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DEF6 expression in ovarian carcinoma correlates with poor patient survival

Background Increased expression of DEF6 is correlated with the malignant behavior of various cancers. Both DEF6 and p16 contribute to the regulation of cell cycle progression, and p53 plays important role in the cell cycle checkpoints. This study was designed to elucidate the prognostic significance of DEF6, p53 and p16 immunoexpressions in different histology subtypes of ovarian carcinoma. Methods Immunohistochemistry results of DEF6, p53 and p16 on ovarian carcinoma were compared with histology subtypes, clinical data, overall survival (OS) and disease-free survival (DFS) by Cox regression and Kaplan-Meier analysis. Results We studied 180 cases of ovarian carcinomas (75 high-grade serous, 41 clear cell, 36 mucinous and 28 endometrioid), including 109 FIGO stage I-II cases and 71 FIGO stage III-IV cases. Ovarian carcinomas positive for both DEF6 and p16 expression were associated with the worst OS (P = 0.027) and DFS (P = 0.023), whereas those negative for both DEF6 and p16 had the best OS and DFS. Aberrant p53 expression combined with positive DEF6 was associated with worst OS (P = 0.031) and DFS (P = 0.028). Kaplan-Meier analysis showed that significantly shorter survival rates were seen in patients with high expressions of DEF6 (P = 0.008) and p16 (P = 0.022). Patients with aberrant p53 expression in high-grade serous carcinoma (P = 0.012) and patients with high DEF6 expression in clear cell carcinoma (P = 0.001) were also associated with shorter overall survival. In univariate analysis, FIGO stage, DEF6 and p16 were associated with poor prognosis. DEF6 expression was the only independent prognostic factor correlated with shorted OS (HR 2.115; P = 0.025) and DFS (HR 2.248; P = 0.016) upon multivariate analysis. Conclusions DEF6 expression may serve as an independent prognostic factor, and interacted positively with p16 toward high tumor stage and shorter survival. Electronic supplementary material The online version of this article (doi:10.1186/s13000-016-0518-y) contains supplementary material, which is available to authorized users.


Background
Ovarian carcinoma is the most lethal malignancy of the female genital tract, mainly due to the failure of early diagnosis, heterogeneous histology subtypes and the limitations for the conventional chemotherapies [1,2]. The important prognostic factors include tumor stage, age at initial diagnosis, tumor morphological subtypes and grade, optimal resection for advanced ovarian cancer, as well as the effect of chemotherapy following primary surgery [3]. Current researches have focused on the study of various molecular signaling reactions or pathways in ovarian carcinoma to explore the molecular markers for early detection, prognosis assessment and hopefully as potential therapeutic targets.
Signaling involving DEF6 has been implicated in tumorigenesis. Increased expression of DEF6 has been shown to be correlated with the malignant behavior of extra-skeletal myxoid chondrosarcoma [18], colorectal cancer [19], breast cancer cells [20], and oral squamous cell carcinoma [21]. DEF6 may serve as a potential target for anti-angiogenic intervention in renal cell carcinoma [22]. DEF6 is also a novel target of tumor suppressor p53 and can suppress cisplatin-mediated apoptosis of breast cancer cells via a negative feedback regulation of the p53 signaling pathway [23]. High levels of DEF6 were found to decrease cisplatin-induced growth suppression and apoptotic cell death, in association with decreased p53 activity and imbalanced expressions of the Bcl-2 family members. Recently, p16 has been shown as a prognostic indicator in ovarian/fallopian tubal highgrade serous carcinoma [24][25][26]. Moreover, both DEF6 and p16 contribute to the regulation of cell cycle progression, and p53 plays important role in the cell cycle checkpoints. Despite their potential close interaction in cell cycle progression, the roles of DEF6, p16 and p53 have not been fully elucidated in ovarian carcinomas.
In the present study, we sought to better understand the differential expression of DEF6, its relation to the expressions of p53 and p16, and the prognostic significance in different histology subtypes of ovarian carcinoma based on a well-defined cohort of ovarian carcinomas. Clinicopathological data and survival curves were compared between patients with different scores of DEF6 to explore the potential of DEF6 as a prognostic marker.

