Stonin 2 Overexpression is Correlated with Unfavorable Prognosis and Tumor Invasion in Epithelial Ovarian Cancer

Stonin 2 (STON2), which functions in adjusting endocytotic complexes, is probably involved in the monitoring of the internalization of dopamine D2 receptors which have an inhibitory action of dopamine on tumor progression. However, its clinical significance in tumor progression and prognosis remains unclear. We explored the association between STON2 and the clinicopathological characteristics of epithelial ovarian cancer (EOC). The STON2 levels in ovarian cancer and normal cell lines and tissues were detected by real-time PCR and Western blot analyses. STON2 protein expression was also detected by an immunohistochemical analysis. The clinical significance of STON2 expression in ovarian cancer was statistically analyzed. STON2 significantly increased in the ovarian cancer cell lines and tissues compared to the normal ones. In the 89 EOC samples tested, STON2 expression was significantly correlated with intraperitoneal metastasis, intestinal metastasis, intraperitoneal recurrence, ascites containing tumor cells, and CA153 level. Moreover, patients with STON2 protein overexpression were more likely to exhibit platinum resistance and to have undergone neoadjuvant chemotherapy. Patients with high STON2 protein expression had a tendency to have a shorter overall survival and a poor prognosis. A multivariate analysis showed that STON2 was an independent prognostic predictor for EOC patients. In conclusion, STON2 plays an important role in the progression and prognosis of ovarian carcinoma, especially in platinum resistance, intraperitoneal metastasis, and recurrence. STON2 can be a novel antitumor drug target and biomarker which predicts an unfavorable prognosis for EOC patients.


Introduction
Ovarian cancer is the leading cause of death from gynecologic malignancy, and is associated with an incidence of approximately 22,280 new cases and an annual mortality rate of 14,240 deaths in the United States in 2016 [1]. Ovarian tumor treatments have been improving over the past few decades in particular, recent studies have explored platinum-containing chemotherapeutic agents as well as neoadjuvant chemotherapy (NAC) [2]. Regardless of these advances, ovarian cancer remains one of the deadliest types of gynecological carcinomas, of which the 5-year survival rate is merely 20-40%, primarily due to the occurrence of tumor metastasis and recurrence [3]. Due to the lack of specific symptoms and the absence of robust early diagnostic methods, epithelial ovarian cancer Obstetrics (FIGO) III or IV), leading to a disappointing prognosis [4,5]. On the other hand, patients with early-stage, localized ovarian cancer were reported to have an average five-year survival rate of 90% [6]. For decades, serum biomarkers such as CA125, HE4, and CA153 have been used to monitor the progress of ovarian cancer, and to detect the recurrence of ovarian cancer in a clinical setting. However, these biomarkers are neither extremely sensitive nor particularly specific for predicting cancer metastasis, recurrence, and prognosis, which may partly explain why their use has not significantly contributed towards improving the survival of EOC patients [7]. Therefore, further studies to identify reliable novel factors that can aid in the detection of tumor metastasis, predict tumor recurrence and survival, and provide personalized prediction for targeted therapy are essential in the prevention of tumor recurrence and for improving the prognosis of patients with EOC.
The STON2 gene located on chromosome 14q in human encodes Stonin 2, which is a clathrin-related sorting protein that functions in adjusting endocytotic complexes. Moreover, many studies have suggested that STON2 may play an important role in schizophrenia as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recirculation [8][9][10][11][12]. More importantly, STON2 probably participates in the monitoring of the internalization of dopamine 2 receptors D2 (D2Rs) [8,13]. It is well known that D2Rs play a significant role in the dopaminergic system and are responsible for the inhibitory action of dopamine on the stimulation of apoptosis, tumor progression, and the maturation of tumor microvessels. However, thus far, no research has specifically investigated the role of STON2 on tumor progression and prognosis in ovarian cancer. Therefore, in this study we aimed to investigate the characteristics of STON2 expression and its clinicopathological implications in ovarian cancer.
Here, we inspected STON2 expression in ovarian cancer cell lines and tissues and in normal control cells and tissues. Correlations between many clinicopathological factors and survival in ovarian cancer, such as age, surgical stage, grade, lymph node metastasis, intraperitoneal metastasis, intraperitoneal recurrence, neoadjuvant chemotherapy, and platinum resistance, were analyzed by employing real-time PCR, Western blot analysis, immunohistochemistry, and statistical analyses.

