Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer

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Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer Xiang Fu , Wei Xie ∗, Xiaoxue Song , Kun Wu , Linkang Xiao , Yongqiang Liu , Lei Zhang Department of General Surgery, Chongqing General Hospital, 400013 Chongqing, People’s Republic of China

Summary Background: Ubiquitin-specific peptidase 9, X-linked (USP9X), a member of deubiquitylating enzymes family, has recently been reported to be associated with a variety of cancer progression. While it functions as either oncogene or tumor suppressor in a context-dependent manner, the expression and role of USP9X in gastric cancer is largely unknown. Methods: Sixty-eight cases of patients with gastric cancer were enrolled in this study. The expression of USP9X and MCL1 were detected by immunohistochemistry. USP9X expression was further analyzed by Western blot. Furthermore, we analyzed the correlation between USP9X and MCL1 expression, as well as USP9X expression and clinicopathologic parameters of gastric cancer. Finally, the significance of USP9X expression in gastric cancer was analyzed by both Kaplan-Meier and Cox regression analysis. Results: USP9X expression significantly increased in gastric cancer tissues compared to matched normal tissues. Moreover, expression of USP9X was positive correlated with MCL1 expression (P = 0.006) and significant associated with lymph node metastasis (P = 0.016), distant metastasis (P = 0.001) and tumor staging (P = 0.013) in gastric cancer. Importantly, the increasing expression of USP9X in gastric cancer reduces overall survival rate and was an independent factor predicts poor prognosis in patients with gastric cancer. Conclusions: In this study, deubiquitylating enzyme USP9X was overexpressed in gastric cancer, suggesting a potential implication as an oncogene, and was significantly associated with a poorer survival. © 2017 Elsevier Masson SAS. All rights reserved.

∗

Corresponding author. E-mail address: [email protected] (W. Xie).

http://dx.doi.org/10.1016/j.clinre.2017.01.008 2210-7401/© 2017 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Fu X, et al. Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.008

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Introduction

Materials and methods

Despite great efforts taken to advance therapeutic measures, gastric cancer (GC) remains the fourth most common cancer world-wide and ranks as the second leading cause of cancer-related death [1]. Since surgery remains the only curative therapy and postoperative outcomes may, to the largest extent, depend on tumor staging, early diagnosis will be the core of the treatment of gastric cancer. However, there is still lack of effective molecular markers for clinical early diagnosis of gastric cancer. As a result, more than half of radically resected gastric cancer patients relapse locally or with distant metastases, or receive the diagnosis of gastric cancer when tumor is disseminated; therefore, median survival rarely exceeds 12 months, and in metastatic setting, 5-years survival is less than 10% [2]. On the other hand, an effective molecular marker will also contribute to develop gene targeting therapy, which may, at least in part, help to improve the prognosis. Therefore, identification of the pathogenic mechanisms involved in GC and development of useful prognostic biomarkers as well as novel targeted therapeutic strategies is an urgent clinical necessity [3]. Ubiquitylating and deubiquitylating are two important regulatory factors for posttranslational modification of cellular proteins, controlling almost all aspects of protein function. In recent years, deubiquitylating enzymes have attracted widespread attention because they have a profound impact on the regulation of multiple biological processes including cell-cycle control, DNA repair, chromatin remodeling and several signaling pathways that are frequently altered in tumor development [4—6]. Ubiquitin-specific peptidase 9, X-linked (USP9X), a member of deubiquitylating enzymes family, has been reported in a variety of diseases, including multiple cancers. In low grade serous ovarian tumours, USP9X was identified as one of the novel candidate driver genes contributors to carcinogenesis [7]. USP9X is an ERG-stabilizing deubiquitinase that promotes prostate cancer progression, inhibition of USP9X with the small molecule WP1130 blocked the growth of cultured prostate cancer cells and prostate tumors that express ERG [8]. USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma [9]. Two reports have identified USP9X mutation in oral squamous cell carcinoma [10,11]. Increase USP9X expression in multiple myeloma patients was associated with poor prognostic outcomes [12]. Knockdown of USP9X reduces the viability of brain cancer cells suggested that USP9X function as an oncogene [13]. However, USP9X has also been reported in pancreatic cancer and acts as a tumor suppressor by limiting K-RAS induced cellular transformation and by suppressing tumor aggressiveness [14]. Altogether, USP9X has been implicated as both an oncogene and tumour suppressor, depending on the type and stage of cancer [15]. In gastric cancer, however, the expression pattern and function of USP9X are largely unknown. Here, we first report the expression and significance of USP9X during gastric carcinogenesis, hope to provide a new target for treatment of gastric cancer.

