Decreased expression of USP9X is associated with poor prognosis in Chinese pancreatic ductal adenocarcinoma patients
- Authors:
- Published online on: April 10, 2018 https://doi.org/10.3892/ol.2018.8452
- Pages: 9287-9292
Abstract
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and fatal types of malignancy, ranking the fourth highest cause of disease-associated mortality worldwide (1). Statistical studies have revealed that the 5-year survival of PDAC is <5%, and ~50% of patients are diagnosed at the advanced stage (2,3). Only <20% of patients are eligible for curative resection, and among these patients, the majority experience recurrence within a year. The identification of reliable clinical markers to predict the prognosis is required for the management of patients with PDAC. It is necessary to identify novel molecular markers for early diagnosis and prediction of prognosis.
Ubiquitin specific peptidase 9X (USP9X), a member of the deubiquitinating protease family, is encoded on the X chromosome and is widely expressed in all tissues (4). Previous studies have demonstrated that the aberrant expression of USP9X is associated with multiple human cancer types, including lung cancer (5), breast cancer (6,7), esophageal carcinoma (8), colorectal carcinoma (9,10), prostate cancer (11), cervical cancer (12), chronic myelogenous leukemia (13), lymphoma and multiple myeloma (14), suggesting the role of USP9X in tumorigenesis, and progression. It has been revealed that USP9X primarily affects proteins that are meant to undergo proteasomal degradation by removing ubiquitin components. Consistently, in a xenograft model with the KRAS wild-type PDAC cell line, tumor volume decreased when USP9X was knockdown (14). However, a report noted that the knockout of USP9X enhances the transformation and protects cancer cells from anoikis in a murine model of PDAC, indicating that USP9X behaves as a tumor suppressor (15). The data of another research argued that USP9X promoted cell growth for established PDAC tumor cells and served a context-dependent role in PDAC (16).
In the present study, the expression level of USP9X was analyzed in Chinese patients with PDAC using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). Then, the association between clinicopathological parameters and USP9X expression of PDAC was explored, and the prognostic value of USP9X expression for the overall survival of Chinese patients with PDAC was evaluated.
Materials and methods
Patients and tissue specimens
The clinicopathological data of 205 patients with PDAC with surgical resection were retrospectively analyzed at the Biliary-Pancreatic Department of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University (Shanghai, China) between January 2002 and June 2014. In this cohort, there were 88 females and 117 males, age ranging from 38 to 90 years old, with a median age of 65 years. The diagnosis was confirmed by pathological examination. Patients who received preoperative chemotherapy, radiotherapy or other anticancer therapies were excluded from this study. An additional 30 fresh frozen cancerous and corresponding non-cancerous tissues of PDAC were also obtained from the same department. The present study was reviewed and approved by the Ethics Committee of the Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent was obtained from all participating patients.
RNA extraction and RT-qPCR. According to the manufacturers' protocols, total RNA from cancerous and corresponding non-cancerous tissue samples was extracted with TRIzol® reagent (Takara Bio, Inc., Otsu Japan), and reverse transcribed using a PrimeScript RT-PCR kit (Takara Bio, Inc.). 1 ug total RNAs were added to each reaction. qPCR reactions were then performed on a 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using the SYBR Premix Ex Taq II (Takara, Japan) in a 10 ul system. The reactions were incubated at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, and 60°C for 34 sec. All quantitative PCR reactions were performed in triplicate. The primer sequences used for USP9X detection were as follows: Forward, 5′-GTAATCCTGAGGAGGAAGAG-3′ and reverse, 5′-ACCACAGGCAGCGAAACAT-3′. The relative levels of USP9X expression were normalized to GAPDH RNA (forward, 5′-GACATCAAGAAGGTGGTGAA-3′ and reverse, 5′-TGTCATACCACGAAATGAGC-3′) using the 2−ΔΔCt method (17).
