, other factors involved in DNA double strand break repair might play a role. For instance, USP47 stabilizes DNA polymerase β, and hence regulates base excision repair after DNA damage [26]. Similarly, USP47 Figure 6. USP47 promotes cell viability in NCI-N87 cells. NCI-N87 cells were seeded in 96 well plates, transfected with control or USP47 siRNA and incubated for 24 h with either with 10 µM Eto or 1 µM CPT. Cell viability was measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assay. * p < 0.05.

Discussion
After cardiovascular diseases, cancer is the second leading cause of death worldwide, and with an increasing and ageing population, it is expected that patient numbers will rise [1]. Chemoresistance constitutes a major problem of chemotherapy, because it promotes relapse and thus increases morbidity [4]. Therefore, it is necessary to improve current chemotherapy regimens and identify new drug targets, especially for malignancies like gastric carcinomas with low cure rates [3]. Deubiquitinylases represent promising novel targets for drug discovery in cancer therapy, due to their involvement in metastasis and their regulatory role in apoptosis pathways [24]. In this study we could identify USP47 as a regulator of the NF-κB pathway. Therapeutic intervention of NF-κB signaling is an intensively studied research field [18,25], because aberrant NF-κB activation in the context of cancer promotes carcinogenesis, tumor progression, metastasis, or chemoresistance [5].
Our results demonstrate that USP47 stabilizes the E3 ligase complex substrate adaptor protein βTrCP, and promotes phosphorylation of RelA at Ser536 (Figure 1). The residual βTrCP levels after USP47 depletion suffice to allow for IκBα degradation. Within the chosen time points, differences between USP47 and control cells did not become apparent, probably due to the rapidness of the process. Nonetheless, nuclear translocation of RelA is diminished after USP47 knockdown (Figure 1), which is consistent with a delayed release from IκBα.
Apart from its regulatory role in the NF-κB pathway, USP47 constitutes an interesting target for gastric cancer chemotherapy, because it promotes gastric carcinoma cell proliferation [19], and its depletion sensitize cancer cells to chemotherapy, probably due to the upregulation of Cdc25a [20]. We therefore addressed the question of whether USP47 inhibition could affect cell survival/proliferation of gastric cancer cells. For instance, USP47 inhibition could be beneficial in attenuating NF-κB-dependent chemoresistance in response to DNA double strand break inducing agents [22]. AGS cells displayed pronounced apoptotic cell death when treated with CPT or Eto ( Figure 3A-D), but no further enhancement was observed after USP47 depletion. It is likely that the already strong response masks additional contributions to the apoptosis caused by the USP47 knockdown. Interestingly, in the CPT-and Eto-resistant gastric carcinoma cell line NCI-N87 ( Figure 5A,B) USP47 depletion resulted in pronounced apoptosis induction after CPT and Eto treatment ( Figure 5C,D and Figure 6). The molecular mechanisms responsible for the drug-resistance are currently unknown. However, it is likely that in addition to the attenuation of NF-κB activity, other factors involved in DNA double strand break repair might play a role. For instance, USP47 stabilizes DNA polymerase β, and hence regulates base excision repair after DNA damage [26]. Similarly, USP47 might stabilize protein factors involved in DNA double strand break repair, and hence, depletion of USP47 could sensitize cancer cells to the action of DNA damaging agents.
USP47 depletion on its own caused no increase in apoptotic cell death (Figures 3-6), suggesting that loss of USP47 activity is not cytotoxic. This together with its potential to overcome chemoresistance in gastric carcinoma cells suggests that USP47 could represent a promising therapeutic target structure. Furthermore, a combination of current chemotherapy regimens together with USP47 inhibition might allow the reduction of the concentration of chemotherapeutic agents without impairing the cytotoxic effect on cancer cells, while reducing side effects. We propose that the development of USP47 inhibitors and their combination with current chemotherapy regimens might reduce relapse after gastrectomy, and extend overall patient survival times.

Conclusions
Here, we could show that USP47 regulates NF-κB activity by promoting the phosphorylation of RelA through the stabilization of βTrCP and subsequent degradation of IκBα. In addition, USP47 contributes to chemoresistance and viability in NCI-N87 gastric carcinoma cells. Inhibition of USP47 activity could represent a viable strategy for gastric cancer chemotherapy by downregulating transcription of NF-κB-regulated pro-survival genes, and overcoming NF-κB-dependent chemoresistance.