Patient specimens
We collected cases with surgically operated ovarian carcinomas from the files of Departments of Pathology of Taipei Medical University Hospital and Wan Fang Medical Center between January 1998 and December 2011. We used the diagnostic criteria of the World Health Organization classification [1] and tumor staging system of the International Federation of Gynecology and Obstetrics (FIGO). The pathological diagnosis was reviewed by at least two pathologists. Cases of secondary metastasis to the ovaries were excluded.
Pathologic variables included histology subtypes (i.e. low-or high-grade serous carcinoma, mucinous carcinoma, endometrioid carcinoma and clear cell carcinoma), stage, therapy, recurrence free interval (if applicable), and site of recurrent disease (if applicable). Debulking surgery was found to be optimal if the maximum diameters of the individual residual tumor deposits were all less than 1.0 cm. Only cases with optimal resection were enrolled and analyzed in this study. Patient information were de-identified and assigned a study number. Tissue microarrays (TMA) with three to four tumor regions were chosen from paraffin embedded blocks. Each tumor core measured 0.3 cm in maximal diameter. This study had been reviewed and approved by the Institutional Review Board of Taipei Medical University (TMU-IRB 99049).

Cell lines and cell lysates
Total cell lysates from six ovarian carcinoma cells, including A2780, ES-2, TOV-21G, TOV-112D, OVCAR3, and HBT75, were used for the detection of DEF6 expression by Western blotting. A2780 (ovarian carcinoma cell line with unknown disease type identification) cell was cultured in RPMI-1640 with 10 % fetal bovine serum (FBS). ES-2 (clear cell carcinoma), TOV-21G (clear cell carcinoma) and TOV-112D (endometrioid carcinoma) were obtained from ATCC bioresource center (Manassas, VA, USA). ES-2 was cultured in McCoy's 5 medium with 10 % FBS. TOV-21G and TOV-112D were cultured in a 50:50 mixture of Medium 199 and MCDB105 with 10 % FBS. Cells were grown to near confluent, washed with PBS, and then lysed by RIPA lysis buffer. The lysates of OVCAR3 (serous carcinoma) and HBT75 (serous carcinoma) cells were kindly provided by Prof. Chao-Lien Liu at the Department of Medical Laboratory Science and Biotechnology, Taipei Medical University.

Western blot assay
Lysates from the six ovarian carcinoma cell lines and two positive control oral carcinoma cells, HSC-3 and SCC25, were separated in a 10 % polyacrylamide gel and transferred onto a PVDF membrane. After blocking, the blot was incubated with indicated antibodies overnight at 4°C. Antibodies against DEF6and β-actin (Genetex, Irvine, CA, USA) were used as the primary antibodies. After washing, the blot was incubated with horseradish peroxidaselabeled goat anti-mouse/rabbit IgG (Jackson Laboratory). The expression profile of the proteins was visualized using a Western Lightening-ECL kit (PerkinElmer, Waltham, MA, USA).

Statistical analyses
We performed statistical analyses by using SPSS for Windows software (SPSS, Chicago, IL). Data were expressed as median (interquartile range), mean (SD), and percentages. Chi-square test, Mann-Whitney U tests, and Student's t-test were used as statistical methods. Spearman rank correlation and multivariate linear regressions with stepwise variable selection were performed to assess the significant associations between ordinal or continuous predictor variables. The Kaplan-Meier method, Cox proportional hazard regression model and multifactorial Cox regression analysis were used to examine all factors found to be prognostic of survival in univariate analysis and the analysis of DFS and OS. A P-value of less than 0.05 was considered statistically significance.

Results
Comparison of DEF6, p53 and p16 expressions in different histological subtypes In our cohort of study, we collected 180 cases of ovarian carcinomas (75 high-grade serous, 41 clear cell, 36 mucinous, and 28 endometrioid carcinomas). We did not enroll cases of low-grade serous carcinoma because only three cases of low-grade serous carcinoma were diagnosed. The patients' ages ranged from 25 to 93 years (mean: 53.8 years). Totally 109 early stage cases (FIGO stage I-II) and 71 advanced stage (FIGO stage III-IV), with a median follow-up time of 33.0 months (mean, 48.3 months; range, 1 to 146 months) were enrolled. The surgical procedures included total hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or para-aortic lymph nodes sampling and omentectomy.
As shown in Table 1, immunoexpressions of DEF6, p53 and p16 showed significant correlation with different histological subtypes of ovarian carcinoma. Score 3 immunoexpression of DEF6 could be assessed in 52 % (39/75) and 64.3 % (18/28) of high-grade serous carcinoma (Figs. 1a, b, c and d) and endometrioid carcinoma, respectively. Aberrant p53 expression was also observed in 82.7 % of high-grade serous carcinoma. The p16 expression was either cytoplasmic or showed a combination of nuclear and cytoplasmic immunoreactivity. The

