NOX4 overexpression is a poor prognostic factor in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma

Surgery xxx (2019) 1e8

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NOX4 overexpression is a poor prognostic factor in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma Yen-Hao Chen, MD, PhDa,b,c, Hung-I. Lu, MDd, Chien-Ming Lo, MDd, Chang-Chun Hsiao, PhDb,e, Shau-Hsuan Li, MDa,* a

Department of Hematology-Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan School of Medicine, Chung Shan Medical University, Taichung, Taiwan d Department of Thoracic & Cardiovascular Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan e Division of Pulmonary and Critical Care Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 14 November 2019 Available online xxx

Background: Nox4 has been associated with tumor progression in various types of malignancies. This study aimed to evaluate the importance of the expression of Nox4 in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma. Methods: We reviewed retrospectively 121 patients with esophageal squamous cell carcinoma who had undergone a curative esophagectomy, including 67 patients with overexpression and 54 patients with low expression of Nox4 as evaluated by immunohistochemical analysis. In addition, 2 esophageal squamous cell carcinoma cell lines, TE11 and KYSE270, were treated with the Nox4 inhibitor GKT-137831 to explore the expression of Nox4, cell proliferative activity, and selected downstream pathways in these esophageal squamous cell carcinoma cell lines by Western blot analysis. Results: Univariate analysis showed that T1-2 status, absence of nodal metastasis, and low Nox4 expression were associated with greater disease-free survival (P ¼ .001) and overall survival (P < .001), and Nox4 overexpression was an independent prognostic factor of worse disease-free survival and overall survival (P ¼ .013 and P ¼ .007, respectively). The esophageal squamous cell carcinoma cell lines treated with the Nox4 inhibitor GKT-137831 showed decreased cell proliferation in a dose-dependent manner (P < .01). Western blot analysis demonstrated that expression of AKT, phosphorylated AKT, mammalian target of rapamycin, and phosphorylated mammalian target of rapamycin were less in esophageal squamous cell carcinoma cells treated with the Nox4 inhibitor (P < .01). Conclusion: This study suggests that Nox4 overexpression is a poor prognostic factor for patients with esophageal squamous cell carcinoma undergoing curative esophagectomy. © 2019 Elsevier Inc. All rights reserved.

Introduction Esophageal squamous cell carcinoma (ESCC) is one of the most common aggressive malignancies with an increasing incidence worldwide and is the ninth leading cause of cancer deaths in

Chang-Chun Hsiao and Shau-Hsuan Li contributed equally to this work. * Reprint requests: Shau-Hsuan Li, MD, Department of Hematology-Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, No.123, Dapi Rd, Niaosong Dist., Kaohsiung City 833, Taiwan (R.O.C.). E-mail address: [email protected] (S.-H. Li). https://doi.org/10.1016/j.surg.2019.11.017 0039-6060/© 2019 Elsevier Inc. All rights reserved.

Taiwan.1 Well-known risk factors for the development of ESCC include a long-term history of alcohol consumption and smoking, chronic mucosal irritation, betel quid chewing, hot beverages, achalasia, esophageal web, and history of upper aerodigestive cancer.2e4 Because symptoms in the early stages of the disease are often not present, the majority of patients with ESCC have locally advanced disease when diagnosed. Despite the more recent improvements in the operative techniques, chemoradiotherapy, and immunotherapy, the outcome of patients with ESCC remains poor.5e7 Therefore, identifying the pathogenesis and the signaling pathways to target that are involved in tumor progression or distant metastasis of ESCC is an important research priority.

