Relationship of common variants in WWOX gene with susceptibility and prognosis of Prostate cancer

The aim of the study was to determine the expression of WW domain containing oxidoreductase (WWOX) in patients with prostate cancer (PCa) and to evaluate the correlation of WWOX expression and patients survival. Immunohistochemistry (IHC) was applied to detect the WWOX expression. Chi-square test was adopted to evaluate the relationship between WWOX expression and clinical features of PCa patients. In addition, Kaplan-Meier curve was made to estimate the survival rate of PCa patients. Multivariate analysis was performed to assess the statistical signicance between WWOX expression and prognosis of PCa patients.


Abstract Background
The aim of the study was to determine the expression of WW domain containing oxidoreductase (WWOX) in patients with prostate cancer (PCa) and to evaluate the correlation of WWOX expression and patients survival.
Methods Immunohistochemistry (IHC) was applied to detect the WWOX expression. Chi-square test was adopted to evaluate the relationship between WWOX expression and clinical features of PCa patients. In addition, Kaplan-Meier curve was made to estimate the survival rate of PCa patients. Multivariate analysis was performed to assess the statistical signi cance between WWOX expression and prognosis of PCa patients.

Results
WWOX was weakly expressed in PCa tissues compared to the paired normal tissues by IHC. WWOX expression was tightly associated with Gleason score, PSA and clinical staging (P < 0.05), but has no relationship with age, tumor column, distant metastasis and ALP (P > 0.05). Survival curve demonstrated that patients with negative WWOX expression had lower survival rate. Finally, Cox analysis illustrated that WWOX was related with the prognosis of PCa patients (P = 0.001, HR = 4.605, 95% CI = 1.814-11.690).

Conclusion
In short, the present study showed strong evidence that WWOX could act as an indicator for prognosis of PCa patients.

Background
Prostate cancer (PCa) is one of the most common non-dermatologic cancers [1][2][3] and mainly occurs in economically developed countries [4]. And it is also one of the leading causes of cancer deaths among men all over the world. The majority of PCa were diagnosed as localized disease [5,6]. Recently, the incidence of PCa has increased and perhaps will remain so in the foreseeable future. PCa is initiated by androgen receptor signaling pathway [7,8]. Therapies for PCa are selected according to such factors as age, body condition and Gleason score, including transurethral prostate resection,androgen deprivation, radiotherapy and endocrine therapy [9,10]. Because most patients are diagnosed with advanced stage, treatments on patients have little effects. Therefore, nding a biomarker for therapy and prognosis of PCa is quite important.
WW-domain containing oxidoreductase (WWOX) locates at chromosome 16q23.3-24.1 and encodes a protein of 46 kD, which is an oxidoreductase containing two WW domains [11][12][13]. It has been determined that WWOX gene could spans the common chromosomal fragile site region FRA16D [14,15]. WWOX is important for UV, TNF, staurosporine and p53-mediated cell deaths. Growing evidence has proved that WWOX played an important role on neurodevelopment, bone metabolism and tumor suppression [16][17][18]. Besides, restored expression of WWOX in lung and breast cancer cells resulted in notable caspase-mediated apoptosis, growth inhibition and blocked tumor development [19,20]. Loss or reduced expression of WWOX was observed in different cancers, such as ovarian cancer, breast cancer, hepatocellular carcinoma, gastric cancer and non-small cell lung cancer [21][22][23]. In the present study, we detected the WWOX expression in PCa tissues to explore the potential biological role of WWOX.