STON2 Expression is Higher in Ovarian Cancer Cell Lines than in Normal Ovarian Cell Lines
Real-time PCR and Western blot analyses were used to determine the expression of STON2 mRNA and protein in ovarian cancer cell lines (CAOV3, COV362, COV504, EFO-27, A2780, OVCAR4, SKOV3, and TOV-21G) and in normal cells (HOSEpiC). The results of the real-time PCR and Western blot analyses revealed that all of the ovarian cancer cell lines overexpressed STON2 protein and mRNA ( Figure 1A,B).

STON2 Expression Significantly Increased in Ovarian Cancer Tissues Compared with Normal Control Tissues
We took advantage of real-time PCR, Western bloting, and immunohistochemical analyses to evaluate STON2 expression in ovarian cancer and in normal control tissues at the mRNA and protein levels. As we predicted, the STON2 mRNA and protein levels were obviously higher in most of the ovarian cancer tissues than in normal ovarian tissues (Figure 2A,B). Simultaneously, the results of the immunohistochemical staining also provided strong evidence that the STON2 protein, which was intensively expressed in the cytoplasm, was upregulated in the ovarian cancer tissues compared to the normal ovarian tissues ( Figure 2C).

STON2 Overexpression Was Related with the Clinical Features of Ovarian Cancer
Considering the high expression of STON2 in ovarian cancer, we further investigated its correlation with the clinical characteristics of ovarian cancer in 89 cases by immunohistochemistry. The percentages of patients with stages I, II, III, and IV tumors were 19.3%, 11.4%, 60.2%, and 9.1%, respectively. Among patients from 16 to 85 years of age, the median age was 50 years, and a total of 89 patients underwent the initial treatment including neoadjuvant therapy, surgery, or post-operation chemotherapy. According to the results of the immunohistochemical analysis, strong staining was detected in the cytoplasm in 44 of the 89 samples (49.4%, Table 1), indicating high STON2 protein expression, while the remaining 45 EOC samples were poorly stained, indicating low STON2 protein expression (50.6%, Table 1). Additionally, the mean optical density of STON2 staining in the ovarian cancer samples was much higher than that in the normal control ovarian tissues ( Figure 3). Furthermore, the results of the statistical analysis revealed a significant relationship between STON2 expression and clinicopathological characteristics of ovarian cancer (Table 2), and a Spearman's correlation analysis verified that high STON2 expression was correlated with the following characteristics: intraperitoneal metastasis (p = 0.011), intestinal metastasis (p = 0.003), intraperitoneal recurrence (p = 0.006), ascites containing tumor cells (p = 0.016), and CA153 level (p = 0.041). Also, patients with STON2 protein overexpression were more likely to exhibit platinum resistance (p = 0.033) and to have undergone neoadjuvant chemotherapy (p = 0.029). In contrast, STON2 expression had no correlation with patient age; histological type; FIGO stage; differentiation grade; lymph node metastasis; serum CA125, CA199, CEA, NSE, and β-HCG levels; and other clinicopathological features (Table 3). Together, these results suggest that STON2 may play an important role in disease development in ovarian cancer.

STON2 Overexpression Was Significantly Associated with a Poor Prognosis
A survival analysis revealed that the cumulative overall survival (OS) and disease-free survival (DFS) rates of ovarian cancer patients decreased with increase in STON2 protein expression (Figure 4), indicating that STON2 overexpression was associated with poor overall survival and survival with a high recurrence rate. In this study, patients with high STON2 expression exhibited a median survival time of 34.36 months and a median progression-free survival time of only 5.1 months, while these values in patients with low STON2 expression were 91.2 and 15.3 months, respectively. We also assessed the prognostic value of STON2 expression in EOC patient subgroups stratified by serum biomarker levels such as CA153, CA199, intraperitoneal metastasis, ascites with tumor cells, neoadjuvant chemotherapy, intraperitoneal recurrence and so on (Figure 4). In a univariate Cox analysis, the STON2 protein level (p < 0.001), CA153 (p = 0.001), CA199 (p = 0.009), intraperitoneal metastasis (p < 0.001), intestinal metastasis (p < 0.001), ascites with tumor cells (p = 0.001), neoadjuvant chemotherapy (p = 0.014), intraperitoneal recurrence (p < 0.001), and platinum resistance (p < 0.001) were significant prognostic factors (Table 4). In addition, multivariate Cox regression analysis further revealed that STON2 protein expression level (p = 0.010), intraperitoneal recurrence (p = 0.011), and platinum resistance (p = 0.003) were indeed independent prognostic factors of ovarian cancer (Table 4). Taken together, these results suggest that as an independent prognostic factor, STON2 may contribute to the prognosis of ovarian cancer.