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The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family of enzymes are involved in the generation of superoxide or hydrogen peroxide. According to various catalytic subunits, the NOXs consist of seven members: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2.8,9 Although they share similar structures with 6 transmembrane and cytoplasmic domains comprising NAPDH- and flavin adenine dinucleotide-binding sites, each member plays a specific biologic role through the generation of superoxides. Prior studies have confirmed a close correlation of NOXs with cancer development and progression; indeed, several studies demonstrated that NOX inhibition suppresses endothelial tumor growth leading to cancer cell apoptosis.10,11 In addition to regulating tumor proliferation, NOXs appear to play a more important role in cancer cell progression. Inhibition of NOX activity downregulates the expression of vascular endothelial growth factor (VEGF) in non-small cell lung cancer (NSCLC) cell lines.12 In addition, inhibition of NOX activity decreases both the generation of reactive oxygen species (ROS) and tumor invasiveness in prostate cancer cells.13 NOX isoforms are expressed aberrantly in several cancer cells, especially Nox4, the most frequently expressed isoform in tumor cells.8 Some previous studies have shown biochemical links between Nox4 and cancer.14 Nox4 increases ROS generation, subsequently promoting tumor cell migration, metastasis, and the protection of cancer cells from apoptosis.15e17 In addition, Nox4 appears to promote tumor angiogenesis and the production of inflammatory cytokines, such as hypoxia-inducible factor 1-a, VEGF, glucose transporter 1, interleukin-6 (IL-6), and IL-8.18e20 Other possible mechanisms by which Nox4 promotes cancer progression include histone modification and pathways involving transforming growth factor-b and epidermal growth factor receptor.21e23 The role of Nox4 in the progression of ESCC remains unclear so far. The aim of our study was to investigate the role of Nox4 in the clinical outcome of patients with ESCC patients who underwent curative esophagectomy and to study the role of Nox4 in 2 ESCC cell lines.

sections were incubated with primary antibodies against Nox4 (ab109225, 1:200, Abcam, Cambridge, MA) for at least 18 h at 4 C overnight after blocking with 1% goat serum at room temperature for 1 hour. Detection of Nox4 expression was determined using the LSAB2 kit (Dako, Carpinteria, CA) followed by hematoxylin used for counterstaining and 3-3’-diaminobenzidine for color development. Incubation without the primary antibody was used as a negative control for Nox4, whereas normal kidney was used as a positive control. Assessment of staining was performed independently by 2 pathologists (C.C.H. and W.T.H.) blinded to any information about the patients’ clinicopathologic features or prognosis. Using a previously published method, the percentage of Nox4-positive tumor cells of all neoplastic cells in the section was scored as follows: low (<10% positive staining tumor cells), intermediate (10%e50% positive staining tumor cells), and high (50% positive staining tumor cells).25 We then considered Nox4 overexpression as staining of 50% of tumor cells, and combined the other 2 groups to be considered as low expression of Nox4.14 Western blotting

Materials and Methods

For protein extraction, samples were homogenized in RIPA lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% sodium dodecyl sulphate). The protein concentration in each sample was estimated by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Immunoblotting was performed according to standard procedures. Antibodies used in this study included polyclonal antibodies from GeneTex, CA, against Nox4 (GTX121929, 1:1000), b-actin from Sigma-Aldrich (St. Louis, MO; A5441, 1:10000), and antibodies from Cell Signaling (Beverly, MA) to AKT (#9272, 1:1000), phosphorylated AKT (p-AKT) (#9271, 1:1000), mammalian target of rapamycin (mTOR) (#2972, 1:1000), and phosphorylated mTOR (p-mTOR) (#2971, 1:1000). The first antibodies were then detected by incubation with secondary antibodies conjugated to horseradish peroxidase (Bio/Can Scientific, Mississauga, ON, Canada) and developed using the Western Lighting Reagent (PerkinElmer, Waltham, MA). Proteins were evaluated using x-ray films.

Patient selection

Cell culture and transfection procedures

Patients with ESCC who underwent operative resection at Kaohsiung Chang Gung Memorial Hospital were reviewed retrospectively. We excluded those with a history of a second primary malignancy, a noncurative R1 or R2 esophagectomy, mortality within 30 postoperative days, and patients receiving preoperative chemotherapy, radiotherapy, or chemoradiotherapy. Finally, 121 patients with available paraffin blocks and follow-up were identified. Patients underwent a radical esophagectomy with cervical esophagogastric anastomosis (McKeown procedure) or an Ivor Lewis type esophagectomy with intrathoracic anastomosis, digestive tract reconstruction with gastric tube, and pylorus drainage procedures. All patients underwent two-field lymph node dissection. The pathologic TNM stage was determined according to the eighth American Joint Committee on Cancer staging system.24