Expression of DEF6 in ovarian carcinoma cell lines
The expression profile of DEF6 was studied in several ovarian carcinoma cell lines by Western blot. Among six ovarian carcinoma cells examined, the expression of DEF6 in TOV-112D (endometrioid carcinoma) and OVCAR3 (serous carcinoma) cells were found to be relatively high when compared to other ovarian cells (Fig. 2). These findings were compatible with immunohistochemical results. This indicated that DEF6 was preferentially overexpressed in ovarian serous and endometrioid carcinomas. The level of DEF6 in A2780 (unknown histology subtype) and TOV-21G (clear cell carcinoma) was comparatively low among the six cells, however, they revealed a doubleband pattern on the DEF6 region as similar to the oral carcinoma cells.
Prognostic values of co-expression DEF6/p16 and DEF6/p53 We analyzed the prognostic significance of DEF6, p16 and p53 in the whole group of 180 cases of ovarian carcinoma. Four subgroups of patients each were formed according to DEF6/p16 and DEF6/p53 co-expressions ( Table 2). The group with positive DEF6 and positive p16 expression was associated with the lowest OS (P = 0.027) Effects of DEF6, p16 and p53 expression on the patient's overall survivals Ovarian carcinoma patients with high DEF6 expression were associated with a poor overall survival compared with the patients with low DEF6 expression (P = 0.008; Fig. 3a). Also, ovarian carcinoma patients with high 16 expression had lower overall survival rate than those with low p16 expression, as determined using the Kaplan-Meier method (P = 0.022; Fig. 3b). Subsequently, we carried out survival analyses according to IHC evaluations in four different histological subtypes of ovarian carcinoma (see Additional file 1). Patients with aberrant p53 expression had shorter survival rate than those with insignificant p53 expression in high-grade serous carcinoma (P = 0.012). More importantly, in ovarian clear cell carcinoma, high expression of DEF6 was associated with shorter overall survival as compared to low DEF6 expression (P = 0.001, Fig. 4).

Prognostic effects of clinicopathological factors and immunohistochemistry
Univariate (Table 3) and multivariate (Table 4) analyses were conducted to analyze the clinicopathological and IHC characteristics of the ovarian cancer patient cohorts to elucidate the crucial prognostic factors. Univariate analysis identified FIGO stage (P = 0.023 for OS and P = 0.034 for DFS), DEF6 (P = 0.013 for OS and P = 0.009 for DFS) and p16 (P = 0.026 for OS and P = 0.031 for DFS) as prognostic factors. Upon multivariate analysis, strong DEF6 was the only independent prognostic factor correlated with shorted OS (HR 2.115; P = 0.025) and DFS (HR 2.248; P = 0.016).