STON2 Overexpression Was Significantly Associated with a Poor Prognosis
A survival analysis revealed that the cumulative overall survival (OS) and disease-free survival (DFS) rates of ovarian cancer patients decreased with increase in STON2 protein expression ( Figure  4), indicating that STON2 overexpression was associated with poor overall survival and survival with a high recurrence rate. In this study, patients with high STON2 expression exhibited a median survival time of 34.36 months and a median progression-free survival time of only 5.1 months, while these values in patients with low STON2 expression were 91.2 and 15.3 months, respectively. We also assessed the prognostic value of STON2 expression in EOC patient subgroups stratified by serum biomarker levels such as CA153, CA199, intraperitoneal metastasis, ascites with tumor cells, neoadjuvant chemotherapy, intraperitoneal recurrence and so on (Figure 4). In a univariate Cox analysis, the STON2 protein level (p < 0.001), CA153 (p = 0.001), CA199 (p = 0.009), intraperitoneal metastasis (p < 0.001), intestinal metastasis (p < 0.001), ascites with tumor cells (p = 0.001), neoadjuvant chemotherapy (p = 0.014), intraperitoneal recurrence (p < 0.001), and platinum resistance (p < 0.001) were significant prognostic factors (Table 4). In addition, multivariate Cox regression analysis further revealed that STON2 protein expression level (p = 0.010), intraperitoneal recurrence (p = 0.011), and platinum resistance (p = 0.003) were indeed independent prognostic factors of ovarian cancer (Table 4). Taken together, these results suggest that as an independent prognostic factor, STON2 may contribute to the prognosis of ovarian cancer.

Discussion
A growing body of evidence has indicated that Stonin 2 may have an important role in endocytosis by encoding a membrane protein that can sort clathrin-related proteins to internalize specific proteins. Furthermore, clathrin-mediated endocytosis represents a significant mechanism for recycling fully fused synaptic vesicles, and it is also involved in dopaminergic signaling, i.e., the attenuation of dopamine D2 receptors (D2Rs) [14,15]. Thus, Stonin 2 is probably involved in