Patients and follow-up A total of 68 cases primary gastric cancer patients (52 males, 16 females, mean age: 56years, range from 28 to 79 years), underwent curative resection and diagnosed pathologically with gastric cancer, were obtained from the Department of general surgery, Chongqing General Hospital, from January 2011 to January 2016. No patients had received chemotherapy or radiotherapy prior to surgery. Tumor stages were determined by TNM classification according to the 2010 International Union against Cancer (UICC) guidelines. All specimens were frozen in liquid nitrogen immediately following surgical resection. Then, frozen specimens were stored at −80 ◦ C until use. Written informed consent was obtained from all patients and this study was approved by the Ethics Committee of Chongqing General Hospital. As to overall survival (OS), patients were followed up from the date of surgery to the date of death or the last observation time. The last follow-up was March, 2016, with a follow-up of 60 months. Regular abdomen ultrasonography or computed tomography (CT) was performed to monitor the relapse and metastasis.

Immunohistochemistry Both fresh gastric cancer and matched normal tissues were collected, PFA-fixed, paraffin-embedded and sectioned at 5 ␮m onto slides. Heat induced epitope retrieval was performed in a microwave oven at < 95 ◦ C for 10 min in 100 mM sodium citrate. The tissues were subsequently incubated with 3% H2 O2 for 30 min. Blocking was performed with 5% BSA for 30 min at 37 ◦ C. The tissues were incubated with rabbit anti-USP9X or anti-MCL1 primary antibody (both are 1:100 dilution, Abcam Biotechnology, Cambridge, UK) overnight at 4 ◦ C. Next day, the tissues were incubated with secondary antibody for 30 min at 37 ◦ C. Then, the tissues were washed with PBS for 5 min and exposed to streptavidinHRP label (Zhongshanjinqiao, Beijing, China) for 30 min. The results were visualized by DAB kit (Zhongshanjinqiao, Beijing, China). Phosphate-buffered saline (PBS) instead of primary antibody was used as a negative control. The results were assessed by two independent investigators according to the immunoreactivity scores (IRS) [16]: staining intensity was categorized as 0, negative (—); 1, weak positive (+); 2, moderate positive (++) and 3, strong positive (+++). The percentage of staining was categorized as 0, no staining (—); 1, < 10% of tumor cell stained (+); 2, 10—40% (++); 3, 40—70% (+++) and 4, > 70% (++++). The values for the staining intensity were multiplied by the values for the percentage area stained to derive a IRS score (ranged from 0 to 12). Patients were separated according to their IRS into 4 groups: 0—1 = negative; 2—4 = weak; 6—8 = moderate; 9—12 = strong.

Western blot analysis Total proteins of each sample were extracted by using RIPA Lysis Buffer (Beyotime, Jiangsu, China) supplemented with

Please cite this article in press as: Fu X, et al. Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.008

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Figure 1 Expression of USP9X and MCL1 in gastric cancers and matched adjacent normal tissues by immunohistochemistry: a: USP9X expression in cancer tissues significantly increase compared with matched normal tissues; b: expression of MCL1 in cancers tissues is higher than that of normal tissues. NC: negative control. Original magnification × 200.

1 mM phenylmethanesulfonyl fluoride (PMSF) for 30 min at ice, followed by centrifugation at 15,000 g for 10 min. The supernatant was collected and protein concentrations were detected by Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). Equal amounts of proteins were fractionated by 8% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk in TBST and then incubated with primary antibody overnight at 4 ◦ C. Next day, the membranes were incubated with a HRP-labeled secondary antibody (goat anti-rabbit IgG) for 2 hours at 37 ◦ C. The results were visualized by an enhanced chemiluminescence kit (Beyontime, Jiangsu, China) on a Bio-Rad imaging system. Primary antibodies used in this study are as follows: USP9X (1:500 dilution, Abcam Biotechnology, Cambridge, UK), ␤actin (1:600 dilution, Beyontime, Jiangsu, China).