The ESCC cell lines, TE11 and KYSE270, were purchased from Riken Cell Bank (Tsukuba, Ibaraki, Japan) and European Collection of Authenticated Cell Cultures (Porton Down, Salisbury, United Kingdom), respectively. The ESCC cell line TE11 was cultured under standard conditions in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 1 MEM nonessential amino acids, 100 U/ mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL Amphotericin B, and 2 mmol/L L-glutamine. The other cell line KYSE-270 was cultured in Roswell Park Memorial Institute 1640/F12 (1:1) medium with 5% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL Amphotericin B, and 2 mmol/L Lglutamine. To examine the cell proliferative activity, an 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (0.5 mg/mL, Sigma) was performed in these cell lines as follows: each cell line (7,000 cells) was incubated and controlled in a 96well, flat-bottomed plate in triplicate. After a 96-hour incubation at 37 C, 100 mL of MTT was added to each well and incubated again for 4 hours. Then, the supernatant was discarded, and the crystal products were eluted with dimethyl sulfoxide (100 mL/well, Sigma). The colorimetric evaluation was tested using a spectrophotometer at 570 nm. To study the role of Nox4 in various malignant properties of both of these ESCC cells, we first determined the expression of Nox4 in

Immunohistochemistry Immunohistochemistry staining was performed using an immunoperoxidase technique. Slides were made from each patient using the 4 mm, formalin-fixed, paraffin-embedded tissue sections After completing deparaffinization and rehydration, slides were treated with 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (95 C) for 20 min to retrieve the antigen. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min. The

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these cell lines by quantifying the expression of Nox4 relative to bactin and found that greater expression was present in the TE11 and KYSE270 cell lines. Then we transfected Nox4 siRNA (sc-41586, Santa Cruz Biotechnology) into ESCC cells to generate Nox4 knockdown cells. The ESCC cells were seeded in a 6-well plate at a density of 5  105 for 24 h before transfection. Nox4 siRNA (40 nM) was transfected into ESCC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions; then the cells were subjected to Western blotting for 48 hours after the transfection. To examine the cell proliferative activity, an MTT assay was performed in the Nox4 knockdown cells. Inhibition Nox4 To determine the role of Nox4 in ESCC cell progression, these ESCC cell lines were treated with or without the Nox4 inhibitor, GKT-137831 (MedChem Express, Monmouth Junction, NJ), a novel and specific dual Nox1/Nox4 inhibitor. Each cell line (2,500 cells) was incubated in 200ml with GKT-137831 (0, 1, 5, 10, 20, 40, 80, and 100 mM) and control cells in a 96-well, flat bottomed plate in triplicate. To examine the effect of the Nox4 inhibitor, cell proliferative activity was evaluated with an MTT assay according to above-mentioned procedures. Statistical analysis Statistical analyses were performed using the SPSS 19 software package (IBM, Armonk, NY). The c2 test, Fisher exact test, and Student’s t test were used to compare data between the 2 groups. Disease-free survival (DFS) was calculated from the date of esophagectomy to the date of local recurrence, distant metastasis, or death from any cause, and overall survival (OS) was calculated from the date of esophageal cancer diagnosis to the date of death from all causes or date of last follow-up. The estimated DFS and OS were calculated using the KaplanMeier method, and between-group differences were assessed using the log-rank test for univariate analysis. The hazard ratio (HR) with 95% confidence interval (CI) and P values were calculated to quantify the strength of associations between prognostic parameters and survival. Ethics statement This retrospective analysis was approved by the Institutional Review Board of the Chang Gung Medical Foundation (201509643B0). All methods were performed in accordance with the approved guidelines, and written informed consent was waived by the Chang Gung Medical Foundation Institutional Review Board. Results Patient characteristics We identified a total of 121 ESCC patients who underwent curative esophagectomy at our institution from January 2001 to December 2012. Participants included 116 male and 5 female patients with a mean age of 55 (range, 29e80) years. The clinicopathologic parameters of these patients are shown in Table I. The tumor T status of these patients were T1 in 35 (28.9%) patients, T2 in 23 (19.1%), T3 in 50 (41.3%), and T4 in 13 (10.7%), whereas the node N status was N0 in 78 (64.4%), N1 in 30 (24.8%), N2 in 7 (5.8%), and N3 in 6 (5.0%) patients. According to American Joint Committee on Cancer eighth staging system, 34 patients (28.1%) were categorized as stage I, 46 (38.0%) as stage II, 22 (18.2%) as stage III, and 19 (15.7%) as stage IVA. Concerning the primary tumor locations, 29

3 Table I Characteristics of 121 patients with ESCC undergoing curative esophagectomy Characteristics Age, y Sex Male Female pT status 1 2 3 4 pN status 0 1 2 3 Tumor stage I II III IVA Location Upper Middle Lower Grade 1 2 3