Discussion
A growing body of evidence has indicated that Stonin 2 may have an important role in endocytosis by encoding a membrane protein that can sort clathrin-related proteins to internalize specific proteins. Furthermore, clathrin-mediated endocytosis represents a significant mechanism for recycling fully fused synaptic vesicles, and it is also involved in dopaminergic signaling, i.e., the attenuation of dopamine D2 receptors (D2Rs) [14,15]. Thus, Stonin 2 is probably involved in regulating the internalization of D2Rs [8,13]. Furthermore, ovarian cancer cells and endothelial cells all carry dopamine receptors, except ovarian cancer cells lacking dopamine receptor 3 (DR3) expression. Dopamine receptor 2 (DR2) is known to play an important role in the inhibitory functions of dopamine on microvessel density (MVD) and tumor growth as well as to have stimulatory functions on apoptosis. Via DR2, dopamine obstructs the vascular permeability factor/vascular endothelial growth factor-A (VPF/VEGF) or norepinephrine mediated invasion of ovarian cancer cells [16]. However, Stonin 2 participated in the internalization of D2Rs. After endocytosis, the D2Rs were shipped to the lysosomal pathway and degraded [17]. The attenuation of D2Rs abolished the dopamine-mediated inhibition of tumor cell invasion. Thus, STON2 may directly or indirectly participate in the invasion of cancers, including ovarian cancer.
Despite the fact that the STON2 protein plays an important role in endocytosis, there was little research investigating the relationship between STON2 and tumors, particularly ovarian cancer, which still remains an important subject to be explored. Therefore, our study is a bold and creative attempt to preliminarily determine the impact of STON2 expression on tumors, particularly in epithelial ovarian carcinoma.
This study has, for the first time, revealed that STON2 protein expression was associated with the prognosis of ovarian cancer. Overexpression of STON2 in patients with EOC was found to be significantly related with intraperitoneal metastasis, intestinal metastasis, intraperitoneal recurrence, ascites with tumor cells, and a high CA153 level. Moreover, patients with STON2 overexpression were more likely to exhibit platinum resistance and to have undergone neoadjuvant chemotherapy. Also, further analysis of STON2 expression showed that the survival time and progression-free survival time of epithelial ovarian carcinoma patients decreased with an increase in STON2 expression. In the light of all the analyses, we concluded that STON2 may be vital in the progression as well as the prognosis of ovarian cancer.
To the best of our knowledge, intraperitoneal metastasis and recurrence are the major risk factors that reduce survival in patients with ovarian cancer; moreover, these factors are of great importance in ovarian cancer staging [3]. In line with the International Federation of Gynecology and Obstetrics (FIGO) 2014, stage III has three substages: IIIA, IIIB, and IIIC. Stage IIIA is further divided into substage IIIA1, which means that patients only have positive retroperitoneal lymph nodes, and substage IIIA2, which means that the patients have microscopic intraperitoneal metastasis. Additionally, patients with macroscopic intraperitoneal metastasis but not parenchymal organ metastasis are usually categorized into stage IIIB or IIIC. The FIGO (2014) stated that patients with positive retroperitoneal lymph nodes alone commonly have a better prognosis than those with intraperitoneal metastasis. Thus, an early diagnosis of intraperitoneal metastasis is important for the survival of EOC patients [18]. However, only a few suitable tumor markers are currently used to predict intraperitoneal metastasis in the clinical setting. Due to all of these data, abnormal STON2 expression was associated with intraperitoneal metastasis and recurrence, as well as a poor OS in ovarian cancer patients, suggesting that STON2 overexpression is associated with a poor prognosis. Thus, STON2 expression can serve as a predictor of intraperitoneal metastasis and recurrence in EOC patients and can be used to provide more precise cancer staging as well as guide a more radical personalized therapy by more effectively reducing the mortality rate of EOC patients.
Although surgery followed by postoperative chemotherapy has been the standard treatment for EOC for decades, patients with ovarian cancer still have a five-year survival rate of only approximately 30% [19]. One of the most significant prognostic factors for patients with advanced ovarian cancer is currently considered to be the capability to perform optimal cytoreduction [20]. If the conditions for optimal primary cytoreductive surgery are not present, neoadjuvant chemotherapy (NACT) is probably administered for patients with advanced ovarian cancer. Compared with primary cytoreductive surgery, neoadjuvant chemotherapy is associated with less blood loss, a superior percentage of optimal cytoreduction, as well as a more favorable quality of life [21]. Moreover, the National Comprehensive Cancer Network (NCCN) 2016 guidelines point out that advanced ovarian cancer patients especially with plenty of ascites and ascites cytology with malignant tumor cells, those with a clear histopathological diagnosis, and those that are evaluated by imaging or laparoscopy defining that the tumor would be troublesome to eliminate should undergo neoadjuvant chemotherapy. Additionally, we found that STON2 overexpression indicated a tendency to have undergone neoadjuvant chemotherapy and the presence of ascites containing tumor cells, which is one of the indications for neoadjuvant chemotherapy. Therefore, STON2 may be used as a NACT predictor for ovarian cancer patients to provide better guidance for individual treatment strategies, indicating that neoadjuvant chemotherapy may be more available for EOC patients with STON2 overexpression.
Currently, ovarian cancer is lethal. Although chemotherapy is used to treat ovarian cancer, 20-30% of patients continue to exhibit platinum resistance [22]. In addition, platinum resistance is one of the most significant obstructions to the successful treatment of ovarian cancer. In our study, when EOC patients were found to have a high expression of STON2, they would more easily get platinum resistance and have a higher risk of worse survival outcomes. Our study is the first to identify the relationship between STON2 and platinum resistance. Further studies with larger cohorts are needed to verify this association in EOC and study its underlying mechanism.
Recently, dopamine treatment has been considered as an effective and promising cancer therapy. Previous studies on mice transplanted with human breast, stomach, and colon tumors found that dopamine treatment could inhibit tumor angiogenesis and progression, as well as extend the life span in these animals [23,24]. Additionally, dopamine, via DR2, has been shown to prevent VPF/VEGF binding, the phosphorylation of receptors, subsequent signaling steps, and inhibit angiogenesis [25]. Moreover, dopamine can act via the D2R-cAMP signaling pathway to block the stimulatory effect of norepinephrine on tumor cell invasion [26]. A few reports have suggested that STON2 may be involved in decreasing the expression of D2Rs by regulating their internalization [8,13]. Another study reported that dopamine treatment can increase tumor angiogenesis and progression on D2 receptor knockout mice [27]. Thus, STON2 has potential for use as a therapeutic target to enhance the dopamine treatment's efficacy in ovarian cancer.