Patients and specimens
A total of 101 patients who were pathologically diagnosed with PCa were selected from Urology Surgery of Beijing Tsinghua Changgung Hospital. Clinical data of patients, including age, PSA, alkaline phosphate (ALP), distant metastasis, clinical staging, Gleason score and tumor column, were obtained from the patients' initial history. Our study was approved by the Ethics Committee of Beijing Tsinghua Changgung Hospital and all patients were asked to sign the con rmed consents before surgery. Immunohistochemistry WWOX protein expression was determined by immunohistochemistry (IHC). The fresh tissues were continuously cut into sections of 2 µm and xed on glass slides. The sections were dewaxed with xylene and rehydrated with graded alcohol after baking at 70℃ for 2 h. Citrate buffer (0.01M, pH = 6.0) was used for antigen retrieval. Then the primary antibody was applied to the sections overnight at 4℃.
Subsequently, the sections were incubated with the second antibody at 37℃ for 2 h. Finally, staining was developed with DAB. The tissues were manually divided into two groups according to the staining percentage of cells (0 to 100%). Sections with cell staining percentages of more than 40% were clari ed as positive, and the others were clari ed as negative.

Statistical analysis
The relationship of clinical features and WWOX expression was evaluated with Chi-square. The overall survival rate of PCa patients was determined according to the Kaplan-Meier curve. Cox regression analysis was adopted to con rm the correlation between WWOX expression and prognosis of PCa patients.

Lowexpression of WWOX in PCa tissues
The expression of WWOX was measured by IHC method in PCa tissues and the paired normal tissues. Low expression of WWOX was observed in the PCa tissues compared to the normal ones. The positive rates of WWOX were 28.7% (29 out of 101) in PCa tissues and 85.1% (86 out of 101) in the paired normal tissues. PCa tissues exhibited signi cantly lower WWOX expression than the paired normal tissues (P < 0.001, Table 1).  Correlation of WWOX expression and the prognosis of PCa patients Survival curve was plotted according to WWOX expression by Kaplan-Meier (Fig. 1). During the follow-up, 43 (59.7%) patients with negative WWOX expression died, while only 6 (20.7%) patients died among those who of positive WWOX expression. Patients with negative WWOX expression were more likely to die than those with positive WWOX expression. Multivariate analysis revealed that WWOX could serve as a statistically signi cant prognostic factor by Cox regression analysis (P = 0.001, HR = 4.605, 95% CI = 1.814-11.690, Table 3).

Discussion
PCa is one of the major causes for cancer deaths of men in Europe and America. The incidence of PCa is positively related with age and is remarkably different in regions and races. The pathogenesy of PCa remains unclear. Studies from such Northern Europe countries as Denmark and Finland demonstrate that PCa derives from gene mutation to a great extent. It is also clari ed that PCa might be associated with the environment.
WWOX is a newly discovered gene that participates in many processes of various cancers. Exon loss, heterozygosity absence and abnormal protein expression of WWOX were frequently present at different cancers, such as breast cancer, prostate cancer, ovarian cancer and esophageal cancer [24,25]. It has been certi ed that WWOX usually behaves as a suppressor of tumor growth. In this study, we assayed its expression in PCa and evaluated the correlation between its expression and the prognosis of PCa patients.
WWOX has been con rmed to be downregulated in different cancers. Jentai Lin et al. studied that downregulation of WWOX was found in RCC [26]. Yachun Huang et al. revealed that WWOX was downregulated in human uroepithelial cells [27]. We rst measured the expression of WWOX in PCa tissues and the paired normal tissues at protein level by HIC in this study. The result showed that the WWOX expression was signi cantly lower in PCa tissues than that in the paired normal tissues, which was in accordance with the existing literature. Then the relationship of WWOX expression and the clinical features of PCa patients was evaluated. Signi cant difference was found from the result, indicating that WWOX might serve as a prognostic factor for PCa patients. Based on the previous presumption, further investigations were performed to estimate the correlation between WWOX expression and the prognosis of PCa patients. The multivariate analysis displayed statistical signi cance between them, suggesting that WWOX was an prognostic indicator for PCa patients.
Though the correlation of WWOX expression and the prognosis was evaluated in this study, the concrete mechanism of WWOX on PCa was still unclear.

Declarations
Ethics approval and consent to participate This study was supported by the Ethics Committee of Beijing Tsinghua Changgung Hospital and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Kaplan-Meier survival curves were made for PCa patients. Patients with negative WWOX expression had higher mortality than those with positive expression. P value was calculated by Log-Rank test.