55 (29e80) 116 (95.1%) 5 (4.9%) 35 23 50 13

(28.9%) (19.1%) (41.3%) (10.7%)

78 (64.4%) 30 (24.8%) 7 (5.8%) 6 (5.0%) 34 46 22 19

(28.1%) (38.0%) (18.2%) (15.7%)

29 (24.0%) 36 (29.8%) 56 (46.2%) 23 (19.1%) 58 (47.9%) 40 (33.0%)

(24.0%) were in the upper third of the esophagus, 36 (29.8%) in the middle third, and 56 (46.2%) in the lower third. Histologically, 23 tumors (19.1%) were grade 1, 58 (47.9%) were grade 2, and 40 (33.0%) were grade 3. Nox4 expression and clinical outcomes Immunohistochemical Nox4 expression in these 121 patients included 67 patients (55.4%) with Nox4 overexpression and 54 (44.6%) with low Nox4 expression (Fig 1). In the DFS analysis, no relevant differences were observed in terms of sex, tumor location, and tumor grade in a univariate analysis. The 75 patients aged <60 years had a greater 5-year DFS rate than the other 46 patients aged 60 years (46.7% vs 18.8%, P ¼ .008). As would be expected , the 58 patients who had T1-2 status had a greater 5-year DFS rate compared with 63 patients who had T3-4 status (80% vs 19%; P < .001), the 78 patients with negative nodal metastasis had superior 5-year DFS rate compared with 43 patients who had positive nodal metastasis (46% vs 19%; P < .001), and the 80 patients who had stage I and II disease had a greater 5-year DFS rate than the 41 patients with stage III and IVA disease (49% vs 12%; P < .001). When evaluating the effect of Nox4 expression, the 54 patients with low Nox4 expression had a greater 5-year DFS rate compared with the 67 patients with Nox4 overexpression (50% vs 25%; P ¼ .001, Fig 2A). Multivariate analysis revealed that T12 status (P < .001, HR, 0.23; 95% CI, 0.11e0.45), negative nodal metastasis (P ¼ .023, HR, 0.58; 95% CI, 0.36e0.93), and low Nox4 expression (P ¼ .013, HR, 0.56; 95% CI, 0.35e0.89) were independent prognostic factors for a greater DFS. With respect to OS, no differences were observed in terms of sex, tumor location, and tumor stage in a univariate analysis. Again as expected, a greater 5-year OS rates were found in 75 patients aged <60 years compared with the other 46 patients aged 60 years (49% vs 26%; P ¼ .031), the 58 patients with

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Fig 1. Immunohistochemical staining of Nox4 in patients with ESCC who underwent curative esophagectomy.

Fig 2. Kaplan-Meier survival curves of DFS and OS among patients with ESCC who underwent curative esophagectomy according to Nox4 expression. (A) DFS and (B) OS.

T1-2 status compared with the 63 patients with T3-4 status (83% vs 23%; P < .001), the 78 patients with negative nodal metastasis compared with the 43 patients with positive nodal metastasis (51% vs 21%; P < .001), and the 80 patients with stage I and II disease compared to the 41 patients with stages III and IVA disease (54% vs 15%; P < .001). The 54 patients with low Nox4 expression also had a greater OS compared to the o 67 patients with Nox4 overexpression (57% vs 27%; P < .001, Fig 2B). According to a multivariate comparison, T1-2 status (P < .001, HR, 0.23; 95% CI, 0.11e0.47), negative nodal metastasis (P ¼ .010, HR, 0.53; 95% CI, 0.32e0.86), and low Nox4

expression (P ¼ .007, HR, 0.52; 95% CI, 0.32e0.84) were independent prognostic factors of greater OS. Univariate and multivariate analyses of DFS and OS in 121 patients with ESCC who underwent curative esophagectomy are shown in Tables II and III. Nox4 knockdown inhibits tumor cell proliferation in vitro In the cell culture study, Western blot analyses were performed to determine Nox4 as well as AKT, P-AKT, mTOR, and P-mTOR expression in the ESCC cell lines TE11 and KYSE270 (Fig 3). Then

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Table II Univariate and multivariate analysis of DFS in 121 patients with ESCC undergoing curative esophagectomy Characteristics

No. of patients

Univariate analysis 5-year DFS rate

Age, y <60 60 Sex Male Female pT status 1þ2 3þ4 pN status 0 1þ2þ3 Tumor stage I þ II III þ IVA Location Upper Middle þ lower Grade 1þ2 3 Nox4 expression High Low