Tissue Specimens and Patient Information
A total of 89 paraffin-embedded ovarian cancer samples from 2002-2010 that had been pathologically confirmed at the Sun Yat-sen University Cancer Center were included in this study. In addition, freshly frozen ovarian cancer tissue samples and freshly frozen noncancerous ovarian biopsies were obtained from surgeries in the Sun Yat-sen University Cancer Center between May 2016 and January 2017 for research purposes. Table 1 summarizes the clinical information of all of the samples analyzed in this study. The follow-up time was between 5.1 and 126.5 months, and the median follow-up time was 49 months.
This trial was obtained approval from the Sun Yat-sen University Cancer Center Institutional Review Board (YB2016-064, 6 April 2016). Written informed consent was obtained from participants who provided the fresh tissue samples.

Immunohistochemistry
The STON2 protein expression levels in the human ovarian cancer tissues were detected by immunohistochemical analysis. Briefly, 4 µm-thick paraffin-embedded sections were baked at 60 • C for 1 h, deparaffinized with xylene, rehydrated, and microwaved in EDTA antigen retrieval buffer. Next, high tension was used for antigen retrieval, and the specimens were treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block nonspecific binding, and incubation with anti-rabbit STON2 polyclonal antibodies (1:1000; Sigma; HPA003086) at 4 • C overnight. Normal goat serum was used as the negative control. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Sigma), then incubated with streptavidin horseradish peroxidase complex (Sigma), immersed in 3-amino-9-ethyl carbazole. The sections were then counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted in Crystal Mount. Two researchers who knew nothing about the histopathological features and patient data of the sections evaluated the degree of immunostaining of each formalin-fixed, paraffin-embedded section. The score was due to both the proportion of positively stained tumor cells and the intensity of staining. The percentage of cancer cells was scored as follows: sections with <10% positive cancer cells were scored as 0; 10-50% positive cancer cells, 1; 50-75% positive cancer cells, 2; and tissue sections with >75% positive cancer cells, 3. The tissues were sorted into four grades based on staining intensity, as follows: 0 indicated no staining; 1 indicated weak staining (light yellow); 2 moderate staining (yellow brown); and 3 strong staining (brown). The staining index (0-9) was calculated as the product of the proportion of positive cells multiplied by the staining intensity score. The best cutoff value was defined as follows: a staining score of ≥6 was considered to have high STON2 expression, and a staining score of ≤4 indicated low STON2 expression.

Statistical Analyses
All of the statistical analyses were carried out with the statistical software package SPSS 20.0. The chi-square test and Fisher's exact test were used to analyze the relationship between STON2 protein expression levels and clinicopathological characteristics. Additionally, bivariate correlations were computed by Spearman's rank correlation coefficients. Patient survival was determined by a Kaplan-Meier analysis, and the differences were counted by the log-rank test. Cox's proportional hazards regression model was applied to the multivariate analysis. A p value of < 0.05 in all of the analyses was considered statistically significant.

Conclusions
Overall, for the first time, the results of this study suggested that STON2 is upregulated in ovarian cancer and its expression is correlated with intraperitoneal metastasis, intestinal metastasis, intraperitoneal recurrence, ascites with tumor cells, CA153 level, and poor prognosis in EOC patients. Additionally, patients in whom STON2 protein was overexpressed tended to be more likely to have undergone neoadjuvant chemotherapy and to exhibit platinum resistance. Therefore, STON2 might play an important role in the invasion of EOC, as well as serve as a novel cancer therapeutic drug target and biomarker that can predict an unfavorable prognosis in EOC patients.