Discussion
DEF6 is a conserved protein associated with the functions of lymphocytes and is highly expressed in T-cells and T-cell homing organs . DEF6 was expressed in breast cancer cells [20], oral squamous cell carcinoma [21], colorectal carcinoma [19], and in tumor vessels of renal cell carcinoma [22]. The role of DEF6 expression in human cancer is unclear, and the prognostic significance of DEF6 expression and the co-expression of p16 and p53 in ovarian carcinomas are largely unknown. In this study of 180 cases of ovarian carcinoma using immunohistochemistry, a high expression of DEF6 was commonly found in ovarian carcinomas, and associated with different histology subtypes, advanced FIGO stage, and reduced overall survival (OS) and disease free survival (DFS). Strong expression of DEF6 was observed in high-grade serous carcinoma and endometrioid carcinoma. These findings were supported by the high expression of DEF6 in two serous, two clear cell and one endometrioid carcinoma cell lines, suggestive of the  There are many molecular markers possessing prognostic value. We compared the clinicopathological parameters and prognostic factors of DEF6, p16 and p53. The p16 is a cyclin-dependent kinase inhibitor which is integral to the retinoblastoma (Rb) genemediated control of the G 1 -S phase transition of the cell cycle [28]. It was also shown that ectopic expression of DEF6 shortened the G 1 interval in the cell cycle, and increased cyclin D1 expression [21]. It is therefore important to explore the cooperation between the two genes in the tumor progression. Although p16 has been widely regarded as a surrogate marker of high-risk human papillomavirus (HPV) in uterine cervical pathology, the role of p16 protein expression in ovarian carcinomas remains limited. In our study (Table 1), we found the highest percentage of score 3 p16 expression (74.7 %) in high-grade serous carcinoma, an ovarian carcinoma with notorious poor outcome, and similarly DEF6 (52 %). Our findings suggest that p16 is also a valuable prognostic marker for ovarian carcinomas. Recent studies have investigated the diagnostic role of p16 and prognostic indicator of p16 with clinical outcome in ovarian/tubal high-grade serous carcinoma. As p16 was expressed in the majority of p53positive and p53-negative serous tubal intraepithelial carcinomas, the addition of p16 helped to compensate the practical limitations of p53 in the diagnosis of serous tubal intraepithelial carcinomas [24]. Moreover, a recent study on ovarian/tubal high grade serous carcinomas revealed three distinct subgroups according to p16 expression and RB1 status (i.e., p16 homogenous stain/RB1-, p16 homogenous stain/RB1+, and p16 heterogeneous stain/RB1+), which possessed clinical relevance for stage and patient outcome upon multivariate analysis [29]. We also assessed the prognostic significance of the status of DEF6/p16 co-expression. Notably, the co-expression of both proteins was associated closely with adverse clinical outcome ( Table 2). The OS and DFS, in particular, were the shortest in the group with strong DEF6 and p16 coexpression, followed by DEF6+/p16-and DEF6-/p16+, while the DEF6-/p16-group had the longest survival rates (P = 0.027 for OS and P = 0.023 for DFS, respectively). These findings suggest that DEF6 and p16 positively interact and contribute to the tumor progression in ovarian carcinoma, and the molecular mechanisms deserve to be further studied. This suggestion is supported by the observations that both proteins play important role in the control of the G 1 -S phase transition of the cell cycle [21,28].
DEF6 is a novel p53 target gene and negatively regulated by p53, it can suppress cisplatin-mediated apoptosis of breast cancer cells [23]. In the present study, we   found that strong DEF6 expression and aberrant p53 expression had the shortest survival, whereas coexpression of insignificant p53 expression and negative DEF6 displayed the longest survival rate, with the other two groups in between (P = 0.031 for OS and P = 0.028 for DFS, Table 3). Thus, the evaluation of the p53 status coupled with DEF6 might be important for risk stratification, which certainly awaits sequencing validation of the p53 status for further clarification.
We further evaluated the overall survival rates of DEF6, p16 and p53 in all patients and four different histology subtypes of ovarian carcinoma by using Kaplan-Meier analysis. Significantly shorter survival rates were seen in patients with high expressions of DEF6 (P = 0.008) and p16 (P = 0.022). Patients with aberrant p53 expression in high-grade serous carcinoma had shorter overall survival (P = 0.012). Surprisingly, high expression of DEF6 in clear cell carcinoma was significantly correlated to shorter overall survival (P = 0.001). We enrolled in total 41 cases of ovarian clear cell carcinoma (30 FIGO stage I, 4 FIGO stage II and 7 FIGO stage III) in this study. Therefore, our study suggests that patients of ovarian clear cell carcinoma with high DEF6 expression deserve a poor prognostic factor compared with other histological subtypes. Our study is the first report on the prognostic impact of DEF6 overexpression in early-stage ovarian clear cell carcinomas, however in-depth investigations are necessary.
Univariate analysis showed that FIGO stage, DEF6 and p16 were associated with shorter survival (Table 3). Upon multivariate analysis, DEF6 remained the significant prognostic value (Table 4). These findings suggest that DEF6 in ovarian carcinomas may facilitate tumor cell growth or proliferation, motility, invasion and metastasis, leading to high tumor stage and hence poor prognosis.

Conclusions
DEF6 was often overexpressed in ovarian carcinomas, particularly in high-grade serous carcinoma and endometrioid carcinoma cells and tissues. DEF6 overexpression in early-stage ovarian clear cell carcinomas may have potential role as a poor prognostic factor. Multivariate analysis showed that DEF6 might serve as independent prognostic biomarker. Our findings suggest that DEF6 contribute cooperatively with p16 and p53, another two important cell cycle regulators, toward high tumor stage and poor OS and DFS in ovarian carcinomas. Hence DEF6 expression deserves further investigation especially its potential as therapeutic target in the dreadful ovarian carcinomas.

Additional file
Additional file 1: Figure S1