Multivariate analysis P value

HR (95% CI)

P value

.23 (.11e.45)

<.001

.58 (.36e.93)

.023

.56 (.35e.89)

.013

.008 75 (62.0%) 46 (38.0%)

46.7% 18.8%

116 (95.1%) 5 (4.9%)

35.2% 60.0%

58 (47.9%) 63 (52.1%)

79.8% 18.6%

78 (64.4%) 43 (33.6%)

45.9% 18.6%

80 (66.1%) 41 (33.9%)

48.5% 12.2%

29 (24.0%) 92 (76.0%)

44.3% 33.7%

81 (77.0%) 40 (33.0%)

35.8% 37.2%

67 (55.4%) 54 (44.6%)

25.4% 49.8%

.21

<.001

<.001

<.001

.31

.41

.001

Table III Univariate and multivariate analysis of OS in 121 patients with ESCC undergoing curative esophagectomy Characteristics

No. of patients

Univariate analysis 5-year OS rate

Age, y <60 60 Sex Male Female pT status 1þ2 3þ4 pN status 0 1þ2þ3 Tumor stage I þ II III þ IVA Location Upper Middle þ lower Grade 1þ2 3 Nox4 expression High Low

Multivariate analysis P value

HR (95% CI)

P value

.23 (.11e.47)

<.001

.53 (.32e.86)

.010

.52 (.32e.84)

.007

.031 75 (62.0%) 46 (38.0%)

49.3% 26.1%

116 (95.1%) 5 (4.9%)

39.6% 60.0%

58 (47.9%) 63 (52.1%)

82.8% 23.3%

78 (64.4%) 43 (33.6%)

51.2% 2.9%

80 (66.1%) 41 (33.9%)

53.6% 14.6%

29 (24.0%) 92 (76.0%)

55.2% 35.9%

81 (77.0%) 40 (33.0%)

40.7% 40.0%

67 (55.4%) 54 (44.6%)

26.9% 57.3%

.26

<.001

<.001

<.001

.11

.59

<.001

these cell lines were stably transfected with Nox4 siRNA to induce Nox4 knockdown. Our data showed that the expression of AKT, PAKT, mTOR, and P-mTOR were decreased in these Nox4-inhibited ESCC cells compared to the control cells (Fig 3). Furthermore, to better understand the biologic role of Nox4 in vitro, these 2 ESCC cell lines were treated with the Nox4 inhibitor GKT-137831 to determine the effect of inhibition of Nox4. GKT-137831 inhibited tumor cell proliferation in a dose-dependent manner at 48 h, 72 h, and 96 h after GKT-137831 treatment (Fig 4).

Discussion In our present study, patients with ESCC with Nox4 overexpression undergoing curative esophagectomy had worse DFS and OS than those with low Nox4 expression in both the univariate and multivariate analyses, indicating Nox4 as an independent prognostic factor of poor prognosis. In addition, inhibiting Nox4 using siRNA or the Nox4 inhibitor GKT-137831 in ESCC cell lines was associated with decreased expression of the AKT/mTOR pathways

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Fig 3. Western blot analysis of expression of Nox4 and the AKT/mTOR pathway in 2 esophageal squamous cell carcinoma cell lines: TE11 and KYSE270.

and inhibition of tumor cell growth. These finding are compatible with those of the previous results in other cancer types, such as NSCLC or pancreatic cancer. The evidence of a close correlation of the expression of the NOXs with cancer development and progression has been increasing. Some studies demonstrated that NOX inhibition suppresses the growth of endothelial tumors and leads to cancer cell apoptosis.10,11 In addition to the regulation of tumor proliferation, NOXs play an important in cancer cell progression. The inhibition of ROS production and NOX activity downregulate VEGF expression in NSCLC cell lines.12 Mitsushita et al demonstrated that siRNAs designed to target Nox1 mRNA effectively blocked Ras transformation, including anchorage-independent growth, morphologic changes, and tumor production.26 Liu et al reported that Nox1 is overexpressed in melanoma cell lines and enhanced cell invasion by upregulation of metaloproteinase 2 and the induction of epithelialmesenchymal transition.27 Furthermore, inhibition of NOX activity decreased both ROS generation and tumor invasiveness in prostate cancer cells.13 In general, NOX isoforms are aberrantly expressed in several cancer cells, especially Nox4, which is the most frequently expressed isoform in tumor cells.8 Some previous studies have shown biochemical associations between Nox4 and cancer. First, increased generation of ROS has been implicated in the pathogenesis in various types of cancer.28,29 ROS produced by Nox4 has been shown to protect pancreatic cancer cells from apoptosis.17 For example, metformin decreases pancreatic cancer cell growth by decreasing ROS production through downregulation the protein expression of Nox4.30