Tissue microarray (TMA) construction
TMAs were assembled using 1.5-mm diameter cores. In all 205 cases, corresponding non-cancerous tissue specimen cores were used as internal controls. After marking the representative area of each tissue, the samples were punched out and fitted into the paraffin array blocks.
IHC staining and scoring
The tissue microarray sections were rehydrated and treated with 3% hydrogen peroxide, followed by antigen retrieval. After being blocked with 10% normal goat serum (Shanghai Long Island Biotec, Co., Ltd., Shanghai, China) at room temperature for 30 min, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation, with a peroxidase-labeled secondary antibody for 30 min at room temperature. Finally, Diaminobenzidine tetrahydrochloride (DAB; Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China) was used for the color-reaction followed by nucleus counterstaining with hematoxylin. The following antibodies were used: rabbit anti-USP9X (1:100, Abcam, Cambridge, UK); Elivision plus Polyer HRP (Mouse/Rabbit) IHC Kit (Fuzhou Maixin Biotech Co., Ltd.). IHC staining was performed on the TMA containing 205 paired PDAC samples by two senior pathologists independently as previously described (18–20). The percentage of positively stained cells were scored using the following scale: 0, 0–5%; 1, 6–35%; 2, 36–70%; and 3, >70% of cells stained. The staining intensity was graded as following: Negative, 0; weakly graded, 1; moderately graded, 2; and strongly graded, 3. The final score was the product of the scores for the positive-staining rate and intensity as follows: ‘−’ for a score of 0–1, ‘+’ for a score of 2–3, ‘++’ for a score of 4–6 and ‘+++’ for a score of >6. A total score <4 in USP9X expression was considered to exhibit low expression and ≥4 as high expression.
Follow-up
The results of clinical and laboratory examinations were followed-up periodically until patients succumbed. Overall survival was defined as the duration between the date of surgery and the date of mortality or the last follow-up.
Statistical analysis
All statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Paired-samples t-tests were used to compare the mRNA levels of USP9X in the tissue samples. The chi-squared test for proportion was used to analyze the comparison of IHC grades in PDAC and normal pancreatic tissues, and the association between clinicopathological characteristics and USP9X expression. The Kaplan-Meier method and the log-rank test were applied in the comparison of the survival curves. Univariate and multivariate Cox proportional hazard analysis was used to investigate the association between clinicopathological variables and USP9X expression on survival. Two-tailed P<0.05 was considered to indicate a statistically significant difference.
Results
USP9X expression in PDAC
RT-qPCR assays were performed using 30 pairs of fresh specimens from patients with PDAC to determine the mRNA levels of USP9X. The USP9X mRNA levels were significantly decreased in PDAC tissue samples (18/30, 60.0%) compared with the matched surrounding non-tumor tissue samples (P=0.039; Fig. 1A). Based on the aforementioned scoring criteria mentioned, the IHC data from the 205 PDAC samples revealed that 73 (35.6%) exhibited high USP9X expression (USP9X ++ or USP9X +++), whereas the remaining 132 cases (64.4%) exhibited low USP9X expression (USP9X- or USP9X +) (Fig. 1B; Tables I and II).
 | Table I.Associations between USP9X expression and clinicopathological features in patients with PDAC. |
| Table II.Comparison of high and low USP9X immunohistochemical expression in PDAC and normal pancreatic tissues. |
Association between USP9X expression and PDAC clinicopathological parameters
The IHC staining scoring of USP9X level was statistically analyzed to explore the association between USP9X expression and clinicopathological parameters in PDAC. The clinicopathological parameters included age, sex, tumor location, tumor size, differentiation status, clinical stage, lymph node metastasis, liver metastasis and vascular invasion. Although the number of patients with PDAC with low USP9X expression was more than that with high expression, USP9X expression was only significantly associated with liver metastasis (P=0.032; Table I).