USP9X expression was significantly increased in gastric cancer tissues compared with the normal tissues (P = 0.006) (Table 1). In addition, due to USP9X play an important role in the stabilization of the anti-apoptotic protein MCL1 [12], we further detected the expression of MCL1 in gastric cancer as well as paired normal tissues. As a result, positive expression of MCL1 in gastric cancer (38/68,55.8%) is also significant higher than paired normal tissues (21/68, 30.8%) (P = 0.003) (Fig. 1b) (Table 1), this result is consistent with previous study [17]. Importantly, we observed that 43 cases of USP9X positive expression samples showed 30 cases of MCL1 positive expression, which showed a positive correlation between expression of USP9X and MCL1 in gastric cancer (r = 0.367, P = 0.002) (Table 1), suggested that USP9X may also, at least in part, interact with MCL1 thus affect the process of gastric cancer.

Statistical analysis

Expression of USP9X in gastric cancer by western blot analysis

All data were analyzed using SPSS 17.0. Data are presented as mean ± SEM. The Student’s two-tailed t-test was used to assess statistically significant differences between experimental and control groups. Categorical data were analyzed using the Chi2 test (2 test). Kaplan-Meier and log-rank test analyses were applied for overall survival. P < 0.05 was considered statistically significant.

To further validate USP9X expression in gastric cancer and adjacent normal tissues, western blot was performed to detect the protein expression of USP9X in these specimens. As expected, we detected more USP9X expression in cancer tissues than adjacent normal tissues (Fig. 2).

Results Increased expression of USP9X in gastric cancer compared with the normal tissues In order to investigate the relationship between USP9X and gastric cancer, we firstly detected the protein expression of USP9X in gastric cancer and matched adjacent normal tissues by using of immunohistochemistry. As shown in Fig. 1a, positive (27/68, 39.7%) and even strong positive (16/68, 23.5%) staining of USP9X was observed in cancer specimens whereas weakly positive (27/68, 39.7%) or negative (41/68, 60.3%) staining in the matched normal tissues.

Figure 2 Western blot analysis for USP9X expression in gastric cancer and matched normal tissues. Compared with matched normal tissues, more USP9X protein expression was detected in gastric cancer. ␤-actin was used as internal control in this study.

Please cite this article in press as: Fu X, et al. Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.008

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X. Fu et al. Table 1 Groups

Cancer Normal

Expression and correlation of USP9X and MCL1 in gastric cancer. USP9X

MCL1

USP9X

+

—

P-value

+

—

P-value

43 27

25 41

0.006

38 21

30 47

0.003

Based on the above results, we demonstrated that USP9X up-regulated in gastric cancer, which implies that USP9X may involve in the occurrence and development of gastric cancer.

Expression of USP9X correlated with clinicopathologic features in gastric cancer The relationships between USP9X expression and clinicopathological parameters of gastric cancer are shown in Table 2. Apparently, expression of USP9X in cancer tissues was not associated with age, gender, tumor size, and differential degree. However, USP9X levels were closely correlated with lymph node metastasis (P = 0.016) and distant metastasis (P = 0.013). In addition, USP9X was more significantly increased in advanced pathologic stages (III and IV), compared to those of earlier pathologic stages (I and II) (P < 0.001), indicated that USP9X expression was also correlated with tumor stage.

USP9X is an independent factor predicts poor prognosis in gastric cancer Since expression of USP9X was up-regulated in gastric cancer and closely associated with tumor stage and metastasis,

Table 2

+ —

MCL +

—

rs

P-value

30 8

13 17

0.367

0.002

it is reasonable to infer that USP9X expression may promote the progress of gastric cancer. It is noteworthy that during 60 months postoperative follow-up period, cases of death due to recurrence or metastasis in USP9X positive staining group (35/43, 81.3%) significantly higher than USP9X negative group (14/25,56%) (P = 0.026), indicated that USP9X expression may be associated with poor prognosis in gastric cancer. To further validate this conclusion, Kaplan-Meier and Cox regression analysis were performed to assess the effect of USP9X expression on overall survival in patients with gastric cancer. As is shown in Fig. 3, high USP9X expression in gastric cancer correlates with poor survival following surgery (log-rank P = 0.003). Cox regression analysis further revealed that USP9X expression, same as distant metastasis, was an independent prognostic factor for overall survival (Table 3).

Discussion Gastric cancer (GC) remains a lethal malignancy despite tremendous progress in its molecular characterization. The most effective therapy currently is radical resection, which may significantly improve the prognosis, however, depend on tumor staging. Since the majority of patients with gastric cancer are detected and diagnosed at a late stage

Correlation between USP9X expression and clinicopathologic parameters in gastric cancer.