Similarly, the downregulation of ROS production inhibited breast cancer invasion by targeting actin nucleators in the TIS21-Akt1Sp1-Nox4 pathway.31 Bauer et al reported that Nox4 expression may be related to tumor recurrence through ROS generation in colon cancer.15 Second, the correlation between the Nox4 and PI3K/ AKT pathway has been documented in several studies. In PANC-1 pancreatic cancer cells, generation of ROS by Nox4 was reported to transmit a cell survival signal, while the inhibition of Nox4 appeared to activate apoptosis via the PI3K/AKT pathway.32 Zhang et al showed that Nox4 interacted with the PI3K/AKT pathway to promote cell proliferation and metastasis in NSCLC.25 Third, Nox4 may induce cancer progression by promoting tumor angiogenesis. In fibrosarcomas of Nox4-knockout mice, vessel density was decreased in tumor vascularization. In addition, a defect in hypoxia signaling was also mentioned in Nox4 knockout mice, leading to the attenuation of expression of hypoxia-inducible factor 1-a, VEGF, glucose transporter 1, and adrenomedullin.19 Fourth, inflammatory cytokines are one of critical mediators in inflammation-associated cancer, especially IL-6. In NSCLC, Nox4 expression was positively correlated with IL-6 expression, and exogenous IL-6 treatment enhanced NOX4 signaling. Nox4 also enhanced IL-6 production and activated IL-6/STAT3 signaling.20 Similar results were also described in renal cell carcinoma cells.18 Other possible mechanisms by which Nox4 may induce cancer progression include histone modification and through transforming growth factor-b, and the epidermal growth factor receptor pathway.21e23 Recently, several studies have reported the correlation between Nox4 and PI3K/AKT pathway. Mochizuki et al reported ROS generated by Nox4 can transmit cell survival signals through the AKT pathway in pancreatic cancer cells. ROS generation via Nox4 stimulated AKT phosphorylation, leading to an antiapoptotic effect, while inhibition of Nox4 activity attenuated the AKT kinase cascade, resulting in apoptosis.32 Another study demonstrated that Nox4 is promoted NSCLC cell proliferation and metastasis through interplaying with the PI3K/AKT pathway.25 Nox4 protein levels were closely correlated with the clinical stage of the disease and survival time, including the tumor size, presence of lymph node metastasis, cancer stage, and overall survival. Nox4 overexpression enhanced the cell proliferation and invasion in vitro and produced larger tumors, lesser survival time, and more lung metastasis in nude mice than in control cells. In contrast, blocking Nox4 activity inhibited the cancer cell aggressiveness in vitro and in vivo.25 Sun et al showed that MiR-99a directly regulated invasion and migration of lung adenocarcinoma by targeting Nox4, ROS, and the downstream PI3K/AKT/mTOR pathway in vitro and in vivo, while downregulation of Nox4 was closely related to low P-AKT expression.33 Our study had several limitations. First, this was a retrospective study and only included patients treated at a single institution; therefore, the sample size was relatively small. Second, the number of female patients in our study was limited, such that the importance of patient sex cannot be determined. Third, the mechanistic investigation of the effects of Nox4 in our cell line experiments was limited, thereby preventing us from really examining the pathways by which Nox4 is involved in cell proliferation, and more experiments are needed in this topic. To the best of our knowledge, however, this is the largest series of patients with ESCC to investigate the role of Nox4 in patients with ESCC who underwent curative esophagectomy. Further studies with a larger population are warranted to validate these findings in this study. In conclusion, our study suggests that Nox4 plays an important role in disease progression of ESCC with Nox4 overexpression being an independent poor prognostic factor for patients with ESCC undergoing curative esophagectomy.

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Fig 4. Nox4 inhibitor (GKT-137831) displays a growth inhibitory effect in a dose-dependent manner in 2 ESCC cell lines: TE11 and KYSE270 (A) TE11 and (B) KYSE270. Columns, mean; bars, standard deviation. Difference in growth inhibition: *P < .05 and yP < .01.