Prognostic significance of USP9X expression in PDAC
Survival analysis of patients was conducted using the Kaplan-Meier curve and log-rank test. As shown in Fig. 2A, patients with a high expression of USP9X exhibited a longer clinical overall survival time compared with those with low USP9X expression (P<0.001). The association between overall survival and USP9X expression level was evaluated according to American Joint Committee on Cancer (AJCC) stage 7th edition (Fig. 2B and C) (21). Similarly patients with stage I–II and stage III–IV with high USP9X expression exhibited a longer overall survival time compared with their low USP9X expression counterparts.
Furthermore, univariate Cox regression analysis was performed in the 205 PDAC cases (Table III). Overall survival was significantly associated with USP9X expression, age, sex, tumor size, tumor differentiation and liver metastasis (P<0.05). Furthermore, following multivariate Cox regression analysis, all six parameters remained significantly associated with overall survival and were identified as independent prognostic factors for PDAC (Table III).
| Table III.Univariate and multivariate analyses of prognostic parameters for survival in patients with PDAC. |
Discussion
Pancreatic cancer is one of the most aggressive and fatal types of malignant tumor in solid tumor oncology in the world. One of the reasons for the poor prognosis of pancreatic cancer is that it is difficult to diagnose at the early stage. In the present study, USP9X expression, and its association with clinicopathological features and clinical prognosis were investigated in Chinese patients with PDAC. It was demonstrated that USP9X may serve as a candidate tumor suppressor and prognostic biomarker in PDAC.
First, USP9X expression was demonstrated to be decreased at the protein level by IHC analysis in 205-paired PDAC sample TMA (132/205, 64.4%). USP9X expression was also significantly decreased in PDAC tissues compared with the matched surrounding non-tumor tissue samples at the mRNA level (P=0.039). Furthermore, liver metastasis is an important negative prognostic indicator of PDAC. In the current study, a potential association was identified between USP9X expression and liver metastasis by statistical analysis with various clinicopathological parameters, which indicated that low USP9X expression suppressed liver metastasis in PDAC (P=0.032). Nevertheless, no statistical significance was identified between USP9X expression and tumor differentiation (P=0.244) or AJCC stage (P=0.238) in PDAC, which may be attributed to the variability in the distribution of different stages across the cohort.
In the further study, it was demonstrated that longer clinical overall survival times occurred in patients with high expression of USP9X when compared with those with low USP9X expression, which verified the suppressor role of USP9X. Perez-Mancera et al (15) reported that knockdown the USP9X expression increased the colony of formation rate of pancreatic cancer cells and protected pancreatic cancer cells from anoikis. The present study revealed that the level of USP9X was significantly reduced in Chinese PDAC tissues compared with the matched surrounding non-tumor tissues. It may be possible that USP9X serves a tumor suppressor role in PDAC. Several previous reports presented that USP9X was a cancer promoter in other malignant solid tumors, including lung cancer (5), breast cancer (6,7), esophageal carcinoma (8), colorectal carcinoma (9,10), prostate cancer (11) and pancreatic cancer (14). Cox et al (16) downregulated the USP9X expression using short hairpin RNA, which resulted in a reduction in the growth of human PDAC cells, indicating that USP9X serves as an oncogene. Notably, it was also reported that the growth of PDAC cell lines was impaired by deubiquitinating protease inhibitor WP1130, which revealed that USP9X may promote PDAC cell growth (16). We hypothesized that USP9X contributes to PDAC by a relatively complicated mechanism involving other participants, including an association with a Kras gene mutation (15), which requires further studies to verify, although Cox et al (16) argued that there is a context-dependent function of USP9X in different stages of PDAC.
In conclusion, the present study demonstrated that USP9X may serve as a candidate tumor suppressor and prognostic biomarker in Chinese patients with PDAC. Further studies are necessary to explore the probable mechanism pathway regarding the regulation of USP9X, which may be a potential target for therapeutic intervention in PDAC.
Acknowledgements
The present study was supported by the National Basic Research Program of the National Natural Science Foundation (grant nos. 81702726, 81702739 and 81702844) and the Cross Research Fund of Biomedical Engineering of Shanghai Jiao Tong University (grant nos. YG2017QN48).
Competing interests
The authors declare that they have no competing interests.
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