Clinicopathologic parameters Age (years) > 60 ≤ 60 Gender Male Female Tumor size ≤ 5 cm > 5 cm Differential degree Poor differentiated Well differentiated Lymph node metastasis Yes No TNM stage I, II III, IV Distant metastasis Yes No

Cases

USP9X (+)

Positive rate (%)

P-value

22 46

12 31

54.5 67.3

0.304

52 16

34 9

65.3 56.2

0.508

41 27

29 14

70.7 51.8

0.114

49 19

33 10

67.3 52.6

0.259

40 28

30 13

75.0 46.4

0.016

27 41

11 32

40.7 78.0

0.001

17 51

15 28

88.2 54.9

0.013

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Figure 3 Kaplan-Meier curve shows the association of overall survival and expression of USP9X in gastric cancer patients. USP9X expression significantly reduces overall survival in patient with gastric cancer. Table 3 Cox regression analysis of clinicopathological parameters associated with overall survival of patients with gastric cancer. Parameters

Age Gender Tumor size Differential degree Lymph node metastasis TNM stage Distant metastasis USP9X expression

Overall survival RR (95% CI)

P-value

1.064 1.180 0.703 1.679 1.391 0.547 2.140 2.463

0.897 0.680 0.306 0.177 0.332 0.079 0.029 0.016

(0.529—2.070) (0.539—2.582) (0.358—1.380) (0.792—3.561) (0.714—2.711) (0.279—1.073) (1.080—4.241) (1.186—5.113)

of cancer progression, early diagnosis plays an important role in treatment of gastric cancer. It is well known to us that carcinogenesis is a complicated multi-step process that involves a series of oncogenes activation and inactivation of tumor suppressor genes. Identification of cancer-associated genes contributes to early diagnosis and treatment. In this study, we reported USP9X, a member of deubiquitylating enzymes family, up-regulated in gastric cancer. Furthermore, over-expression of USP9X correlated with lymph node metastasis, distant metastasis and tumor stage. Importantly, USP9X expression predicts poor outcome in patient with gastric cancer. As a deubiquitylating enzyme, USP9X is implicated in a number of essential cellular processes, and knockout of this gene is embryonic lethal in mice [18]. In addition, USP9X has been reported to regulate cell adhesion molecules like ␤-catenin and E-cadherin [19], mTOR [20], TGF␤ signaling [21], p53 and anti-apoptotic protein MCL1 [12]. All of these molecules are significantly associated with cancer progression, making USP9X aroused widespread interest in recent years. Several researches have reported the role of USP9X in cancer. Interestingly, while most studies have identified USP9X as an oncogene in carcinogenesis [8,9,12,13,22,23], USP9X functioned as a tumor suppressor

has also been reported in pancreatic ductal adenocarcinoma (PDA) [11,14,18]. Accordingly, USP9X acts as an oncogene or as a tumor suppressor in a context-dependent manner [24]. However, the relationship between USP9X and gastric cancer is largely unknown. To address this problem, we explored the expression of USP9X in gastric cancer by immunohistochemistry and western blot analysis. All results indicated that compared to match normal tissues, USP9X expression in gastric cancer significantly increased. Therefore, USP9X may be related to the progress of gastric cancer. Meanwhile, we detected a significantly increased expression of MCL1, an anti-apoptotic protein regulated by USP9X [12,25], in gastric cancer. Interestingly, the expression of MCL1 is positive correlated with USP9X, suggested that the regulation of MCL1 by USP9X is also applicable in gastric cancer. In addition, Kaplan-Meier and Cox regression analysis revealed that USP9X expression was significantly inversely correlation with survival rates and was an independent factor predicts poor prognosis in gastric cancer. In recent years, increasing evidences indicated that USP9X is associated with chemotherapy response. Loss of USP9X function prevented tamoxifen-induced proliferation arrest in oestrogen receptor a-positive breast cancer cells [26]. Low USP9X expression exhibit increased sensitivity to therapeutic doses of 5-FU in colorectal cancer cells [27]. Inhibition of USP9X by WP1130 enhanced the chemosensitivity to doxorubicin in hepatocellular carcinoma cells [28]. Base on these results, we attempt to investigate the effects of USP9X on chemotherapy response in patient with gastric cancer.

Conclusions In this study, deubiquitylating enzyme USP9X was overexpressed in gastric cancer, suggesting a potential implication as an oncogene, and was significantly associated with a poorer survival. Further in-depth research is required for that it may provide a new target for the treatment of gastric cancer.

Disclosure of interest The authors declare that they have no competing interest.

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Please cite this article in press as: Fu X, et al. Aberrant expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.008