Funding/Support This work was supported by grants from the National Science Council of Taiwan, Taiwan (MOST 105e2314-B-182A-029, MOST 106e2314-B-182A-159-MY3, MOST 106e2320-B-182A-015, and MOST 107e2314-B-182A-156-MY3) and Chang Gung Memorial Hospital, Taiwan (CMRPG8J0401, CMRPG8G0892, CMRPG8I0201, and CMRPG8J1061). Conflict of interest/Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. Acknowledgments The authors thank Drs Chao-Cheng Huang and Wan-Ting Huang in the department of pathology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, for assessing the immunohistochemical staining and the Kaohsiung Chang Gung Memorial Hospital Tissue Bank Core Laboratory of the Chang Gung Medical Foundation for excellent technical support. References 1. National Department of Health, Republic of China. Cancer Registry Annual Report 2015. 2. Chen CH, Lu HI, Wang YM, et al. Areca nut is associated with younger age of diagnosis, poor chemoradiotherapy response, and shorter overall survival in esophageal squamous cell carcinoma. PLoS One. 2017;12:e0172752. 3. Chen YH, Lu HI, Chien CY, et al. Treatment outcomes of patients with locally advanced synchronous esophageal and head/neck squamous cell carcinoma receiving curative concurrent chemoradiotherapy. Sci Rep. 2017;7:41785. 4. Hu J, Nyren O, Wolk A, et al. Risk factors for oesophageal cancer in northeast China. Int J Cancer. 1994;57:38e46.

5. Burmeister BH, Smithers BM, Gebski V, et al. Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial. Lancet Oncol. 2005;6:659e668. 6. Kelsen DP, Winter KA, Gunderson LL, et al. Long-term results of RTOG trial 8911 (USA Intergroup 113): a random assignment trial comparison of chemotherapy followed by surgery compared with surgery alone for esophageal cancer. J Clin Oncol. 2007;25:3719e3725. 7. Medical Research Council Oesophageal Cancer Working G. Surgical resection with or without preoperative chemotherapy in oesophageal cancer: a randomised controlled trial. Lancet. 2002;359:1727e1733. 8. Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene. 2001;269:131e140. 9. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004;4:181e189. 10. Bhandarkar SS, Jaconi M, Fried LE, et al. Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice. J Clin Invest. 2009;119:2359e2365. 11. Brar SS, Corbin Z, Kennedy TP, et al. NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells. Am J Physiol Cell Physiol. 2003;285:C353eC369. 12. Chen J, Liu B, Yuan J, et al. Atorvastatin reduces vascular endothelial growth factor (VEGF) expression in human non-small cell lung carcinomas (NSCLCs) via inhibition of reactive oxygen species (ROS) production. Mol Oncol. 2012;6:62e72. 13. Jajoo S, Mukherjea D, Watabe K, Ramkumar V. Adenosine A(3) receptor suppresses prostate cancer metastasis by inhibiting NADPH oxidase activity. Neoplasia. 2009;11:1132e1145. 14. Chen YH, Chien CY, Fang FM, et al. Nox4 overexpression as a poor prognostic factor in patients with oral tongue squamous cell carcinoma receiving surgical resection. J Clin Med. 2018;7. 15. Bauer KM, Watts TN, Buechler S, Hummon AB. Proteomic and functional investigation of the colon cancer relapse-associated genes NOX4 and ITGA3. J Proteome Res. 2014;13:4910e4918. 16. Paletta-Silva R, Rocco-Machado N, Meyer-Fernandes JR. NADPH oxidase biology and the regulation of tyrosine kinase receptor signaling and cancer drug cytotoxicity. Int J Mol Sci. 2013;14:3683e3704. 17. Vaquero EC, Edderkaoui M, Pandol SJ, Gukovsky I, Gukovskaya AS. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells. J Biol Chem. 2004;279:34643e34654. 18. Fitzgerald JP, Nayak B, Shanmugasundaram K, et al. Nox4 mediates renal cell carcinoma cell invasion through hypoxia-induced interleukin 6- and 8- production. PLoS One. 2012;7:e30712. 19. Helfinger V, Henke N, Harenkamp S, et al. The NADPH Oxidase Nox4 mediates tumour angiogenesis. Acta Physiol (Oxf). 2016;216:435e446.

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